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Expression of factor V by resident macrophages boosts host defense in the peritoneal cavity

Expression of factor V by resident macrophages boosts host defense in the peritoneal cavity BRIEF DEFINITIVE REPORT Expression of factor V by resident macrophages boosts host defense in the peritoneal cavity 1 1 1 1 1 1 3 Nan Zhang , Rafael S. Czepielewski , Nicholas N. Jarjour , Emma C. Erlich , Ekaterina Esaulova ,Brian T. Saunders , Steven P. Grover , 4 2 1 3 1 1 Audrey C. Cleuren ,George J. Broze , Brian T. Edelson , Nigel Mackman , Bernd H. Zinselmeyer , and Gwendalyn J. Randolph  Macrophages resident in different organs express distinct genes, but understanding how this diversity fits into tissue-specific features is limited. Here, we show that selective expression of coagulation factor V (FV) by resident peritoneal macrophages in mice promotes bacterial clearance in the peritoneal cavity and serves to facilitate the well-known but poorly understood “macrophage disappearance reaction.” Intravital imaging revealed that resident macrophages were nonadherent in peritoneal fluid during homeostasis. Bacterial entry into the peritoneum acutely induced macrophage adherence and associated bacterial phagocytosis. However, optimal control of bacterial expansion in the peritoneum also required expression of FV by the macrophages to form local clots that effectively brought macrophages and bacteria in proximity and out of the fluid phase. Thus, acute cellular adhesion and resident macrophage–induced coagulation operate independently and cooperatively to meet the challenges of a unique, open tissue environment. These events collectively account for the macrophage disappearance reaction in the peritoneal cavity. Introduction Our understanding of the biology of tissue-resident macrophages Results and discussion A prominent example of tissue-restricted gene expression in has evolved greatly in the past decade. Resident macrophages in macrophages is the selective detection in LPMs of mRNA for most organs arrive during embryogenesis, maintaining them- coagulation factors, including factor V (FV; F5), FVII (F7), and FX selves for long periods via local proliferation (Ginhoux and (F10; Fig. 1 A). The latter two factors were also expressed by lung Guilliams, 2016). These resident macrophages express special- macrophages, whereas FV was unique to peritoneal macro- ized sets of genes that are distinct between different organs phages. Activated FV is a key cofactor in the common pathway (Gautier et al., 2012). Even when monocytes are recruited to for activated FX, forming the prothrombinase complex that, these organs during inflammation, the inflammatory macro- during coagulation, converts prothrombin to thrombin that in phages they become are reticent to take up the specialized res- turn converts fibrinogen to fibrin to form clots. Indeed, the co- ident phenotype, if they do at all (Gautier et al., 2013; Guilliams agulation pathway was among top pathways selectively en- and Scott, 2017; Misharin et al., 2017). It is assumed that spe- riched in LPMs (Gautier et al., 2012). The unique expression of cialized genes expressed by particular resident macrophages FV, in particular, among tissue-resident macrophages caused us encode products tailored to the specific physiological needs or to consider the possibility that coagulation may have a key role constraints of that tissue, but illustration of direct links often in peritoneal macrophage function. The expression of FVII, remain unexplored. Transcription factors that regulate special- ized macrophage gene sets in different organs have, however, important for initiating the extrinsic pathway of coagulation in been identified. One such transcription factor is Gata6, which complex with tissue factor (TF; F3) that is acutely induced after selectively governs the life cycle of murine resident peritoneal tissue damage, led us to consider this pathway of coagulation in macrophages, often called large peritoneal macrophages (LPMs; particular. Gautier et al., 2012, 2014; Okabe and Medzhitov, 2014; Rosas A classic response to inflammation exhibited by LPMs is et al., 2014). In this study, we focused on understanding how known as the “macrophage disappearance reaction” (MDR), first the transcriptional profile of resident peritoneal macrophages described decades ago (Nelson, 1963). In this reaction, LPMs could be linked to the specialized function of these cells. become irretrievable from lavage just hours after introduction of ............................................................................................................................................................................. 1 2 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO; Department of Medicine, Washington University School of 3 4 Medicine, St. Louis, MO; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC; Life Sciences Institute, University of Michigan, Ann Arbor, MI. Correspondence to Gwendalyn J. Randolph: gjrandolph@wustl.edu. © 2019 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.20182024 1291 J. Exp. Med. 2019 Vol. 216 No. 6 1291–1300 Figure 1. Coagulation and adhesion additively cooperate to account for the MDR in response to inflammation. (A) Gene array analysis (from ImmGen; n = 3 separate pools) of classical coagulation factors in major tissue resident macrophages, including those from the spleen, central nervous system (CNS), lung, and peritoneum. (B) Quantification of LPMs in peritoneal lavage 3 h after zymosan injection i.p. when clotting and/or adhesion was inhibited. (C) Aggregates retrieved from the peritoneum 5 h after zymosan injection. (D–G) Immunofluorescence staining of the aggregates for fibrin(ogen) and macrophage markers. D and G are stained frozen sections of the clots; E and F are whole-mount preparations. Scale bars represent 100 µm (D and G), 50 µm (E), and 10 µm (F). (H) Flow cytometry on peritoneal exudate cells from untreated mice (left), 3 h after zymosan i.p. (middle), and clots 3 h after zymosan i.p. (right). (I) Cre fl/fl Quantification of LPMs 3 h after zymosan injection in clots and omenta in WT and Lyz2 ;Tln1 mice. One-way ANOVA was used to test statistical signif- icance. Symbols represent individual mice studied. Error bars represent ± SEM. All experiments were repeated at least two or three times. **, P < 0.01; ***, P < 0.001. inflammatory stimuli like the bacillus Calmette-Guerin vaccine, inflammation otherwise appears to have resolved (Gautier et al., lipopolysaccharide, zymosan, or thioglycollate (Nelson, 1963; 2013). In the 1960s, Nelson proposed a role for coagulation in the Barth et al., 1995; Davies et al., 2013; Gautier et al., 2013; Meza- MDR, because he could fully reverse it by administering hepa- Perez and Randall, 2017). Repopulation of resident macrophages rin, which can block coagulation or adhesion; MDR was also is slow following the MDR. LPMs are not always restored after reversed to an appreciable but lesser extent by warfarin, which Zhang et al. Journal of Experimental Medicine 1292 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 Table 1. Peritoneal fluid volume, peritoneal cell density, and total cell anti-Ly6G mAb (Fig. S1 A) demonstrated the neutrophils were and macrophage numbers in 6-wk-old C57BL/6J mice (comparison of dispensable as a cause for MDR (Fig. S1 B). Furthermore, neu- lavage and collection of undiluted fluid) trophil recruitment was unaffected by Tln1 deficiency in this model (Fig. S1, C and D), which is consistent with previous re- Volume Cell density Total cells Gata6 resident port (Lim and Su, 2018). We conclude that neutrophil infiltration 4 6 (μl± (×10 /µl ± (×10 ± macrophages does not play a role in MDR in this model. However, integrin– SEM) SEM) SEM) (×10 ±SEM) mediated adhesion is relevant to MDR, but such adhesion cannot Peritoneal 52.14 ± 6.50 ± 0.85 (6) 3.26 ± 0.27 (6) 1.73 ± 0.11 (6) account for a distinct, prominent role of the coagulation cascade fluid 4.02 (7) in affecting macrophage removal. Lavage N/A N/A 3.20 ± 0.15 (12) 1.81 ± 0 09 (12) A close physical examination of the peritoneal cavity after MDR revealed millimeter-sized aggregates within the peritoneal Numbers in parentheses indicate the numbers of mice used. N/A, not cavity 3–5 h after i.p. injection of zymosan (Fig. 1 C). These ag- applicable. gregates stained positively for fibrin(ogen) (Fig. 1, D–F), indi- cating that they were clots, with such staining most easily would more specifically target coagulation (Nelson, 1963). In the appreciated in whole-mount preparations where strands of fi- ensuing years, with recognition that fibrin(ogen) participates in brin were observed between clumps of zymosan particles and adhesion, the view developed that coagulation factors support cells (Fig. 