Get 20M+ Full-Text Papers For Less Than $1.50/day. Subscribe now for You or Your Team.

Learn More →

LYVE1+ macrophages of murine peritoneal mesothelium promote omentum-independent ovarian tumor growth

LYVE1+ macrophages of murine peritoneal mesothelium promote omentum-independent ovarian tumor growth ARTICLE LYVE1 macrophages of murine peritoneal mesothelium promote omentum-independent ovarian tumor growth 1 2 1,3 1 1 2 1 Nan Zhang *,Seung Hyeon Kim *, Anastasiia Gainullina **, Emma C. Erlich **, Emily J. Onufer ,Jiseon Kim , Rafael S. Czepielewski , 5 2 1 2 1 4 6,7 Beth A. Helmink ,JosephR. Dominguez , Brian T. Saunders ,Jie Ding , Jesse W. Williams ,JeanX. Jiang , Brahm H. Segal , 1 1 1,2 Bernd H. Zinselmeyer , Gwendalyn J. Randolph ,and Ki-WookKim  Two resident macrophage subsets reside in peritoneal fluid. Macrophages also reside within mesothelial membranes lining the hi lo-hi peritoneal cavity, but they remain poorly characterized. Here, we identified two macrophage populations (LYVE1 MHC II lo/− lo/− hi hi CX CR1gfp and LYVE1 MHC II CX CR1gfp subsets) in the mesenteric and parietal mesothelial linings of the 3 3 peritoneum. These macrophages resembled LYVE1 macrophages within surface membranes of numerous organs. Fate- hi mapping approaches and analysis of newborn mice showed that LYVE1 macrophages predominantly originated from embryonic-derived progenitors and were controlled by CSF1 made by Wt1 stromal cells. Their gene expression profile closely overlapped with ovarian tumor-associated macrophages previously described in the omentum. Indeed, syngeneic hi epithelial ovarian tumor growth was strongly reduced following in vivo ablation of LYVE1 macrophages, including in mice that received omentectomy to dissociate the role from omental macrophages. These data reveal that the peritoneal compartment hi contains at least four resident macrophage populations and that LYVE1 mesothelial macrophages drive tumor growth independently of the omentum. Introduction Serous membranes line the peritoneal cavity as they generate a following tissue injury. However, whether these membrane- functional border for visceral organs. A major serosal surface associated macrophages are related to those in the peritoneal includes the gut-associated mesentery that anatomically bridges fluid in phenotype or origin is unknown. Indeed, the full phe- the intestines and mesenteric lymph nodes (MLNs). The mes- notypic and gene expression profile of peritoneal membrane– entery anchors the small intestine and colon and facilitates blood associated macrophages has not been reported. circulation and interstitial fluid flow through the mesenteric The peritoneal cavity contains serous fluid that hosts two lymphatic vessels to maintain tissue homeostasis. The serous types of resident macrophages that have been well characterized membranes of the mesentery are enriched in stromal cells such in recent years, the Gata6-dependent large peritoneal macro- as fibroblasts and mesothelial cells that produce vitamin A me- phages (LPMs; Gautier et al., 2014; Gautier et al., 2012; Ghosn tabolites that sustain peritoneal fluid macrophages (Buechler et al., 2010; Okabe and Medzhitov, 2014; Rosas et al., 2014)and et al., 2019), as well as collagens, elastin, laminin, and glyco- the IRF4-dependent small peritoneal macrophages (Ghosn et al., proteins that form a complex extracellular matrix (Jackson-Jones 2010; Kim et al., 2016). The LPMs float freely in peritoneal fluid et al., 2020). Resident macrophages on the serosal surface of the and participate critically in the entrapment and clearance of liver (David et al., 2016) and within parietal peritoneal mem- microorganisms that might gain entry to the cavity after breach branes (Uderhardt et al., 2019) have been described, and both of the intestinal boundary (Zhang et al., 2019a). The biol- populations play a role in governing recruitment of neutrophils ogy of the peritoneal macrophage has long been linked to the ............................................................................................................................................................................. 1 2 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO; Department of Pharmacology and Regenerative Medicine, 3 4 University of Illinois College of Medicine, Chicago, IL; Computer Technologies Department, ITMO University, St. Petersburg, Russia; Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX; Department of Surgery, Section of Surgical Oncology, Washington 6 7 University School of Medicine, St. Louis, MO; Departments of Internal Medicine and Immunology, Roswell Park Comprehensive Cancer Center, Buffalo, NY; Department of Medicine, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY. *N. Zhang and S.H. Kim contributed equally to this paper; **A. Gainullina and E.C. Erlich contributed equally to this paper; Correspondence to Gwendalyn J. Randolph: gjrandolph@wustl.edu; Ki-Wook Kim: kiwook@uic.edu; Nan Zhang: nzhang@wistar.org; N. Zhang’s present address is Wistar Institute, Philadelphia, PA. © 2021 Zhang et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Rockefeller University Press https://doi.org/10.1084/jem.20210924 1of 17 J. Exp. Med. 2021 Vol. 218 No. 12 e20210924 immunologic properties of the omental adipose tissue located in the absence of the omentum. Since omentectomy is standard within the peritoneal cavity. The omentum is home to collections in primary debulking surgery for ovarian cancer, our results hi of organized immune cells called milky spots or fat-associated raise the possibility that embryonic-derived mesenteric LYVE1 lymphoid clusters containing memory T cells (Han et al., 2017), macrophages may drive tumor progression and potentially Bcells (Wu et al., 2019), natural killer T cells (Ben ´ ez ´ ech et al., 2015), disease recurrence after initial therapy for metastatic ovarian dendritic cells, and innate lymphoid cells (Moro et al., 2010). Fur- cancer. thermore, the peritoneum can be affected by pathologies that in- clude postsurgical adhesions, endometriosis, and metastases of tumors most commonly arising from the colon, appendix, ovaries, Results or stomach. Experimental mouse models have begun to explore the Mesenteric membrane–associated macrophages are role of macrophages in these conditions (Hogg et al., 2021; Weiss phenotypically distinct from macrophages in the liver capsule et al., 2018; Xia et al., 2020; Zindel et al., 2021). and peritoneal cavity Epithelial ovarian cancer is the deadliest gynecological ma- The vascular and lymphatic tracts that connect the intestine lignancy, with high-grade serous ovarian cancer (HGSOC) being with the draining MLNs in mice are joined by avascular sheets of the most common subtype. HGSOC can originate from both tissue known to be lined by mesothelium and accompanying fallopian tube and ovarian surface epithelium (Zhang et al., fibroblasts (Fig. 1 A). Taking advantage of two myeloid-specific CreERT2 LSL-tdTomato Cre 2019b) and is commonly associated with widespread perito- reporter strains, Csf1r :Rosa26 and Lyz2 : LSL-tdTomato + neal carcinomatosis (Lengyel, 2010). More than 60% of patients Rosa26 mice, we observed elongated Tomato cells in with ovarian cancers are diagnosed at an advanced stage with these mesenteric membranes (Fig. 1, B and C). The cells were EYFP Cre peritoneal metastases (Siegel et al., 2018). HGSOC metastasis negative for enhanced YFP (EYFP) in CD11c mice (Lyz2 : LSL-tdTomato EYFP often leads to malignant ascites that is predominantly composed Rosa26 :CD11c ), distinguishing them from liver of inflammatory cells that include macrophages, neutrophils, capsule macrophages (David et al., 2016)thatwere positive for lymphocytes (Robinson-Smith et al., 2007; Singel et al., 2019), both EYFP and Tomato reporters in the respective strains (Fig. cancer-associated fibroblasts (Gao et al., 2019), and tumor cells. S1 A). We did, however, observe a few round-shaped Tomato HGOSC metastasis typically involves the peritoneal cavity, in- EYFP cells sparsely scattered on the mesenteric sheet (Fig. S1 cluding relevant adjacent tissues such as the omentum. In ad- B), and they were in close contact with Tomato cells (Video 1). + + dition to the omentum, peritoneal serosa and mesentery are also In flow cytometric analysis of gut mesentery, CD11b EYFP cells lo + common metastatic sites (Steinkamp et al., 2013); distant met- corresponded to the previously described F4/80 CD226 MHC + + − astatic seeding to abdominal organs and to the lungs and pleura II macrophages (Kim et al., 2016), but most CD11b EYFP cells also occurs. Contributions of different immune cells to this were F4/80 and did not express CD226 (Fig. S1 C). Instead, they metastatic evolution of cancer cells in the peritoneal environ- expressed high levels of CD206, which is absent on peritoneal ment remain understudied. Recently, Lawrence and colleagues macrophages (Fig. S1 D). ICAM2, which marks peritoneal mac- (Etzerodt et al., 2020) characterized omental macrophages and rophages (Gautier et al., 2012), was observed only on a few concluded that a major subset of omental macrophages could round-shaped Tomato cells sitting atop the mesenteric sheet hi lo account for the tumor-promoting role of the omentum. (Fig. S1 D). Flow cytometric analysis of F4/80 and F4/80 Here, we profiled mesenteric membrane–associated macro- peritoneal macrophage subsets and mesenteric macrophages in hi phages and identified two distinct populations (LYVE1 MHC the same mice confirmed that mesenteric macrophages are lo-hi lo/− lo/− hi hi II CX CR1gfp and LYVE1 MHC II CX CR1gfp subsets) positive for CD206 and MHC II. They were negative for ICAM2 3 3 that coexist in the mesothelial layer, generally resembling in- and CD226 (Fig. S1, E and F). The mean fluorescence intensity for terstitial macrophages described in the lung previously MHC II expression in mesenteric macrophages was relatively hi lo (Chakarov et al., 2019; Gibbings et al., 2017). Mesenteric LYVE1 lower than that of F4/80 peritoneal macrophages (Fig. S1 F). macrophages were derived from embryonic precursors and Overall, it appears that mesenteric membrane–associated mac- were controlled by colony stimulating factor 1 (CSF1) derived rophages are phenotypically distinct from the two well-established from local stromal cells. These macrophages did not depend on serous fluid peritoneal macrophage populations, which only GATA6 or IRF4 and maintained a life cycle distinct from that of infrequently attach to the mesenteric membrane in unper- peritoneal fluid macrophages. Bulk RNA sequencing (RNA-seq) turbed mice. An estimate of their number (see Materials and hi 6 analysis of LYVE1 membrane-associated macrophages and methods) at 10 within the peritoneal cavity suggests that their its comparison to single-cell RNA-seq (scRNA-seq) of omental total numbers in the peritoneal cavity are approximately sim- hi macrophages in ovarian tumors showed that LYVE1 macro- ilar to the number of resident macrophages in the peritoneal phages had a specialized gene expression pattern that correlated fluid (1–2× 10 ; Zhang et al., 2019a). By comparison, we esti- hi with the genes expressed by LYVE1 omental macrophages mated the number of macrophages in the omentum to be far during ovarian cancer progression (Etzerodt et al., 2020). Using lower, at 2 × 10 per omentum. in vivo ablation approaches in omentectomized mice, we un- hi raveled the relationship between LYVE1 macrophages, the Mesenteric membrane–associated macrophages consist of omentum, and ovarian tumor progression. Our data indicate that two distinct subsets hi LYVE1 macrophages within peritoneal membranes like those Some macrophage populations constitutively express GFP re- gfp/+ associated with the mesentery promote tumor progression even porter in CX CR1 mice (Bain et al., 2014; Gibbings et al., 2017; Zhang et al. Journal of Experimental Medicine 2of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Figure 1. Two distinct macrophage populations coexist in the avascular regions of mesenteric membranes. (A) Whole-mount images of gut mesentery in adult WT mice. Square box indicates a region of avascular mesenteric membrane. (B and C) Whole-mount images of the avascular mesenteric membrane CreERT2 LSL-tdTomato Cre LSL-tdTomato from tamoxifen-induced Csf1r :R26 mice (B) and Lyz2 :R26 mice (C). Scale bar, 50 µm. (D) Whole-mount images of mesenteric CreERT2 LSL-tdTomato gfp + membrane from tamoxifen-induced Csf1r :R26 :CX CR1 mice. CX CR1-GFP cells (left), Csf1r-expressing Tomato cells (middle), and merged 3 3 pictures (right) for two distinct macrophage populations. Scale bar, 50 µm. (E) Immunohistochemistry analysis of a whole-mount mesenteric membrane from gfp/+ CX CR1 mouse stained for LYVE1. CX CR1gfp expression (left), LYVE1 (middle), and merged pictures (right) for two distinct macrophage subsets. Scale bar, 3 3 hi lo/− lo/− hi 50 µm. (F) Quantification of LYVE1 CX CR1gfp macrophages and LYVE1 CX CR1gfp macrophages in mesenteric membranes. Data are representative of 3 3 three independent experiments (n = 3; mean ± SEM). Macrophages were quantified in two different regions of mesenteric membrane per mouse. Unpaired Cre LSL-tdTomato Student’s t test: ****, P < 0.0001. (G) Immunohistochemistry analysis of a whole-mount mesenteric membrane from Lyz2 :R26 mice stained with LYVE1 and MHCII. Scale bar, 50 µm. (H) Flow cytometric analysis of membrane-associated macrophages isolated from gut mesentery with CD45, F4/80, CD64, hi lo/− LYVE1, and MHC II staining. SSC, side scatter. (I) Frequency of LYVE1 membrane-associated macrophages and LYVE1 membrane-associated macrophages from flow cytometric analysis (H). Data are pooled from two independent experiments (n = 9; mean ± SEM). Unpaired Student’s t test: ****, P < 0.0001. All imaging data are representative of at least three independent experiments. Stamatiades et al., 2016; Williams et al., 2020; Zigmond et al., (Chakarov et al., 2019; Etzerodt et al., 2020; Lacerda Mariano gfp/+ 2014; Zigmond et al., 2012), while other tissue-resident macro- et al., 2020; Lim et al., 2018). In CX CR1 mice, we observed lo/− phages do not (Yona et al., 2013). To determine whether mes- that most CX CR1gfp macrophages highly expressed LYVE1, hi enteric membrane–associated macrophages express GFP reporter in while CX CR1gfp macrophages were negative or had low ex- gfp/+ CreERT2 LSL-tdTomato hi CX CR1 mice, Csf1r :Rosa26 mice were crossed pression for LYVE1 (Fig. 1 E). LYVE1 macrophages were the gfp CreERT2 LSL-tdTomato hi with CX CR1 mice to generate Csf1r :Rosa26 : dominant population, compared with CX CR1gfp macrophages 3 3 gfp/+ CX CR1 mice. In the mesenteric membranes of these dual re- (Fig. 1 F). hi lo/− porter mice, we observed two distinct macrophage subsets: To further characterize LYVE1 and LYVE1 macrophages, + lo/− + hi Tomato CX CR1gfp macrophages and Tomato CX CR1gfp membrane-associated macrophages were stained for MHC II in 3 3 Cre LSL-tdTomato hi lo/− macrophages (Fig. 1 D). To characterize these subsets further, Lyz2 :Rosa26 mice. Both LYVE1 and LYVE1 mesenteric membranes were stained for LYVE1, as it was re- membrane-associated macrophages expressed MHC II, albeit hi lo hi cently reported that two distinct LYVE1 and LYVE1 inter- more weakly in the LYVE1 macrophages, in images from stitial macrophage subsets are present together in some tissues confocal microscopy (Fig. 1 G). Through flow cytometric analysis Zhang et al. Journal of Experimental Medicine 3of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 of single-cell suspensions generated from gut mesentery, we resident in dura mater and pia mater as vascular-associated + + confirmed that F4/80 CD64 macrophages could be divided into macrophages (Kim et al., 2021), we found Tomato-labeled hi lo-hi lo/− two major populations: LYVE1 MHC II and LYVE1 MHC meningeal perivascular macrophages near the blood vascu- hi + II macrophages (Fig. 1 H). The frequency of the two macro- lature underneath the skull (Fig. 2 F and Video 2). Tomato phage subsets did not differ depending on whether they were macrophages were also concentrated in the collagen-enriched examined by confocal microscopy or flow cytometry (Fig. 1, F barrier surface of the pancreas (Fig. 2 G)and theparietal and I). peritoneal membrane (Fig. 2 H and Video 3), indicating that hi lo/− Both LYVE1 and LYVE1 membrane-associated macro- LYVE1-expressing macrophages were enriched at a number of phages were intact in the absence of Gata6 expression within barrier surfaces, including multiple mesothelial linings of the cre fl/fl + macrophages (Lyz2 :Gata6 mice; Fig. S2 A), whereas peri- peritoneal cavity. However, Tomato macrophageswereab- toneal macrophages were highly reduced (Fig. S2 B), indicating sent from the liver capsule (Fig. 2 I, left). At the liver surface, + gfp/+ that the membranous macrophages were maintained inde- we detected only GFP cells in CX CR1 mice (Fig. 2 I, pendently from peritoneal fluid macrophages. Taken together, right). mesenteric membrane–associated macrophages consist of two hi lo/− lo-hi hi distinct subsets: LYVE1 CX CR1 MHC II macrophages LYVE1 membrane-associated macrophages constitutively hi lo/− (henceforth termed LYVE1 macrophages) and LYVE1 display an alternatively activated macrophage (AAM) gene hi hi lo/− CX CR1 MHC II macrophages (henceforth termed LYVE1 expression profile macrophages). After bulk RNA-seq, principal component analysis (PCA) hi showed that LYVE1 macrophages cluster together as repli- hi LYVE1 membrane-associated macrophages are located on the cates. They clustered distinctly from other tissue-resident barrier surfaces of many organs macrophages including resident macrophages from peritoneal hi In the mesentery, LYVE1 macrophages, probed initially by lavage, lung, spleen, and brain or two blood monocyte subsets immunostaining for LYVE1, were rather evenly distributed (Fig. 3 A). Among the most highly up-regulated genes (≥16-fold across the mesenteric surface in both the avascular and vascular compared with the other macrophage populations) in the hi areas of the mesentery (Fig. 2 A). The vascularized area was rich mesenteric LYVE1 macrophages were Mgl2 (CD301b), Mmp9, in adipose tissue housing the nerves, blood vessels, and lym- Lyve1, C1qtnf1, Folr2, Cbr2, and AAM-related genes such as Retnla CreERT2 phatic vessels, marked using tamoxifen-treated Prox1 : (RELMα)and Mrc1 (CD206; Fig. 3 B). In addition to the up-regulation LSL-tdTomato R26 mice (Fig. 2 A). of canonical AAM-related genes, pathway analysis implemented LYVE1 is transiently expressed in erythroid-myeloid pro- by fast gene set enrichment analysis (fast GSEA) showed that genitors (EMPs) during embryogenesis and in hematopoietic pathways related to extracellular matrix organization such as stem cells (HSCs) in adult hematopoiesis (Lee et al., 2016). As collagen formation and degradation were enriched in mesenteric hi such, 50–70% of blood leukocytes expressed the Tomato reporter LYVE1 macrophages (Fig. 3 C). Cre LSL-tdTomato in Lyve1 :Rosa26 mice due to this embryonic history To further investigate and validate these findings, we rean- (Fig. S3 A). Thus, to visualize LYVE1-expressing macrophages alyzed an scRNA-seq dataset that examined whole mesenteric selectively using reporter mice, we designed a bone marrow cells (GEO accession no. GSE102665; Koga et al., 2018), produc- (BM) chimeric mouse model in which CD45.2 Tomato BM cells ing 16 clusters based on cell-specific gene expression repre- Cre LSL-tdTomato were first isolated from Lyve1 :Rosa26 mice by senting a range of cell types (Figs. 3 D and S4 A). We defined FACS (Fig. 2 B). They were then transplanted into lethally ir- clusters 3 and 8 of this t-distributed stochastic neighbor em- radiated CD45.1 congenic mice. 8–10 wk later, BM chimeric bedding (t-SNE) plot as macrophage populations due to ex- mice were analyzed by flow cytometry and microscopy. In pression of Cd68, Lyz2, Mrc1, Cd14,and Mgl2 (Fig. S4, A and B)and Cre LSL-tdTomato contrast to unmanipulated Lyve1 :Rosa26 mice, cluster 14 as a dendritic cell population due to coenrichment in Tomato reporter–expressing cells were not found among blood genes such as Cd209, Flt3,and Itgae (CD103; Fig. S4 C). The Lyve1, leukocytes in mice receiving the Tomato BM transplant (Fig. Mmp9,and Folr2 mRNA transcripts that were up-regulated in hi S3 B). Other tissue-resident macrophages such as microglia, red bulk RNA-seq of LYVE1 membrane-associated macrophages pulp macrophages, and alveolar macrophages also were not were selectively detected in cluster 3 (Fig. 3 E). Overall, cluster 3 hi lo labeled by the Tomato reporter due to marked radioresistance corresponded to both LYVE1 and LYVE1 mesenteric macro- and lack of LYVE1 expression (microglia) or radiosensitivity phage populations, whereas other macrophages corresponded to and lack of LYVE1 expression (e.g., spleen or alveolar macro- cluster 8 (Fig. 3, D and E;and Fig. S4 B). Folate receptor 2 ex- hi phages; Fig. 2, C and D). By contrast, mesenteric macrophages pression, encoded by Folr2 mRNA, was observed in LYVE1 lo were mainly radiosensitive, and >70% were highly labeled by and LYVE1 macrophages through flow cytometric analysis the Tomato reporter in flow cytometric analysis and imaging (Fig. 3 F), confirming that LYVE1 macrophages, including both hi lo (Fig. 2, C–E; and Fig. S3 C). LYVE1 and LYVE1 subsets, belong to cluster 3 (Fig. 3, D–F). Cre LSL-tdTomato Across a range of organs, Lyve1 :Rosa26 BM- Other AAM-associated genes, Mrc1 and Retnla, characterized transplanted mice were visualized by two-photon microscopy LYVE1-enriched macrophages in both datasets (Fig. 3 G), with immediately after intravenous injection of Alexa Fluor 488– many AAM genes including Retnla and Mrc1 also highly en- lo/− conjugated lectins to label blood vasculature. Consistent with riched in LYVE1 mesenteric macrophages. Thus, mesenteric hi the recent report that LYVE1 perivascular macrophages are barrier membrane macrophages are oriented toward support of Zhang et al. Journal of Experimental Medicine 4of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 hi Figure 2. Lyve1 membrane-associated macrophages are located in serous membrane of tissue parenchyma. (A) Whole-mount, confocal microscopy hi CreER LSL-tdTomato tile-scan reconstructions to examine the location and distribution of LYVE1 mesenteric macrophages in tamoxifen-induced Prox1 :R26 mice (red, Prox1-expressing lymphatic collector; white, LYVE1-expressing macrophages and lymphatic capillaries; blue, nuclei). Scale bar, 400 µm. Images are representative of two independent experiments with scanning of a large region of tissue. (B) Schemes for Tomato BM transplantation to whole-body-ir- − cre LSL-tdTomato radiated mice (created with BioRender.com). Sorted CD45.2 Tomato BM cells from Lyve1 :R26 mice were transplanted to irradiated congenic CD45.1 WT mice. Tomato reporter will label only adult macrophages with an active LYVE1 promoter in adulthood, bypassing embryo-restricted activity at this promoter. (C) Histogram showing Tomato reporter expression of tissue-resident macrophages in brain, spleen, lung, and gut mesentery of Tomato BM transplanted CD45.1 recipient mice. (D) Quantification of Tomato expression in donor-derived tissue-resident macrophages of Tomato BM transplanted CD45.1 mice. (C-D) Data are pooled from at least two independent experiments (n = 6, mean ± SEM). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple-comparison test: ****, P < 0.0001. (E) Whole mount images of the mesenteric membrane in Tomato BM transplanted chimeric mice (red, + − Tomato macrophages; blue, collagens imaged by second harmonic generation [SHG]). Scale bar, 20 µm. (F) Whole mount images of the meninge in Tomato BM transplanted chimeric mice (red, Tomato macrophages; green, Alexa488-conjugated lectin injected blood vessels; blue, skull imaged by SHG). Note + − Tomato perivascular macrophages underneath skull. Scale bar, 20 µm. (G) Whole mount images of pancreas from Tomato BM transplanted chimeric mice (red, Tomato macrophages; green, Alexa488-conjugated lectin injected blood vessels; blue, collagen bundles shown as SHG) Scale bar, 50 µm. (H) Whole- − + mount images of the parietal peritoneal membrane from Tomato BM transplanted chimeric mice (red, Tomato macrophages; green, Alexa488-conjugated lectin injected blood vessels; blue, collagens shown by SHG). Scale bar, 50 µm, (E, G, and H) Note Tomato membrane-associated macrophages in collagen- − gfp/+ enriched serosa membrane. (I) Comparison of liver capsular macrophages between Tomato BM-transplanted chimeric mice (left) and CX CR1 mice (right); + + red, Tomato macrophages; green, CX CR1gfp macrophages; blue, collagen. Scale bar, 50 µm. All two-photon microscopic images are representative of at least two independent experiments. Zhang et al. Journal of Experimental Medicine 5of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 hi + Figure 3. LYVE1 macrophages have their own gene expression patterns. (A) PCA of tissue-resident macrophages (alveolar macrophages, F4/80 hi hi lo peritoneal macrophages, microglia, splenic red pulp macrophages, and LYVE1 membrane-associated macrophages) and blood monocytes (Ly6C and Ly6C hi monocytes) obtained from RNA-seq dataset. PC, principal component. (B) Heatmap analysis of top 50 up-regulated genes of 12,000 genes expressed in LYVE1 hi membrane-associated macrophages. Heatmap depicts mean expression intensity of mRNA transcripts for genes differentially expressed between LYVE1 hi macrophages and other macrophages, including monocytes. (C) Pathway analysis of genes differentially expressed in LYVE1 membrane-associated mac- rophages implemented by fast GSEA, showing top 10 enriched pathways from Reactome database and Molecular Signatures Database. NES, normalized enrichment score. (D) t-SNE plot displaying reanalyzed scRNA-seq of whole mesentery cells (accession no. GSE102665). (E) Expression of Lyve1, MMP9,and hi lo Folr2 on the t-SNE plot of scRNA-seq described in D. (F) Flow cytometric analysis showing FOLR2 expression of LYVE1 and LYVE1 mesenteric macrophages. Data are representative of three mice. (G) Violin plot of Retnla and Mrc1 expression obtained from scRNA-seq described in D. extracellular matrix remodeling, likely needed to build and are established from CX CR1-expressing precursor cells dur- hi maintain the barrier in which the cells reside. ing embryogenesis (Yona et al., 2013). LYVE1 macrophages were already established in the mesenteric membrane of these hi hi LYVE1 membrane-associated macrophages originate from newborn mice (Fig. 4 A). Unlike LYVE1 macrophages from gfp/+ embryonic precursors adult CX CR1 mice, which harbored low expression of GFP hi Many peripheral tissue macrophages develop from embryonic reporter (Fig. 1, D and E), LYVE1 macrophages in the mes- precursors (EMPs or fetal liver monocytes; Gomez Perdiguero entery of newborn mice highly expressed GFP in >90% of lo/− + et al., 2015; Hoeffel et al., 2015; Yona et al., 2013). However, liver all mesenteric macrophages (Fig. 4 B). LYVE1 CX CR1gfp capsular macrophages are derived from circulating mono- macrophages (Fig. 4 A, arrows) accounted for <10% of total cytes, not embryonic precursors (Sierro et al., 2017). Thus, we macrophages in newborn mice (Fig. 4 C). In newborn mice, hi hi lo/− wondered whether LYVE1 mesenteric membrane–associated both LYVE1 and LYVE1 macrophagesweremore amoe- macrophages were derived from embryonic precursors or boid in morphology and rarely expressed MHC II, although were replenished from circulating monocytes. We first ex- MHC II was highly expressed in cells in the MLN. By P6, these gfp/+ lo/− amined membrane-associated macrophages in newborn CX CR1 macrophages elongated, and MHC II expression in some LYVE1 mice (postnatal day 0 [P0]), as most tissue-resident macrophages macrophages began to emerge within 2 wk after birth (Fig. 4 D). Zhang et al. Journal of Experimental Medicine 6of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 gfp/+ Figure 4. Membrane-associated macrophages originate from embryonic precursors. (A) Whole-mount images of mesentery in newborn (P0) CX CR1 lo/− + pups stained with LYVE1. Right-most image is the enlargement of the boxed area in the adjacent image. Arrows indicate LYVE1 CX CR1-GFP macrophages. Images are representative of two independent experiments. Scale bar, 100 µm; 30 µm (higher-magnification image). (B) CX CR1-GFP expression within the hi hi Lyve1 macrophage pool in the mesenteric membrane of newborn mice. GFP expression is quantified within total LYVE1 macrophage population. (C) The hi lo/− frequency of LYVE1 macrophages versus LYVE1 macrophages in the mesenteric membrane of newborn mice. In B and C, data are representative of two independent experiments (n = 4; mean ± SEM). Membrane-associated macrophages were quantified in one to three different regions of mesenteric membrane per mouse. Unpaired Student’s t test: ****, P < 0.0001. (D) Whole-mount images of MLNs and mesenteric membranes of P1, P6, and P14 neonatal mice stained with LYVE1 and MHCII. White, LYVE1; green, MHCII; blue, DAPI. The yellow line in the P1 panel indicates the border of mesenteric vessels and mesenteric membrane. Images are representative of at least two independent experiments per time point. Scale bar, 50 µm. (E) Representative whole-mount images of CreERT2 LSL-Tomato adult CX CR1 :R26 mice in which tamoxifen was injected on P1 (white, LYVE1; red, Tomato reporter). Scale bar, 50 µm. (F) Tomato expression in hi hi microglia, LYVE1 mesenteric membrane–associated macrophages and blood monocyte subsets. LYVE1 membrane-associated macrophages were quantified in two different regions of membrane per mouse from confocal microscopy images. Microglia and blood monocytes were quantified via flow cytometric analysis. In E and F, data are pooled from two independent experiments (n = 6; mean ± SEM). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple-comparison test: ****, P < 0.0001. hi We conclude that mesenteric membrane–associated macrophages are likely long-lived or self-renewed from P1-labeled LYVE1 are established during embryogenesis but undergo postnatal macrophages (Fig. 4 F). Collectively, these data suggest that most hi adaptations in phenotype and morphology. LYVE1 macrophages originate from embryonic precursors and hi To determine if embryonic derived LYVE1 macrophages are are then maintained locally for long periods. maintained through self-renewal, we performed tamoxifen- hi pulse labeling to track the LYVE1 macrophages after birth. Membrane-associated macrophages are controlled by CSF1 Accordingly, tamoxifen was injected i.p. into P1 pups of produced in Wt1-expressing stromal cells CreERT2 LSL-tdTomato CX CR1 :R26 mice. 8–10 wk later, Tomato re- It is well established that CSF1 receptor signaling is crucial for hi porter was visualized in LYVE1 macrophages (Fig. 4 E). Tomato the generation, differentiation, and survival of most tissue- hi reporter remained high in LYVE1 macrophages (80.08 ± 1.78%) resident macrophages (Cecchini et al., 1994; Dai et al., 2002; and microglia (96.28 ± 1.39%), while it was negative at these time Ivanov et al., 2019; Williams et al., 2020). Thus, we investigated hi lo hi points in blood Ly6C monocytes (0.09 ± 0.02%) and Ly6C whether LYVE1 macrophages required CSF1 receptor signaling. hi Cre fl/fl ΔCsf1r hi monocytes (2.19 ± 0.32%), suggesting that LYVE1 macrophages Indeed, in Lyve1 :Csf1r mice (Lyve1 mice), LYVE1 Zhang et al. Journal of Experimental Medicine 7of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Figure 5. Membrane-associated macrophages are controlled by CSF1 produced by stromal cells of serous membranes. (A) Whole-mount images of the Cre fl/fl fl/fl two distinct membrane-associated macrophages and their quantification in Lyve1 :Csf1r mice and littermate Csf1r control mice. Scale bar, 50 µm. Data are representative of three independent experiments (n = 3 per genotype; mean ± SEM). Macrophages were quantified in multiple regions of mesenteric membrane per mouse. Unpaired Student’s t test: ****, P < 0.0001. (B) Expression pattern of Csf1, Il34,and Wt1 depicted on the t-SNE plot derived from scRNA- cre LSL-tdTomato seq of whole mesenteric cells shown in Fig. 3 D. (C) Whole-mount images stained for LYVE1 in mesenteric membranes from WT1 :R26 mice. lo/− Representative images of three independent experiments. Arrows indicate LYVE1 macrophages (red, Wt1 Tomato reporter; green, LYVE1; blue, DAPI). Scale Cre fl/fl fl/fl bar, 20 µm. (D) Whole-mount images of mesenteric membranes from Wt1 :Csf1 mice and littermate Csf1 control mice. Representative images of two hi lo independent experiments. Scale bar, 100 µm. (E) Quantification of LYVE1 and LYVE1 membrane-associated macrophages obtained from whole-mount Cre fl/fl fl/fl fl/fl Cre fl/fl images of Wt1 :Csf1 mice and littermate Csf1 control mice. Data are pooled from two independent experiments (Csf1 mice, n =4; Wt1 :Csf1 mice, n = 6; mean ± SEM). Macrophages were quantified from multiple regions of mesenteric membrane per mouse. Unpaired Student’s t test: **, P < 0.01; ****, P < + fl/fl 0.0001. (F) Quantification of ICAM2 macrophages in peritoneal cavity. Data are representative of at least three independent experiments (Csf1 mice, n =4; Cre fl/fl hi Wt1 :Csf1 mice, n = 5; mean ± SEM). Unpaired Student’s t test: ****, P < 0.0001. (G) Quantification of Ly6C monocytes and neutrophils in blood. Data are fl/fl Cre fl/fl representative of three independent experiments (Csf1 mice, n =5; Wt1 :Csf1 mice, n = 4; mean ± SEM). Unpaired Student’s t test. (H) Confocal image cre LSL-DTA from the mesenteric membrane of Adiponectin :R26 mice and controls. Images are representative of two independent experiments. Scale bar, 50 µm. lo/− macrophages were reduced by ∼80%. The number of LYVE1 necessary for persistence of macrophages that might arise from hi MHC II macrophages was comparable to that of littermate Cre-recombinase activated HSC/EMP progenitors, such that controls (Fig. 5 A). Additionally, the number of other tissue- there would evolve a natural selection bias to greatly favor the resident macrophages known to lack LYVE1 expression resid- seeding of tissue macrophages arising from Cre-recombinase ΔCsf1r ing in peritoneum, spleen, lung, and brain of Lyve1 mice nonactivated HSC/EMP progenitors (30–50% of populations). was not significantly changed compared with standard WT mice Then, expression of LYVE1 by tissue-resident macrophages (Fig. S5, A–D), and Csf1 receptor is normally expressed in peri- would trigger selective loss of Csf1R, such that only LYVE1 ΔCsf1r toneal fluid macrophages of Lyve1 mice (Fig. S5 E). These macrophages would be substantially impacted. results imply that Csf1 receptor deficiency in the stage of EMP or CSF1 and IL34 are the ligands of CSF1 receptor (Wang et al., HSC has no impact on the pool of tissue-resident macrophages, 2012), so we examined mRNA for CSF1 and IL34 in the scRNA- hi and Lyve1 membrane-associated macrophages are selectively seq data from the whole mesentery. Csf1 in particular is highly depleted by the lack of Csf1 receptor–mediated signaling in up-regulated in Wilms tumor 1 homologue (Wt1)–expressing ΔCsf1r Lyve1 mice. This outcome may arise because Csf1R would be mesenteric fibroblasts and mesothelial cells (Fig. 5 B), and both Zhang et al. Journal of Experimental Medicine 8of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 lo/− hi hi LYVE1 macrophages (yellow arrows) and LYVE1 macro- expressed in cluster 10 were highly matched to the LYVE1 phages were located in proximity to Tomato-labeled stromal cells macrophages in GSEA (Fig. 6 D). We conclude that macro- cre LSL-tdTomato within the mesothelial membranes from Wt1 :R26 phages constitutively present in the mesenteric membrane mice (Fig. 5 C). To test whether membrane-associated macro- express a pattern of genes previously associated with omental phages were controlled by Csf1 produced by local stromal cells, macrophages that drive tumor progression. cre fl/fl ΔCsf1 we generated Wt1 :Csf1 mice (Wt1 mice) that would hi delete Csf1 in WT1-expressing fibroblasts and mesothelial cells. LYVE1 mesothelial macrophages enhance omentum- hi Both LYVE1 macrophages in particular and, to a lesser extent, independent ovarian tumor progression lo/− LYVE1 macrophages were significantly reduced in the ab- Given the overlap between omental macrophage gene expres- sence of Csf1 produced in stromal cells, with residual cells ap- sion and that of other macrophages in peritoneal membranes pearing rounded in morphology (Fig. 5, D and E). Consistent with lining the cavity where the tumor was implanted, we wondered + + a previous finding (Bellomo et al., 2020), F4/80 ICAM2 peri- if the previous correlation in reduced tumor progression be- ΔCsf1 hi toneal macrophages were highly reduced in Wt1 mice tween removal of the omentum and the lack of LYVE1 mac- hi (Fig. 5 F). However, blood Ly6C monocytes and neutrophils of rophages (Etzerodt et al., 2020) meant de facto that only omental ΔCsf1 Wt1 mice were comparable in number to their littermate macrophages inside the peritoneal cavity were implicated in controls (Fig. 5 G). tumor progression. In particular, we wondered if membrane- hi Some visceral white adipose tissues (WATs) are generated associated LYVE1 macrophages beyond those in the omentum from Wt1-expressing cells (Chau et al., 2014). To test whether were relevant drivers of tumor progression. To examine these membrane-associated macrophages are controlled by WAT- questions, luciferase/GFP-labeled murine epithelial ovarian tu- cre LSL-DTA hi derived CSF1, we studied Adiponectin :R26 mice lacking mor cells (ID8) were injected i.p. into LYVE1 macrophage– hi lo/− ΔCsf1r fl/fl adipocytes. LYVE1 and LYVE1 macrophages in mesenteric ablated mice (Lyve1 ) or their littermate controls (Csf1r ) membranes were intact in these mice, implying that membrane- to model intraperitoneal metastatic HGSOC (Leinster et al., 2012; associated macrophages did not depend on adipocytes for main- Lengyel et al., 2014). Tumor burden was monitored biweekly tenance (Fig. 5 H). Altogether, we conclude that CSF1 locally through noninvasive bioluminescence imaging. In the first 4 wk, ΔCsf1r produced by fibroblasts and/or mesothelial cells within serous peritoneal tumor burden was comparable between Lyve1 membranes controls the development and maintenance of mice and littermate controls (Fig. 7 A). During this period, the macrophages within the membranes themselves and in the tumor was particularly localized to the omental fat region of adjacent fluid cavities. the peritoneal cavity, spreading into the greater cavity space thereafter (Etzerodt et al., 2020). By 6 wk after implantation, hi hi Gene expression patterns in LYVE1 mesenteric tumor progression was significantly delayed if LYVE1 macro- membrane–associated macrophages resemble those phages were genetically ablated, compared with littermate expressed by omental macrophages in ovarian tumors controls (Fig. 7, A and B). These findings were similar to those It was reported that tissue-resident macrophages play important previously reported (Etzerodt et al., 2020). roles in tumor progression by expressing profibrotic factors The key question was whether the role of macrophages in (Zhu et al., 2017). More recently, Etzerodt et al. (2020) showed driving tumor progression was restricted and required the + hi that TIM4 LYVE1 omental macrophages promote ovarian tu- omentum. To investigate this issue, we performed surgical mor progression through scRNA-seq and a cell ablation model. omentectomy and compared tumor progression between hi ΔCsf1r We suspected that the LYVE1 macrophages we identified lining Lyve1 mice and littermate controls, designing two sets of hi the mesenteric membranes of the peritoneal cavity were related experiments to rigorously test whether LYVE1 macrophages to the tumor-associated macrophages in the omentum. To test promoted ovarian tumors independently of the omentum. First, hi this possibility, we compared the RNA-seq data from LYVE1 we used the ID8-Luc-GFP cell line to model intraperitoneal cre fl/fl membrane-associated macrophages to the scRNA-seq data metastasis after omentectomy. Both Lyve1 :Csf1r mice and (ArrayExpress accession no. E-MTAB-8593) from omental their littermates developed significantly less intraperitoneal macrophages performed in mice bearing experimental ovar- metastasis of ovarian tumors than mice without omentectomy ian tumors. Tumor-associated omental macrophages were re- (Fig. 7, A and C), confirming the critical role of the omentum in analyzed and divided into 21 different clusters based on their ovarian tumor progression. However, in the absence of ΔCsf1r gene expression patterns (Fig. 6 A). Timd4 was up-regulated in omentum, Lyve1 mice still developed less intraperitoneal clusters 10 and 16 and Lyve1 in clusters 6, 8, 10, 15, and 16, with metastasis than littermate controls (Fig. 7 C), demonstrating an hi highest expression in cluster 10 among these five clusters. This independent role for the LYVE1 macrophages outside of the + + analysis placed the tumor-promoting TIM4 LYVE1 omental omentum. Second, as previously pioneered (Etzerodt et al., macrophages in cluster 10 of our reanalysis (Fig. 6 B). Next, the 2020), we developed omentum-primed ID8 cells by condition- hi top 100 enriched genes expressed by LYVE1 membrane- ing ID8 cells in WT mice for 12 wk. We termed recovered cells associated macrophages were compared with genes expressed ID8-A12. ID8-A12 cells were inoculated into omentectomized ΔCsf1r in the scRNA-seq that we reanalyzed. As shown in our heatmap Lyve1 mice or littermate controls. Ovarian tumors were analysis, we confirmed that the gene expression pattern of rapidly expanded in both genotypes within 4 wk after inocu- hi LYVE1 mesenteric membrane macrophages most closely matched lation. However, ID8-A12 cells expanded significantly more ΔCsf1r genes expressed in cluster 10 (Fig. 6 C). Conversely, genes slowly in Lyve1 mice than in control mice (Fig. 7 D). The Zhang et al. Journal of Experimental Medicine 9of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 hi hi Figure 6. Comparison of gene expressions between RNA-seq of Lyve1 membrane-associated macrophages and scRNA-seq of Lyve1 omental macrophages in ovarian tumor progression. (A) Reanalyzed uniform manifold approximation and projection (UMAP) plot of scRNA-seq data (ArrayExpress + + accession no. E-MTAB-8593) of F4/80 CD64 omental macrophages isolated 10 wk after ID8 ovarian tumor cell injection. (B) Violin plot of Timd4 and Lyve1 hi expression from the scRNA-seq dataset in A. (C) UMAP plot of scRNA-seq showing top 100 genes up-regulated in bulk RNA-seq datasets of LYVE1 membrane- hi associated macrophages. (D) GSEA of RNA-seq data from Lyve1 membrane-associated macrophages showing select genes enriched in Cluster 10 of scRNA- seq. NES, normalized enrichment score. tumor within ascites rather than the mesentery accounted for Discussion the differences in tumor burden between the two genotypes The complexity of resting macrophage heterogeneity and spe- hi (Fig. 7 E). Taken together, these data underscore that LYVE1 cialization across and within given organs continues to evolve macrophages promote intraperitoneal expansion of ovarian and grow. The present focus on the peritoneal cavity in this tumors independent of the omentum. body of work highlights the intricate network of resident hi Figure 7. Deficiency in LYVE1 macrophages delays intraperitoneal expansion of ovarian cancer in an omentum-independent manner. (A) Quanti- fl/fl ΔCsf1r fication of bioluminescence signals at different time points after tumor implantation (Csf1r mice, n = 12; Lyve1 mice, n =13; mean ±SEM). (B) Bio- luminescence images of tumor-bearing mice at 6 wk after inoculation. (C) Quantification of bioluminescence signal at different time points after inoculation in fl/fl ΔCsf1r omentectomized (OMX) mice (Csf1r mice, n = 11; Lyve1 mice, n = 11; mean ± SEM). (D) Quantification of bioluminescence signal at different time points fl/fl ΔCsf1r after inoculation of the omentum-primed ID8-A12 cells in OMX mice (Csf1r mice, n = 10; Lyve1 mice, n = 12; mean ± SEM). (E) Quantification of bi- oluminescence signal of ascites and mesenteries 4 wk after inoculation of the omentum-primed ID8-A12 cells in OMX mice (mean ± SEM). Unpaired Student’s t test: *, P < 0.05; ***, P < 0.001. Statistical analysis was performed using one-way ANOVA (A, C, and D) and Student’s t test (E). Zhang et al. Journal of Experimental Medicine 10 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 macrophages that occupy the body cavity. We and others have cells that serve as the crucial source of CSF1 that these mac- recognized the existence of two resident peritoneal macro- rophages require for persistence. phage subsets in the serosal fluid of the peritoneum, GATA6- With the clear evidence that the phenotype of interstitial dependent LPMs (Gautier et al., 2014; Okabe and Medzhitov, macrophage subsets lining the mesenteric and peritoneal 2014; Rosas et al., 2014) and monocyte-derived, IRF4-dependent membranes is distinct from the phenotype and life cycle of the small peritoneal macrophages (Kim et al., 2016). Evidence of fluid-borne macrophage subsets of the peritoneum, it is rea- the existence of more than these two resident macrophages in sonable to regard the peritoneal cavity space as being influ- the body cavity is found in the literature, particularly in the enced by four different types of resident macrophages. One studies of Uderhardt et al. (2019) identifying fixed macrophages begins to wonder, when this point is taken into account, how in the peritoneal lining membranes. However, the basic phe- the invasion into injured organs by fluid-borne peritoneal notype of these fixed macrophages has not been reported or macrophages (Wang and Kubes, 2016), and their influence on compared with the fluid-borne macrophages, and thus even surgical adhesions (Zindel et al., 2021), is impacted by these very recent reviews (Liu et al., 2021) and papers in the field interstitial macrophages. For example, cell deletion schemes focused on peritoneal macrophages (Louwe et al., 2021)have that have targeted LPMs of the serous fluid are broad enough in not placed these fixed macrophages into context. Instead, their mechanism of action to have also deleted these interstitial the omental fat-associated macrophages are typically the macrophages in relevant anatomic spaces, but this point was only macrophages routinely considered beyond the serous not considered, as the presence of these macrophages was not fluid macrophages within the peritoneal compartment (Liu clear at the time of the studies. It may also be the case that et al., 2021). Here, we show that macrophages resembling deletions of specific but still rather broadly expressed genes omental macrophages are situated as fixed macrophages such as Rxra affected outcomes such as peritoneal cancer in the mesothelial linings of the peritoneum, including the progression (Casanova-Acebes et al., 2020)due to deletions vascularized and avascular parts of the mesentery, the pa- of the LYVE1 serosal macrophages, as their status was not rietal peritoneal membrane, and the surface of organs such checked. On the other hand, it very well may be that serous as the pancreas. fluid peritoneal macrophages and the LYVE1 interstitial Strikingly, phenotypes of the fixed macrophages observed macrophages we describe here each have requisite roles in were characterized by high expression of LYVE1 and low peritoneal tumor progression (Casanova-Acebes et al., 2020; expression of MHC II in the first population and lower ex- Xia et al., 2020). pression of LYVE1 but higher expression of MHC II in the With respect to cancer progression, a recent study published hi second population. Indeed, it is striking that almost all organ compelling evidence that LYVE1 macrophages were critical surfaces including the meninges of the central nervous sys- mediators of ovarian tumor cell expansion in the peritoneal tem are characterized by the presence of LYVE1 macrophages, cavity (Etzerodt et al., 2020). We show here that the macro- even when the major organ parenchymal macrophage is devoid phages we describe are a similar population to those studied by of this marker. We note that the liver stands out as the ex- Etzerodt et al. (2020). Because the same authors reproduced that ception to the pattern, having a phenotypically distinct mac- the presence of the omental fat tissue drove tumor progression rophage type at the border surface (Sierro et al., 2017), which and because they were able to identify interstitial peritoneal hi lo/− hi our findings confirmed. The LYVE1 and LYVE1 macro- macrophages with the LYVE1 phenotype in the omentum, they phages within the serous membranes are likely counterparts to concluded that omental macrophages in particular were neces- the interstitial macrophages previously described in the lungs sary for ovarian tumor progression in the peritoneal cavity. In (Chakarov et al., 2019; Gibbings et al., 2017), artery wall (Lim our study, we posited that the role of these macrophages in af- et al., 2018), and mammary gland (Wang et al., 2020). Indeed, fecting tumor expansion might not be restricted to the omen- although it is clear that organs have unique resident macro- tum. To address this issue, we surgically removed the omentum phages, it is also emerging that some macrophage phenotypes in two different experimental scenarios, with one scenario in- are found more broadly across all organs. We argue that the volving tumors that were allowed to be conditioned by omental cells we have characterized here should be called interstitial factors that enhance the aggressiveness of the tumor but then peritoneal macrophages to help distinguish them from serous reimplanted into mice wherein the omentum had been surgi- fluid-borne macrophages of the peritoneal cavity and to si- cally removed. We could not remove the lesser omentum due to multaneously underscore their potential common features with its key role in maintaining viability of portions of the stomach the interstitial macrophages of other organs (Chakarov et al., and spleen, so we cannot eliminate a role for lesser omental 2019; Gibbings et al., 2017). As noted in these past studies and macrophages. However, we underscore that the number of recapitulated in our present study in the peritoneal cavity, their omental macrophages is much smaller than those lining the phenotype is oriented toward an alternatively activated, or M2, peritoneal compartment overall. Furthermore, although it was state and to the maintenance and remodeling of extracellular not directly stated in the previous publication, it is highly un- matrix, perhaps especially relevant in light of the collagen- and likely that Etzerodt et al. (2020) removed the lesser omentum, matrix-rich environment these cells live within and their due to its importance in physiology. Finally, patients with possible role in maintaining the exterior barrier of the associ- ovarian cancer metastasis to the peritoneum undergo resection ated membranes and organ surfaces. We show that they appear of the omentum as routine debulking; thus, this study high- hi to be in a state of interdependence with neighboring stromal lighting a role for LYVE1 macrophages in tumor progression Zhang et al. Journal of Experimental Medicine 11 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 hi beyond the role of the greater omentum represents a clinically LYVE1 macrophage population. We intend to investigate this important finding (National Comprehensive Cancer Network, question in the future. 