1, E and F). Clots were also enriched in LPMs identified macrophage disappearance by promoting adhesion and migra- by coexpression of Gata6 and ICAM2 (Fig. 1 G), but very few tion (Szaba and Smiley, 2002) to locations like the omentum peritoneal B cells were incorporated into the clots (Fig. S1 E), during the MDR (Meza-Perez and Randall, 2017). Thus, a clas- despite that peritoneal B cells are almost as abundant as mac- sical role for coagulation in peritoneal host defense was largely rophages in the steady state peritoneal fluid. Some macrophages overlooked. Our intravital imaging of the peritoneal cavity in clots were loaded with zymosan particles, but other zymosan through an intact abdominal wall indicated that a distinct fea- particles appeared outside of cells (Fig. 1, E and F), suggesting ture of LPMs, relative to resident macrophages in other organs, that clots were efficient at entrapping debris not yet phagocy- is that they are free floating in peritoneal fluid (Video 1). LPMs tized. Clots also incorporated S100A9 neutrophils, which are only adhered to the walls of the peritoneal cavity covering vis- known to be rapidly recruited to the peritoneum after zymosan ceral organs after inflammatory stimuli (heat-killed bacteria) is injected i.p. (Fig. 1 D). Flow cytometry confirmed enrichment were introduced (Video 1). Cell density in peritoneal fluid was of LPMs in the clots, while they were largely absent in the lavage high, at ∼6.5 × 10 /µl (Table 1), ∼20× the concentration of leu- 3 h after i.p. zymosan (Fig. 1 H). Approximately three times more kocytes in blood (Mouse Phenome Database; The Jackson LPMs were recovered in clots compared with the omentum Laboratory). (Fig. 1 I), indicating that clots are prominent sites for macro- Given this literature and the striking expression of coag- phage “disappearance” in response to inflammatory triggers. ulation factors in LPMs, we wondered whether coagulation Consistent with the integrin independence of macrophage in- might drive important processes in the peritoneum. First, we clusion in clots, macrophage recovery from clots was unaffected confirmed that heparin fully reversed the MDR induced by by Tln1 deficiency (Fig. 1 I), whereas recovery from the omentum 1 mg zymosan i.p. (Fig. 1 B). The very selective inhibitor of trended downward, consistent with a role for adhesion in re- thrombin hirudin partially (∼50%) inhibited MDR (Fig. 1 B), taining macrophages in the omentum. reminiscent of the effect of warfarin relative to heparin Work beginning in the 1980s revealed that LPMs produce FV (Nelson, 1963). Heparin has both anticoagulant and anti- in culture (Osterud et al., 1981), including production of a adhesive activity (Woods et al., 1986), and this may explain functional prothrombinase complex (activated FV/activated FX; why it is more effective at inhibiting MDR compared with Pejler et al., 2000) long before it was recognized that F5 mRNA either warfarin or hirudin. expression was selective to LPMs. Although the liver is a known To directly address the role of integrin–mediated adhesion in source of FV, expression of FV by LPMs led us to hypothesize Cre fl/fl MDR, in the same experiments we employed Lyz2 ;Tln1 mice these macrophages maintain FV levels in peritoneal fluid in the (Fig. 1 B) to prevent en bloc activation of integrins through loss steady state. We thus analyzed FV activity in peritoneal fluid of the integrin activation adaptor talin-1 (Tln1) in lysozyme- using a one-stage clotting assay. In WT C57BL/6J mice, FV ac- expressing cells (Yago et al., 2015), which includes LPMs and tivity in peritoneal interstitial fluid was ∼13% of that in plasma other myeloid cells like neutrophils (Faust et al., 2000). The (Fig. 2 A), consistent with the documented poor entry of plasma absence of integrin signaling also partially inhibited MDR, proteins as large as FV (a ∼300-kD protein) into interstitial fluid similar to the inhibition achieved with hirudin alone (∼50%). of molecules (Yang et al., 1998; Michel et al., 2015). Peritoneal −/− However, rather than being redundant, the combination of fluid was thus analyzed from F5 ;AlbF5Tg mice, engineered hirudin and genetic loss of Tln1 had additive effects, such that such that the liver sustains expression of FV from the albumin when the two interventions were combined, MDR was fully promoter but all other tissues, including LPMs, lack FV (Sun reversed, mirroring heparin (Fig. 1 B). In addition, because et al., 2003). These mice displayed a ∼75% reduction in FV ac- neutrophil infiltration almost always correlates with MDR after tivity compared with WT peritoneal fluid (Fig. 2 A), raising the zymosan injection, we tested the hypothesis that infiltrated possibility that the major source of FV in the peritoneal fluid was neutrophils cause MDR. Results from depleting neutrophils with not the liver. Zhang et al. Journal of Experimental Medicine 1293 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 Figure 2. FV and TF from resident peritoneal macrophages are responsible for peritoneal fluid clotting. (A) Quantification of FV activity in plasma or peritoneal fluid of various genotypes of mice or after bone marrow transplant of indicated donor genotypes into irradiated WT recipients (last bars onthe right). Zym, zymosan. (B) Quantification of LPMs 3 h after zymosan injection. (C) Quantification of LPMs 3 h after zymosan injection. One-way ANOVA was +/+ fl/fl used to test statistical significance, except for the bone marrow chimera result in B and the hF groups and F3 groups in C, which used a two-tailed t test. Symbols represent individual mice studied. Error bars represent ± SEM. All experiments were repeated at least two or three times. *, P < 0.05; **, P < 0.01; ***, P<0.001. Demonstrating that a major local source of FV was LPMs, FV extrinsic coagulation (Parry et al., 1998), while their control Cre fl/fl activity in peritoneal fluid of the Lyz2 ;Gata6 mouse, which counterparts from a sister line expressing the human transgene lack most LPMs (Gautier et al., 2014; Okabe and Medzhitov, as well as murine F3 exhibited a complete MDR in response to −/− 2014; Rosas et al., 2014), mirrored the very low level in F5 ; zymosan (Fig. 2 C). Likewise, the MDR was partially suppressed Cre fl/fl fl/fl AlbF5Tg mice (Fig. 2 A). LPMs are the only immune cell in the in Lyz2 ;F3 mice (Pawlinski et al., 2010) compared with F3 mouse that expresses Gata6 (Gautier et al., 2014). Thus, while control mice (Fig. 2 C). Even though F3 mRNA is not detectable in Cre Cre fl/fl Lyz2 is not completely selective to LPMs, Lyz2 ;Gata6 mice resting LPMs (Fig. 1 A), TF can be rapidly (within 1 h) up- exhibit a highly selective defect in LPMs (Gautier et al., 2014). As regulated on the LPM surface and is capable of initiating clot- an additional evaluation, we performed bone marrow trans- ting in vitro (data not shown). Thus, it is likely that LPMs −/− plantation using WT or F5 ;AlbF5Tg marrow as donors for ir- quickly generate enough TF to initiate the extrinsic coagulation radiated WT recipients that would have normal liver FV. With cascade during the MDR. 80–85% reconstitution of resident peritoneal macrophages with Finally, we wondered whether clotting of interstitial fluid −/− donor marrow (data not shown), mice receiving F5 ;AlbF5Tg had a critical functional role. While clotting of interstitial fluid marrow showed significantly lower FV activity in peritoneal would seem irrelevant to hemostasis, we wondered if interstitial fluid than mice receiving WT marrow (Fig. 2 A,bars on right). clotting might serve to entrap both free-floating macrophages We then hypothesized that this LPM–derived FV is crucial to and microbes that invade the peritoneum. Indeed, a previous promote early local clotting in the peritoneal fluid. Indeed, the study revealed that a single dose of heparin or hirudin caused −/− zymosan-induced MDR in F5 ;AlbF5Tg mice reduced the MDR increased mortality after mice were subjected to cecal-ligation to a similar level as the hirudin-treated group (Fig. 2 B), sug- puncture (Echtenacher et al., 2001), although the role of local gesting that FV from LPMs is critical for the peritoneal clotting cells and factors remained unclear. To investigate whether LPM- in this model. Thus, we conclude that LPMs produce FV in the dependent local peritoneal coagulation controlled peritoneal steady state that in turn promotes clotting of local interstitial infection, we measured the thrombin–antithrombin complex fluid during the MDR (Sun et al., 2003). (TAT), the standard assay to track occurrence of coagulation, in In the extrinsic coagulation cascade, TF (FIII; F3) initiates the peritoneal fluid and plasma over time after i.p. infection clotting and has in other systems been demonstrated to be with live E. coli (Fig. 3 A), normalizing the data as a percentage of rapidly expressed by macrophages upon activation. We ob- maximal TAT in the serum from exogenously clotted plasma. served rescue of MDR in response to zymosan-injected i.p. in Both peritoneal and blood TAT were below detection in the mice deficient for murine TF that express very low human TF steady state using the assay. However, peritoneal but not blood −/− +/+ levels (hF3; ∼1%; mF ;LowhF3 ), in contrast to control mice TAT complex was detected within 2 h of infection, lasting for at +/+ +/+ MeriCreMer fl/fl with normal TF levels (mF ;LowhF3 ; Fig. 2 C). The 1% of least 6 h (Fig. 3 A). In tamoxifen-treated Csf1r ;Gata6 Cre fl/fl normal TF levels in the hF3 transgenic mouse can restore via- or Lyz2 ;Gata6 mice, which exhibit selectively and greatly bility of the line but has a markedly impaired capacity to initiate reduced LPMs (Gautier et al., 2014), TAT was an average of 48% Zhang et al. Journal of Experimental Medicine 1294 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 Figure 3. Macrophage adhesion and macrophage-driven clotting cooperatively promote peritoneal bacterial clearance. (A) Quantification of TAT complex in peritoneal fluid and plasma in the steady state or after E. coli infection. (B) Quantification of TAT complex in peritoneal fluid 4 h after E. coli infection. Cre fl/fl Cre fl/fl Ctrl, control. (C) CFUs per microliter peritoneal fluid from untreated, hirudin-treated WT, Lyz2 ;Talin mice, hirudin-treated Lyz2 ;Talin mice, and fl/fl Cre fl/fl clodronate liposome-treated mice 4 h after E. coli infection. (D) CFUs per microliter peritoneal fluid from Gata6 and Lyz2 ;Gata6 mice 4 h after E. coli infection. (E) CFUs per microliter peritoneal fluid from C57BL/6J treated with or without hirudin 4 h after E. coli infection. (F) CFUs per microliter peritoneal −/− fluid from control and F5 ;AlbF5Tg mice 4 h after E. coli infection. One-way ANOVA was used to test statistical significance, except for C, D, and F and the −/− F5 ;AlbF5Tg groups in B, which were examined by a two-tailed t test. Symbols represent individual mice studied. Error bars represent ± SEM. All experiments were repeated at least two or three times. *, P < 0.05; **, P < 0.01; ***, P < 0.001. and 57% lower, respectively, than in control mice upon infection. an increase in permeability might account for why TAT levels −/− Furthermore, F5 ;AlbF5Tg mice showed comparably reduced are not as low as the basal level of FV observed in resting mice. peritoneal TAT after infection (Fig. 3 B). As expected, Tln1 de- We next investigated the functional impact of MDR on local ficiency in macrophages did not affect TAT levels (Fig. 3 B), and bacterial clearance during a 4-h period following infection. In- neither did neutrophil depletion (data not shown). These data hibition of both clotting and macrophage adhesion to collectively indicate that Gata6 resident macrophages contribute substan- block the MDR dramatically increased CFUs in the peritoneal tially to peritoneal coagulation in an FV-dependent manner fluid compared with untreated mice, clotting-inhibited mice, or early after infection. Even though it would be anticipated that adhesion-blocked mice (Fig. 3 C), strongly suggesting that both the infectious state would increase vascular permeability and clotting and adhesion are required for optimal bacterial clear- allow for increased influx of plasma–derived proteins, the data ance. By examining bacterial numbers that we could retrieve indicate that any role for permeability in bringing in plasma FV from the peritoneal cavity 1 min after their installation (Fig. 3 C, in the few hours after infection is not sufficient to raise TAT to right column) and comparing with all data obtained at 4 h, it was levels observed in mice that can generate local FV. Nonetheless, clear that all CFUs recovered at 4 h were reduced from the Zhang et al. Journal of Experimental Medicine 1295 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 numbers instilled. Thus, CFUs assessed at 4 h served primarily to blockade of β1and β2 integrins would be expected to interfere assess how efficiently bacteria were cleared rather than to assess with adhesion of peritoneal macrophages to mesothelial walls bacterial growth. In the absence of any interference to the MDR, (Bellingan et al., 2002; Cao et al., 2005) and, fortunately, neu- an average of 98% of bacteria were cleared in 4 h compared tralizing mAbs to these molecules had a neutral impact on E. coli with the number of CFUs retrieved at 1 min. When macrophages CFUs in the spleen when E. coli was administered to blood (Fig. 4 had earlier been depleted from the peritoneal cavity with B). These data thus indicate that the most optimal tools in hand clodronate-loaded liposomes without affecting blood monocytes, to next evaluate the impact of macrophage–mediated coagula- as per previous protocol (Wang and Kubes, 2016), clearance of tion and adhesion on E. coli dissemination from the peritoneum −/− bacteria was much less efficient, at only 49%, indicating the were F5 ;AlbF5Tg mice, heparin, or hirudin to block coagula- pivotal role of local macrophages in bacterial removal. With use tion and combined anti-β1and -β2 mAbs to block adhesion. of hirudin alone or loss of Tln1 alone, clearance of bacteria av- When we next instilled E. coli into the peritoneal cavity to eraged 93% and 86%, respectively. Remarkably, when both co- study its dissemination, mice with a complete loss of MDR agulation and adhesion were inhibited, clearance at 4 h through the use of heparin exhibited markedly elevated CFUs in decreased to an average of 64%, approaching the impact that the spleen at 16 h after E. coli i.p. instillation (Fig. 4 D). Fur- −/− complete macrophage depletion achieved (Fig. 3 C). Thus, these thermore, hematopoietic FV-deficient (F5 ;AlbF5Tg)mice data strongly suggest that both coagulation and adhesion opti- treated with blocking antibodies against both β1and β2 integrins mize bacterial clearance from the peritoneal cavity. showed significantly increased CFUs in the spleen when E. coli We then wondered if LPMs or LPM-dependent clotting played were administered i.p. (Fig. 4 E). Mice treated with both hirudin a significant role in accounting for the overall role played by and blocking antibodies against β1and β2 integrins showed macrophages in bacterial clearance as revealed from the use of the significantly increased bacterial burden in the spleen at 16 h clodronate-loaded liposomes. Mice genetically lacking the major- after E. coli i.p. instillation compared with those treated with Cre fl/fl ity of LPMs (Lyz2 ;Gata6 ) showed CFUs approximately seven anti-β1and β2 mAbs alone (Fig. 4 F), demonstrating that clotting times higher than those of control mice (Fig. 3 D). Deficiency in indeed impacted bacterial dissemination, beyond the impact of −/− hematopoietic FV (F5 ;AlbF5Tg) also reduced CFU by two- to impaired adhesion. Tln1 deficiency in myeloid cells, combined threefold (Fig. 3 F), confirming that local FV accounted for the role with hirudin, did not affect dissemination (Fig. 4 G), but this is of coagulation in local bacterial clearance. Intravital imaging of likely due to the conflicting roles of Tln1 deficiency in the peri- clots ex vivo showed Escherichia coli within lysozyme cells at the toneum (Fig. 1 B) versus the blood (Fig. 4 A). Together, these data time of imaging and other motile bacteria not yet engulfed by strongly suggest that the two processes that cooperatively and phagocytes but nonetheless trapped within the clot (Video 2). nonredundantly account for the MDR, coagulation and adhesion, Lastly, we investigated whether the local control of bacteria also cooperatively and nonredundantly function to optimize by the MDR or clotting had any impact on bacterial dissemina- bacterial clearance from the peritoneal cavity, which is essential tion from the peritoneal cavity to the blood and ultimately the to contain dissemination. spleen. Dissemination is a complex evaluation because there can These studies, in summary, illustrate how specialized gene be roles for leukocytes or coagulation beyond the peritoneum expression in a particular macrophage is intimately tied to the within the vasculature itself. The most ideal experimental sce- unique characteristics and physiology of the organ. Specifi- nario would be one in which the tools applied to the question of cally, to support high-quality host defense in the capture of dissemination from the peritoneum would have neutral effects microorganisms that might gain access to the peritoneal on CFU in the spleen after E. coli administration in the blood so cavity through the digestive tract, resident peritoneal mac- that the effects from the peritoneum could be more selectively rophages constitutively produce FV, along with other clotting