2021). Multiple distinct tumor immune microenvironments In closing, we have defined the phenotype of fixed tissue can coexist within ovarian cancer from the same patient and macrophages in the membranes, especially the membranes of likely contribute to heterogeneous responses of metastatic le- the mesentery, lining the peritoneal cavity. These findings in sions to therapy (Jimene ´ z-Sanc ´ hez et al., 2017). Seen in this light, turn reveal the complexity of the resident macrophage pool that hi our results raise the potential that mesothelial LYVE1 macro- can influence the peritoneal space, with at least two macro- phages could influence the growth of microscopic nonresected phages in the fluid and two in the membranes with distinct hi residual tumor and affect the response to adjuvant chemother- phenotypes. Genetic loss of LYVE1 macrophages through an apy and cancer recurrence. approach that takes advantage of their dependence on CSF1 and hi Our findings clearly point to a role for LYVE1 macrophages CSF1R allows us to demonstrate that, beyond the omentum, hi in promoting tumor progression under conditions when LYVE1 macrophages promote ovarian tumor growth in the omental macrophages are not relevant due to omental resection. peritoneal space. Future studies focused on the biology of resi- hi This approach allows us to reveal the role of LYVE1 macro- dent peritoneal macrophages must take into account all of the phages beyond the omentum, but the design does not allow us to relevant macrophages in the compartment. hi independently address whether the LYVE1 macrophages of the omentum also contribute to the tumor expansion. We assume that, in presence of the omentum, they do. The question then Materials and methods turns to how these macrophages contribute to tumor progres- Mice sion. In general, M2-type macrophages are thought to drive Mice were maintained in specific pathogen–free (SPF) barrier tumor expansion, and one way they might do so is through facilities with 12-h light–dark cycle by the Division of Compar- extracellular matrix remodeling (DeNardo and Ruffell, 2019; ative Medicine, Washington University School of Medicine Noy and Pollard, 2014), highly consistent with the phenotypic (WUSM), or the Biological Resource Laboratory, University of hi orientation of the LYVE1 macrophages. However, one puzzle is Illinois at Chicago (UIC). All animal experiments and procedures that the ovarian tumor presence in peritoneal fluid rather than were approved by the Institutional Animal Care and Use Commit- hi CreERT2 on the membrane is most impacted by the loss of LYVE1 tees at WUSM and UIC. Csf1r mice (FVB-Tg(Csf1r-cre/Esr1*) cre tm1(cre)Ifo macrophages. A future direction will be to turn toward under- 1Jwp/J; Qian et al., 2011), Lyz2 mice (B6.129P2-Lyz2 /J; hi LSL-tdTomato standing how the LYVE1 macrophages orchestrate an altered Clausen et al., 1999), R26 mice (B6.Cg-Gt(ROSA) tm9(CAG-tdTomato)Hze YFP tumor response and whether they are directly involved in se- 26Sor /J; Madisen et al., 2010), CD11c creting relevant factors or act in other ways, such as condi- transgenic mice (B6.Cg-Tg(Itgax-Venus)1Mnz/J; Lindquist et al., gfp/+ tm1Litt tioning the stromal cells nearby through cell–cell contact. A 2004), CX CR1 mice (B6.129P2(Cg)-Cx3cr1 /J; Jung et al., CreERT2 tm3(cre/ERT2)Gco fl/fl limitation of our study, and a common limitation affecting the 2000), Prox1 mice (Prox1 /J), Gata6 mice tm2.1Sad cre previous study and many others in the field, is that while it is (Gata6 /J; Sodhi et al., 2006), Lyve1 mice (B6;129P2- hi tm1.1(EGFP/cre)Cys fl/fl likely that the deletion of local peritoneal-lining LYVE1 Lyve1 /J; Pham et al., 2010), Csf1r mice tm1.2Jwp a macrophages in the omentectomized mice accounts for the (B6.Cg-Csf1r /J; Li et al., 2006), CD45.1 mice (B6.SJL-Ptprc hi b cre tm1(EGFP/cre)Wtp reduced tumor growth,wecannot besurethatthe LYVE1 Pepc /BoyJ), and WT1 mice (Wt1 /J; Zhou et al., CreERT2 body cavity macrophages per se are the ones at play in con- 2008) were purchased from The Jackson Laboratory. Csf1r fl/fl trolling tumor growth. It remains possible, albeit perhaps mice and Gata6 mice were backcrossed to C57BL6 background hi unlikely, that LYVE1 macrophages resident in distal tissues using the Speed Congenics mouse genetics core at WUSM. CreERT2 play a key role. CX CR1 mice were reconstituted from the cryopreserved hi lo LYVE1 and LYVE1 interstitial peritoneal macrophages ex- sperm provided from S. Jung (Weizmann Institute of Science, cre LSL-DTA press many common genes, including some signature genes, but Rehovot, Israel; Yona et al., 2013). Adiponectin :R26 ΔCsf1r fl/fl others are not shared. In our depletion system, Lyve1 mice mice and Csf1 mice were kindly provided by Charles A. Harris hi lose LYVE1 macrophages in the peritoneal lining mesentery (WUSM, St. Louis, MO) and Jean Jiang (University of Texas lo/− + and other membranes. However, LYVE1 MHC II membrane- Health Science Center, San Antonio, TX; Harris et al., 2012), associated macrophages and other tissue-resident macrophages respectively. in peritoneum, spleen, lung, and brain are not deleted. In ΔCsf1r Lyve1 mice, we nonetheless observed a reduction in tumor Tamoxifen treatment lo/− expansion, indicating that LYVE1 macrophages are not Tamoxifen diet (500 mg/kg; Envigo) was fed ad libitum to adult hi CreERT2 LSL-tdTomato CreERT2 LSL-tdTomato functionally able to stand in for the LYVE1 macrophages, at Csf1r :Rosa26 ,Csf1r :Rosa26 : gfp/+ CreERT2 LSL-tdTomato least when it comes to supporting tumor expansion, despite CX CR1 , and Prox1 :Rosa26 mice for 3 wk. sharing location and some phenotypic similarity. It is interesting For fate mapping, 40 µg tamoxifen (Sigma-Aldrich) was injected hi CreERT2 LSL-tdTomato that, at birth, the LYVE1 macrophages appear almost exclu- i.p. into P1 pups of CX CR1 :Rosa26 mice. sively present, only to give way over time to sharing the space lo with the LYVE1 population that also is prone to inducing MHC Cell isolation and staining for flow cytometry lo/− II. We suspect that the LYVE1 macrophages are monocyte Blood cells were collected by puncture of submandibular cheek derived and can develop, if the right conditions exist, into the vessels into 2 mM EDTA–containing tubes. RBCs were removed Zhang et al. Journal of Experimental Medicine 12 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 by lysis buffer (BD) in accordance with the manufacturer’sin- Project Consortium (ImmGen; https://www.immgen.org/ structions. Blood leukocytes were then stained to detect surface ImmGenProtocols.html). Ly6G, CD11b, CD115, Siglec F, and Ly6C. Peritoneal cells were collected after injection of 5 ml HBSS containing 2 mM EDTA Ultra-low input (ULI) RNA-seq and 2% FBS into the peritoneal cavity. After blood and peritoneal Library preparation, quality controls and generation of data cells were collected, mice were perfused with PBS. Mesenteric were performed by ImmGen according to the standard operating membranes were isolated from gut mesentery, including WAT. procedure for ULI RNA-seq. Data can be accessed through GEO Brain, spleen, lung, and mesenteric membranes were enzy- accession no. GSE122108. Reads were aligned to the mouse ge- matically digested with collagenase types I and X (Sigma-Al- nome GRCm38/mm10 primary assembly (GENCODE) and gene drich), hyaluronidase (Sigma-Aldrich), and DNase (Roche) in a annotation Ver.M16 with STAR 2.5.4a. The raw read counts were gentle-shaking incubator (250 rpm, 37°C, 30 min), and then cells generated by featureCounts (http://subread.sourceforge.net/) were filtered through 70-µm cell strainers. For analysis of mi- and normalized with DESeq2 package from Bioconductor. The croglia of the brain, cells were resuspended in 40% Percoll and top 12,000 genes ranked by average gene expression were se- subjected to density centrifugation (2,000 g, 20 min at 20°C lected for differential expression analysis using the DESeq2. Top hi with no break/acceleration). To process spleens, isolated cells 100 differentially expressed genes in Lyve1 macrophage sam- were lysed with lysis buffer (BD). Total peritoneal cells, blood ples were used as gene signatures for the ensuing analysis. leukocytes, and tissue-resident macrophages were counted using Heatmaps and PCA plots were generated using the Phantasus an automated cell counter (Nexcelom). For quantification, these online service (Artyomov, 2021b). + + numbers were multiplied by the percentage of CD11b CD115 For the analysis of the open source scRNA-seq datasets (ac- + + + Ly6C monocytes and CD11b Ly6G neutrophils in blood, cession nos. GSE102665 and E-MTAB-8593), the Seurat package + + + lo CD45 F4/80 CD64 CD11b for red pulp macrophages in (Butler et al., 2018) was used. Raw reads in each cell were first + hi + lo spleen, CD45 CD11c CD64 CD11b for alveolar macrophages scaled by library size and then log-transformed. To improve + lo hi in lung, CD64 CD45 CD11b for microglia in brain, and downstream dimensionality reduction and clustering, any un- + + hi CD11b CD115 macrophages stained with F4/80 for LPM and wanted source of variation arising from the number of de- lo F4/80 for small peritoneal macrophages in peritoneum. tected molecules was first regressed out. Highly variable Single-cell suspensions collected from each tissue were genes were then identified and selected for PCA reduction of maintained on ice for staining. Dead cells were identified by high-dimensional data. The top 10 principal components were propidium iodide staining during flow cytometry. Antibodies selected for unsupervised clustering of cells. Clustering results purchased from BioLegend/Invitrogen or BD Biosciences were are shown in a t-SNE plot from Single Cell Navigator online used as follows; CD45 (30F11), CD45.1 (A20), CD45.2 (104), CD11b service (Artyomov, 2021a). GSEA was performed to test for the (M1/70), CD115 (AFS98), CD102 (ICAM2; 3C4(MIC2/4)), MHC II enrichment of cluster-specific gene sets at the top of the bulk (I-A/I-E; M5/114.15.2), Ly6C (HK1.4), Ly6G (1A8), CD11c (N418), RNA-seq genes ranked according to their differential expression hi CD170 (Siglec F; E50-2440), CD64 (FcRγI; X54-5/7.1), MerTK significance (Lyve1 macrophages versus all others). Violin and (DS5MMER), F4/80 (BM8), CD206 (Mrc1;C068C2), LYVE1 feature plots were generated using Seurat package. (ALY7), FOLR2 (10/FR2), and isotype controls (IgG2a κ chain, IgG1a, and IgG2b κ chain). Whole-mount imaging by confocal microscope Mesenteries of adult mice were detached from the associated gut Cell sorting and fixed with 4% paraformaldehyde (Thermo Fisher Scientific) To remove dead cells, cells stained with propidium iodide were containing 30% sucrose (Fisher) overnight at 4°C. Gut mesen- gated out during cell sorting on a BD Aria II instrument. CD3ε, teries still attached to the intestine of neonatal mice (P0–14) CD19, and Ly6G staining was used to exclude lymphocytes and were pinned on SYLGARD184 (Ellsworth; 4019862)-coated plates neutrophils in some tissues. For selection of brain microglia by and fixed with 4% paraformaldehyde overnight at 4°C. After sorting, cells were stained with CD45, CD11b, CD64, F4/80, and fixation, samples were stored at 4°C in PBS containing 0.01% CD206. For splenic red pulp macrophages, cells were stained sodium azide. For whole-mount imaging by confocal micro- with CD45, CD64, MerTK, F4/80, and CD11b. For lung alveolar scope, gut mesenteries were blocked in a solution containing 5% macrophages, cells were stained with CD45, CD11c, Siglec F, goat serum (Sigma-Aldrich; D9663) or 5% BSA (Sigma-Aldrich; hi CD64, and CD11b. For membrane-associated Lyve1 macro- A9576) overnight at 4°C. Samples were stained with rabbit-anti phages of gut mesentery, cells were stained with CD45, F4/80, LYVE1 (Abcam; 14917), rat-anti MHC II (Invitrogen; M5/ CD64, and LYVE1. For peritoneal macrophages, peritoneal cells 114.15.2), rat-Folr2 (BioLegend; 10/FR2), rat-anti CD206 (Bio- hi were stained with CD11b, CD115, MHCII, and ICAM2. For Ly6C Rad; MR5D3), and rat-anti-ICAM2 (Invitrogen; 3C4(mIC2/4), lo and Ly6C monocytes in blood, blood leukocytes were stained Alexa Fluor 488) diluted in 1% BSA and incubated overnight 4°C with CD11b, CD115, and Ly6C. For bulk RNA-seq, we double- with gentle agitation. Samples were washed with PBS then in- sorted tissue-resident macrophages (1,000 cells/replicate), cubated with secondary antibodies conjugated with Alexa Fluor and the sorted cells were directly collected into LoBind tubes 488, Cy3, or Alexa Fluor 647/Cy5 (Jackson ImmunoResearch) containing 5 µl of TCL lysis buffer (Qiagen) containing 1% overnight 4°C. After nuclei were further stained with bis Ben- β-mercaptoethanol, based on the Cell Preparation and Sorting zimide H 3342 (Sigma-Aldrich), macrophages in mesenteric Standard Operating Procedures of the Immunological Genome membranes and mesenteric fat tissues were visualized on a Zhang et al. Journal of Experimental Medicine 13 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 confocal microscope (Leica SPE/inverted Leica SP8 or Zeiss 880). (50 mM), penicillin-streptomycin (100 U/ml), and nonessen- All images were collected using Leica LAS X software, and tial amino acids (Godoy et al., 2013). ID8-A12 ascites cells were analysis was performed using Imaris software (Bitplane). generated by harvesting total ascites cells 12 wk after implanting 6 6 10 ID8 cells i.p. in C57BL6 mice. 10 ID8 or ID8-A12 cells were Estimation of serous membrane macrophage numbers in the injected i.p. into mice of different genotypes with or without peritoneal cavity and omentum omentectomy. Biweekly bioluminescence imaging was per- We estimated the approximate area of the parietal membrane formed to noninvasively quantify tumor burden in the perito- 2 2 (∼6cm ), back of peritoneal wall (∼6cm ), peritoneal mem- neal cavity. brane at the bottom of the peritoneal cavity (∼4cm ), and sur- faces of serous tissues (pancreas, ∼4cm ;gut mesentery, ∼8-12 IVIS imaging 2 2 cm ; and diaphragm, ∼4cm ). This area in total added up to In vivo bioluminescence imaging was performed on an IVIS 50 2 hi 32–36 cm . Our data show that Lyve1 macrophages ranged (PerkinElmer; Living Image 4.3.1), with exposures of 1 s to 1 min, from ∼245–380 cells/mm (Fig. 5 A). Therefore, we estimate that binning 2–8, field of view 12.5 cm, f/stop 1, and open filter. hi the total number of LYVE1 macrophages may be ∼0.78–1.36 × D-Luciferin (150 mg/kg in PBS; Gold Biotechnology) was injected 6 6 hi 10 cells per mouse (∼10 cells). For estimating LYVE1 mac- into the mice i.p. and imaged ventrally using isoflurane anes- rophages in the omentum, we first made a single-cell suspension thesia (2% vaporized in O ). The total photon flux (photons/s) hi and counted the yield of LYVE1 macrophages per mouse was measured from regions of interest using the Living Image omentum, deriving 0.5–1×10 . We then stained greater 2.6 program. omentum tissue from three mice for LYVE1 macrophages. By Cells and mesenteries were imaged using the IVIS 50 with 4 hi immunostaining, we estimated there were 2 × 10 LYVE1 (PerkinElmer; Living Image 4.3.1) 1-s to 1-min exposure, bin 4–8, macrophages, far less than in the peritoneal surfaces and field of view 12 cm, f/stop 1, and open filter after addition of mesenteries. 150 µg/ml D-luciferin (Gold Biotechnology). For analysis, a grid was placed over the plate, and total photon flux (photons/s) was BM transplantation measured using Living Image 2.6. Cre BM cells were isolated from the tibia and femur of LYVE1 : LSL-tdTomato R26 mice on a CD45.2 background. After lysis of RBC Omentectomy in BM cells, Tomato cells were sorted using a FACS Aria II Operative omentectomy in mice was accomplished under gen- system (BD). Sorted BM cells (>95% purity) were injected i.v. eral anesthesia by continuous inhalation of 2–3% isoflurane in into lethally irradiated (950 rad) CD45.1 congenic mice (1.5–3.0 × 60% oxygen using a veterinary vaporizer, and then mice were 10 cells/mouse). Recipient mice were euthanized and analyzed placed on a heating pad in a supine position. Through a midline 8–10 wk after BM transplantation. incision in the region of the stomach, the greater omentum was carefully exposed and removed via electrocautery. The midline Whole-mount imaging using two-photon microscopy incision was then closed with absorbable sutures in two layers. Cre LSL-tdTomato cEYP Lyz2 :R26 :CD11 mice and DyLight488-conjugated Mice were resuscitated with an i.p. injection of saline, given a lectin (50 µg/mouse; Vector Laboratories; DL1174)–injected BM- local injection of analgesia, and then allowed to recover in a transplanted mice were used for live imaging. Immediately warmed incubator. Removal of both the entire greater and lesser after sacrificing BM-transplanted mice, Tomato reporter– omentum resulted in malperfusion of the stomach and spleen labeled macrophages and DyLight488-labeled blood vascula- and thus was not feasible. tures of brain (including the skull), liver, pancreas, peri- toneum, and gut mesenteries were visualized through the Online supplemental material customized Leica SP8 two-photon microscope with a Mai Tai HP Figs. S1 and S2 accompany Fig. 1 and add additional information DeepSee laser (Spectra-Physics) and a 25×, 0.95-NA water- on characterization of mesenteric membrane macrophages, in- immersion objective. All images including the 3D video were col- cluding comparison to the liver surface macrophages (Fig. S1) lected by Leica LAS X software and generated by Imaris (Bitplane) and demonstration of independence from LPM (Fig. S2). Fig. S3 Cre LSL-tdTomato software. Liver and gut mesentery of Lyz2 :R26 : accompanies Fig. 2 andillustrates theneed touseaspecialized cEYP gfp/+ CD11 and CX CR1 mice were fixed with 4% paraformalde- BM transplant strategy to obtain faithful reporting of fluores- hi hyde (Affymetrix) overnight at 4°C, and tissues were stored in PBS cent tags to LYVE1 macrophages in adult mice. Fig. S4 supplies containing 0.01% sodium azide at overnight at 4°C until ready additional information to Fig. 3 on the computational analysis of to image. mesenteric membrane macrophages from a previously pub- lished dataset. Fig. S5 accompanies Fig. 5 and shows the lack cre fl/fl Tumor implantation of impact of the LYVE1 :Csf1r genotype on resident The original ID8 cell line, derived from spontaneous in vitro macrophage numbers in organs where macrophages do not malignant transformation of C57BL6 mouse ovarian surface express LYVE1. Video 1 illustrates contact between mesen- epithelial cells (Roby et al., 2000), was modified to express GFP teric membrane macrophages and peritoneal fluid macro- and firefly luciferase. These ID8 cells were cultured in RPMI phages. Video 2 and Video 3 show 3D rotational views of hi 1640 with heat-inactivated FBS (10%), L-glutamine (2 mM), LYVE1 macrophages in the meninges covering the brain Hepes (25 mM), sodium pyruvate (1 mM), 2-mercaptoethanol and in the parietal peritoneal membrane to support the Zhang et al. Journal of Experimental Medicine 14 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Maintains and Replenishes Splenic Red Pulp Macrophages. Immunity. conclusion that they are preferentially positioned on the 53:127–142.e7. https://doi.org/10.1016/j.immuni.2020.06.008 border of the tissue. Ben ´ ezec ´ h, C., N.T. Luu, J.A. Walker, A.A. Kruglov, Y. Loo, K. Nakamura, Y. Zhang, S. Nayar, L.H. Jones, A. Flores-Langarica, et al. 2015. Inflammation-induced formation of fat-associated lymphoid clusters. Nat. Immunol. 16:819–828. https://doi.org/10.1038/ni.3215 Acknowledgments Buechler, M.B., K.W. Kim, E.J. Onufer, J.W. Williams, C.C. Little, C.X. Dom- We thank Dr. Steffen Jung (Weizmann Institute of Science, Is- inguez, Q. Li, W. Sandoval, J.E. Cooper, C.A. Harris, et al. 2019. A Stromal Niche Defined by Expression of the Transcription Factor WT1 rael) and Charles Harris (WUSM) for providing mouse strains. Mediates Programming and Homeostasis of Cavity-Resident Macro- We also thank all the members of the Randolph laboratory at phages. Immunity. 51:119–130.e5. https://doi.org/10.1016/j.immuni.2019 WUSM and the Kim laboratory at UIC for helpful discussion or .05.010 Butler, A., P. Hoffman, P. Smibert, E. Papalexi, and R. Satija. 2018. Integrating reading the manuscript. We thank Christophe Benoist and col- single-cell transcriptomic data across different conditions, technolo- leagues at ImmGen for collecting samples and generating data gies, and species. Nat. Biotechnol. 36:411–420. https://doi.org/10.1038/ from ULI-RNAseq. We also thank WUSM and UIC Flow Cy- nbt.4096 ´ ´ Casanova-Acebes, M., M.P. Menendez-Gutierrez, J. Porcuna, D. Alvarez-Er- tometry Core. We are grateful for technical support from Julie ´ ´ rico, Y. Lavin, A. Garcıa, S. Kobayashi, J. Le Berichel, V. Nuñez, F. Were, Prior and Kathleen Duncan from the Molecular Imaging Center et al. 2020. RXRs control serous macrophage neonatal expansion and at WUSM. identity and contribute to ovarian cancer progression. Nat. Commun. 11: 1655. https://doi.org/10.1038/s41467-020-15371-0 This study was supported by National Institutes of Health Cecchini, M.G., M.G. Dominguez, S. Mocci, A. Wetterwald, R. Felix, H. grants R37 AI049653 (to G.J. Randolph); R01DK119147, DP1DK126190, Fleisch, O. Chisholm, W. Hofstetter, J.W. Pollard, and E.R. Stanley. and R01DK126753 (to K-W. Kim); R01AG045040 (to J.X. Jiang); 1994. Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of R01CA188900 (to B.H. Segal); R00HL138163 (to J.W. Williams); the mouse. Development. 120:1357–1372. https://doi.org/10.1242/dev T32DK077653 (to E.C. Erlich and E.J. Onufer); P50CA094056 (to .120.6.1357 WUSM Molecular Imaging Center); P30AR0737752 (to WUSM Chakarov, S., H.Y. Lim, L. Tan, S.Y. Lim, P. See, J. Lum, X.M. Zhang, S. Foo, S. Nakamizo, K. Duan, et al. 2019. Two distinct interstitial macrophage Rheumatic Disease Research Center); and P30CA091842 (to WUSM populations coexist across tissues in specific subtissular niches. Science. Siteman Cancer Center Small Animal Cancer Imaging shared re- 363:eaau0964. https://doi.org/10.1126/science.aau0964 source); and Welch Foundation grant AQ-1507 (to J.X. Jiang). R.S. Chau, Y.Y., R. Bandiera, A. Serrels, O.M. Mart´ ınez-Estrada, W. Qing, M. Lee, J. Slight, A. Thornburn, R. Berry, S. McHaffie, et al. 2014. Visceral and Czepielewski received support from the Lawrence C. Pakula, MD, subcutaneous fat have different origins and evidence supports a mes- IBD Research Fellowship (FA-2020-01-IBD-1). othelial source. Nat. Cell Biol. 16:367–375. https://doi.org/10.1038/ Author contributions: Conceptualization: K-W. Kim, N. ncb2922 Clausen, B.E., C. Burkhardt, W. Reith, R. Renkawitz, and I. Fors ¨ ter. 1999. Zhang, G.J. Randolph; Investigation: K-W. Kim, N. Zhang, S.H. Conditional gene targeting in macrophages and granulocytes using Kim, E.C. Erlich, E.J. Onufer, J. Kim, J. Ding, B.T. Saunders, J.R. LysMcre mice. Transgenic Res. 8:265–277. https://doi.org/10.1023/A: Dominguez, R.S. Czepielewski, B.A. Helmink, J.W. Williams; Dai, X.M., G.R. Ryan, A.J. Hapel, M.G. Dominguez, R.G. Russell, S. Kapp, V. Resources: G.J. Randolph, K-W. Kim, J.X. Jiang, B.H. Segal; Sylvestre, and E.R. Stanley. 