evaluated. Thus, we first evaluated whether the tools we used in factors. This production makes up for the minimal access that the peritoneal cavity that regulate adhesion, including the liver–derived FV has to the peritoneum. Expression of FV al- Cre fl/fl Lyz2 ;Tln1 mouse strain and heparin, altered CFUs in the lows macrophages to generate clots that trap microorganisms spleen after i.v. delivery, as any effects in this arm of the ex- even before they have a chance to be phagocytosed. This form periment could confound interpretation of dissemination from of host defense is especially relevant in a fast-flowing envi- Cre fl/fl the peritoneum. Indeed, and surprisingly, Lyz2 ;Tln1 mice ronment of fluid that comprises a challenging space for effi- displayed fewer CFUs in the spleen after E. coli delivery i.v. cient phagocytosis. While the clots collect microorganisms in (Fig. 4 A). This protective effect of Tln1 deficiency in blood large numbers, additional macrophages adhering within the myeloid cells might be expected to offset our predicted increase omentum and upon visceral organ surfaces undoubtedly in dissemination of E. coli from the peritoneum. By contrast, promote capture of organisms that avoided entrapment in heparin administered i.p. did not show any statistically signifi- clots. These two mechanisms collectively account for the vast cant effect on CFU in the spleen after i.v. administration of E. coli majority of macrophage disappearance in the context of per- −/− (Fig. 4 B). F5 ;AlbF5Tg mice and hirudin administered i.p. also itoneal inflammation, and our data indicate that both arms of had neutral effects on E. coli CFUs in the spleen when E. coli was the MDR, adhesion and coagulation, are required for optimal administered i.v. (Fig. 4, B and C). With respect to adhesion, host defense. Finally, in many clinical scenarios, therapeutic because Tln1 deficiency was not neutral in the blood–spleen agents are used to limit coagulation to minimize its occur- compartment (Fig. 4 A), we sought to block macrophage adhe- rence in blood. The present work, however, illustrates that in sion using antibodies rather than Tln1 deficiency. Combined the particular tissue microenvironment of the peritoneum, Zhang et al. Journal of Experimental Medicine 1296 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 Figure 4. MDR restricts bacterial dissemina- tion out of the peritoneal cavity to the spleen. fl/fl Cre (A) CFUs per spleen from Tln1 mice or Lyz2 ; fl/fl Tln1 mice at 16 h after i.v. E. coli infection. (B) CFUs per spleen from mice untreated or treated with heparin, anti-integrin β1and β2 blocking antibodies, or hirudin at 16 h after i.v. E. coli infection. Differences between groups are not statistically significant. (C) CFU per spleen −/− from F5 littermates and F5 ;AlbF5Tg mice at 16 h after i.v. E. coli infection. Differences are not statistically significant. Ctrl, control. (D) CFUs per spleen from untreated and heparin-treated mice at 16 h after i.p. E. coli infection. (E) CFUs −/− per spleen from F5 littermates and F5 ;AlbF5Tg mice treated with anti-integrin β1and β2block- ing antibodies at 16 h after i.p. E. coli infection. (F) CFUs per spleen from mice treated with anti- integrin β1and β2 blocking antibodies and mice treated with hirudin and anti-integrin β1and β2 blocking antibodies at 16 h after i.p. E. coli fl/fl infection. (G) CFUs per spleen from Tln1 mice Cre fl/fl or Lyz2 ;Tln1 mice treated with hirudin at 16 h after i.p. E. coli infection. Differences are not statistically significant. Two-tailed t tests were used to test statistical significance for A and C–G. One-way ANOVA was used to test statis- tical significance for B. Symbols represent indi- vidual mice studied. Error bars represent ± SEM. All experiments were repeated at least one to three times. *, P < 0.05; **, P < 0.01; ***, P < 0.001. resident macrophage–mediated coagulation produces impor- polyclonal antibody was purchased from R&D Systems tant beneficial effects. (AF2065-SP). Anti-Gata6 monoclonal antibody was pur- chased from Cell Signaling Technology (5851). Anti- fibrin(ogen) polyclonal antibody was purchased from Agilent Dako (A0080). The Alexa Fluor 594–conjugated heat-killed Materials and methods bacterial particles were purchased from Thermo Fisher Sci- Mice entific (E23370). Clodronate-loaded liposome was purchased All C57BL/6J mice were purchased at the age of 8 wk from The Cre fl/fl from ClodronateLiposomes.org. Recombinant hirudin was Jackson Laboratory. Lyz2 ;Tln1 mice were a kind gift from purchased from Aniara Diagnostica (ARE120A). Zymosan was Dr. Rodger P. McEver (Oklahoma Medical Research Foundation, Cre fl/fl purchased from Sigma-Aldrich (Z4250). GFP E. coli was pur- Oklahoma City, OK). Low human TF mice, Lyz2 ;F3 mice, −/− Cre fl/fl chased from ATCC (ATCC 25922GFP). Human FV–deficient F5 ;AlbF5Tg mice, Lyz2 ;Gata6 mice (now backcrossed to Cre LSL-Tdtomato GFP plasma was purchased from Haematologic Technologies. C57BL/6 background), Lyz2 ;R26 mice, and Bhlhe40 Rabbit thromboplastin (44213), Liberase, hyaluronidase, mice were described previously (Parry et al., 1998; Sun et al., DNase I, streptokinase, and collagenase D were purchased 2003; Pawlinski et al., 2010; Gautier et al., 2014; Lin et al., 2016; MeriCreMer fl/fl from Sigma-Aldrich. Plasminogen was purchased from Lee Wang and Kubes, 2016). Csf1r ;Gata6 mice (fully Biosolutions. backcrossed to C57BL/6 background) were generated by MeriCreMer fl/fl crossing Csf1r mice with Gata6 mice. Mouse studies were approved by animal use oversight committees at Wash- Flow cytometry ington University School of Medicine and the University of Flow cytometry was performed as described previously (Gautier North Carolina (studies on TF). et al., 2014). Briefly, peritoneal exudate cells were collected by flushing the peritoneum with 6 ml HBSS with 2.5 mM EDTA and Reagents 0.2% BSA. Peritoneal clots were collected 3 h after zymosan Pac Blue anti-CD45 mAb, allophycocyanin anti-ICAM2 mAb, injection and digested using a cocktail of 1 mg/ml collagenase D, allophycocyanin/Cy7 anti-CD11b mAb, PE anti-Ly6G mAb, 50 U/ml streptokinase, 4 U/ml plasminogen, 100 µg/ml Lib- PerCP/Cy5.5 anti-Ly6C mAb, PE/Cy7 anti-F4/80 mAb, and erase, 100 µg/ml DNase I, and 0.5 mg/ml hyaluronidase in RPMI anti-ICAM2 mAb were all purchased from BioLegend. Anti-S100A9 with 1% FBS. The digestion was incubated at 37°C for 30 min and Zhang et al. Journal of Experimental Medicine 1297 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 mixed by pipetting every 5 min. It was then passed through FV activity measurement 70-µm cell strainers to collect single-cell suspension for anti- Whole blood from C57BL/6J mice was drawn from inferior vena body staining. All antibodies were incubated with cells on ice cava with one-tenth volume 3.8% sodium citrate. Plasma was and diluted 1:200. Peritoneal cells and clot digestions were in- collected by centrifuging at 2,000 g for 10 min. Peritoneal fluid cubated for 30 min with Pac Blue anti-CD45 mAb, allophyco- was centrifuged at 5,000 g for 10 min, aliquoted, and stored in cyanin anti-ICAM2 mAb, allophycocyanin/Cy7 anti-CD11b mAb, a −80°C freezer. FV activity was determined using a one-stage PE anti-Ly6G mAb, PerCP/Cy5.5 anti-Ly6C mAb, and PE/Cy7 clotting assay with human FV–deficient plasma, as previously anti-F4/80 mAb and then washed, resuspended, and analyzed on described, with a Coatron M1 machine. The mouse plasma was a BD FACSCanto II (BD Biosciences) using FlowJo software. used as standard and set as 100%. Peritonitis models Collection of peritoneal interstitial fluid Briefly, 1 mg zymosan or 2 × 10 E. coli ATCC 25922 was injected Pure peritoneal interstitial fluid was collected as described by i.p. E. coli was washed with sterile PBS before injection. Peri- Hartveit and Thunold (1966). Briefly, it was collected by as- toneal exudate cells, peritoneal fluid, blood, or spleens were pirating with a 10-µl pipette tip without any anticoagulant, collected at different time points after injection. The cell num- centrifuged at 2,000 g for 10 min, and stored in a −80°C bers were counted using a Nexcelom cell counter. freezer. i.v. E. coli infection model Bone marrow transplantation Briefly, 2 × 10 E. coli ATCC 25922 was injected retro-orbitally. Bone marrow transplantation was performed as described pre- E. coli was washed with sterile PBS before injection. Spleens viously (Zhang et al., 2016). Briefly, bone marrow cells were −/− 6 were collected at 16 h after injection. CFUs were counted and isolated from CD45.2 or F5 ;AlbF5Tg mice. The cells (2 × 10 ) calculated as below. were injected i.v. into lethally irradiated 6-8-wk-old CD45.1/ CD45.2 mice (1,100 rad). 6 wk after transplantation, reconsti- E. coli burden measurement tution was determined by quantitating percentages of CD45.2 E. coli ATCC 25922 was cultured in LB medium with 100 µg/ml resident peritoneal macrophages with flow cytometry. ampicillin overnight. CFUs were calculated based on OD and confirmed by plating the serial dilution and culturing TAT ELISA overnightonLBplates withampicillin. 2×10 E. coli was in- TAT ELISA-paired antibodies were purchased from Cedarlane jected i.p., based on this dose as the LD from previous (CL20018K). The ELISA was performed as per the manu- studies (Xiang et al., 2013). 4 h after infection, peritoneal fluid facturer’s instructions. Mouse plasma was fully clotted by re- was collected, diluted accordingly, and cultured overnight on calcification to generate serum that has a maximal amount of LB plates with ampicillin to count and calculate CFU on the TAT. This serum was aliquoted and used as the standard. 10 µl of next day. Bar graphs were plotted as CFUs per microliter of the peritoneal fluid was diluted 1:10 into the plate. 10 µl of the peritoneal fluid, which subsequently was used to measure serum was diluted 1:10 and set as 100%. Bar graphs were plotted TAT concentration. as the percentages of the serum. Intravital two-photon microscopy Confocal microscopy GFP Bhlhe40 mice were anesthetized and placed in a supine Peritoneal clots were collected 3–5 h after zymosan injection, position. The abdominal skin was opened and separated from fixed in 4% paraformaldehyde overnight at 4°C, transferred into the muscle, leaving the peritoneal cavity intact. Some mice 20% sucrose overnight at 4°C, embedded in Tissue-Tek O.C.T. were injected with AF594-conjugated heat-killed E. coli from Compound (Triangle Biomedical Sciences), and processed into the left side of the peritoneal cavity (away from the imaging 10-µm sections. After fixation and permeabilization in 4% par- region) using a 30G needle while taking images. Images were aformaldehyde for 5 min, cryosections were rinsed with PBS, collected using a customized Leica SP8 two-photon micro- incubated in PBS with 3% BSA and 1% Triton at room temper- scope equipped with a 25×, 0.95 NA water-immersion objec- ature for 60 min, and then incubated with primary antibodies tive and a Mai Tai HP DeepSee Laser (Spectra-Physics) tuned (α-ICAM2, α-fibrin(ogen), α-Gata6, and α-S100A9) overnight at to 900 nm. Fluorescence emission was guided directly to hy- 4°C, washed with PBS, and incubated with secondary antibodies brid photodetectors. For signal separation, three dichroic (Cy2, Cy3, and Cy5) at room temperature for 1 h. After washing, beam splitters (Semrock) were used at 458, 495, and 560 nm mounting medium was added to the slides. Images were col- (FF458-Di02, FF495-Di03, and FF560-Di01). Clots from GFP lected using a Leica SP8 confocal microscope. Whole-mount E. coli–infected mice were immediately placed into an imaging imaging followed a similar protocol but extended antibody in- chamber between 50-µm mesh and a quartz coverslip to keep cubations to overnight. it at physiological temperature and was examined for up to 1 h. Most experiments focused on the 30-min time period. Gene expression analysis Clots were imaged with 20 to 35 optical sections at 2.5 µm, Gene expression analysis in macrophage herein used a previ- each taken at regular time intervals (typically 30 s), in order ously described database created by the Immunological Genome to capture cell dynamics within the clot. Project (detailed by Gautier et al., 2014). Zhang et al. Journal of Experimental Medicine 1298 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 marrow-derived and tissue-resident macrophage lineages proliferate at Statistics key stages during inflammation. Nat. Commun. 4:1886. https://doi.org/ Statistical analysis was performed using the Student’s t test for 10.1038/ncomms2877 unpaired samples or one-way ANOVA with a post-hoc Tukey’s Echtenacher, B., K. Weigl, N. Lehn, and D.N. Mannel. 2001. Tumor necrosis multiple comparisons test. Results were considered significant factor-dependent adhesions as a major protective mechanism early in septic peritonitis in mice. Infect. Immun. 69:3550–3555. https://doi.org/ at P < 0.05. Results display all replicated experiments, and val- 10.1128/IAI.69.6.3550-3555.2001 ues are mean ± SEM. Faust, N., F. Varas, L.M. Kelly, S. Heck, and T. Graf. 2000. Insertion of en- hanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood. 96: Online supplemental material 719–726. Fig. S1 shows coagulation and adhesion additively cooperate to Gautier, E.L., T. Shay, J. Miller, M. Greter, C. Jakubzick, S. Ivanov, J. Helft, A. account for the MDR in response to inflammation. Video 1 shows Chow, K.G. Elpek, S. Gordonov, et al; Immunological Genome Consor- tium. 2012. Gene-expression profiles and transcriptional regulatory intravital imaging of peritoneal macrophages through the intact pathways that underlie the identity and diversity of mouse tissue abdominal wall. Video 2 shows intravital imaging of a clot after macrophages. Nat. Immunol. 13:1118–1128. https://doi.org/10.1038/ni removal from a mouse 3 h after injection of GFP–E. coli. .2419 Gautier, E.L., S. Ivanov, P. Lesnik, and G.J. Randolph. 2013. Local apoptosis mediates clearance of macrophages from resolving inflammation in mice. Blood. 122:2714–2722. https://doi.org/10.1182/blood-2013-01 Acknowledgments -478206 Cre fl/fl Gautier, E.L., S. Ivanov, J.W. Williams, S.C. Huang, G. Marcelin, K. Fairfax, We thank R.P. McEver for providing Lyz2 ;Tln1 mice, M. P.L. Wang, J.S. Francis, P. Leone, D.B. Wilson, et al 2014. Gata6 regulates Wohltmann for assistance with mouse breeding and care, and aspartoacylase expression in resident peritoneal macrophages and T.J. Girard for technical assistance. controls their survival. J. Exp. Med. 211:1525–1531. https://doi.org/10 .1084/jem.20140570 This research was supported in large part by a National In- Ginhoux, F., and M. Guilliams. 2016. Tissue-Resident Macrophage Ontogeny stitutes of Heath grant (5R37AI049653 to G.J. Randolph) and a and Homeostasis. Immunity. 44:439–449. https://doi.org/10.1016/j National Institute of Diabetes and Digestive and Kidney Diseases .immuni.2016.02.024 Guilliams, M., and C.L. Scott. 2017. Does niche competition determine the pilot and feasibility grant (P30 DK052574 to B.H. Zinselmeyer), origin of tissue-resident macrophages? Nat. Rev. Immunol. 17:451–460. with additional support from the National Institutes of Heath https://doi.org/10.1038/nri.2017.42 (grants DP1DK109668 and R01HL118206 to G.J. Randolph), the Hartveit, F., and S. Thunold. 1966. Peritoneal fluid volume and the oestrus cycle in mice. Nature. 210:1123–1125. https://doi.org/10.1038/2101123a0 American Heart Association (grant 16SDG30480008 to B.H. Lim, T.J.F., and I.H. Su. 2018. Talin1 Methylation Is Required for Neutrophil Zinselmeyer), National Institutes of Heath (grant R01AI113118 to Infiltration and Lipopolysaccharide-Induced Lethality. J. Immunol. 201: B.T. Edelson), and a John C. Parker Endowed Professorship (to N. 3651–3661. https://doi.org/10.4049/jimmunol.1800567 Mackman). Lin, C.C., T.R. Bradstreet, E.A. Schwarzkopf, N.N. Jarjour, C. Chou, A.S. Archambault, J. Sim, B.H. Zinselmeyer, J.A. Carrero, G.F. Wu, et al 2016. The authors declare no competing financial interests. IL-1-induced Bhlhe40 identifies pathogenic T helper cells in a model of Author contributions: N. Zhang, R.S. Czepielewski, E.C. autoimmune neuroinflammation. J. Exp. Med. 213:251–271. https://doi Erlich, S.P. Grover, and B.T. Saunders performed experiments .org/10.1084/jem.20150568 Meza-Perez, S., and T.D. Randall. 2017. Immunological Functions of the and analyzed data. N.N. Jarjour, B.T. Edelson, and E.C. Erlich Omentum. Trends Immunol. 38:526–536. https://doi.org/10.1016/j.it contributed key reagents and associated key pilot data. A.C. .2017.03.002 Cleuren and N. Mackman contributed established, key materi- Michel, C.C., M.N. Nanjee, W.L. Olszewski, and N.E. Miller. 2015. LDL and HDL transfer rates across peripheral microvascular endothelium agree als. G.J. Broza provided training and guidance. B.H. Zinselmeyer with those predicted for passive ultrafiltration in humans. J. Lipid Res. developed the intravital imaging prep, pilot data, and funding. 56:122–128. https://doi.org/10.1194/jlr.M055053 N. Zhang, N. Mackman, and G.J. Randolph provided conceptual Misharin, A.V., L. Morales-Nebreda, P.A. Reyfman, C.M. Cuda, J.M. Walter, A.C. McQuattie-Pimentel, C.I. Chen, K.R. Anekalla, N. Joshi, K.J.N. guidance. N. Zhang and G.J. Randolph designed the project and Williams, et al 2017. Monocyte-derived alveolar macrophages drive wrote the manuscript. lung fibrosis and persist in the lung over the life span. J. Exp. Med. 214: 2387–2404. https://doi.org/10.1084/jem.20162152 Nelson, D.S. 1963. Reaction to antigens in vivo of the peritoneal macrophages Submitted: 29 October 2018 of guinea-pigs with delayed type hypersensitivity. 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Expression of factor V by resident macrophages boosts host defense in the peritoneal cavity