2002. Targeted disruption of the mouse Formal analysis and visualization: N. Zhang, K-W. Kim, S.H. colony-stimulating factor 1 receptor gene results in osteopetrosis, Kim, J. Kim, J. Ding, A. Gainullina, B.T. Saunders, B.H. Zinsel- mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood. 99:111–120. https://doi meyer; Writing: K-W. Kim, N. Zhang, S.H. Kim, G.J. Randolph; .org/10.1182/blood.V99.1.111 Supervision: K-W. Kim, G.J. Randolph, N. Zhang. All authors David, B.A., R.M. Rezende, M.M. Antunes, M.M. Santos, M.A. Freitas Lopes, edited the manuscript. A.B. Diniz, R.V. Sousa Pereira, S.C. Marchesi, D.M. Alvarenga, B.N. Nakagaki, et al. 2016. Combination of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation Disclosures: J.W. Williams reported grants from American of Liver Myeloid Cells in Mice. Gastroenterology. 151:1176–1191. https:// Heart Association and grants from NIH NHLBI outside the doi.org/10.1053/j.gastro.2016.08.024 DeNardo, D.G., and B. Ruffell. 2019. Macrophages as regulators of tumour submitted work. No other disclosures were reported. immunity and immunotherapy. Nat. Rev. Immunol. 19:369–382. https:// doi.org/10.1038/s41577-019-0127-6 Submitted: 30 April 2021 Etzerodt, A., M. Moulin, T.K. Doktor, M. Delfini, N. Mossadegh-Keller, M. Bajenoff, M.H. Sieweke, S.K. Moestrup, N. Auphan-Anezin, and T. Revised: 13 September 2021 Lawrence. 2020. Tissue-resident macrophages in omentum promote Accepted: 14 October 2021 metastatic spread of ovarian cancer. J. Exp. Med. 217:e20191869. https:// doi.org/10.1084/jem.20191869 Gao, Q., Z. Yang, S. Xu, X. Li, X. Yang, P. Jin, Y. Liu, X. Zhou, T. Zhang, C. Gong, et al. 2019. Heterotypic CAF-tumor spheroids promote early References peritoneal metastatis of ovarian cancer. J. Exp. Med. 216:688–703. Artyomov, M. 2021a. Single Cell Navigator. https://artyomovlab.wustl.edu/ https://doi.org/10.1084/jem.20180765 scn/ (accessed October 26, 2021) Gautier, E.L., T. Shay, J. Miller, M. Greter, C. Jakubzick, S. Ivanov, J. Helft, A. Artyomov, M. 2021b. Phantasus. https://artyomovlab.wustl.edu/phantasus/ Chow, K.G. Elpek, S. Gordonov, et al. Immunological Genome Consor- (accessed October 26, 2021) tium. 2012. Gene-expression profiles and transcriptional regulatory Bain, C.C., A. Bravo-Blas, C.L. Scott, E.G. Perdiguero, F. Geissmann, S. Henri, pathways that underlie the identity and diversity of mouse tissue B. Malissen, L.C. Osborne, D. Artis, and A.M. Mowat. 2014. Constant macrophages. Nat. Immunol. 13:1118–1128. https://doi.org/10.1038/ni replenishment from circulating monocytes maintains the macrophage .2419 pool in the intestine of adult mice. Nat. Immunol. 15:929–937. https://doi Gautier, E.L., S. Ivanov, J.W. Williams, S.C. Huang, G. Marcelin, K. Fairfax, .org/10.1038/ni.2967 P.L. Wang, J.S. Francis, P. Leone, D.B. Wilson, et al. 2014. Gata6 regu- Bellomo, A., I. Mondor, L. Spinelli, M. Lagueyrie, B.J. Stewart, N. Brouilly, B. lates aspartoacylase expression in resident peritoneal macrophages and Malissen, M.R. Clatworthy, and M. Bajeno ´ ff. 2020. Reticular Fibroblasts controls their survival. J. Exp. Med. 211:1525–1531. https://doi.org/10 Expressing the Transcription Factor WT1 Define a Stromal Niche that .1084/jem.20140570 Zhang et al. Journal of Experimental Medicine 15 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Ghosn, E.E., A.A. Cassado, G.R. Govoni, T. Fukuhara, Y. Yang, D.M. Monack, Lacerda Mariano, L., M. Rousseau, H. Varet, R. Legendre, R. Gentek, J. Saenz K.R. Bortoluci, S.R. Almeida, L.A. Herzenberg, and L.A. Herzenberg. Coronilla, M. Bajenoff, E. Gomez Perdiguero, and M.A. Ingersoll. 2020. 2010. Two physically, functionally, and developmentally distinct peri- Functionally distinct resident macrophage subsets differentially shape toneal macrophage subsets. Proc. Natl. Acad. Sci. USA. 107:2568–2573. responses to infection in the bladder. Sci. Adv. 6:eabc5739. https://doi https://doi.org/10.1073/pnas.0915000107 .org/10.1126/sciadv.abc5739 Gibbings, S.L., S.M. Thomas, S.M. Atif, A.L. McCubbrey, A.N. Desch, T. Lee, L.K., Y. Ghorbanian, W. Wang, Y. Wang, Y.J. Kim, I.L. Weissman, M.A. Danhorn, S.M. Leach, D.L. Bratton, P.M. Henson, W.J. Janssen, and C.V. Inlay, and H.K.A. Mikkola. 2016. LYVE1 Marks the Divergence of Yolk Jakubzick. 2017. Three Unique Interstitial Macrophages in the Murine Sac Definitive Hemogenic Endothelium from the Primitive Erythroid Lung at Steady State. Am. J. Respir. Cell Mol. Biol. 57:66–76. https://doi Lineage. Cell Rep. 17:2286–2298. https://doi.org/10.1016/j.celrep.2016.10 .org/10.1165/rcmb.2016-0361OC .080 Godoy, H.E., A.N. Khan, R.R. Vethanayagam, M.J. Grimm, K.L. Singel, N. Leinster, D.A., H. Kulbe, G. Everitt, R. Thompson, M. Perretti, F.N. Gavins, D. Kolomeyevskaya, K.J. Sexton, A. Parameswaran, S.I. Abrams, K. Odunsi, Cooper, D. Gould, D.P. Ennis, M. Lockley, et al. 2012. The peritoneal and B.H. Segal. 2013. Myeloid-derived suppressor cells modulate im- tumour microenvironment of high-grade serous ovarian cancer. J. Pathol. mune responses independently of NADPH oxidase in the ovarian tumor 227:136–145. https://doi.org/10.1002/path.4002 microenvironment in mice. PLoS One. 8:e69631. https://doi.org/10.1371/ Lengyel, E. 2010. Ovarian cancer development and metastasis. Am. J. Pathol. journal.pone.0069631 177:1053–1064. https://doi.org/10.2353/ajpath.2010.100105 Gomez Perdiguero, E., K. Klapproth, C. Schulz, K. Busch, E. Azzoni, L. Crozet, Lengyel, E., J.E. Burdette, H.A. Kenny, D. Matei, J. Pilrose, P. Haluska, K.P. H. Garner, C. Trouillet, M.F. de Bruijn, F. Geissmann, and H.R. Rode- Nephew, D.B. Hales, and M.S. Stack. 2014. Epithelial ovarian cancer wald. 2015. Tissue-resident macrophages originate from yolk-sac-de- experimental models. Oncogene. 33:3619–3633. https://doi.org/10.1038/ rived erythro-myeloid progenitors. Nature. 518:547–551. https://doi onc.2013.321 .org/10.1038/nature13989 Li, J., K. Chen, L. Zhu, and J.W. Pollard. 2006. Conditional deletion of the Han, S.J., A. Glatman Zaretsky, V. Andrade-Oliveira, N. Collins, A. Dzutsev, J. colony stimulating factor-1 receptor (c-fms proto-oncogene) in mice. Shaik, D. Morais da Fonseca, O.J. Harrison, S. Tamoutounour, A.L. Byrd, Genesis. 44:328–335. https://doi.org/10.1002/dvg.20219 et al. 2017. White Adipose Tissue Is a Reservoir for Memory T Cells and Lim, H.Y., S.Y. Lim, C.K. Tan, C.H. Thiam, C.C. Goh, D. Carbajo, S.H.S. Chew, Promotes Protective Memory Responses to Infection. Immunity. 47: P. See, S. Chakarov, X.N. Wang, et al. 2018. Hyaluronan Receptor LYVE- 1154–1168.e6. https://doi.org/10.1016/j.immuni.2017.11.009 1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan- Harris, S.E., M. MacDougall, D. Horn, K. Woodruff, S.N. Zimmer, V.I. Rebel, Mediated Regulation of Smooth Muscle Cell Collagen. Immunity. 49:1191. R. Fajardo, J.Q. Feng, J. Gluhak-Heinrich, M.A. Harris, and S. Abboud https://doi.org/10.1016/j.immuni.2018.12.009 Werner. 2012. Meox2Cre-mediated disruption of CSF-1 leads to osteo- Lindquist, R.L., G. Shakhar, D. Dudziak, H. Wardemann, T. Eisenreich, M.L. petrosis and osteocyte defects. Bone. 50:42–53. https://doi.org/10.1016/j Dustin, and M.C. Nussenzweig. 2004. Visualizing dendritic cell net- .bone.2011.09.038 works in vivo. Nat. Immunol. 5:1243–1250. https://doi.org/10.1038/ Hoeffel, G., J. Chen, Y. Lavin, D. Low, F.F. Almeida, P. See, A.E. Beaudin, J. ni1139 Lum, I. Low, E.C. Forsberg, et al. 2015. C-Myb(+) erythro-myeloid Liu, M., A. Silva-Sanchez, T.D. Randall, and S. Meza-Perez. 2021. Specialized progenitor-derived fetal monocytes give rise to adult tissue-resident immune responses in the peritoneal cavity and omentum. J. Leukoc. Biol. macrophages. Immunity. 42:665–678. https://doi.org/10.1016/j.immuni 109:717–729. https://doi.org/10.1002/JLB.5MIR0720-271RR .2015.03.011 Louwe, P.A., L. Badiola Gomez, H. Webster, G. Perona-Wright, C.C. Bain, S.J. Hogg, C., K. Panir, P. Dhami, M. Rosser, M. Mack, D. Soong, J.W. Pollard, S.J. Forbes, and S.J. Jenkins. 2021. Recruited macrophages that colonize the Jenkins, A.W. Horne, and E. Greaves. 2021. Macrophages inhibit and post-inflammatory peritoneal niche convert into functionally divergent enhance endometriosis depending on their origin. Proc. Natl. Acad. Sci. resident cells. Nat. Commun. 12:1770. https://doi.org/10.1038/s41467-021 USA. 118:e2013776118. https://doi.org/10.1073/pnas.2013776118 -21778-0 Ivanov, S., A. Gallerand, M. Gros, M.I. Stunault, J. Merlin, N. Vaillant, L. Madisen, L., T.A. Zwingman, S.M. Sunkin, S.W. Oh, H.A. Zariwala, H. Gu, L.L. Yvan-Charvet, and R.R. Guinamard. 2019. Mesothelial cell CSF1 sustains Ng, R.D. Palmiter, M.J. Hawrylycz, A.R. Jones, et al. 2010. A robust and peritoneal macrophage proliferation. Eur. J. Immunol. 49:2012–2018. high-throughput Cre reporting and characterization system for the https://doi.org/10.1002/eji.201948164 whole mouse brain. Nat. Neurosci. 13:133–140. https://doi.org/10.1038/ Jackson-Jones, L.H., P. Smith, J.R. Portman, M.S. Magalhaes, K.J. Mylonas, nn.2467 M.M. Vermeren, M. Nixon, B.E.P. Henderson, R. Dobie, S. Vermeren, Moro, K., T. Yamada, M. Tanabe, T. Takeuchi, T. Ikawa, H. Kawamoto, J. et al. 2020. Stromal Cells Covering Omental Fat-Associated Lymphoid Furusawa, M. Ohtani, H. Fujii, and S. Koyasu. 2010. Innate production Clusters Trigger Formation of Neutrophil Aggregates to Capture Peri- of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lym- toneal Contaminants. Immunity. 52:700–715.e6. https://doi.org/10.1016/ phoid cells. Nature. 463:540–544. https://doi.org/10.1038/nature08636 j.immuni.2020.03.011 National Comprehensive Cancer Network. 2021. National Comprehensive ´ ´ Jimenez-Sanchez, A., D. Memon, S. Pourpe, H. Veeraraghavan, Y. Li, H.A. Cancer Network Guidelines 2021. https://www.nccn.org (accessed Oc- Vargas, M.B. Gill, K.J. Park, O. Zivanovic, J. Konner, et al. 2017. Heter- tober 26, 2021) ogeneous Tumor-Immune Microenvironments among Differentially Noy, R., and J.W. Pollard. 2014. Tumor-associated macrophages: from Growing Metastases in an Ovarian Cancer Patient. Cell. 170: mechanisms to therapy. Immunity. 41:49–61. https://doi.org/10.1016/j 927–938.e20. https://doi.org/10.1016/j.cell.2017.07.025 .immuni.2014.06.010 Jung, S., J. Aliberti, P. Graemmel, M.J. Sunshine, G.W. Kreutzberg, A. Sher, Okabe, Y., and R. Medzhitov. 2014. Tissue-specific signals control reversible and D.R. Littman. 2000. Analysis of fractalkine receptor CX(3)CR1 program of localization and functional polarization of macrophages. function by targeted deletion and green fluorescent protein reporter Cell. 157:832–844. https://doi.org/10.1016/j.cell.2014.04.016 gene insertion. Mol. Cell. Biol. 20:4106–4114. https://doi.org/10.1128/ Pham, T.H., P. Baluk, Y. Xu, I. Grigorova, A.J. Bankovich, R. Pappu, S.R. MCB.20.11.4106-4114.2000 Coughlin, D.M. McDonald, S.R. Schwab, and J.G. Cyster. 2010. Lym- Kim, K.W., J.W. Williams, Y.T. Wang, S. Ivanov, S. Gilfillan, M. Colonna, H.W. phatic endothelial cell sphingosine kinase activity is required for lym- Virgin, E.L. Gautier, and G.J. Randolph. 2016. MHC II+ resident peri- phocyte egress and lymphatic patterning. J. Exp. Med. 207:17–27. https:// toneal and pleural macrophages rely on IRF4 for development from doi.org/10.1084/jem.20091619 circulating monocytes. J. Exp. Med. 213:1951–1959. https://doi.org/10 Qian, B.Z., J. Li, H. Zhang, T. Kitamura, J. Zhang, L.R. Campion, E.A. Kaiser, .1084/jem.20160486 L.A. Snyder, and J.W. Pollard. 2011. CCL2 recruits inflammatory mon- Kim, J.S., M. Kolesnikov, S. Peled-Hajaj, I. Scheyltjens, Y. Xia, S. Trzebanski, ocytes to facilitate breast-tumour metastasis. Nature. 475:222–225. Z. Haimon, A. Shemer, A. Lubart, H. Van Hove, et al. 2021. A Binary Cre https://doi.org/10.1038/nature10138 Transgenic Approach Dissects Microglia and CNS Border-Associated Robinson-Smith, T.M., I. Isaacsohn, C.A. Mercer, M. Zhou, N. Van Rooijen, N. Macrophages. Immunity. 54:176–190.e7. https://doi.org/10.1016/j Husseinzadeh, M.M. McFarland-Mancini, and A.F. Drew. 2007. Mac- .immuni.2020.11.007 rophages mediate inflammation-enhanced metastasis of ovarian tu- Koga, S., K. Hozumi, K.I. Hirano, M. Yazawa, T. Terooatea, A. Minoda, T. mors in mice. Cancer Res. 67:5708–5716. https://doi.org/10.1158/0008 + + Nagasawa, S. Koyasu, and K. Moro. 2018. Peripheral PDGFRα gp38 -5472.CAN-06-4375 mesenchymal cells support the differentiation of fetal liver-derived Roby, K.F., C.C. Taylor, J.P. Sweetwood, Y. Cheng, J.L. Pace, O. Tawfik, D.L. ILC2. J. Exp. Med. 215:1609–1626. https://doi.org/10.1084/jem.20172310 Persons, P.G. Smith, and P.F. Terranova. 2000. Development of a Zhang et al. Journal of Experimental Medicine 16 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 syngeneic mouse model for events related to ovarian cancer. Carcino- Williams, J.W., K. Zaitsev, K.W. Kim, S. Ivanov, B.T. Saunders, P.R. Schrank, genesis. 21:585–591. https://doi.org/10.1093/carcin/21.4.585 K. Kim, A. Elvington, S.H. Kim, C.G. Tucker, et al. 2020. Limited pro- Rosas, M., L.C. Davies, P.J. Giles, C.T. Liao, B. Kharfan, T.C. Stone, V.B. liferation capacity of aortic intima resident macrophages requires O’Donnell, D.J. Fraser, S.A. Jones, and P.R. Taylor. 2014. The tran- monocyte recruitment for atherosclerotic plaque progression. Nat. scription factor Gata6 links tissue macrophage phenotype and prolif- Immunol. 21:1194–1204. https://doi.org/10.1038/s41590-020-0768-4 erative renewal. Science. 344:645–648. https://doi.org/10.1126/science Wu, Z., J. Xu, J. Tan, Y. Song, L. Liu, F. Zhang, Y. Zhang, X. Li, Y. Chi, and Y. .1251414 Liu. 2019. Mesenteric adipose tissue B lymphocytes promote local and Siegel, R.L., K.D. Miller, and A. Jemal. 2018. Cancer statistics, 2018. CA Cancer hepatic inflammation in non-alcoholic fatty liver disease mice. J. Cell. J. Clin. 68:7–30. https://doi.org/10.3322/caac.21442 Mol. Med. 23:3375–3385. https://doi.org/10.1111/jcmm.14232 Sierro, F., M. Evrard, S. Rizzetto, M. Melino, A.J. Mitchell, M. Florido, L. Xia, H., S. Li, X. Li, W. Wang, Y. Bian, S. Wei, S. Grove, W. Wang, L. Vatan, J.R. Beattie, S.B. Walters, S.S. Tay, B. Lu, et al. 2017. A Liver Capsular Liu, et al. 2020. Autophagic adaptation to oxidative stress alters peri- Network of Monocyte-Derived Macrophages Restricts Hepatic Dis- toneal residential macrophage survival and ovarian cancer metastasis. semination of Intraperitoneal Bacteria by Neutrophil Recruitment. JCI Insight. 5:e141115. https://doi.org/10.1172/jci.insight.141115 Immunity. 47:374–388.e6. https://doi.org/10.1016/j.immuni.2017.07.018 Yona, S., K.W. Kim, Y. Wolf, A. Mildner, D. Varol, M. Breker, D. Strauss-Ayali, Singel, K.L., T.R. Emmons, A.N.H. Khan, P.C. Mayor, S. Shen, J.T. Wong, K. S. Viukov, M. Guilliams, A. Misharin, et al. 2013. Fate mapping reveals Morrell, K.H. Eng, J. Mark, R.B. Bankert, et al. 2019. Mature neutrophils origins and dynamics of monocytes and tissue macrophages under suppress T cell immunity in ovarian cancer microenvironment. JCI homeostasis. Immunity. 38:79–91. https://doi.org/10.1016/j.immuni Insight. 4:e122311. https://doi.org/10.1172/jci.insight.122311 .2012.12.001 Sodhi, C.P., J. Li, and S.A. Duncan. 2006. Generation of mice harbouring a Zhang, N., R.S. Czepielewski, N.N. Jarjour, E.C. Erlich, E. Esaulova, B.T. Sa- conditional loss-of-function allele of Gata6. BMC Dev. Biol. 6:19. https:// unders, S.P. Grover, A.C. Cleuren, G.J. Broze, B.T. Edelson, et al. 2019a. doi.org/10.1186/1471-213X-6-19 Expression of factor V by resident macrophages boosts host defense in Stamatiades, E.G., M.E. Tremblay, M. Bohm, L. Crozet, K. Bisht, D. Kao, C. the peritoneal cavity. J. Exp. Med. 216:1291–1300. https://doi.org/10 Coelho, X. Fan, W.T. Yewdell, A. Davidson, et al. 2016. Immune Moni- .1084/jem.20182024 toring of Trans-endothelial Transport by Kidney-Resident Macro- Zhang, S., I. Dolgalev, T. Zhang, H. Ran, D.A. Levine, and B.G. Neel. 2019b. Both fallopian tube and ovarian surface epithelium are cells-of-origin phages. Cell. 166:991–1003. https://doi.org/10.1016/j.cell.2016.06.058 Steinkamp, M.P., K.K. Winner, S. Davies, C. Muller, Y. Zhang, R.M. Hoffman, for high-grade serous ovarian carcinoma. Nat. Commun. 10:5367. A. Shirinifard, M. Moses, Y. Jiang, and B.S. Wilson. 2013. Ovarian tumor https://doi.org/10.1038/s41467-019-13116-2 attachment, invasion, and vascularization reflect unique microenviron- Zhou, B., Q. Ma, S. Rajagopal, S.M. Wu, I. Domian, J. Rivera-Feliciano, D. ments in the peritoneum: insights from xenograft and mathematical Jiang, A. von Gise, S. Ikeda, K.R. Chien, and W.T. Pu. 2008. Epicardial models. Front. Oncol. 3:97. https://doi.org/10.3389/fonc.2013.00097 progenitors contribute to the cardiomyocyte lineage in the developing Uderhardt, S., A.J. Martins, J.S. Tsang, T. Lamm ¨ ermann, and R.N. Germain. heart. Nature. 454:109–113. https://doi.org/10.1038/nature07060 2019. Resident Macrophages Cloak Tissue Microlesions to Prevent Zhu, Y., J.M. Herndon, D.K. Sojka, K.W. Kim, B.L. Knolhoff, C. Zuo, D.R. Neutrophil-Driven Inflammatory Damage. Cell. 177:541–555.e17. https:// Cullinan, J. Luo, A.R. Bearden, K.J. Lavine, et al. 2017. Tissue-Resident doi.org/10.1016/j.cell.2019.02.028 Macrophages in Pancreatic Ductal Adenocarcinoma Originate from Wang, J., and P. Kubes. 2016. A Reservoir of Mature Cavity Macrophages that Embryonic Hematopoiesis and Promote Tumor Progression. Im- Can Rapidly Invade Visceral Organs to Affect Tissue Repair. Cell. 165: munity. 47:323–338.e6. https://doi.org/10.1016/j.immuni.2017.07 668–678. https://doi.org/10.1016/j.cell.2016.03.009 .014 Wang, Y., K.J. Szretter, W. Vermi, S. Gilfillan, C. Rossini, M. Cella, A.D. Zigmond, E., C. Varol, J. Farache, E. Elmaliah, A.T. Satpathy, G. Friedlander, Barrow, M.S. Diamond, and M. Colonna. 2012. IL-34 is a tissue- M. Mack, N. Shpigel, I.G. Boneca, K.M. Murphy, et al. 2012. Ly6C hi restricted ligand of CSF1R required for the development of Langer- monocytes in the inflamed colon give rise to proinflammatory effector hans cells and microglia. Nat. Immunol. 13:753–760. https://doi.org/10 cells and migratory antigen-presenting cells. Immunity. 37:1076–1090. .1038/ni.2360 https://doi.org/10.1016/j.immuni.2012.08.026 Wang, Y., T.S. Chaffee, R.S. LaRue, D.N. Huggins, P.M. Witschen, A.M. Zigmond, E., B. Bernshtein, G. Friedlander, C.R. Walker, S. Yona, K.W. Kim, Ibrahim, A.C. Nelson, H.L. Machado, and K.L. Schwertfeger. 2020. O. Brenner, R. Krauthgamer, C. Varol, W. Müller, and S. Jung. 2014. Tissue-resident macrophages promote extracellular matrix homeosta- Macrophage-restricted interleukin-10 receptor deficiency, but not IL- sis in the mammary gland stroma of nulliparous mice. eLife. 9:e57438. 10 deficiency, causes severe spontaneous colitis. Immunity. 40:720–733. https://doi.org/10.7554/eLife.57438 https://doi.org/10.1016/j.immuni.2014.03.012 Weiss, J.M., L.C. Davies, M. Karwan, L. Ileva, M.K. Ozaki, R.Y. Cheng, L.A. Zindel, J., M. Peiseler, M. Hossain, C. Deppermann, W.Y. Lee, B. Haenni, B. Ridnour, C.M. Annunziata, D.A. Wink, and D.W. McVicar. 2018. Ita- Zuber, J.F. Deniset, B.G.J. Surewaard, D. Candinas, and P. Kubes. 2021. conic acid mediates crosstalk between macrophage metabolism and Primordial GATA6 macrophages function as extravascular platelets in peritoneal tumors. J. Clin. Invest. 128:3794–3805. https://doi.org/10.1172/ sterile injury. Science. 371:eabe0595. https://doi.org/10.1126/science JCI99169 .abe0595 Zhang et al. Journal of Experimental Medicine 17 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Supplemental material Zhang et al. Journal of Experimental Medicine S1 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 cre LSL-tdTomato EYFP Figure S1. Characterization of membrane-associated macrophages. (A) Two-photon images of liver capsule from Lyz2 :R26 :CD11c mice. cre Tomao EYFP Scale bar, 70 µm. (B) Two-photon images of mesenteric membrane from Lyz2 :R26 :CD11c mice. Scale bar, 40 µm. (C) Representative flow cy- EFYP EYFP + lo-to-hi + − tometric analysis of gut mesentery in CD11c mice. CD11c and CD11b gating of CD45 MHCII mesenteric cells (left). Overlay of CD11b EYFP and + + CreER Tomato CD11b EYFP cells (right; n =6). (D) Whole-mount confocal images of Csf1r :R26 mice with CD206 and ICAM2 staining. Imaging data are repre- hi sentative of at least two independent experiments. Scale bar, 40 µm. (E) Flow cytometric analysis for ICAM2, CD206, MHC II, and CD226 expression in F4/80 lo hi LPMs, F4/80 small peritoneal macrophages (SPM) and F4/80 mesenteric macrophages. Data are representative of at least two independent experiments. (F) Mean fluorescent intensity (MFI) values of ICAM2, CD206, MHC II, and CD226, which are normalized by isotype controls. Data are pooled from at least two independent experiments (n =3–6 mice). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple-comparison test: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Zhang et al. Journal of Experimental Medicine S2 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Cre fl/fl Figure S2. Mesenteric membrane–associated macrophages in Lyz2 :Gata6 mice. (A) Whole-mount images and quantification of membrane- cre fl/fl + associated macrophages of Lyz2 :Gata6 mice and littermate controls. Scale bar, 50 µm. (B) Quantification of ICAM2 peritoneal macrophages in cre fl/fl Lyz2 :Gata6 mice and littermate controls. Imaging data and flow cytometric analysis are representative of two independent experiments (n = 3 per genotype; mean ± SEM). Unpaired Student’s t test: ***, P < 0.001. cre LSL-tdTomato − Figure S3. Comparison of blood leukocytes between naive Lyve1 :R26 mice and Tomato BM-transplanted mice. (A) Tomato expression cre LSL-tdTomato − of blood leukocytes in naive Lyve1 :R26 mice (n =5). (B) Tomato expression of blood leukocytes from Tomato BM-transplanted mice (n = 4). Data are representative of at least two independent experiments. (C) Images of avascular region of a mesenteric membrane and the region containing adipose tissue in the BM-transplanted mice. Scale bars, 100 and 30 µm. Zhang et al. Journal of Experimental Medicine S3 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Figure S4. t-SNE plot identifying different cell populations in scRNA-seq of whole mesentery cells (accession no. GSE102665). (A) Signature genes that represent different cell populations of whole mesenteric cells. (B) t-SNE-plot for macrophage populations. (C) t-SNE-plot for dendritic cell populations. Zhang et al. Journal of Experimental Medicine S4 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Cre fl/fl Figure S5. Quantification of tissue-resident macrophages of Lyve1 :Csf1r and control mice. (A) Gating strategy of two peritoneal macrophage Cre fl/fl subsets and their quantification in Lyve1 :Csf1r mice and control mice. FSC, forward scatter; SSC, side scatter. (B) Gating strategy of splenic red pulp Cre fl/fl macrophages and their quantification in Lyve1 :Csf1r mice and control mice. (C) Gating strategy of alveolar macrophages and their quantification of in Cre fl/fl + Cre fl/fl Lyve1 :Csf1r mice and control mice. (D) Gating strategy and percentage of microglia in CD45 brain leukocytes of Lyve1 :Csf1r mice and control mice. + + (E) Representative histogram of CSF1R expression in ICAM2 and MHC II peritoneal macrophage subsets analyzed in A. In A–E, data were pooled from two independent experiments (n =5–8 mice). Statistical analysis was performed by unpaired Student’s t test. Zhang et al. Journal of Experimental Medicine S5 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Video 1. Video reconstruction of interactions of Tomato-expressing macrophages under Lyz2 promoter with CD11cEYFP-expressing peritoneal macrophages. Two-photon intravital microscope visualized Tomato-expressing mesenteric membrane macrophage in contact with CD11c EYFP-expressing lo F4/80 peritoneal macrophages. Video 2. Video reconstruction of Tomato-expressing macrophages driven by Lyve1 promoter in pia/dura mater of brain shown in Fig. 2 E. Tomato cre LSL-tdTomato BM cells of Lyve1 :R26 mice were transplanted into irradiated WT mice. The mice were used to visualize meningeal perivascular macrophages. Tomato-expressing macrophages were associated with blood vasculature underneath the skull. Video 3. Video reconstruction of Tomato-expressing macrophages driven by LYVE1 promoter in the peritoneal parietal membrane shown in Fig. 2 G. − cre LSL-tdTomato Tomato BM cells of Lyve1 :R26 mice were transplanted into irradiated WT mice. 10 wk later, the mice were injected with Alexa Fluor 488– conjugated lectin, and the parietal peritoneal membrane was visualized. Tomato-expressing macrophages were mainly located in the collagen-enriched serosal membrane of the parietal peritoneum. Zhang et al. Journal of Experimental Medicine S6 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

LYVE1+ macrophages of murine peritoneal mesothelium promote omentum-independent ovarian tumor growth

Loading next page...