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Abstract

BRIEF DEFINITIVE REPORT Expression of factor V by resident macrophages boosts host defense in the peritoneal cavity 1 1 1 1 1 1 3 Nan Zhang , Rafael S. Czepielewski , Nicholas N. Jarjour , Emma C. Erlich , Ekaterina Esaulova ,Brian T. Saunders , Steven P. Grover , 4 2 1 3 1 1 Audrey C. Cleuren ,George J. Broze , Brian T. Edelson , Nigel Mackman , Bernd H. Zinselmeyer , and Gwendalyn J. Randolph  Macrophages resident in different organs express distinct genes, but understanding how this diversity fits into tissue-specific features is limited. Here, we show that selective expression of coagulation factor V (FV) by resident peritoneal macrophages in mice promotes bacterial clearance in the peritoneal cavity and serves to facilitate the well-known but poorly understood “macrophage disappearance reaction.” Intravital imaging revealed that resident macrophages were nonadherent in peritoneal fluid during homeostasis. Bacterial entry into the peritoneum acutely induced macrophage adherence and associated bacterial phagocytosis. However, optimal control of bacterial expansion in the peritoneum also required expression of FV by the macrophages to form local clots that effectively brought macrophages and bacteria in proximity and out of the fluid phase. Thus, acute cellular adhesion and resident macrophage–induced coagulation operate independently and cooperatively to meet the challenges of a unique, open tissue environment. These events collectively account for the macrophage disappearance reaction in the peritoneal cavity. Introduction Our understanding of the biology of tissue-resident macrophages Results and discussion A prominent example of tissue-restricted gene expression in has evolved greatly in the past decade. Resident macrophages in macrophages is the selective detection in LPMs of mRNA for most organs arrive during embryogenesis, maintaining them- coagulation factors, including factor V (FV; F5), FVII (F7), and FX selves for long periods via local proliferation (Ginhoux and (F10; Fig. 1 A). The latter two factors were also expressed by lung Guilliams, 2016). These resident macrophages express special- macrophages, whereas FV was unique to peritoneal macro- ized sets of genes that are distinct between different organs phages. Activated FV is a key cofactor in the common pathway (Gautier et al., 2012). Even when monocytes are recruited to for activated FX, forming the prothrombinase complex that, these organs during inflammation, the inflammatory macro- during coagulation, converts prothrombin to thrombin that in phages they become are reticent to take up the specialized res- turn converts fibrinogen to fibrin to form clots. Indeed, the co- ident phenotype, if they do at all (Gautier et al., 2013; Guilliams agulation pathway was among top pathways selectively en- and Scott, 2017; Misharin et al., 2017). It is assumed that spe- riched in LPMs (Gautier et al., 2012). The unique expression of cialized genes expressed by particular resident macrophages FV, in particular, among tissue-resident macrophages caused us encode products tailored to the specific physiological needs or to consider the possibility that coagulation may have a key role constraints of that tissue, but illustration of direct links often in peritoneal macrophage function. The expression of FVII, remain unexplored. Transcription factors that regulate special- ized macrophage gene sets in different organs have, however, important for initiating the extrinsic pathway of coagulation in been identified. One such transcription factor is Gata6, which complex with tissue factor (TF; F3) that is acutely induced after selectively governs the life cycle of murine resident peritoneal tissue damage, led us to consider this pathway of coagulation in macrophages, often called large peritoneal macrophages (LPMs; particular. Gautier et al., 2012, 2014; Okabe and Medzhitov, 2014; Rosas A classic response to inflammation exhibited by LPMs is et al., 2014). In this study, we focused on understanding how known as the “macrophage disappearance reaction” (MDR), first the transcriptional profile of resident peritoneal macrophages described decades ago (Nelson, 1963). In this reaction, LPMs could be linked to the specialized function of these cells. become irretrievable from lavage just hours after introduction of ............................................................................................................................................................................. 1 2 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO; Department of Medicine, Washington University School of 3 4 Medicine, St. Louis, MO; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC; Life Sciences Institute, University of Michigan, Ann Arbor, MI. Correspondence to Gwendalyn J. Randolph: gjrandolph@wustl.edu. © 2019 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.20182024 1291 J. Exp. Med. 2019 Vol. 216 No. 6 1291–1300 Figure 1. Coagulation and adhesion additively cooperate to account for the MDR in response to inflammation. (A) Gene array analysis (from ImmGen; n = 3 separate pools) of classical coagulation factors in major tissue resident macrophages, including those from the spleen, central nervous system (CNS), lung, and peritoneum. (B) Quantification of LPMs in peritoneal lavage 3 h after zymosan injection i.p. when clotting and/or adhesion was inhibited. (C) Aggregates retrieved from the peritoneum 5 h after zymosan injection. (D–G) Immunofluorescence staining of the aggregates for fibrin(ogen) and macrophage markers. D and G are stained frozen sections of the clots; E and F are whole-mount preparations. Scale bars represent 100 µm (D and G), 50 µm (E), and 10 µm (F). (H) Flow cytometry on peritoneal exudate cells from untreated mice (left), 3 h after zymosan i.p. (middle), and clots 3 h after zymosan i.p. (right). (I) Cre fl/fl Quantification of LPMs 3 h after zymosan injection in clots and omenta in WT and Lyz2 ;Tln1 mice. One-way ANOVA was used to test statistical signif- icance. Symbols represent individual mice studied. Error bars represent ± SEM. All experiments were repeated at least two or three times. **, P < 0.01; ***, P < 0.001. inflammatory stimuli like the bacillus Calmette-Guerin vaccine, inflammation otherwise appears to have resolved (Gautier et al., lipopolysaccharide, zymosan, or thioglycollate (Nelson, 1963; 2013). In the 1960s, Nelson proposed a role for coagulation in the Barth et al., 1995; Davies et al., 2013; Gautier et al., 2013; Meza- MDR, because he could fully reverse it by administering hepa- Perez and Randall, 2017). Repopulation of resident macrophages rin, which can block coagulation or adhesion; MDR was also is slow following the MDR. LPMs are not always restored after reversed to an appreciable but lesser extent by warfarin, which Zhang et al. Journal of Experimental Medicine 1292 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 Table 1. Peritoneal fluid volume, peritoneal cell density, and total cell anti-Ly6G mAb (Fig. S1 A) demonstrated the neutrophils were and macrophage numbers in 6-wk-old C57BL/6J mice (comparison of dispensable as a cause for MDR (Fig. S1 B). Furthermore, neu- lavage and collection of undiluted fluid) trophil recruitment was unaffected by Tln1 deficiency in this model (Fig. S1, C and D), which is consistent with previous re- Volume Cell density Total cells Gata6 resident port (Lim and Su, 2018). We conclude that neutrophil infiltration 4 6 (μl± (×10 /µl ± (×10 ± macrophages does not play a role in MDR in this model. However, integrin– SEM) SEM) SEM) (×10 ±SEM) mediated adhesion is relevant to MDR, but such adhesion cannot Peritoneal 52.14 ± 6.50 ± 0.85 (6) 3.26 ± 0.27 (6) 1.73 ± 0.11 (6) account for a distinct, prominent role of the coagulation cascade fluid 4.02 (7) in affecting macrophage removal. Lavage N/A N/A 3.20 ± 0.15 (12) 1.81 ± 0 09 (12) A close physical examination of the peritoneal cavity after MDR revealed millimeter-sized aggregates within the peritoneal Numbers in parentheses indicate the numbers of mice used. N/A, not cavity 3–5 h after i.p. injection of zymosan (Fig. 1 C). These ag- applicable. gregates stained positively for fibrin(ogen) (Fig. 1, D–F), indi- cating that they were clots, with such staining most easily would more specifically target coagulation (Nelson, 1963). In the appreciated in whole-mount preparations where strands of fi- ensuing years, with recognition that fibrin(ogen) participates in brin were observed between clumps of zymosan particles and adhesion, the view developed that coagulation factors support cells (Fig. 1, E and F). Clots were also enriched in LPMs identified macrophage disappearance by promoting adhesion and migra- by coexpression of Gata6 and ICAM2 (Fig. 1 G), but very few tion (Szaba and Smiley, 2002) to locations like the omentum peritoneal B cells were incorporated into the clots (Fig. S1 E), during the MDR (Meza-Perez and Randall, 2017). Thus, a clas- despite that peritoneal B cells are almost as abundant as mac- sical role for coagulation in peritoneal host defense was largely rophages in the steady state peritoneal fluid. Some macrophages overlooked. Our intravital imaging of the peritoneal cavity in clots were loaded with zymosan particles, but other zymosan through an intact abdominal wall indicated that a distinct fea- particles appeared outside of cells (Fig. 1, E and F), suggesting ture of LPMs, relative to resident macrophages in other organs, that clots were efficient at entrapping debris not yet phagocy- is that they are free floating in peritoneal fluid (Video 1). LPMs tized. Clots also incorporated S100A9 neutrophils, which are only adhered to the walls of the peritoneal cavity covering vis- known to be rapidly recruited to the peritoneum after zymosan ceral organs after inflammatory stimuli (heat-killed bacteria) is injected i.p. (Fig. 1 D). Flow cytometry confirmed enrichment were introduced (Video 1). Cell density in peritoneal fluid was of LPMs in the clots, while they were largely absent in the lavage high, at ∼6.5 × 10 /µl (Table 1), ∼20× the concentration of leu- 3 h after i.p. zymosan (Fig. 1 H). Approximately three times more kocytes in blood (Mouse Phenome Database; The Jackson LPMs were recovered in clots compared with the omentum Laboratory). (Fig. 1 I), indicating that clots are prominent sites for macro- Given this literature and the striking expression of coag- phage “disappearance” in response to inflammatory triggers. ulation factors in LPMs, we wondered whether coagulation Consistent with the integrin independence of macrophage in- might drive important processes in the peritoneum. First, we clusion in clots, macrophage recovery from clots was unaffected confirmed that heparin fully reversed the MDR induced by by Tln1 deficiency (Fig. 1 I), whereas recovery from the omentum 1 mg zymosan i.p. (Fig. 1 B). The very selective inhibitor of trended downward, consistent with a role for adhesion in re- thrombin hirudin partially (∼50%) inhibited MDR (Fig. 1 B), taining macrophages in the omentum. reminiscent of the effect of warfarin relative to heparin Work beginning in the 1980s revealed that LPMs produce FV (Nelson, 1963). Heparin has both anticoagulant and anti- in culture (Osterud et al., 1981), including production of a adhesive activity (Woods et al., 1986), and this may explain functional prothrombinase complex (activated FV/activated FX; why it is more effective at inhibiting MDR compared with Pejler et al., 2000) long before it was recognized that F5 mRNA either warfarin or hirudin. expression was selective to LPMs. Although the liver is a known To directly address the role of integrin–mediated adhesion in source of FV, expression of FV by LPMs led us to hypothesize Cre fl/fl MDR, in the same experiments we employed Lyz2 ;Tln1 mice these macrophages maintain FV levels in peritoneal fluid in the (Fig. 1 B) to prevent en bloc activation of integrins through loss steady state. We thus analyzed FV activity in peritoneal fluid of the integrin activation adaptor talin-1 (Tln1) in lysozyme- using a one-stage clotting assay. In WT C57BL/6J mice, FV ac- expressing cells (Yago et al., 2015), which includes LPMs and tivity in peritoneal interstitial fluid was ∼13% of that in plasma other myeloid cells like neutrophils (Faust et al., 2000). The (Fig. 2 A), consistent with the documented poor entry of plasma absence of integrin signaling also partially inhibited MDR, proteins as large as FV (a ∼300-kD protein) into interstitial fluid similar to the inhibition achieved with hirudin alone (∼50%). of molecules (Yang et al., 1998; Michel et al., 2015). Peritoneal −/− However, rather than being redundant, the combination of fluid was thus analyzed from F5 ;AlbF5Tg mice, engineered hirudin and genetic loss of Tln1 had additive effects, such that such that the liver sustains expression of FV from the albumin when the two interventions were combined, MDR was fully promoter but all other tissues, including LPMs, lack FV (Sun reversed, mirroring heparin (Fig. 1 B). In addition, because et al., 2003). These mice displayed a ∼75% reduction in FV ac- neutrophil infiltration almost always correlates with MDR after tivity compared with WT peritoneal fluid (Fig. 2 A), raising the zymosan injection, we tested the hypothesis that infiltrated possibility that the major source of FV in the peritoneal fluid was neutrophils cause MDR. Results from depleting neutrophils with not the liver. Zhang et al. Journal of Experimental Medicine 1293 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 Figure 2. FV and TF from resident peritoneal macrophages are responsible for peritoneal fluid clotting. (A) Quantification of FV activity in plasma or peritoneal fluid of various genotypes of mice or after bone marrow transplant of indicated donor genotypes into irradiated WT recipients (last bars onthe right). Zym, zymosan. (B) Quantification of LPMs 3 h after zymosan injection. (C) Quantification of LPMs 3 h after zymosan injection. One-way ANOVA was +/+ fl/fl used to test statistical significance, except for the bone marrow chimera result in B and the hF groups and F3 groups in C, which used a two-tailed t test. Symbols represent individual mice studied. Error bars represent ± SEM. All experiments were repeated at least two or three times. *, P < 0.05; **, P < 0.01; ***, P<0.001. Demonstrating that a major local source of FV was LPMs, FV extrinsic coagulation (Parry et al., 1998), while their control Cre fl/fl activity in peritoneal fluid of the Lyz2 ;Gata6 mouse, which counterparts from a sister line expressing the human transgene lack most LPMs (Gautier et al., 2014; Okabe and Medzhitov, as well as murine F3 exhibited a complete MDR in response to −/− 2014; Rosas et al., 2014), mirrored the very low level in F5 ; zymosan (Fig. 2 C). Likewise, the MDR was partially suppressed Cre fl/fl fl/fl AlbF5Tg mice (Fig. 2 A). LPMs are the only immune cell in the in Lyz2 ;F3 mice (Pawlinski et al., 2010) compared with F3 mouse that expresses Gata6 (Gautier et al., 2014). Thus, while control mice (Fig. 2 C). Even though F3 mRNA is not detectable in Cre Cre fl/fl Lyz2 is not completely selective to LPMs, Lyz2 ;Gata6 mice resting LPMs (Fig. 1 A), TF can be rapidly (within 1 h) up- exhibit a highly selective defect in LPMs (Gautier et al., 2014). As regulated on the LPM surface and is capable of initiating clot- an additional evaluation, we performed bone marrow trans- ting in vitro (data not shown). Thus, it is likely that LPMs −/− plantation using WT or F5 ;AlbF5Tg marrow as donors for ir- quickly generate enough TF to initiate the extrinsic coagulation radiated WT recipients that would have normal liver FV. With cascade during the MDR. 80–85% reconstitution of resident peritoneal macrophages with Finally, we wondered whether clotting of interstitial fluid −/− donor marrow (data not shown), mice receiving F5 ;AlbF5Tg had a critical functional role. While clotting of interstitial fluid marrow showed significantly lower FV activity in peritoneal would seem irrelevant to hemostasis, we wondered if interstitial fluid than mice receiving WT marrow (Fig. 2 A,bars on right). clotting might serve to entrap both free-floating macrophages We then hypothesized that this LPM–derived FV is crucial to and microbes that invade the peritoneum. Indeed, a previous promote early local clotting in the peritoneal fluid. Indeed, the study revealed that a single dose of heparin or hirudin caused −/− zymosan-induced MDR in F5 ;AlbF5Tg mice reduced the MDR increased mortality after mice were subjected to cecal-ligation to a similar level as the hirudin-treated group (Fig. 2 B), sug- puncture (Echtenacher et al., 2001), although the role of local gesting that FV from LPMs is critical for the peritoneal clotting cells and factors remained unclear. To investigate whether LPM- in this model. Thus, we conclude that LPMs produce FV in the dependent local peritoneal coagulation controlled peritoneal steady state that in turn promotes clotting of local interstitial infection, we measured the thrombin–antithrombin complex fluid during the MDR (Sun et al., 2003). (TAT), the standard assay to track occurrence of coagulation, in In the extrinsic coagulation cascade, TF (FIII; F3) initiates the peritoneal fluid and plasma over time after i.p. infection clotting and has in other systems been demonstrated to be with live E. coli (Fig. 3 A), normalizing