 
/lp/pubmed-central/lyve1-macrophages-of-murine-peritoneal-mesothelium-promote-omentum-Cle4YY0is3

References (152)

Publisher
Pubmed Central
Copyright
© 2021 Zhang et al.
ISSN
0022-1007
eISSN
1540-9538
DOI
10.1084/jem.20210924
Publisher site
See Article on Publisher Site

Abstract

ARTICLE LYVE1 macrophages of murine peritoneal mesothelium promote omentum-independent ovarian tumor growth 1 2 1,3 1 1 2 1 Nan Zhang *,Seung Hyeon Kim *, Anastasiia Gainullina **, Emma C. Erlich **, Emily J. Onufer ,Jiseon Kim , Rafael S. Czepielewski , 5 2 1 2 1 4 6,7 Beth A. Helmink ,JosephR. Dominguez , Brian T. Saunders ,Jie Ding , Jesse W. Williams ,JeanX. Jiang , Brahm H. Segal , 1 1 1,2 Bernd H. Zinselmeyer , Gwendalyn J. Randolph ,and Ki-WookKim  Two resident macrophage subsets reside in peritoneal fluid. Macrophages also reside within mesothelial membranes lining the hi lo-hi peritoneal cavity, but they remain poorly characterized. Here, we identified two macrophage populations (LYVE1 MHC II lo/− lo/− hi hi CX CR1gfp and LYVE1 MHC II CX CR1gfp subsets) in the mesenteric and parietal mesothelial linings of the 3 3 peritoneum. These macrophages resembled LYVE1 macrophages within surface membranes of numerous organs. Fate- hi mapping approaches and analysis of newborn mice showed that LYVE1 macrophages predominantly originated from embryonic-derived progenitors and were controlled by CSF1 made by Wt1 stromal cells. Their gene expression profile closely overlapped with ovarian tumor-associated macrophages previously described in the omentum. Indeed, syngeneic hi epithelial ovarian tumor growth was strongly reduced following in vivo ablation of LYVE1 macrophages, including in mice that received omentectomy to dissociate the role from omental macrophages. These data reveal that the peritoneal compartment hi contains at least four resident macrophage populations and that LYVE1 mesothelial macrophages drive tumor growth independently of the omentum. Introduction Serous membranes line the peritoneal cavity as they generate a following tissue injury. However, whether these membrane- functional border for visceral organs. A major serosal surface associated macrophages are related to those in the peritoneal includes the gut-associated mesentery that anatomically bridges fluid in phenotype or origin is unknown. Indeed, the full phe- the intestines and mesenteric lymph nodes (MLNs). The mes- notypic and gene expression profile of peritoneal membrane– entery anchors the small intestine and colon and facilitates blood associated macrophages has not been reported. circulation and interstitial fluid flow through the mesenteric The peritoneal cavity contains serous fluid that hosts two lymphatic vessels to maintain tissue homeostasis. The serous types of resident macrophages that have been well characterized membranes of the mesentery are enriched in stromal cells such in recent years, the Gata6-dependent large peritoneal macro- as fibroblasts and mesothelial cells that produce vitamin A me- phages (LPMs; Gautier et al., 2014; Gautier et al., 2012; Ghosn tabolites that sustain peritoneal fluid macrophages (Buechler et al., 2010; Okabe and Medzhitov, 2014; Rosas et al., 2014)and et al., 2019), as well as collagens, elastin, laminin, and glyco- the IRF4-dependent small peritoneal macrophages (Ghosn et al., proteins that form a complex extracellular matrix (Jackson-Jones 2010; Kim et al., 2016). The LPMs float freely in peritoneal fluid et al., 2020). Resident macrophages on the serosal surface of the and participate critically in the entrapment and clearance of liver (David et al., 2016) and within parietal peritoneal mem- microorganisms that might gain entry to the cavity after breach branes (Uderhardt et al., 2019) have been described, and both of the intestinal boundary (Zhang et al., 2019a). The biol- populations play a role in governing recruitment of neutrophils ogy of the peritoneal macrophage has long been linked to the ............................................................................................................................................................................. 1 2 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO; Department of Pharmacology and Regenerative Medicine, 3 4 University of Illinois College of Medicine, Chicago, IL; Computer Technologies Department, ITMO University, St. Petersburg, Russia; Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX; Department of Surgery, Section of Surgical Oncology, Washington 6 7 University School of Medicine, St. Louis, MO; Departments of Internal Medicine and Immunology, Roswell Park Comprehensive Cancer Center, Buffalo, NY; Department of Medicine, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY. *N. Zhang and S.H. Kim contributed equally to this paper; **A. Gainullina and E.C. Erlich contributed equally to this paper; Correspondence to Gwendalyn J. Randolph: gjrandolph@wustl.edu; Ki-Wook Kim: kiwook@uic.edu; Nan Zhang: nzhang@wistar.org; N. Zhang’s present address is Wistar Institute, Philadelphia, PA. © 2021 Zhang et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Rockefeller University Press https://doi.org/10.1084/jem.20210924 1of 17 J. Exp. Med. 2021 Vol. 218 No. 12 e20210924 immunologic properties of the omental adipose tissue located in the absence of the omentum. Since omentectomy is standard within the peritoneal cavity. The omentum is home to collections in primary debulking surgery for ovarian cancer, our results hi of organized immune cells called milky spots or fat-associated raise the possibility that embryonic-derived mesenteric LYVE1 lymphoid clusters containing memory T cells (Han et al., 2017), macrophages may drive tumor progression and potentially Bcells (Wu et al., 2019), natural killer T cells (Ben ´ ez ´ ech et al., 2015), disease recurrence after initial therapy for metastatic ovarian dendritic cells, and innate lymphoid cells (Moro et al., 2010). Fur- cancer. thermore, the peritoneum can be affected by pathologies that in- clude postsurgical adhesions, endometriosis, and metastases of tumors most commonly arising from the colon, appendix, ovaries, Results or stomach. Experimental mouse models have begun to explore the Mesenteric membrane–associated macrophages are role of macrophages in these conditions (Hogg et al., 2021; Weiss phenotypically distinct from macrophages in the liver capsule et al., 2018; Xia et al., 2020; Zindel et al., 2021). and peritoneal cavity Epithelial ovarian cancer is the deadliest gynecological ma- The vascular and lymphatic tracts that connect the intestine lignancy, with high-grade serous ovarian cancer (HGSOC) being with the draining MLNs in mice are joined by avascular sheets of the most common subtype. HGSOC can originate from both tissue known to be lined by mesothelium and accompanying fallopian tube and ovarian surface epithelium (Zhang et al., fibroblasts (Fig. 1 A). Taking advantage of two myeloid-specific CreERT2 LSL-tdTomato Cre 2019b) and is commonly associated with widespread perito- reporter strains, Csf1r :Rosa26 and Lyz2 : LSL-tdTomato + neal carcinomatosis (Lengyel, 2010). More than 60% of patients Rosa26 mice, we observed elongated Tomato cells in with ovarian cancers are diagnosed at an advanced stage with these mesenteric membranes (Fig. 1, B and C). The cells were EYFP Cre peritoneal metastases (Siegel et al., 2018). HGSOC metastasis negative for enhanced YFP (EYFP) in CD11c mice (Lyz2 : LSL-tdTomato EYFP often leads to malignant ascites that is predominantly composed Rosa26 :CD11c ), distinguishing them from liver of inflammatory cells that include macrophages, neutrophils, capsule macrophages (David et al., 2016)thatwere positive for lymphocytes (Robinson-Smith et al., 2007; Singel et al., 2019), both EYFP and Tomato reporters in the respective strains (Fig. cancer-associated fibroblasts (Gao et al., 2019), and tumor cells. S1 A). We did, however, observe a few round-shaped Tomato HGOSC metastasis typically involves the peritoneal cavity, in- EYFP cells sparsely scattered on the mesenteric sheet (Fig. S1 cluding relevant adjacent tissues such as the omentum. In ad- B), and they were in close contact with Tomato cells (Video 1). + + dition to the omentum, peritoneal serosa and mesentery are also In flow cytometric analysis of gut mesentery, CD11b EYFP cells lo + common metastatic sites (Steinkamp et al., 2013); distant met- corresponded to the previously described F4/80 CD226 MHC + + − astatic seeding to abdominal organs and to the lungs and pleura II macrophages (Kim et al., 2016), but most CD11b EYFP cells also occurs. Contributions of different immune cells to this were F4/80 and did not express CD226 (Fig. S1 C). Instead, they metastatic evolution of cancer cells in the peritoneal environ- expressed high levels of CD206, which is absent on peritoneal ment remain understudied. Recently, Lawrence and colleagues macrophages (Fig. S1 D). ICAM2, which marks peritoneal mac- (Etzerodt et al., 2020) characterized omental macrophages and rophages (Gautier et al., 2012), was observed only on a few concluded that a major subset of omental macrophages could round-shaped Tomato cells sitting atop the mesenteric sheet hi lo account for the tumor-promoting role of the omentum. (Fig. S1 D). Flow cytometric analysis of F4/80 and F4/80 Here, we profiled mesenteric membrane–associated macro- peritoneal macrophage subsets and mesenteric macrophages in hi phages and identified two distinct populations (LYVE1 MHC the same mice confirmed that mesenteric macrophages are lo-hi lo/− lo/− hi hi II CX CR1gfp and LYVE1 MHC II CX CR1gfp subsets) positive for CD206 and MHC II. They were negative for ICAM2 3 3 that coexist in the mesothelial layer, generally resembling in- and CD226 (Fig. S1, E and F). The mean fluorescence intensity for terstitial macrophages described in the lung previously MHC II expression in mesenteric macrophages was relatively hi lo (Chakarov et al., 2019; Gibbings et al., 2017). Mesenteric LYVE1 lower than that of F4/80 peritoneal macrophages (Fig. S1 F). macrophages were derived from embryonic precursors and Overall, it appears that mesenteric membrane–associated mac- were controlled by colony stimulating factor 1 (CSF1) derived rophages are phenotypically distinct from the two well-established from local stromal cells. These macrophages did not depend on serous fluid peritoneal macrophage populations, which only GATA6 or IRF4 and maintained a life cycle distinct from that of infrequently attach to the mesenteric membrane in unper- peritoneal fluid macrophages. Bulk RNA sequencing (RNA-seq) turbed mice. An estimate of their number (see Materials and hi 6 analysis of LYVE1 membrane-associated macrophages and methods) at 10 within the peritoneal cavity suggests that their its comparison to single-cell RNA-seq (scRNA-seq) of omental total numbers in the peritoneal cavity are approximately sim- hi macrophages in ovarian tumors showed that LYVE1 macro- ilar to the number of resident macrophages in the peritoneal phages had a specialized gene expression pattern that correlated fluid (1–2× 10 ; Zhang et al., 2019a). By comparison, we esti- hi with the genes expressed by LYVE1 omental macrophages mated the number of macrophages in the omentum to be far during ovarian cancer progression (Etzerodt et al., 2020). Using lower, at 2 × 10 per omentum. in vivo ablation approaches in omentectomized mice, we un- hi raveled the relationship between LYVE1 macrophages, the Mesenteric membrane–associated macrophages consist of omentum, and ovarian tumor progression. Our data indicate that two distinct subsets hi LYVE1 macrophages within peritoneal membranes like those Some macrophage populations constitutively express GFP re- gfp/+ associated with the mesentery promote tumor progression even porter in CX CR1 mice (Bain et al., 2014; Gibbings et al., 2017; Zhang et al. Journal of Experimental Medicine 2of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Figure 1. Two distinct macrophage populations coexist in the avascular regions of mesenteric membranes. (A) Whole-mount images of gut mesentery in adult WT mice. Square box indicates a region of avascular mesenteric membrane. (B and C) Whole-mount images of the avascular mesenteric membrane CreERT2 LSL-tdTomato Cre LSL-tdTomato from tamoxifen-induced Csf1r :R26 mice (B) and Lyz2 :R26 mice (C). Scale bar, 50 µm. (D) Whole-mount images of mesenteric CreERT2 LSL-tdTomato gfp + membrane from tamoxifen-induced Csf1r :R26 :CX CR1 mice. CX CR1-GFP cells (left), Csf1r-expressing Tomato cells (middle), and merged 3 3 pictures (right) for two distinct macrophage populations. Scale bar, 50 µm. (E) Immunohistochemistry analysis of a whole-mount mesenteric membrane from gfp/+ CX CR1 mouse stained for LYVE1. CX CR1gfp expression (left), LYVE1 (middle), and merged pictures (right) for two distinct macrophage subsets. Scale bar, 3 3 hi lo/− lo/− hi 50 µm. (F) Quantification of LYVE1 CX CR1gfp macrophages and LYVE1 CX CR1gfp macrophages in mesenteric membranes. Data are representative of 3 3 three independent experiments (n = 3; mean ± SEM). Macrophages were quantified in two different regions of mesenteric membrane per mouse. Unpaired Cre LSL-tdTomato Student’s t test: ****, P < 0.0001. (G) Immunohistochemistry analysis of a whole-mount mesenteric membrane from Lyz2 :R26 mice stained with LYVE1 and MHCII. Scale bar, 50 µm. (H) Flow cytometric analysis of membrane-associated macrophages isolated from gut mesentery with CD45, F4/80, CD64, hi lo/− LYVE1, and MHC II staining. SSC, side scatter. (I) Frequency of LYVE1 membrane-associated macrophages and LYVE1 membrane-associated macrophages from flow cytometric analysis (H). Data are pooled from two independent experiments (n = 9; mean ± SEM). Unpaired Student’s t test: ****, P < 0.0001. All imaging data are representative of at least three independent experiments. Stamatiades et al., 2016; Williams et al., 2020; Zigmond et al., (Chakarov et al., 2019; Etzerodt et al., 2020; Lacerda Mariano gfp/+ 2014; Zigmond et al., 2012), while other tissue-resident macro- et al., 2020; Lim et al., 2018). In CX CR1 mice, we observed lo/− phages do not (Yona et al., 2013). To determine whether mes- that most CX CR1gfp macrophages highly expressed LYVE1, hi enteric membrane–associated macrophages express GFP reporter in while CX CR1gfp macrophages were negative or had low ex- gfp/+ CreERT2 LSL-tdTomato hi CX CR1 mice, Csf1r :Rosa26 mice were crossed pression for LYVE1 (Fig. 1 E). LYVE1 macrophages were the gfp CreERT2 LSL-tdTomato hi with CX CR1 mice to generate Csf1r :Rosa26 : dominant population, compared with CX CR1gfp macrophages 3 3 gfp/+ CX CR1 mice. In the mesenteric membranes of these dual re- (Fig. 1 F). hi lo/− porter mice, we observed two distinct macrophage subsets: To further characterize LYVE1 and LYVE1 macrophages, + lo/− + hi Tomato CX CR1gfp macrophages and Tomato CX CR1gfp membrane-associated macrophages were stained for MHC II in 3 3 Cre LSL-tdTomato hi lo/− macrophages (Fig. 1 D). To characterize these subsets further, Lyz2 :Rosa26 mice. Both LYVE1 and LYVE1 mesenteric membranes were stained for LYVE1, as it was re- membrane-associated macrophages expressed MHC II, albeit hi lo hi cently reported that two distinct LYVE1 and LYVE1 inter- more weakly in the LYVE1 macrophages, in images from stitial macrophage subsets are present together in some tissues confocal microscopy (Fig. 1 G). Through flow cytometric analysis Zhang et al. Journal of Experimental Medicine 3of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 of single-cell suspensions generated from gut mesentery, we resident in dura mater and pia mater as vascular-associated + + confirmed that F4/80 CD64 macrophages could be divided into macrophages (Kim et al., 2021), we found Tomato-labeled hi lo-hi lo/− two major populations: LYVE1 MHC II and LYVE1 MHC meningeal perivascular macrophages near the blood vascu- hi + II macrophages (Fig. 1 H). The frequency of the two macro- lature underneath the skull (Fig. 2 F and Video 2). Tomato phage subsets did not differ depending on whether they were macrophages were also concentrated in the collagen-enriched examined by confocal microscopy or flow cytometry (Fig. 1, F barrier surface of the pancreas (Fig. 2 G)and theparietal and I). peritoneal membrane (Fig. 2 H and Video 3), indicating that hi lo/− Both LYVE1 and LYVE1 membrane-associated macro- LYVE1-expressing macrophages were enriched at a number of phages were intact in the absence of Gata6 expression within barrier surfaces, including multiple mesothelial linings of the cre fl/fl + macrophages (Lyz2 :Gata6 mice; Fig. S2 A), whereas peri- peritoneal cavity. However, Tomato macrophageswereab- toneal macrophages were highly reduced (Fig. S2 B), indicating sent from the liver capsule (Fig. 2 I, left). At the liver surface, + gfp/+ that the membranous macrophages were maintained inde- we detected only GFP cells in CX CR1 mice (Fig. 2 I, pendently from peritoneal fluid macrophages. Taken together, right). mesenteric membrane–associated macrophages consist of two hi lo/− lo-hi hi distinct subsets: LYVE1 CX CR1 MHC II macrophages LYVE1 membrane-associated macrophages constitutively hi lo/− (henceforth termed LYVE1 macrophages) and LYVE1 display an alternatively activated macrophage (AAM) gene hi hi lo/− CX CR1 MHC II macrophages (henceforth termed LYVE1 expression profile macrophages). After bulk RNA-seq, principal component analysis (PCA) hi showed that LYVE1 macrophages cluster together as repli- hi LYVE1 membrane-associated macrophages are located on the cates. They clustered distinctly from other tissue-resident barrier surfaces of many organs macrophages including resident macrophages from peritoneal hi In the mesentery, LYVE1 macrophages, probed initially by lavage, lung, spleen, and brain or two blood monocyte subsets immunostaining for LYVE1, were rather evenly distributed (Fig. 3 A). Among the most highly up-regulated genes (≥16-fold across the mesenteric surface in both the avascular and vascular compared with the other macrophage populations) in the hi areas of the mesentery (Fig. 2 A). The vascularized area was rich mesenteric LYVE1 macrophages were Mgl2 (CD301b), Mmp9, in adipose tissue housing the nerves, blood vessels, and lym- Lyve1, C1qtnf1, Folr2, Cbr2, and AAM-related genes such as Retnla CreERT2 phatic vessels, marked using tamoxifen-treated Prox1 : (RELMα)and Mrc1 (CD206; Fig. 3 B). In addition to the up-regulation LSL-tdTomato R26 mice (Fig. 2 A). of canonical AAM-related genes, pathway analysis implemented LYVE1 is transiently expressed in erythroid-myeloid pro- by fast gene set enrichment analysis (fast GSEA) showed that genitors (EMPs) during embryogenesis and in hematopoietic pathways related to extracellular matrix organization such as stem cells (HSCs) in adult hematopoiesis (Lee et al., 2016). As collagen formation and degradation were enriched in mesenteric hi such, 50–70% of blood leukocytes expressed the Tomato reporter LYVE1 macrophages (Fig. 3 C). Cre LSL-tdTomato in Lyve1 :Rosa26 mice due to this embryonic history To further investigate and validate these findings, we rean- (Fig. S3 A). Thus, to visualize LYVE1-expressing macrophages alyzed an scRNA-seq dataset that examined whole mesenteric selectively using reporter mice, we designed a bone marrow cells (GEO accession no. GSE102665; Koga et al., 2018), produc- (BM) chimeric mouse model in which CD45.2 Tomato BM cells ing 16 clusters based on cell-specific gene expression repre- Cre LSL-tdTomato were first isolated from Lyve1 :Rosa26 mice by senting a range of cell types (Figs. 3 D and S4 A). We defined FACS (Fig. 2 B). They were then transplanted into lethally ir- clusters 3 and 8 of this t-distributed stochastic neighbor em- radiated CD45.1 congenic mice. 8–10 wk later, BM chimeric bedding (t-SNE) plot as macrophage populations due to ex- mice were analyzed by flow cytometry and microscopy. In pression of Cd68, Lyz2, Mrc1, Cd14,and Mgl2 (Fig. S4, A and B)and Cre LSL-tdTomato contrast to unmanipulated Lyve1 :Rosa26 mice, cluster 14 as a dendritic cell population due to coenrichment in Tomato reporter–expressing cells were not found among blood genes such as Cd209, Flt3,and Itgae (CD103; Fig. S4 C). The Lyve1, leukocytes in mice receiving the Tomato BM transplant (Fig. Mmp9,and Folr2 mRNA transcripts that were up-regulated in hi S3 B). Other tissue-resident macrophages such as microglia, red bulk RNA-seq of LYVE1 membrane-associated macrophages pulp macrophages, and alveolar macrophages also were not were selectively detected in cluster 3 (Fig. 3 E). Overall, cluster 3 hi lo labeled by the Tomato reporter due to marked radioresistance corresponded to both LYVE1 and LYVE1 mesenteric macro- and lack of LYVE1 expression (microglia) or radiosensitivity phage populations, whereas other macrophages corresponded to and lack of LYVE1 expression (e.g., spleen or alveolar macro- cluster 8 (Fig. 3, D and E;and Fig. S4 B). Folate receptor 2 ex- hi phages; Fig. 2, C and D). By contrast, mesenteric macrophages pression, encoded by Folr2 mRNA, was observed in LYVE1 lo were mainly radiosensitive, and >70% were highly labeled by and LYVE1 macrophages through flow cytometric analysis the Tomato reporter in flow cytometric analysis and imaging (Fig. 3 F), confirming that LYVE1 macrophages, including both hi lo (Fig. 2, C–E; and Fig. S3 C). LYVE1 and LYVE1 subsets, belong to cluster 3 (Fig. 3, D–F). Cre LSL-tdTomato Across a range of organs, Lyve1 :Rosa26 BM- Other AAM-associated genes, Mrc1 and Retnla, characterized transplanted mice were visualized by two-photon microscopy LYVE1-enriched macrophages in both datasets (Fig. 3 G), with immediately after intravenous injection of Alexa Fluor 488– many AAM genes including Retnla and Mrc1 also highly en- lo/− conjugated lectins to label blood vasculature. Consistent with riched in LYVE1 mesenteric macrophages. Thus, mesenteric hi the recent report that LYVE1 perivascular macrophages are barrier membrane macrophages are oriented toward support of Zhang et al. Journal of Experimental Medicine 4of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 hi Figure 2. Lyve1 membrane-associated macrophages are located in serous membrane of tissue parenchyma. (A) Whole-mount, confocal microscopy hi CreER LSL-tdTomato tile-scan reconstructions to examine the location and distribution of LYVE1 mesenteric macrophages in tamoxifen-induced Prox1 :R26 mice (red, Prox1-expressing lymphatic collector; white, LYVE1-expressing macrophages and lymphatic capillaries; blue, nuclei). Scale bar, 400 µm. Images are representative of two independent experiments with scanning of a large region of tissue. (B) Schemes for Tomato BM transplantation to whole-body-ir- − cre LSL-tdTomato radiated mice (created with BioRender.com). Sorted CD45.2 Tomato BM cells from Lyve1 :R26 mice were transplanted to irradiated congenic CD45.1 WT mice. Tomato reporter will label only adult macrophages with an active LYVE1 promoter in adulthood, bypassing embryo-restricted activity at this promoter. (C) Histogram showing Tomato reporter expression of tissue-resident macrophages in brain, spleen, lung, and gut mesentery of Tomato BM transplanted CD45.1 recipient mice. (D) Quantification of Tomato expression in donor-derived tissue-resident macrophages of Tomato BM transplanted CD45.1 mice. (C-D) Data are pooled from at least two independent experiments (n = 6, mean ± SEM). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple-comparison test: ****, P < 0.0001. (E) Whole mount images of the mesenteric membrane in Tomato BM transplanted chimeric mice (red, + − Tomato macrophages; blue, collagens imaged by second harmonic generation [SHG]). Scale bar, 20 µm. (F) Whole mount images of the meninge in Tomato BM transplanted chimeric mice (red, Tomato macrophages; green, Alexa488-conjugated lectin injected blood vessels; blue, skull imaged by SHG). Note + − Tomato perivascular macrophages underneath skull. Scale bar, 20 µm. (G) Whole mount images of pancreas from Tomato BM transplanted chimeric mice (red, Tomato macrophages; green, Alexa488-conjugated lectin injected blood vessels; blue, collagen bundles shown as SHG) Scale bar, 50 µm. (H) Whole- − + mount images of the parietal peritoneal membrane from Tomato BM transplanted chimeric mice (red, Tomato macrophages; green, Alexa488-conjugated lectin injected blood vessels; blue, collagens shown by SHG). Scale bar, 50 µm, (E, G, and H) Note Tomato membrane-associated macrophages in collagen- − gfp/+ enriched serosa membrane. (I) Comparison of liver capsular macrophages between Tomato BM-transplanted chimeric mice (left) and CX CR1 mice (right); + + red, Tomato macrophages; green, CX CR1gfp macrophages; blue, collagen. Scale bar, 50 µm. All two-photon microscopic images are representative of at least two independent experiments. Zhang et al. Journal of Experimental Medicine 5of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 hi + Figure 3. LYVE1 macrophages have their own gene expression patterns. (A) PCA of tissue-resident macrophages (alveolar macrophages, F4/80 hi hi lo peritoneal macrophages, microglia, splenic red pulp macrophages, and LYVE1 membrane-associated macrophages) and blood monocytes (Ly6C and Ly6C hi monocytes) obtained from RNA-seq dataset. PC, principal component. (B) Heatmap analysis of top 50 up-regulated genes of 12,000 genes expressed in LYVE1 hi membrane-associated macrophages. Heatmap depicts mean expression intensity of mRNA transcripts for genes differentially expressed between LYVE1 hi macrophages and other macrophages, including monocytes. (C) Pathway analysis of genes differentially expressed in LYVE1 membrane-associated mac- rophages implemented by fast GSEA, showing top 10 enriched pathways from Reactome database and Molecular Signatures Database. NES, normalized enrichment score. (D) t-SNE plot displaying reanalyzed scRNA-seq of whole mesentery cells (accession no. GSE102665). (E) Expression of Lyve1, MMP9,and hi lo Folr2 on the t-SNE plot of scRNA-seq described in D. (F) Flow cytometric analysis showing FOLR2 expression of LYVE1 and LYVE1 mesenteric macrophages. Data are representative of three mice. (G) Violin plot of Retnla and Mrc1 expression obtained from scRNA-seq described in D. extracellular matrix remodeling, likely needed to build and are established from CX CR1-expressing precursor cells dur- hi maintain the barrier in which the cells reside. ing embryogenesis (Yona et al., 2013). LYVE1 macrophages were already established in the mesenteric membrane of these hi hi LYVE1 membrane-associated macrophages originate from newborn mice (Fig. 4 A). Unlike LYVE1 macrophages from gfp/+ embryonic precursors adult CX CR1 mice, which harbored low expression of GFP hi Many peripheral tissue macrophages develop from embryonic reporter (Fig. 1, D and E), LYVE1 macrophages in the mes- precursors (EMPs or fetal liver monocytes; Gomez Perdiguero entery of newborn mice highly expressed GFP in >90% of lo/− + et al., 2015; Hoeffel et al., 2015; Yona et al., 2013). However, liver all mesenteric macrophages (Fig. 4 B). LYVE1 CX CR1gfp capsular macrophages are derived from circulating mono- macrophages (Fig. 4 A, arrows) accounted for <10% of total cytes, not embryonic precursors (Sierro et al., 2017). Thus, we macrophages in newborn mice (Fig. 4 C). In newborn mice, hi hi lo/− wondered whether LYVE1 mesenteric membrane–associated both LYVE1 and LYVE1 macrophagesweremore amoe- macrophages were derived from embryonic precursors or boid in morphology and rarely expressed MHC II, although were replenished from circulating monocytes. We first ex- MHC II was highly expressed in cells in the MLN. By P6, these gfp/+ lo/− amined membrane-associated macrophages in newborn CX CR1 macrophages elongated, and MHC II expression in some LYVE1 mice (postnatal day 0 [P0]), as most tissue-resident macrophages macrophages began to emerge within 2 wk after birth (Fig. 4 D). Zhang et al. Journal of Experimental Medicine 6of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 gfp/+ Figure 4. Membrane-associated macrophages originate from embryonic precursors. (A) Whole-mount images of mesentery in newborn (P0) CX CR1 lo/− + pups stained with LYVE1. Right-most image is the enlargement of the boxed area in the adjacent image. Arrows indicate LYVE1 CX CR1-GFP macrophages. Images are representative of two independent experiments. Scale bar, 100 µm; 30 µm (higher-magnification image). (B) CX CR1-GFP expression within the hi hi Lyve1 macrophage pool in the mesenteric membrane of newborn mice. GFP expression is quantified within total LYVE1 macrophage population. (C) The hi lo/− frequency of LYVE1 macrophages versus LYVE1 macrophages in the mesenteric membrane of newborn mice. In B and C, data are representative of two independent experiments (n = 4; mean ± SEM). Membrane-associated macrophages were quantified in one to three different regions of mesenteric membrane per mouse. Unpaired Student’s t test: ****, P < 0.0001. (D) Whole-mount images of MLNs and mesenteric membranes of P1, P6, and P14 neonatal mice stained with LYVE1 and MHCII. White, LYVE1; green, MHCII; blue, DAPI. The yellow line in the P1 panel indicates the border of mesenteric vessels and mesenteric membrane. Images are representative of at least two independent experiments per time point. Scale bar, 50 µm. (E) Representative whole-mount images of CreERT2 LSL-Tomato adult CX CR1 :R26 mice in which tamoxifen was injected on P1 (white, LYVE1; red, Tomato reporter). Scale bar, 50 µm. (F) Tomato expression in hi hi microglia, LYVE1 mesenteric membrane–associated macrophages and blood monocyte subsets. LYVE1 membrane-associated macrophages were quantified in two different regions of membrane per mouse from confocal microscopy images. Microglia and blood monocytes were quantified via flow cytometric analysis. In E and F, data are pooled from two independent experiments (n = 6; mean ± SEM). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple-comparison test: ****, P < 0.0001. hi We conclude that mesenteric membrane–associated macrophages are likely long-lived or self-renewed from P1-labeled LYVE1 are established during embryogenesis but undergo postnatal macrophages (Fig. 4 F). Collectively, these data suggest that most hi adaptations in phenotype and morphology. LYVE1 macrophages originate from embryonic precursors and hi To determine if embryonic derived LYVE1 macrophages are are then maintained locally for long periods. maintained through self-renewal, we performed tamoxifen- hi pulse labeling to track the LYVE1 macrophages after birth. Membrane-associated macrophages are controlled by CSF1 Accordingly, tamoxifen was injected i.p. into P1 pups of produced in Wt1-expressing stromal cells CreERT2 LSL-tdTomato CX CR1 :R26 mice. 8–10 wk later, Tomato re- It is well established that CSF1 receptor signaling is crucial for hi porter was visualized in LYVE1 macrophages (Fig. 4 E). Tomato the generation, differentiation, and survival of most tissue- hi reporter remained high in LYVE1 macrophages (80.08 ± 1.78%) resident macrophages (Cecchini et al., 1994; Dai et al., 2002; and microglia (96.28 ± 1.39%), while it was negative at these time Ivanov et al., 2019; Williams et al., 2020). Thus, we investigated hi lo hi points in blood Ly6C monocytes (0.09 ± 0.02%) and Ly6C whether LYVE1 macrophages required CSF1 receptor signaling. hi Cre fl/fl ΔCsf1r hi monocytes (2.19 ± 0.32%), suggesting that LYVE1 macrophages Indeed, in Lyve1 :Csf1r mice (Lyve1 mice), LYVE1 Zhang et al. Journal of Experimental Medicine 7of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Figure 5. Membrane-associated macrophages are controlled by CSF1 produced by stromal cells of serous membranes. (A) Whole-mount images of the Cre fl/fl fl/fl two distinct membrane-associated macrophages and their quantification in Lyve1 :Csf1r mice and littermate Csf1r control mice. Scale bar, 50 µm. Data are representative of three independent experiments (n = 3 per genotype; mean ± SEM). Macrophages were quantified in multiple regions of mesenteric membrane per mouse. Unpaired Student’s t test: ****, P < 0.0001. (B) Expression pattern of Csf1, Il34,and Wt1 depicted on the t-SNE plot derived from scRNA- cre LSL-tdTomato seq of whole mesenteric cells shown in Fig. 3 D. (C) Whole-mount images stained for LYVE1 in mesenteric membranes from WT1 :R26 mice. lo/− Representative images of three independent experiments. Arrows indicate LYVE1 macrophages (red, Wt1 Tomato reporter; green, LYVE1; blue, DAPI). Scale Cre fl/fl fl/fl bar, 20 µm. (D) Whole-mount images of mesenteric membranes from Wt1 :Csf1 mice and littermate Csf1 control mice. Representative images of two hi lo independent experiments. Scale bar, 100 µm. (E) Quantification of LYVE1 and LYVE1 membrane-associated macrophages obtained from whole-mount Cre fl/fl fl/fl fl/fl Cre fl/fl images of Wt1 :Csf1 mice and littermate Csf1 control mice. Data are pooled from two independent experiments (Csf1 mice, n =4; Wt1 :Csf1 mice, n = 6; mean ± SEM). Macrophages were quantified from multiple regions of mesenteric membrane per mouse. Unpaired Student’s t test: **, P < 0.01; ****, P < + fl/fl 0.0001. (F) Quantification of ICAM2 macrophages in peritoneal cavity. Data are representative of at least three independent experiments (Csf1 mice, n =4; Cre fl/fl hi Wt1 :Csf1 mice, n = 5; mean ± SEM). Unpaired Student’s t test: ****, P < 0.0001. (G) Quantification of Ly6C monocytes and neutrophils in blood. Data are fl/fl Cre fl/fl representative of three independent experiments (Csf1 mice, n =5; Wt1 :Csf1 mice, n = 4; mean ± SEM). Unpaired Student’s t test. (H) Confocal image cre LSL-DTA from the mesenteric membrane of Adiponectin :R26 mice and controls. Images are representative of two independent experiments. Scale bar, 50 µm. lo/− macrophages were reduced by ∼80%. The number of LYVE1 necessary for persistence of macrophages that might arise from hi MHC II macrophages was comparable to that of littermate Cre-recombinase activated HSC/EMP progenitors, such that controls (Fig. 5 A). Additionally, the number of other tissue- there would evolve a natural selection bias to greatly favor the resident macrophages known to lack LYVE1 expression resid- seeding of tissue macrophages arising from Cre-recombinase ΔCsf1r ing in peritoneum, spleen, lung, and brain of Lyve1 mice nonactivated HSC/EMP progenitors (30–50% of populations). was not significantly changed compared with standard WT mice Then, expression of LYVE1 by tissue-resident macrophages (Fig. S5, A–D), and Csf1 receptor is normally expressed in peri- would trigger selective loss of Csf1R, such that only LYVE1 ΔCsf1r toneal fluid macrophages of Lyve1 mice (Fig. S5 E). These macrophages would be substantially impacted. results imply that Csf1 receptor deficiency in the stage of EMP or CSF1 and IL34 are the ligands of CSF1 receptor (Wang et al., HSC has no impact on the pool of tissue-resident macrophages, 2012), so we examined mRNA for CSF1 and IL34 in the scRNA- hi and Lyve1 membrane-associated macrophages are selectively seq data from the whole mesentery. Csf1 in particular is highly depleted by the lack of Csf1 receptor–mediated signaling in up-regulated in Wilms tumor 1 homologue (Wt1)–expressing ΔCsf1r Lyve1 mice. This outcome may arise because Csf1R would be mesenteric fibroblasts and mesothelial cells (Fig. 5 B), and both Zhang et al. Journal of Experimental Medicine 8of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 lo/− hi hi LYVE1 macrophages (yellow arrows) and LYVE1 macro- expressed in cluster 10 were highly matched to the LYVE1 phages were located in proximity to Tomato-labeled stromal cells macrophages in GSEA (Fig. 6 D). We conclude that macro- cre LSL-tdTomato within the mesothelial membranes from Wt1 :R26 phages constitutively present in the mesenteric membrane mice (Fig. 5 C). To test whether membrane-associated macro- express a pattern of genes previously associated with omental phages were controlled by Csf1 produced by local stromal cells, macrophages that drive tumor progression. cre fl/fl ΔCsf1 we generated Wt1 :Csf1 mice (Wt1 mice) that would hi delete Csf1 in WT1-expressing fibroblasts and mesothelial cells. LYVE1 mesothelial macrophages enhance omentum- hi Both LYVE1 macrophages in particular and, to a lesser extent, independent ovarian tumor progression lo/− LYVE1 macrophages were significantly reduced in the ab- Given the overlap between omental macrophage gene expres- sence of Csf1 produced in stromal cells, with residual cells ap- sion and that of other macrophages in peritoneal membranes pearing rounded in morphology (Fig. 5, D and E). Consistent with lining the cavity where the tumor was implanted, we wondered + + a previous finding (Bellomo et al., 2020), F4/80 ICAM2 peri- if the previous correlation in reduced tumor progression be- ΔCsf1 hi toneal macrophages were highly reduced in Wt1 mice tween removal of the omentum and the lack of LYVE1 mac- hi (Fig. 5 F). However, blood Ly6C monocytes and neutrophils of rophages (Etzerodt et al., 2020) meant de facto that only omental ΔCsf1 Wt1 mice were comparable in number to their littermate macrophages inside the peritoneal cavity were implicated in controls (Fig. 5 G). tumor progression. In particular, we wondered if membrane- hi Some visceral white adipose tissues (WATs) are generated associated LYVE1 macrophages beyond those in the omentum from Wt1-expressing cells (Chau et al., 2014). To test whether were relevant drivers of tumor progression. To examine these membrane-associated macrophages are controlled by WAT- questions, luciferase/GFP-labeled murine epithelial ovarian tu- cre LSL-DTA hi derived CSF1, we studied Adiponectin :R26 mice lacking mor cells (ID8) were injected i.p. into LYVE1 macrophage– hi lo/− ΔCsf1r fl/fl adipocytes. LYVE1 and LYVE1 macrophages in mesenteric ablated mice (Lyve1 ) or their littermate controls (Csf1r ) membranes were intact in these mice, implying that membrane- to model intraperitoneal metastatic HGSOC (Leinster et al., 2012; associated macrophages did not depend on adipocytes for main- Lengyel et al., 2014). Tumor burden was monitored biweekly tenance (Fig. 5 H). Altogether, we conclude that CSF1 locally through noninvasive bioluminescence imaging. In the first 4 wk, ΔCsf1r produced by fibroblasts and/or mesothelial cells within serous peritoneal tumor burden was comparable between Lyve1 membranes controls the development and maintenance of mice and littermate controls (Fig. 7 A). During this period, the macrophages within the membranes themselves and in the tumor was particularly localized to the omental fat region of adjacent fluid cavities. the peritoneal cavity, spreading into the greater cavity space thereafter (Etzerodt et al., 2020). By 6 wk after implantation, hi hi Gene expression patterns in LYVE1 mesenteric tumor progression was significantly delayed if LYVE1 macro- membrane–associated macrophages resemble those phages were genetically ablated, compared with littermate expressed by omental macrophages in ovarian tumors controls (Fig. 7, A and B). These findings were similar to those It was reported that tissue-resident macrophages play important previously reported (Etzerodt et al., 2020). roles in tumor progression by expressing profibrotic factors The key question was whether the role of macrophages in (Zhu et al., 2017). More recently, Etzerodt et al. (2020) showed driving tumor progression was restricted and required the + hi that TIM4 LYVE1 omental macrophages promote ovarian tu- omentum. To investigate this issue, we performed surgical mor progression through scRNA-seq and a cell ablation model. omentectomy and compared tumor progression between hi ΔCsf1r We suspected that the LYVE1 macrophages we identified lining Lyve1 mice and littermate controls, designing two sets of hi the mesenteric membranes of the peritoneal cavity were related experiments to rigorously test whether LYVE1 macrophages to the tumor-associated macrophages in the omentum. To test promoted ovarian tumors independently of the omentum. First, hi this possibility, we compared the RNA-seq data from LYVE1 we used the ID8-Luc-GFP cell line to model intraperitoneal cre fl/fl membrane-associated macrophages to the scRNA-seq data metastasis after omentectomy. Both Lyve1 :Csf1r mice and (ArrayExpress accession no. E-MTAB-8593) from omental their littermates developed significantly less intraperitoneal macrophages performed in mice bearing experimental ovar- metastasis of ovarian tumors than mice without omentectomy ian tumors. Tumor-associated omental macrophages were re- (Fig. 7, A and C), confirming the critical role of the omentum in analyzed and divided into 21 different clusters based on their ovarian tumor progression. However, in the absence of ΔCsf1r gene expression patterns (Fig. 6 A). Timd4 was up-regulated in omentum, Lyve1 mice still developed less intraperitoneal clusters 10 and 16 and Lyve1 in clusters 6, 8, 10, 15, and 16, with metastasis than littermate controls (Fig. 7 C), demonstrating an hi highest expression in cluster 10 among these five clusters. This independent role for the LYVE1 macrophages outside of the + + analysis placed the tumor-promoting TIM4 LYVE1 omental omentum. Second, as previously pioneered (Etzerodt et al., macrophages in cluster 10 of our reanalysis (Fig. 6 B). Next, the 2020), we developed omentum-primed ID8 cells by condition- hi top 100 enriched genes expressed by LYVE1 membrane- ing ID8 cells in WT mice for 12 wk. We termed recovered cells associated macrophages were compared with genes expressed ID8-A12. ID8-A12 cells were inoculated into omentectomized ΔCsf1r in the scRNA-seq that we reanalyzed. As shown in our heatmap Lyve1 mice or littermate controls. Ovarian tumors were analysis, we confirmed that the gene expression pattern of rapidly expanded in both genotypes within 4 wk after inocu- hi LYVE1 mesenteric membrane macrophages most closely matched lation. However, ID8-A12 cells expanded significantly more ΔCsf1r genes expressed in cluster 10 (Fig. 6 C). Conversely, genes slowly in Lyve1 mice than in control mice (Fig. 7 D). The Zhang et al. Journal of Experimental Medicine 9of17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 hi hi Figure 6. Comparison of gene expressions between RNA-seq of Lyve1 membrane-associated macrophages and scRNA-seq of Lyve1 omental macrophages in ovarian tumor progression. (A) Reanalyzed uniform manifold approximation and projection (UMAP) plot of scRNA-seq data (ArrayExpress + + accession no. E-MTAB-8593) of F4/80 CD64 omental macrophages isolated 10 wk after ID8 ovarian tumor cell injection. (B) Violin plot of Timd4 and Lyve1 hi expression from the scRNA-seq dataset in A. (C) UMAP plot of scRNA-seq showing top 100 genes up-regulated in bulk RNA-seq datasets of LYVE1 membrane- hi associated macrophages. (D) GSEA of RNA-seq data from Lyve1 membrane-associated macrophages showing select genes enriched in Cluster 10 of scRNA- seq. NES, normalized enrichment score. tumor within ascites rather than the mesentery accounted for Discussion the differences in tumor burden between the two genotypes The complexity of resting macrophage heterogeneity and spe- hi (Fig. 7 E). Taken together, these data underscore that LYVE1 cialization across and within given organs continues to evolve macrophages promote intraperitoneal expansion of ovarian and grow. The present focus on the peritoneal cavity in this tumors independent of the omentum. body of work highlights the intricate network of resident hi Figure 7. Deficiency in LYVE1 macrophages delays intraperitoneal expansion of ovarian cancer in an omentum-independent manner. (A) Quanti- fl/fl ΔCsf1r fication of bioluminescence signals at different time points after tumor implantation (Csf1r mice, n = 12; Lyve1 mice, n =13; mean ±SEM). (B) Bio- luminescence images of tumor-bearing mice at 6 wk after inoculation. (C) Quantification of bioluminescence signal at different time points after inoculation in fl/fl ΔCsf1r omentectomized (OMX) mice (Csf1r mice, n = 11; Lyve1 mice, n = 11; mean ± SEM). (D) Quantification of bioluminescence signal at different time points fl/fl ΔCsf1r after inoculation of the omentum-primed ID8-A12 cells in OMX mice (Csf1r mice, n = 10; Lyve1 mice, n = 12; mean ± SEM). (E) Quantification of bi- oluminescence signal of ascites and mesenteries 4 wk after inoculation of the omentum-primed ID8-A12 cells in OMX mice (mean ± SEM). Unpaired Student’s t test: *, P < 0.05; ***, P < 0.001. Statistical analysis was performed using one-way ANOVA (A, C, and D) and Student’s t test (E). Zhang et al. Journal of Experimental Medicine 10 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 macrophages that occupy the body cavity. We and others have cells that serve as the crucial source of CSF1 that these mac- recognized the existence of two resident peritoneal macro- rophages require for persistence. phage subsets in the serosal fluid of the peritoneum, GATA6- With the clear evidence that the phenotype of interstitial dependent LPMs (Gautier et al., 2014; Okabe and Medzhitov, macrophage subsets lining the mesenteric and peritoneal 2014; Rosas et al., 2014) and monocyte-derived, IRF4-dependent membranes is distinct from the phenotype and life cycle of the small peritoneal macrophages (Kim et al., 2016). Evidence of fluid-borne macrophage subsets of the peritoneum, it is rea- the existence of more than these two resident macrophages in sonable to regard the peritoneal cavity space as being influ- the body cavity is found in the literature, particularly in the enced by four different types of resident macrophages. One studies of Uderhardt et al. (2019) identifying fixed macrophages begins to wonder, when this point is taken into account, how in the peritoneal lining membranes. However, the basic phe- the invasion into injured organs by fluid-borne peritoneal notype of these fixed macrophages has not been reported or macrophages (Wang and Kubes, 2016), and their influence on compared with the fluid-borne macrophages, and thus even surgical adhesions (Zindel et al., 2021), is impacted by these very recent reviews (Liu et al., 2021) and papers in the field interstitial macrophages. For example, cell deletion schemes focused on peritoneal macrophages (Louwe et al., 2021)have that have targeted LPMs of the serous fluid are broad enough in not placed these fixed macrophages into context. Instead, their mechanism of action to have also deleted these interstitial the omental fat-associated macrophages are typically the macrophages in relevant anatomic spaces, but this point was only macrophages routinely considered beyond the serous not considered, as the presence of these macrophages was not fluid macrophages within the peritoneal compartment (Liu clear at the time of the studies. It may also be the case that et al., 2021). Here, we show that macrophages resembling deletions of specific but still rather broadly expressed genes omental macrophages are situated as fixed macrophages such as Rxra affected outcomes such as peritoneal cancer in the mesothelial linings of the peritoneum, including the progression (Casanova-Acebes et al., 2020)due to deletions vascularized and avascular parts of the mesentery, the pa- of the LYVE1 serosal macrophages, as their status was not rietal peritoneal membrane, and the surface of organs such checked. On the other hand, it very well may be that serous as the pancreas. fluid peritoneal macrophages and the LYVE1 interstitial Strikingly, phenotypes of the fixed macrophages observed macrophages we describe here each have requisite roles in were characterized by high expression of LYVE1 and low peritoneal tumor progression (Casanova-Acebes et al., 2020; expression of MHC II in the first population and lower ex- Xia et al., 2020). pression of LYVE1 but higher expression of MHC II in the With respect to cancer progression, a recent study published hi second population. Indeed, it is striking that almost all organ compelling evidence that LYVE1 macrophages were critical surfaces including the meninges of the central nervous sys- mediators of ovarian tumor cell expansion in the peritoneal tem are characterized by the presence of LYVE1 macrophages, cavity (Etzerodt et al., 2020). We show here that the macro- even when the major organ parenchymal macrophage is devoid phages we describe are a similar population to those studied by of this marker. We note that the liver stands out as the ex- Etzerodt et al. (2020). Because the same authors reproduced that ception to the pattern, having a phenotypically distinct mac- the presence of the omental fat tissue drove tumor progression rophage type at the border surface (Sierro et al., 2017), which and because they were able to identify interstitial peritoneal hi lo/− hi our findings confirmed. The LYVE1 and LYVE1 macro- macrophages with the LYVE1 phenotype in the omentum, they phages within the serous membranes are likely counterparts to concluded that omental macrophages in particular were neces- the interstitial macrophages previously described in the lungs sary for ovarian tumor progression in the peritoneal cavity. In (Chakarov et al., 2019; Gibbings et al., 2017), artery wall (Lim our study, we posited that the role of these macrophages in af- et al., 2018), and mammary gland (Wang et al., 2020). Indeed, fecting tumor expansion might not be restricted to the omen- although it is clear that organs have unique resident macro- tum. To address this issue, we surgically removed the omentum phages, it is also emerging that some macrophage phenotypes in two different experimental scenarios, with one scenario in- are found more broadly across all organs. We argue that the volving tumors that were allowed to be conditioned by omental cells we have characterized here should be called interstitial factors that enhance the aggressiveness of the tumor but then peritoneal macrophages to help distinguish them from serous reimplanted into mice wherein the omentum had been surgi- fluid-borne macrophages of the peritoneal cavity and to si- cally removed. We could not remove the lesser omentum due to multaneously underscore their potential common features with its key role in maintaining viability of portions of the stomach the interstitial macrophages of other organs (Chakarov et al., and spleen, so we cannot eliminate a role for lesser omental 2019; Gibbings et al., 2017). As noted in these past studies and macrophages. However, we underscore that the number of recapitulated in our present study in the peritoneal cavity, their omental macrophages is much smaller than those lining the phenotype is oriented toward an alternatively activated, or M2, peritoneal compartment overall. Furthermore, although it was state and to the maintenance and remodeling of extracellular not directly stated in the previous publication, it is highly un- matrix, perhaps especially relevant in light of the collagen- and likely that Etzerodt et al. (2020) removed the lesser omentum, matrix-rich environment these cells live within and their due to its importance in physiology. Finally, patients with possible role in maintaining the exterior barrier of the associ- ovarian cancer metastasis to the peritoneum undergo resection ated membranes and organ surfaces. We show that they appear of the omentum as routine debulking; thus, this study high- hi to be in a state of interdependence with neighboring stromal lighting a role for LYVE1 macrophages in tumor progression Zhang et al. Journal of Experimental Medicine 11 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 hi beyond the role of the greater omentum represents a clinically LYVE1 macrophage population. We intend to investigate this important finding (National Comprehensive Cancer Network, question in the future. 2021). Multiple distinct tumor immune microenvironments In closing, we have defined the phenotype of fixed tissue can coexist within ovarian cancer from the same patient and macrophages in the membranes, especially the membranes of likely contribute to heterogeneous responses of metastatic le- the mesentery, lining the peritoneal cavity. These findings in sions to therapy (Jimene ´ z-Sanc ´ hez et al., 2017). Seen in this light, turn reveal the complexity of the resident macrophage pool that hi our results raise the potential that mesothelial LYVE1 macro- can influence the peritoneal space, with at least two macro- phages could influence the growth of microscopic nonresected phages in the fluid and two in the membranes with distinct hi residual tumor and affect the response to adjuvant chemother- phenotypes. Genetic loss of LYVE1 macrophages through an apy and cancer recurrence. approach that takes advantage of their dependence on CSF1 and hi Our findings clearly point to a role for LYVE1 macrophages CSF1R allows us to demonstrate that, beyond the omentum, hi in promoting tumor progression under conditions when LYVE1 macrophages promote ovarian tumor growth in the omental macrophages are not relevant due to omental resection. peritoneal space. Future studies focused on the biology of resi- hi This approach allows us to reveal the role of LYVE1 macro- dent peritoneal macrophages must take into account all of the phages beyond the omentum, but the design does not allow us to relevant macrophages in the compartment. hi independently address whether the LYVE1 macrophages of the omentum also contribute to the tumor expansion. We assume that, in presence of the omentum, they do. The question then Materials and methods turns to how these macrophages contribute to tumor progres- Mice sion. In general, M2-type macrophages are thought to drive Mice were maintained in specific pathogen–free (SPF) barrier tumor expansion, and one way they might do so is through facilities with 12-h light–dark cycle by the Division of Compar- extracellular matrix remodeling (DeNardo and Ruffell, 2019; ative Medicine, Washington University School of Medicine Noy and Pollard, 2014), highly consistent with the phenotypic (WUSM), or the Biological Resource Laboratory, University of hi orientation of the LYVE1 macrophages. However, one puzzle is Illinois at Chicago (UIC). All animal experiments and procedures that the ovarian tumor presence in peritoneal fluid rather than were approved by the Institutional Animal Care and Use Commit- hi CreERT2 on the membrane is most impacted by the loss of LYVE1 tees at WUSM and UIC. Csf1r mice (FVB-Tg(Csf1r-cre/Esr1*) cre tm1(cre)Ifo macrophages. A future direction will be to turn toward under- 1Jwp/J; Qian et al., 2011), Lyz2 mice (B6.129P2-Lyz2 /J; hi LSL-tdTomato standing how the LYVE1 macrophages orchestrate an altered Clausen et al., 1999), R26 mice (B6.Cg-Gt(ROSA) tm9(CAG-tdTomato)Hze YFP tumor response and whether they are directly involved in se- 26Sor /J; Madisen et al., 2010), CD11c creting relevant factors or act in other ways, such as condi- transgenic mice (B6.Cg-Tg(Itgax-Venus)1Mnz/J; Lindquist et al., gfp/+ tm1Litt tioning the stromal cells nearby through cell–cell contact. A 2004), CX CR1 mice (B6.129P2(Cg)-Cx3cr1 /J; Jung et al., CreERT2 tm3(cre/ERT2)Gco fl/fl limitation of our study, and a common limitation affecting the 2000), Prox1 mice (Prox1 /J), Gata6 mice tm2.1Sad cre previous study and many others in the field, is that while it is (Gata6 /J; Sodhi et al., 2006), Lyve1 mice (B6;129P2- hi tm1.1(EGFP/cre)Cys fl/fl likely that the deletion of local peritoneal-lining LYVE1 Lyve1 /J; Pham et al., 2010), Csf1r mice tm1.2Jwp a macrophages in the omentectomized mice accounts for the (B6.Cg-Csf1r /J; Li et al., 2006), CD45.1 mice (B6.SJL-Ptprc hi b cre tm1(EGFP/cre)Wtp reduced tumor growth,wecannot besurethatthe LYVE1 Pepc /BoyJ), and WT1 mice (Wt1 /J; Zhou et al., CreERT2 body cavity macrophages per se are the ones at play in con- 2008) were purchased from The Jackson Laboratory. Csf1r fl/fl trolling tumor growth. It remains possible, albeit perhaps mice and Gata6 mice were backcrossed to C57BL6 background hi unlikely, that LYVE1 macrophages resident in distal tissues using the Speed Congenics mouse genetics core at WUSM. CreERT2 play a key role. CX CR1 mice were reconstituted from the cryopreserved hi lo LYVE1 and LYVE1 interstitial peritoneal macrophages ex- sperm provided from S. Jung (Weizmann Institute of Science, cre LSL-DTA press many common genes, including some signature genes, but Rehovot, Israel; Yona et al., 2013). Adiponectin :R26 ΔCsf1r fl/fl others are not shared. In our depletion system, Lyve1 mice mice and Csf1 mice were kindly provided by Charles A. Harris hi lose LYVE1 macrophages in the peritoneal lining mesentery (WUSM, St. Louis, MO) and Jean Jiang (University of Texas lo/− + and other membranes. However, LYVE1 MHC II membrane- Health Science Center, San Antonio, TX; Harris et al., 2012), associated macrophages and other tissue-resident macrophages respectively. in peritoneum, spleen, lung, and brain are not deleted. In ΔCsf1r Lyve1 mice, we nonetheless observed a reduction in tumor Tamoxifen treatment lo/− expansion, indicating that LYVE1 macrophages are not Tamoxifen diet (500 mg/kg; Envigo) was fed ad libitum to adult hi CreERT2 LSL-tdTomato CreERT2 LSL-tdTomato functionally able to stand in for the LYVE1 macrophages, at Csf1r :Rosa26 ,Csf1r :Rosa26 : gfp/+ CreERT2 LSL-tdTomato least when it comes to supporting tumor expansion, despite CX CR1 , and Prox1 :Rosa26 mice for 3 wk. sharing location and some phenotypic similarity. It is interesting For fate mapping, 40 µg tamoxifen (Sigma-Aldrich) was injected hi CreERT2 LSL-tdTomato that, at birth, the LYVE1 macrophages appear almost exclu- i.p. into P1 pups of CX CR1 :Rosa26 mice. sively present, only to give way over time to sharing the space lo with the LYVE1 population that also is prone to inducing MHC Cell isolation and staining for flow cytometry lo/− II. We suspect that the LYVE1 macrophages are monocyte Blood cells were collected by puncture of submandibular cheek derived and can develop, if the right conditions exist, into the vessels into 2 mM EDTA–containing tubes. RBCs were removed Zhang et al. Journal of Experimental Medicine 12 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 by lysis buffer (BD) in accordance with the manufacturer’sin- Project Consortium (ImmGen; https://www.immgen.org/ structions. Blood leukocytes were then stained to detect surface ImmGenProtocols.html). Ly6G, CD11b, CD115, Siglec F, and Ly6C. Peritoneal cells were collected after injection of 5 ml HBSS containing 2 mM EDTA Ultra-low input (ULI) RNA-seq and 2% FBS into the peritoneal cavity. After blood and peritoneal Library preparation, quality controls and generation of data cells were collected, mice were perfused with PBS. Mesenteric were performed by ImmGen according to the standard operating membranes were isolated from gut mesentery, including WAT. procedure for ULI RNA-seq. Data can be accessed through GEO Brain, spleen, lung, and mesenteric membranes were enzy- accession no. GSE122108. Reads were aligned to the mouse ge- matically digested with collagenase types I and X (Sigma-Al- nome GRCm38/mm10 primary assembly (GENCODE) and gene drich), hyaluronidase (Sigma-Aldrich), and DNase (Roche) in a annotation Ver.M16 with STAR 2.5.4a. The raw read counts were gentle-shaking incubator (250 rpm, 37°C, 30 min), and then cells generated by featureCounts (http://subread.sourceforge.net/) were filtered through 70-µm cell strainers. For analysis of mi- and normalized with DESeq2 package from Bioconductor. The croglia of the brain, cells were resuspended in 40% Percoll and top 12,000 genes ranked by average gene expression were se- subjected to density centrifugation (2,000 g, 20 min at 20°C lected for differential expression analysis using the DESeq2. Top hi with no break/acceleration). To process spleens, isolated cells 100 differentially expressed genes in Lyve1 macrophage sam- were lysed with lysis buffer (BD). Total peritoneal cells, blood ples were used as gene signatures for the ensuing analysis. leukocytes, and tissue-resident macrophages were counted using Heatmaps and PCA plots were generated using the Phantasus an automated cell counter (Nexcelom). For quantification, these online service (Artyomov, 2021b). + + numbers were multiplied by the percentage of CD11b CD115 For the analysis of the open source scRNA-seq datasets (ac- + + + Ly6C monocytes and CD11b Ly6G neutrophils in blood, cession nos. GSE102665 and E-MTAB-8593), the Seurat package + + + lo CD45 F4/80 CD64 CD11b for red pulp macrophages in (Butler et al., 2018) was used. Raw reads in each cell were first + hi + lo spleen, CD45 CD11c CD64 CD11b for alveolar macrophages scaled by library size and then log-transformed. To improve + lo hi in lung, CD64 CD45 CD11b for microglia in brain, and downstream dimensionality reduction and clustering, any un- + + hi CD11b CD115 macrophages stained with F4/80 for LPM and wanted source of variation arising from the number of de- lo F4/80 for small peritoneal macrophages in peritoneum. tected molecules was first regressed out. Highly variable Single-cell suspensions collected from each tissue were genes were then identified and selected for PCA reduction of maintained on ice for staining. Dead cells were identified by high-dimensional data. The top 10 principal components were propidium iodide staining during flow cytometry. Antibodies selected for unsupervised clustering of cells. Clustering results purchased from BioLegend/Invitrogen or BD Biosciences were are shown in a t-SNE plot from Single Cell Navigator online used as follows; CD45 (30F11), CD45.1 (A20), CD45.2 (104), CD11b service (Artyomov, 2021a). GSEA was performed to test for the (M1/70), CD115 (AFS98), CD102 (ICAM2; 3C4(MIC2/4)), MHC II enrichment of cluster-specific gene sets at the top of the bulk (I-A/I-E; M5/114.15.2), Ly6C (HK1.4), Ly6G (1A8), CD11c (N418), RNA-seq genes ranked according to their differential expression hi CD170 (Siglec F; E50-2440), CD64 (FcRγI; X54-5/7.1), MerTK significance (Lyve1 macrophages versus all others). Violin and (DS5MMER), F4/80 (BM8), CD206 (Mrc1;C068C2), LYVE1 feature plots were generated using Seurat package. (ALY7), FOLR2 (10/FR2), and isotype controls (IgG2a κ chain, IgG1a, and IgG2b κ chain). Whole-mount imaging by confocal microscope Mesenteries of adult mice were detached from the associated gut Cell sorting and fixed with 4% paraformaldehyde (Thermo Fisher Scientific) To remove dead cells, cells stained with propidium iodide were containing 30% sucrose (Fisher) overnight at 4°C. Gut mesen- gated out during cell sorting on a BD Aria II instrument. CD3ε, teries still attached to the intestine of neonatal mice (P0–14) CD19, and Ly6G staining was used to exclude lymphocytes and were pinned on SYLGARD184 (Ellsworth; 4019862)-coated plates neutrophils in some tissues. For selection of brain microglia by and fixed with 4% paraformaldehyde overnight at 4°C. After sorting, cells were stained with CD45, CD11b, CD64, F4/80, and fixation, samples were stored at 4°C in PBS containing 0.01% CD206. For splenic red pulp macrophages, cells were stained sodium azide. For whole-mount imaging by confocal micro- with CD45, CD64, MerTK, F4/80, and CD11b. For lung alveolar scope, gut mesenteries were blocked in a solution containing 5% macrophages, cells were stained with CD45, CD11c, Siglec F, goat serum (Sigma-Aldrich; D9663) or 5% BSA (Sigma-Aldrich; hi CD64, and CD11b. For membrane-associated Lyve1 macro- A9576) overnight at 4°C. Samples were stained with rabbit-anti phages of gut mesentery, cells were stained with CD45, F4/80, LYVE1 (Abcam; 14917), rat-anti MHC II (Invitrogen; M5/ CD64, and LYVE1. For peritoneal macrophages, peritoneal cells 114.15.2), rat-Folr2 (BioLegend; 10/FR2), rat-anti CD206 (Bio- hi were stained with CD11b, CD115, MHCII, and ICAM2. For Ly6C Rad; MR5D3), and rat-anti-ICAM2 (Invitrogen; 3C4(mIC2/4), lo and Ly6C monocytes in blood, blood leukocytes were stained Alexa Fluor 488) diluted in 1% BSA and incubated overnight 4°C with CD11b, CD115, and Ly6C. For bulk RNA-seq, we double- with gentle agitation. Samples were washed with PBS then in- sorted tissue-resident macrophages (1,000 cells/replicate), cubated with secondary antibodies conjugated with Alexa Fluor and the sorted cells were directly collected into LoBind tubes 488, Cy3, or Alexa Fluor 647/Cy5 (Jackson ImmunoResearch) containing 5 µl of TCL lysis buffer (Qiagen) containing 1% overnight 4°C. After nuclei were further stained with bis Ben- β-mercaptoethanol, based on the Cell Preparation and Sorting zimide H 3342 (Sigma-Aldrich), macrophages in mesenteric Standard Operating Procedures of the Immunological Genome membranes and mesenteric fat tissues were visualized on a Zhang et al. Journal of Experimental Medicine 13 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 confocal microscope (Leica SPE/inverted Leica SP8 or Zeiss 880). (50 mM), penicillin-streptomycin (100 U/ml), and nonessen- All images were collected using Leica LAS X software, and tial amino acids (Godoy et al., 2013). ID8-A12 ascites cells were analysis was performed using Imaris software (Bitplane). generated by harvesting total ascites cells 12 wk after implanting 6 6 10 ID8 cells i.p. in C57BL6 mice. 10 ID8 or ID8-A12 cells were Estimation of serous membrane macrophage numbers in the injected i.p. into mice of different genotypes with or without peritoneal cavity and omentum omentectomy. Biweekly bioluminescence imaging was per- We estimated the approximate area of the parietal membrane formed to noninvasively quantify tumor burden in the perito- 2 2 (∼6cm ), back of peritoneal wall (∼6cm ), peritoneal mem- neal cavity. brane at the bottom of the peritoneal cavity (∼4cm ), and sur- faces of serous tissues (pancreas, ∼4cm ;gut mesentery, ∼8-12 IVIS imaging 2 2 cm ; and diaphragm, ∼4cm ). This area in total added up to In vivo bioluminescence imaging was performed on an IVIS 50 2 hi 32–36 cm . Our data show that Lyve1 macrophages ranged (PerkinElmer; Living Image 4.3.1), with exposures of 1 s to 1 min, from ∼245–380 cells/mm (Fig. 5 A). Therefore, we estimate that binning 2–8, field of view 12.5 cm, f/stop 1, and open filter. hi the total number of LYVE1 macrophages may be ∼0.78–1.36 × D-Luciferin (150 mg/kg in PBS; Gold Biotechnology) was injected 6 6 hi 10 cells per mouse (∼10 cells). For estimating LYVE1 mac- into the mice i.p. and imaged ventrally using isoflurane anes- rophages in the omentum, we first made a single-cell suspension thesia (2% vaporized in O ). The total photon flux (photons/s) hi and counted the yield of LYVE1 macrophages per mouse was measured from regions of interest using the Living Image omentum, deriving 0.5–1×10 . We then stained greater 2.6 program. omentum tissue from three mice for LYVE1 macrophages. By Cells and mesenteries were imaged using the IVIS 50 with 4 hi immunostaining, we estimated there were 2 × 10 LYVE1 (PerkinElmer; Living Image 4.3.1) 1-s to 1-min exposure, bin 4–8, macrophages, far less than in the peritoneal surfaces and field of view 12 cm, f/stop 1, and open filter after addition of mesenteries. 150 µg/ml D-luciferin (Gold Biotechnology). For analysis, a grid was placed over the plate, and total photon flux (photons/s) was BM transplantation measured using Living Image 2.6. Cre BM cells were isolated from the tibia and femur of LYVE1 : LSL-tdTomato R26 mice on a CD45.2 background. After lysis of RBC Omentectomy in BM cells, Tomato cells were sorted using a FACS Aria II Operative omentectomy in mice was accomplished under gen- system (BD). Sorted BM cells (>95% purity) were injected i.v. eral anesthesia by continuous inhalation of 2–3% isoflurane in into lethally irradiated (950 rad) CD45.1 congenic mice (1.5–3.0 × 60% oxygen using a veterinary vaporizer, and then mice were 10 cells/mouse). Recipient mice were euthanized and analyzed placed on a heating pad in a supine position. Through a midline 8–10 wk after BM transplantation. incision in the region of the stomach, the greater omentum was carefully exposed and removed via electrocautery. The midline Whole-mount imaging using two-photon microscopy incision was then closed with absorbable sutures in two layers. Cre LSL-tdTomato cEYP Lyz2 :R26 :CD11 mice and DyLight488-conjugated Mice were resuscitated with an i.p. injection of saline, given a lectin (50 µg/mouse; Vector Laboratories; DL1174)–injected BM- local injection of analgesia, and then allowed to recover in a transplanted mice were used for live imaging. Immediately warmed incubator. Removal of both the entire greater and lesser after sacrificing BM-transplanted mice, Tomato reporter– omentum resulted in malperfusion of the stomach and spleen labeled macrophages and DyLight488-labeled blood vascula- and thus was not feasible. tures of brain (including the skull), liver, pancreas, peri- toneum, and gut mesenteries were visualized through the Online supplemental material customized Leica SP8 two-photon microscope with a Mai Tai HP Figs. S1 and S2 accompany Fig. 1 and add additional information DeepSee laser (Spectra-Physics) and a 25×, 0.95-NA water- on characterization of mesenteric membrane macrophages, in- immersion objective. All images including the 3D video were col- cluding comparison to the liver surface macrophages (Fig. S1) lected by Leica LAS X software and generated by Imaris (Bitplane) and demonstration of independence from LPM (Fig. S2). Fig. S3 Cre LSL-tdTomato software. Liver and gut mesentery of Lyz2 :R26 : accompanies Fig. 2 andillustrates theneed touseaspecialized cEYP gfp/+ CD11 and CX CR1 mice were fixed with 4% paraformalde- BM transplant strategy to obtain faithful reporting of fluores- hi hyde (Affymetrix) overnight at 4°C, and tissues were stored in PBS cent tags to LYVE1 macrophages in adult mice. Fig. S4 supplies containing 0.01% sodium azide at overnight at 4°C until ready additional information to Fig. 3 on the computational analysis of to image. mesenteric membrane macrophages from a previously pub- lished dataset. Fig. S5 accompanies Fig. 5 and shows the lack cre fl/fl Tumor implantation of impact of the LYVE1 :Csf1r genotype on resident The original ID8 cell line, derived from spontaneous in vitro macrophage numbers in organs where macrophages do not malignant transformation of C57BL6 mouse ovarian surface express LYVE1. Video 1 illustrates contact between mesen- epithelial cells (Roby et al., 2000), was modified to express GFP teric membrane macrophages and peritoneal fluid macro- and firefly luciferase. These ID8 cells were cultured in RPMI phages. Video 2 and Video 3 show 3D rotational views of hi 1640 with heat-inactivated FBS (10%), L-glutamine (2 mM), LYVE1 macrophages in the meninges covering the brain Hepes (25 mM), sodium pyruvate (1 mM), 2-mercaptoethanol and in the parietal peritoneal membrane to support the Zhang et al. Journal of Experimental Medicine 14 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Maintains and Replenishes Splenic Red Pulp Macrophages. Immunity. conclusion that they are preferentially positioned on the 53:127–142.e7. https://doi.org/10.1016/j.immuni.2020.06.008 border of the tissue. Ben ´ ezec ´ h, C., N.T. Luu, J.A. Walker, A.A. Kruglov, Y. Loo, K. Nakamura, Y. Zhang, S. Nayar, L.H. Jones, A. Flores-Langarica, et al. 2015. Inflammation-induced formation of fat-associated lymphoid clusters. Nat. Immunol. 16:819–828. https://doi.org/10.1038/ni.3215 Acknowledgments Buechler, M.B., K.W. Kim, E.J. Onufer, J.W. Williams, C.C. Little, C.X. Dom- We thank Dr. Steffen Jung (Weizmann Institute of Science, Is- inguez, Q. Li, W. Sandoval, J.E. Cooper, C.A. Harris, et al. 2019. A Stromal Niche Defined by Expression of the Transcription Factor WT1 rael) and Charles Harris (WUSM) for providing mouse strains. Mediates Programming and Homeostasis of Cavity-Resident Macro- We also thank all the members of the Randolph laboratory at phages. Immunity. 51:119–130.e5. https://doi.org/10.1016/j.immuni.2019 WUSM and the Kim laboratory at UIC for helpful discussion or .05.010 Butler, A., P. Hoffman, P. Smibert, E. Papalexi, and R. Satija. 2018. Integrating reading the manuscript. We thank Christophe Benoist and col- single-cell transcriptomic data across different conditions, technolo- leagues at ImmGen for collecting samples and generating data gies, and species. Nat. Biotechnol. 36:411–420. https://doi.org/10.1038/ from ULI-RNAseq. We also thank WUSM and UIC Flow Cy- nbt.4096 ´ ´ Casanova-Acebes, M., M.P. Menendez-Gutierrez, J. Porcuna, D. Alvarez-Er- tometry Core. We are grateful for technical support from Julie ´ ´ rico, Y. Lavin, A. Garcıa, S. Kobayashi, J. Le Berichel, V. Nuñez, F. Were, Prior and Kathleen Duncan from the Molecular Imaging Center et al. 2020. RXRs control serous macrophage neonatal expansion and at WUSM. identity and contribute to ovarian cancer progression. Nat. Commun. 11: 1655. https://doi.org/10.1038/s41467-020-15371-0 This study was supported by National Institutes of Health Cecchini, M.G., M.G. Dominguez, S. Mocci, A. Wetterwald, R. Felix, H. grants R37 AI049653 (to G.J. Randolph); R01DK119147, DP1DK126190, Fleisch, O. Chisholm, W. Hofstetter, J.W. Pollard, and E.R. Stanley. and R01DK126753 (to K-W. Kim); R01AG045040 (to J.X. Jiang); 1994. Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of R01CA188900 (to B.H. Segal); R00HL138163 (to J.W. Williams); the mouse. Development. 120:1357–1372. https://doi.org/10.1242/dev T32DK077653 (to E.C. Erlich and E.J. Onufer); P50CA094056 (to .120.6.1357 WUSM Molecular Imaging Center); P30AR0737752 (to WUSM Chakarov, S., H.Y. Lim, L. Tan, S.Y. Lim, P. See, J. Lum, X.M. Zhang, S. Foo, S. Nakamizo, K. Duan, et al. 2019. Two distinct interstitial macrophage Rheumatic Disease Research Center); and P30CA091842 (to WUSM populations coexist across tissues in specific subtissular niches. Science. Siteman Cancer Center Small Animal Cancer Imaging shared re- 363:eaau0964. https://doi.org/10.1126/science.aau0964 source); and Welch Foundation grant AQ-1507 (to J.X. Jiang). R.S. Chau, Y.Y., R. Bandiera, A. Serrels, O.M. Mart´ ınez-Estrada, W. Qing, M. Lee, J. Slight, A. Thornburn, R. Berry, S. McHaffie, et al. 2014. Visceral and Czepielewski received support from the Lawrence C. Pakula, MD, subcutaneous fat have different origins and evidence supports a mes- IBD Research Fellowship (FA-2020-01-IBD-1). othelial source. Nat. Cell Biol. 16:367–375. https://doi.org/10.1038/ Author contributions: Conceptualization: K-W. Kim, N. ncb2922 Clausen, B.E., C. Burkhardt, W. Reith, R. Renkawitz, and I. Fors ¨ ter. 1999. Zhang, G.J. Randolph; Investigation: K-W. Kim, N. Zhang, S.H. Conditional gene targeting in macrophages and granulocytes using Kim, E.C. Erlich, E.J. Onufer, J. Kim, J. Ding, B.T. Saunders, J.R. LysMcre mice. Transgenic Res. 8:265–277. https://doi.org/10.1023/A: Dominguez, R.S. Czepielewski, B.A. Helmink, J.W. Williams; Dai, X.M., G.R. Ryan, A.J. Hapel, M.G. Dominguez, R.G. Russell, S. Kapp, V. Resources: G.J. Randolph, K-W. Kim, J.X. Jiang, B.H. Segal; Sylvestre, and E.R. Stanley. 2002. Targeted disruption of the mouse Formal analysis and visualization: N. Zhang, K-W. Kim, S.H. colony-stimulating factor 1 receptor gene results in osteopetrosis, Kim, J. Kim, J. Ding, A. Gainullina, B.T. Saunders, B.H. Zinsel- mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood. 99:111–120. https://doi meyer; Writing: K-W. Kim, N. Zhang, S.H. Kim, G.J. Randolph; .org/10.1182/blood.V99.1.111 Supervision: K-W. Kim, G.J. Randolph, N. Zhang. All authors David, B.A., R.M. Rezende, M.M. Antunes, M.M. Santos, M.A. Freitas Lopes, edited the manuscript. A.B. Diniz, R.V. Sousa Pereira, S.C. Marchesi, D.M. Alvarenga, B.N. Nakagaki, et al. 2016. Combination of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation Disclosures: J.W. Williams reported grants from American of Liver Myeloid Cells in Mice. Gastroenterology. 151:1176–1191. https:// Heart Association and grants from NIH NHLBI outside the doi.org/10.1053/j.gastro.2016.08.024 DeNardo, D.G., and B. Ruffell. 2019. Macrophages as regulators of tumour submitted work. No other disclosures were reported. immunity and immunotherapy. Nat. Rev. Immunol. 19:369–382. https:// doi.org/10.1038/s41577-019-0127-6 Submitted: 30 April 2021 Etzerodt, A., M. Moulin, T.K. Doktor, M. Delfini, N. Mossadegh-Keller, M. Bajenoff, M.H. Sieweke, S.K. Moestrup, N. Auphan-Anezin, and T. Revised: 13 September 2021 Lawrence. 2020. Tissue-resident macrophages in omentum promote Accepted: 14 October 2021 metastatic spread of ovarian cancer. J. Exp. Med. 217:e20191869. https:// doi.org/10.1084/jem.20191869 Gao, Q., Z. Yang, S. Xu, X. Li, X. Yang, P. Jin, Y. Liu, X. Zhou, T. Zhang, C. Gong, et al. 2019. Heterotypic CAF-tumor spheroids promote early References peritoneal metastatis of ovarian cancer. J. Exp. Med. 216:688–703. Artyomov, M. 2021a. Single Cell Navigator. https://artyomovlab.wustl.edu/ https://doi.org/10.1084/jem.20180765 scn/ (accessed October 26, 2021) Gautier, E.L., T. Shay, J. Miller, M. Greter, C. Jakubzick, S. Ivanov, J. Helft, A. Artyomov, M. 2021b. Phantasus. https://artyomovlab.wustl.edu/phantasus/ Chow, K.G. Elpek, S. Gordonov, et al. Immunological Genome Consor- (accessed October 26, 2021) tium. 2012. Gene-expression profiles and transcriptional regulatory Bain, C.C., A. Bravo-Blas, C.L. Scott, E.G. Perdiguero, F. Geissmann, S. Henri, pathways that underlie the identity and diversity of mouse tissue B. Malissen, L.C. Osborne, D. Artis, and A.M. Mowat. 2014. Constant macrophages. Nat. Immunol. 13:1118–1128. https://doi.org/10.1038/ni replenishment from circulating monocytes maintains the macrophage .2419 pool in the intestine of adult mice. Nat. Immunol. 15:929–937. https://doi Gautier, E.L., S. Ivanov, J.W. Williams, S.C. Huang, G. Marcelin, K. Fairfax, .org/10.1038/ni.2967 P.L. Wang, J.S. Francis, P. Leone, D.B. Wilson, et al. 2014. Gata6 regu- Bellomo, A., I. Mondor, L. Spinelli, M. Lagueyrie, B.J. Stewart, N. Brouilly, B. lates aspartoacylase expression in resident peritoneal macrophages and Malissen, M.R. Clatworthy, and M. Bajeno ´ ff. 2020. Reticular Fibroblasts controls their survival. J. Exp. Med. 211:1525–1531. https://doi.org/10 Expressing the Transcription Factor WT1 Define a Stromal Niche that .1084/jem.20140570 Zhang et al. Journal of Experimental Medicine 15 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Ghosn, E.E., A.A. Cassado, G.R. Govoni, T. Fukuhara, Y. Yang, D.M. Monack, Lacerda Mariano, L., M. Rousseau, H. Varet, R. Legendre, R. Gentek, J. Saenz K.R. Bortoluci, S.R. Almeida, L.A. Herzenberg, and L.A. Herzenberg. Coronilla, M. Bajenoff, E. Gomez Perdiguero, and M.A. Ingersoll. 2020. 2010. Two physically, functionally, and developmentally distinct peri- Functionally distinct resident macrophage subsets differentially shape toneal macrophage subsets. Proc. Natl. Acad. Sci. USA. 107:2568–2573. responses to infection in the bladder. Sci. Adv. 6:eabc5739. https://doi https://doi.org/10.1073/pnas.0915000107 .org/10.1126/sciadv.abc5739 Gibbings, S.L., S.M. Thomas, S.M. Atif, A.L. McCubbrey, A.N. Desch, T. Lee, L.K., Y. Ghorbanian, W. Wang, Y. Wang, Y.J. Kim, I.L. Weissman, M.A. Danhorn, S.M. Leach, D.L. Bratton, P.M. Henson, W.J. Janssen, and C.V. Inlay, and H.K.A. Mikkola. 2016. LYVE1 Marks the Divergence of Yolk Jakubzick. 2017. Three Unique Interstitial Macrophages in the Murine Sac Definitive Hemogenic Endothelium from the Primitive Erythroid Lung at Steady State. Am. J. Respir. Cell Mol. Biol. 57:66–76. https://doi Lineage. Cell Rep. 17:2286–2298. https://doi.org/10.1016/j.celrep.2016.10 .org/10.1165/rcmb.2016-0361OC .080 Godoy, H.E., A.N. Khan, R.R. Vethanayagam, M.J. Grimm, K.L. Singel, N. Leinster, D.A., H. Kulbe, G. Everitt, R. Thompson, M. Perretti, F.N. Gavins, D. Kolomeyevskaya, K.J. Sexton, A. Parameswaran, S.I. Abrams, K. Odunsi, Cooper, D. Gould, D.P. Ennis, M. Lockley, et al. 2012. The peritoneal and B.H. Segal. 2013. Myeloid-derived suppressor cells modulate im- tumour microenvironment of high-grade serous ovarian cancer. J. Pathol. mune responses independently of NADPH oxidase in the ovarian tumor 227:136–145. https://doi.org/10.1002/path.4002 microenvironment in mice. PLoS One. 8:e69631. https://doi.org/10.1371/ Lengyel, E. 2010. Ovarian cancer development and metastasis. Am. J. Pathol. journal.pone.0069631 177:1053–1064. https://doi.org/10.2353/ajpath.2010.100105 Gomez Perdiguero, E., K. Klapproth, C. Schulz, K. Busch, E. Azzoni, L. Crozet, Lengyel, E., J.E. Burdette, H.A. Kenny, D. Matei, J. Pilrose, P. Haluska, K.P. H. Garner, C. Trouillet, M.F. de Bruijn, F. Geissmann, and H.R. Rode- Nephew, D.B. Hales, and M.S. Stack. 2014. Epithelial ovarian cancer wald. 2015. Tissue-resident macrophages originate from yolk-sac-de- experimental models. Oncogene. 33:3619–3633. https://doi.org/10.1038/ rived erythro-myeloid progenitors. Nature. 518:547–551. https://doi onc.2013.321 .org/10.1038/nature13989 Li, J., K. Chen, L. Zhu, and J.W. Pollard. 2006. Conditional deletion of the Han, S.J., A. Glatman Zaretsky, V. Andrade-Oliveira, N. Collins, A. Dzutsev, J. colony stimulating factor-1 receptor (c-fms proto-oncogene) in mice. Shaik, D. Morais da Fonseca, O.J. Harrison, S. Tamoutounour, A.L. Byrd, Genesis. 44:328–335. https://doi.org/10.1002/dvg.20219 et al. 2017. White Adipose Tissue Is a Reservoir for Memory T Cells and Lim, H.Y., S.Y. Lim, C.K. Tan, C.H. Thiam, C.C. Goh, D. Carbajo, S.H.S. Chew, Promotes Protective Memory Responses to Infection. Immunity. 47: P. See, S. Chakarov, X.N. Wang, et al. 2018. Hyaluronan Receptor LYVE- 1154–1168.e6. https://doi.org/10.1016/j.immuni.2017.11.009 1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan- Harris, S.E., M. MacDougall, D. Horn, K. Woodruff, S.N. Zimmer, V.I. Rebel, Mediated Regulation of Smooth Muscle Cell Collagen. Immunity. 49:1191. R. Fajardo, J.Q. Feng, J. Gluhak-Heinrich, M.A. Harris, and S. Abboud https://doi.org/10.1016/j.immuni.2018.12.009 Werner. 2012. Meox2Cre-mediated disruption of CSF-1 leads to osteo- Lindquist, R.L., G. Shakhar, D. Dudziak, H. Wardemann, T. Eisenreich, M.L. petrosis and osteocyte defects. Bone. 50:42–53. https://doi.org/10.1016/j Dustin, and M.C. Nussenzweig. 2004. Visualizing dendritic cell net- .bone.2011.09.038 works in vivo. Nat. Immunol. 5:1243–1250. https://doi.org/10.1038/ Hoeffel, G., J. Chen, Y. Lavin, D. Low, F.F. Almeida, P. See, A.E. Beaudin, J. ni1139 Lum, I. Low, E.C. Forsberg, et al. 2015. C-Myb(+) erythro-myeloid Liu, M., A. Silva-Sanchez, T.D. Randall, and S. Meza-Perez. 2021. Specialized progenitor-derived fetal monocytes give rise to adult tissue-resident immune responses in the peritoneal cavity and omentum. J. Leukoc. Biol. macrophages. Immunity. 42:665–678. https://doi.org/10.1016/j.immuni 109:717–729. https://doi.org/10.1002/JLB.5MIR0720-271RR .2015.03.011 Louwe, P.A., L. Badiola Gomez, H. Webster, G. Perona-Wright, C.C. Bain, S.J. Hogg, C., K. Panir, P. Dhami, M. Rosser, M. Mack, D. Soong, J.W. Pollard, S.J. Forbes, and S.J. Jenkins. 2021. Recruited macrophages that colonize the Jenkins, A.W. Horne, and E. Greaves. 2021. Macrophages inhibit and post-inflammatory peritoneal niche convert into functionally divergent enhance endometriosis depending on their origin. Proc. Natl. Acad. Sci. resident cells. Nat. Commun. 12:1770. https://doi.org/10.1038/s41467-021 USA. 118:e2013776118. https://doi.org/10.1073/pnas.2013776118 -21778-0 Ivanov, S., A. Gallerand, M. Gros, M.I. Stunault, J. Merlin, N. Vaillant, L. Madisen, L., T.A. Zwingman, S.M. Sunkin, S.W. Oh, H.A. Zariwala, H. Gu, L.L. Yvan-Charvet, and R.R. Guinamard. 2019. Mesothelial cell CSF1 sustains Ng, R.D. Palmiter, M.J. Hawrylycz, A.R. Jones, et al. 2010. A robust and peritoneal macrophage proliferation. Eur. J. Immunol. 49:2012–2018. high-throughput Cre reporting and characterization system for the https://doi.org/10.1002/eji.201948164 whole mouse brain. Nat. Neurosci. 13:133–140. https://doi.org/10.1038/ Jackson-Jones, L.H., P. Smith, J.R. Portman, M.S. Magalhaes, K.J. Mylonas, nn.2467 M.M. Vermeren, M. Nixon, B.E.P. Henderson, R. Dobie, S. Vermeren, Moro, K., T. Yamada, M. Tanabe, T. Takeuchi, T. Ikawa, H. Kawamoto, J. et al. 2020. Stromal Cells Covering Omental Fat-Associated Lymphoid Furusawa, M. Ohtani, H. Fujii, and S. Koyasu. 2010. Innate production Clusters Trigger Formation of Neutrophil Aggregates to Capture Peri- of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lym- toneal Contaminants. Immunity. 52:700–715.e6. https://doi.org/10.1016/ phoid cells. Nature. 463:540–544. https://doi.org/10.1038/nature08636 j.immuni.2020.03.011 National Comprehensive Cancer Network. 2021. National Comprehensive ´ ´ Jimenez-Sanchez, A., D. Memon, S. Pourpe, H. Veeraraghavan, Y. Li, H.A. Cancer Network Guidelines 2021. https://www.nccn.org (accessed Oc- Vargas, M.B. Gill, K.J. Park, O. Zivanovic, J. Konner, et al. 2017. Heter- tober 26, 2021) ogeneous Tumor-Immune Microenvironments among Differentially Noy, R., and J.W. Pollard. 2014. Tumor-associated macrophages: from Growing Metastases in an Ovarian Cancer Patient. Cell. 170: mechanisms to therapy. Immunity. 41:49–61. https://doi.org/10.1016/j 927–938.e20. https://doi.org/10.1016/j.cell.2017.07.025 .immuni.2014.06.010 Jung, S., J. Aliberti, P. Graemmel, M.J. Sunshine, G.W. Kreutzberg, A. Sher, Okabe, Y., and R. Medzhitov. 2014. Tissue-specific signals control reversible and D.R. Littman. 2000. Analysis of fractalkine receptor CX(3)CR1 program of localization and functional polarization of macrophages. function by targeted deletion and green fluorescent protein reporter Cell. 157:832–844. https://doi.org/10.1016/j.cell.2014.04.016 gene insertion. Mol. Cell. Biol. 20:4106–4114. https://doi.org/10.1128/ Pham, T.H., P. Baluk, Y. Xu, I. Grigorova, A.J. Bankovich, R. Pappu, S.R. MCB.20.11.4106-4114.2000 Coughlin, D.M. McDonald, S.R. Schwab, and J.G. Cyster. 2010. Lym- Kim, K.W., J.W. Williams, Y.T. Wang, S. Ivanov, S. Gilfillan, M. Colonna, H.W. phatic endothelial cell sphingosine kinase activity is required for lym- Virgin, E.L. Gautier, and G.J. Randolph. 2016. MHC II+ resident peri- phocyte egress and lymphatic patterning. J. Exp. Med. 207:17–27. https:// toneal and pleural macrophages rely on IRF4 for development from doi.org/10.1084/jem.20091619 circulating monocytes. J. Exp. Med. 213:1951–1959. https://doi.org/10 Qian, B.Z., J. Li, H. Zhang, T. Kitamura, J. Zhang, L.R. Campion, E.A. Kaiser, .1084/jem.20160486 L.A. Snyder, and J.W. Pollard. 2011. CCL2 recruits inflammatory mon- Kim, J.S., M. Kolesnikov, S. Peled-Hajaj, I. Scheyltjens, Y. Xia, S. Trzebanski, ocytes to facilitate breast-tumour metastasis. Nature. 475:222–225. Z. Haimon, A. Shemer, A. Lubart, H. Van Hove, et al. 2021. A Binary Cre https://doi.org/10.1038/nature10138 Transgenic Approach Dissects Microglia and CNS Border-Associated Robinson-Smith, T.M., I. Isaacsohn, C.A. Mercer, M. Zhou, N. Van Rooijen, N. Macrophages. Immunity. 54:176–190.e7. https://doi.org/10.1016/j Husseinzadeh, M.M. McFarland-Mancini, and A.F. Drew. 2007. Mac- .immuni.2020.11.007 rophages mediate inflammation-enhanced metastasis of ovarian tu- Koga, S., K. Hozumi, K.I. Hirano, M. Yazawa, T. Terooatea, A. Minoda, T. mors in mice. Cancer Res. 67:5708–5716. https://doi.org/10.1158/0008 + + Nagasawa, S. Koyasu, and K. Moro. 2018. Peripheral PDGFRα gp38 -5472.CAN-06-4375 mesenchymal cells support the differentiation of fetal liver-derived Roby, K.F., C.C. Taylor, J.P. Sweetwood, Y. Cheng, J.L. Pace, O. Tawfik, D.L. ILC2. J. Exp. Med. 215:1609–1626. https://doi.org/10.1084/jem.20172310 Persons, P.G. Smith, and P.F. Terranova. 2000. Development of a Zhang et al. Journal of Experimental Medicine 16 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 syngeneic mouse model for events related to ovarian cancer. Carcino- Williams, J.W., K. Zaitsev, K.W. Kim, S. Ivanov, B.T. Saunders, P.R. Schrank, genesis. 21:585–591. https://doi.org/10.1093/carcin/21.4.585 K. Kim, A. Elvington, S.H. Kim, C.G. Tucker, et al. 2020. Limited pro- Rosas, M., L.C. Davies, P.J. Giles, C.T. Liao, B. Kharfan, T.C. Stone, V.B. liferation capacity of aortic intima resident macrophages requires O’Donnell, D.J. Fraser, S.A. Jones, and P.R. Taylor. 2014. The tran- monocyte recruitment for atherosclerotic plaque progression. Nat. scription factor Gata6 links tissue macrophage phenotype and prolif- Immunol. 21:1194–1204. https://doi.org/10.1038/s41590-020-0768-4 erative renewal. Science. 344:645–648. https://doi.org/10.1126/science Wu, Z., J. Xu, J. Tan, Y. Song, L. Liu, F. Zhang, Y. Zhang, X. Li, Y. Chi, and Y. .1251414 Liu. 2019. Mesenteric adipose tissue B lymphocytes promote local and Siegel, R.L., K.D. Miller, and A. Jemal. 2018. Cancer statistics, 2018. CA Cancer hepatic inflammation in non-alcoholic fatty liver disease mice. J. Cell. J. Clin. 68:7–30. https://doi.org/10.3322/caac.21442 Mol. Med. 23:3375–3385. https://doi.org/10.1111/jcmm.14232 Sierro, F., M. Evrard, S. Rizzetto, M. Melino, A.J. Mitchell, M. Florido, L. Xia, H., S. Li, X. Li, W. Wang, Y. Bian, S. Wei, S. Grove, W. Wang, L. Vatan, J.R. Beattie, S.B. Walters, S.S. Tay, B. Lu, et al. 2017. A Liver Capsular Liu, et al. 2020. Autophagic adaptation to oxidative stress alters peri- Network of Monocyte-Derived Macrophages Restricts Hepatic Dis- toneal residential macrophage survival and ovarian cancer metastasis. semination of Intraperitoneal Bacteria by Neutrophil Recruitment. JCI Insight. 5:e141115. https://doi.org/10.1172/jci.insight.141115 Immunity. 47:374–388.e6. https://doi.org/10.1016/j.immuni.2017.07.018 Yona, S., K.W. Kim, Y. Wolf, A. Mildner, D. Varol, M. Breker, D. Strauss-Ayali, Singel, K.L., T.R. Emmons, A.N.H. Khan, P.C. Mayor, S. Shen, J.T. Wong, K. S. Viukov, M. Guilliams, A. Misharin, et al. 2013. Fate mapping reveals Morrell, K.H. Eng, J. Mark, R.B. Bankert, et al. 2019. Mature neutrophils origins and dynamics of monocytes and tissue macrophages under suppress T cell immunity in ovarian cancer microenvironment. JCI homeostasis. Immunity. 38:79–91. https://doi.org/10.1016/j.immuni Insight. 4:e122311. https://doi.org/10.1172/jci.insight.122311 .2012.12.001 Sodhi, C.P., J. Li, and S.A. Duncan. 2006. Generation of mice harbouring a Zhang, N., R.S. Czepielewski, N.N. Jarjour, E.C. Erlich, E. Esaulova, B.T. Sa- conditional loss-of-function allele of Gata6. BMC Dev. Biol. 6:19. https:// unders, S.P. Grover, A.C. Cleuren, G.J. Broze, B.T. Edelson, et al. 2019a. doi.org/10.1186/1471-213X-6-19 Expression of factor V by resident macrophages boosts host defense in Stamatiades, E.G., M.E. Tremblay, M. Bohm, L. Crozet, K. Bisht, D. Kao, C. the peritoneal cavity. J. Exp. Med. 216:1291–1300. https://doi.org/10 Coelho, X. Fan, W.T. Yewdell, A. Davidson, et al. 2016. Immune Moni- .1084/jem.20182024 toring of Trans-endothelial Transport by Kidney-Resident Macro- Zhang, S., I. Dolgalev, T. Zhang, H. Ran, D.A. Levine, and B.G. Neel. 2019b. Both fallopian tube and ovarian surface epithelium are cells-of-origin phages. Cell. 166:991–1003. https://doi.org/10.1016/j.cell.2016.06.058 Steinkamp, M.P., K.K. Winner, S. Davies, C. Muller, Y. Zhang, R.M. Hoffman, for high-grade serous ovarian carcinoma. Nat. Commun. 10:5367. A. Shirinifard, M. Moses, Y. Jiang, and B.S. Wilson. 2013. Ovarian tumor https://doi.org/10.1038/s41467-019-13116-2 attachment, invasion, and vascularization reflect unique microenviron- Zhou, B., Q. Ma, S. Rajagopal, S.M. Wu, I. Domian, J. Rivera-Feliciano, D. ments in the peritoneum: insights from xenograft and mathematical Jiang, A. von Gise, S. Ikeda, K.R. Chien, and W.T. Pu. 2008. Epicardial models. Front. Oncol. 3:97. https://doi.org/10.3389/fonc.2013.00097 progenitors contribute to the cardiomyocyte lineage in the developing Uderhardt, S., A.J. Martins, J.S. Tsang, T. Lamm ¨ ermann, and R.N. Germain. heart. Nature. 454:109–113. https://doi.org/10.1038/nature07060 2019. Resident Macrophages Cloak Tissue Microlesions to Prevent Zhu, Y., J.M. Herndon, D.K. Sojka, K.W. Kim, B.L. Knolhoff, C. Zuo, D.R. Neutrophil-Driven Inflammatory Damage. Cell. 177:541–555.e17. https:// Cullinan, J. Luo, A.R. Bearden, K.J. Lavine, et al. 2017. Tissue-Resident doi.org/10.1016/j.cell.2019.02.028 Macrophages in Pancreatic Ductal Adenocarcinoma Originate from Wang, J., and P. Kubes. 2016. A Reservoir of Mature Cavity Macrophages that Embryonic Hematopoiesis and Promote Tumor Progression. Im- Can Rapidly Invade Visceral Organs to Affect Tissue Repair. Cell. 165: munity. 47:323–338.e6. https://doi.org/10.1016/j.immuni.2017.07 668–678. https://doi.org/10.1016/j.cell.2016.03.009 .014 Wang, Y., K.J. Szretter, W. Vermi, S. Gilfillan, C. Rossini, M. Cella, A.D. Zigmond, E., C. Varol, J. Farache, E. Elmaliah, A.T. Satpathy, G. Friedlander, Barrow, M.S. Diamond, and M. Colonna. 2012. IL-34 is a tissue- M. Mack, N. Shpigel, I.G. Boneca, K.M. Murphy, et al. 2012. Ly6C hi restricted ligand of CSF1R required for the development of Langer- monocytes in the inflamed colon give rise to proinflammatory effector hans cells and microglia. Nat. Immunol. 13:753–760. https://doi.org/10 cells and migratory antigen-presenting cells. Immunity. 37:1076–1090. .1038/ni.2360 https://doi.org/10.1016/j.immuni.2012.08.026 Wang, Y., T.S. Chaffee, R.S. LaRue, D.N. Huggins, P.M. Witschen, A.M. Zigmond, E., B. Bernshtein, G. Friedlander, C.R. Walker, S. Yona, K.W. Kim, Ibrahim, A.C. Nelson, H.L. Machado, and K.L. Schwertfeger. 2020. O. Brenner, R. Krauthgamer, C. Varol, W. Müller, and S. Jung. 2014. Tissue-resident macrophages promote extracellular matrix homeosta- Macrophage-restricted interleukin-10 receptor deficiency, but not IL- sis in the mammary gland stroma of nulliparous mice. eLife. 9:e57438. 10 deficiency, causes severe spontaneous colitis. Immunity. 40:720–733. https://doi.org/10.7554/eLife.57438 https://doi.org/10.1016/j.immuni.2014.03.012 Weiss, J.M., L.C. Davies, M. Karwan, L. Ileva, M.K. Ozaki, R.Y. Cheng, L.A. Zindel, J., M. Peiseler, M. Hossain, C. Deppermann, W.Y. Lee, B. Haenni, B. Ridnour, C.M. Annunziata, D.A. Wink, and D.W. McVicar. 2018. Ita- Zuber, J.F. Deniset, B.G.J. Surewaard, D. Candinas, and P. Kubes. 2021. conic acid mediates crosstalk between macrophage metabolism and Primordial GATA6 macrophages function as extravascular platelets in peritoneal tumors. J. Clin. Invest. 128:3794–3805. https://doi.org/10.1172/ sterile injury. Science. 371:eabe0595. https://doi.org/10.1126/science JCI99169 .abe0595 Zhang et al. Journal of Experimental Medicine 17 of 17 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Supplemental material Zhang et al. Journal of Experimental Medicine S1 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 cre LSL-tdTomato EYFP Figure S1. Characterization of membrane-associated macrophages. (A) Two-photon images of liver capsule from Lyz2 :R26 :CD11c mice. cre Tomao EYFP Scale bar, 70 µm. (B) Two-photon images of mesenteric membrane from Lyz2 :R26 :CD11c mice. Scale bar, 40 µm. (C) Representative flow cy- EFYP EYFP + lo-to-hi + − tometric analysis of gut mesentery in CD11c mice. CD11c and CD11b gating of CD45 MHCII mesenteric cells (left). Overlay of CD11b EYFP and + + CreER Tomato CD11b EYFP cells (right; n =6). (D) Whole-mount confocal images of Csf1r :R26 mice with CD206 and ICAM2 staining. Imaging data are repre- hi sentative of at least two independent experiments. Scale bar, 40 µm. (E) Flow cytometric analysis for ICAM2, CD206, MHC II, and CD226 expression in F4/80 lo hi LPMs, F4/80 small peritoneal macrophages (SPM) and F4/80 mesenteric macrophages. Data are representative of at least two independent experiments. (F) Mean fluorescent intensity (MFI) values of ICAM2, CD206, MHC II, and CD226, which are normalized by isotype controls. Data are pooled from at least two independent experiments (n =3–6 mice). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple-comparison test: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Zhang et al. Journal of Experimental Medicine S2 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Cre fl/fl Figure S2. Mesenteric membrane–associated macrophages in Lyz2 :Gata6 mice. (A) Whole-mount images and quantification of membrane- cre fl/fl + associated macrophages of Lyz2 :Gata6 mice and littermate controls. Scale bar, 50 µm. (B) Quantification of ICAM2 peritoneal macrophages in cre fl/fl Lyz2 :Gata6 mice and littermate controls. Imaging data and flow cytometric analysis are representative of two independent experiments (n = 3 per genotype; mean ± SEM). Unpaired Student’s t test: ***, P < 0.001. cre LSL-tdTomato − Figure S3. Comparison of blood leukocytes between naive Lyve1 :R26 mice and Tomato BM-transplanted mice. (A) Tomato expression cre LSL-tdTomato − of blood leukocytes in naive Lyve1 :R26 mice (n =5). (B) Tomato expression of blood leukocytes from Tomato BM-transplanted mice (n = 4). Data are representative of at least two independent experiments. (C) Images of avascular region of a mesenteric membrane and the region containing adipose tissue in the BM-transplanted mice. Scale bars, 100 and 30 µm. Zhang et al. Journal of Experimental Medicine S3 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Figure S4. t-SNE plot identifying different cell populations in scRNA-seq of whole mesentery cells (accession no. GSE102665). (A) Signature genes that represent different cell populations of whole mesenteric cells. (B) t-SNE-plot for macrophage populations. (C) t-SNE-plot for dendritic cell populations. Zhang et al. Journal of Experimental Medicine S4 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Cre fl/fl Figure S5. Quantification of tissue-resident macrophages of Lyve1 :Csf1r and control mice. (A) Gating strategy of two peritoneal macrophage Cre fl/fl subsets and their quantification in Lyve1 :Csf1r mice and control mice. FSC, forward scatter; SSC, side scatter. (B) Gating strategy of splenic red pulp Cre fl/fl macrophages and their quantification in Lyve1 :Csf1r mice and control mice. (C) Gating strategy of alveolar macrophages and their quantification of in Cre fl/fl + Cre fl/fl Lyve1 :Csf1r mice and control mice. (D) Gating strategy and percentage of microglia in CD45 brain leukocytes of Lyve1 :Csf1r mice and control mice. + + (E) Representative histogram of CSF1R expression in ICAM2 and MHC II peritoneal macrophage subsets analyzed in A. In A–E, data were pooled from two independent experiments (n =5–8 mice). Statistical analysis was performed by unpaired Student’s t test. Zhang et al. Journal of Experimental Medicine S5 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924 Video 1. Video reconstruction of interactions of Tomato-expressing macrophages under Lyz2 promoter with CD11cEYFP-expressing peritoneal macrophages. Two-photon intravital microscope visualized Tomato-expressing mesenteric membrane macrophage in contact with CD11c EYFP-expressing lo F4/80 peritoneal macrophages. Video 2. Video reconstruction of Tomato-expressing macrophages driven by Lyve1 promoter in pia/dura mater of brain shown in Fig. 2 E. Tomato cre LSL-tdTomato BM cells of Lyve1 :R26 mice were transplanted into irradiated WT mice. The mice were used to visualize meningeal perivascular macrophages. Tomato-expressing macrophages were associated with blood vasculature underneath the skull. Video 3. Video reconstruction of Tomato-expressing macrophages driven by LYVE1 promoter in the peritoneal parietal membrane shown in Fig. 2 G. − cre LSL-tdTomato Tomato BM cells of Lyve1 :R26 mice were transplanted into irradiated WT mice. 10 wk later, the mice were injected with Alexa Fluor 488– conjugated lectin, and the parietal peritoneal membrane was visualized. Tomato-expressing macrophages were mainly located in the collagen-enriched serosal membrane of the parietal peritoneum. Zhang et al. Journal of Experimental Medicine S6 Mesothelium-associated macrophages https://doi.org/10.1084/jem.20210924

Journal

The Journal of Experimental MedicinePubmed Central

Published: Oct 29, 2021

There are no references for this article.