the data as a percentage of rapidly expressed by macrophages upon activation. We ob- maximal TAT in the serum from exogenously clotted plasma. served rescue of MDR in response to zymosan-injected i.p. in Both peritoneal and blood TAT were below detection in the mice deficient for murine TF that express very low human TF steady state using the assay. However, peritoneal but not blood −/− +/+ levels (hF3; ∼1%; mF ;LowhF3 ), in contrast to control mice TAT complex was detected within 2 h of infection, lasting for at +/+ +/+ MeriCreMer fl/fl with normal TF levels (mF ;LowhF3 ; Fig. 2 C). The 1% of least 6 h (Fig. 3 A). In tamoxifen-treated Csf1r ;Gata6 Cre fl/fl normal TF levels in the hF3 transgenic mouse can restore via- or Lyz2 ;Gata6 mice, which exhibit selectively and greatly bility of the line but has a markedly impaired capacity to initiate reduced LPMs (Gautier et al., 2014), TAT was an average of 48% Zhang et al. Journal of Experimental Medicine 1294 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 Figure 3. Macrophage adhesion and macrophage-driven clotting cooperatively promote peritoneal bacterial clearance. (A) Quantification of TAT complex in peritoneal fluid and plasma in the steady state or after E. coli infection. (B) Quantification of TAT complex in peritoneal fluid 4 h after E. coli infection. Cre fl/fl Cre fl/fl Ctrl, control. (C) CFUs per microliter peritoneal fluid from untreated, hirudin-treated WT, Lyz2 ;Talin mice, hirudin-treated Lyz2 ;Talin mice, and fl/fl Cre fl/fl clodronate liposome-treated mice 4 h after E. coli infection. (D) CFUs per microliter peritoneal fluid from Gata6 and Lyz2 ;Gata6 mice 4 h after E. coli infection. (E) CFUs per microliter peritoneal fluid from C57BL/6J treated with or without hirudin 4 h after E. coli infection. (F) CFUs per microliter peritoneal −/− fluid from control and F5 ;AlbF5Tg mice 4 h after E. coli infection. One-way ANOVA was used to test statistical significance, except for C, D, and F and the −/− F5 ;AlbF5Tg groups in B, which were examined by a two-tailed t test. Symbols represent individual mice studied. Error bars represent ± SEM. All experiments were repeated at least two or three times. *, P < 0.05; **, P < 0.01; ***, P < 0.001. and 57% lower, respectively, than in control mice upon infection. an increase in permeability might account for why TAT levels −/− Furthermore, F5 ;AlbF5Tg mice showed comparably reduced are not as low as the basal level of FV observed in resting mice. peritoneal TAT after infection (Fig. 3 B). As expected, Tln1 de- We next investigated the functional impact of MDR on local ficiency in macrophages did not affect TAT levels (Fig. 3 B), and bacterial clearance during a 4-h period following infection. In- neither did neutrophil depletion (data not shown). These data hibition of both clotting and macrophage adhesion to collectively indicate that Gata6 resident macrophages contribute substan- block the MDR dramatically increased CFUs in the peritoneal tially to peritoneal coagulation in an FV-dependent manner fluid compared with untreated mice, clotting-inhibited mice, or early after infection. Even though it would be anticipated that adhesion-blocked mice (Fig. 3 C), strongly suggesting that both the infectious state would increase vascular permeability and clotting and adhesion are required for optimal bacterial clear- allow for increased influx of plasma–derived proteins, the data ance. By examining bacterial numbers that we could retrieve indicate that any role for permeability in bringing in plasma FV from the peritoneal cavity 1 min after their installation (Fig. 3 C, in the few hours after infection is not sufficient to raise TAT to right column) and comparing with all data obtained at 4 h, it was levels observed in mice that can generate local FV. Nonetheless, clear that all CFUs recovered at 4 h were reduced from the Zhang et al. Journal of Experimental Medicine 1295 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 numbers instilled. Thus, CFUs assessed at 4 h served primarily to blockade of β1and β2 integrins would be expected to interfere assess how efficiently bacteria were cleared rather than to assess with adhesion of peritoneal macrophages to mesothelial walls bacterial growth. In the absence of any interference to the MDR, (Bellingan et al., 2002; Cao et al., 2005) and, fortunately, neu- an average of 98% of bacteria were cleared in 4 h compared tralizing mAbs to these molecules had a neutral impact on E. coli with the number of CFUs retrieved at 1 min. When macrophages CFUs in the spleen when E. coli was administered to blood (Fig. 4 had earlier been depleted from the peritoneal cavity with B). These data thus indicate that the most optimal tools in hand clodronate-loaded liposomes without affecting blood monocytes, to next evaluate the impact of macrophage–mediated coagula- as per previous protocol (Wang and Kubes, 2016), clearance of tion and adhesion on E. coli dissemination from the peritoneum −/− bacteria was much less efficient, at only 49%, indicating the were F5 ;AlbF5Tg mice, heparin, or hirudin to block coagula- pivotal role of local macrophages in bacterial removal. With use tion and combined anti-β1and -β2 mAbs to block adhesion. of hirudin alone or loss of Tln1 alone, clearance of bacteria av- When we next instilled E. coli into the peritoneal cavity to eraged 93% and 86%, respectively. Remarkably, when both co- study its dissemination, mice with a complete loss of MDR agulation and adhesion were inhibited, clearance at 4 h through the use of heparin exhibited markedly elevated CFUs in decreased to an average of 64%, approaching the impact that the spleen at 16 h after E. coli i.p. instillation (Fig. 4 D). Fur- −/− complete macrophage depletion achieved (Fig. 3 C). Thus, these thermore, hematopoietic FV-deficient (F5 ;AlbF5Tg)mice data strongly suggest that both coagulation and adhesion opti- treated with blocking antibodies against both β1and β2 integrins mize bacterial clearance from the peritoneal cavity. showed significantly increased CFUs in the spleen when E. coli We then wondered if LPMs or LPM-dependent clotting played were administered i.p. (Fig. 4 E). Mice treated with both hirudin a significant role in accounting for the overall role played by and blocking antibodies against β1and β2 integrins showed macrophages in bacterial clearance as revealed from the use of the significantly increased bacterial burden in the spleen at 16 h clodronate-loaded liposomes. Mice genetically lacking the major- after E. coli i.p. instillation compared with those treated with Cre fl/fl ity of LPMs (Lyz2 ;Gata6 ) showed CFUs approximately seven anti-β1and β2 mAbs alone (Fig. 4 F), demonstrating that clotting times higher than those of control mice (Fig. 3 D). Deficiency in indeed impacted bacterial dissemination, beyond the impact of −/− hematopoietic FV (F5 ;AlbF5Tg) also reduced CFU by two- to impaired adhesion. Tln1 deficiency in myeloid cells, combined threefold (Fig. 3 F), confirming that local FV accounted for the role with hirudin, did not affect dissemination (Fig. 4 G), but this is of coagulation in local bacterial clearance. Intravital imaging of likely due to the conflicting roles of Tln1 deficiency in the peri- clots ex vivo showed Escherichia coli within lysozyme cells at the toneum (Fig. 1 B) versus the blood (Fig. 4 A). Together, these data time of imaging and other motile bacteria not yet engulfed by strongly suggest that the two processes that cooperatively and phagocytes but nonetheless trapped within the clot (Video 2). nonredundantly account for the MDR, coagulation and adhesion, Lastly, we investigated whether the local control of bacteria also cooperatively and nonredundantly function to optimize by the MDR or clotting had any impact on bacterial dissemina- bacterial clearance from the peritoneal cavity, which is essential tion from the peritoneal cavity to the blood and ultimately the to contain dissemination. spleen. Dissemination is a complex evaluation because there can These studies, in summary, illustrate how specialized gene be roles for leukocytes or coagulation beyond the peritoneum expression in a particular macrophage is intimately tied to the within the vasculature itself. The most ideal experimental sce- unique characteristics and physiology of the organ. Specifi- nario would be one in which the tools applied to the question of cally, to support high-quality host defense in the capture of dissemination from the peritoneum would have neutral effects microorganisms that might gain access to the peritoneal on CFU in the spleen after E. coli administration in the blood so cavity through the digestive tract, resident peritoneal mac- that the effects from the peritoneum could be more selectively rophages constitutively produce FV, along with other clotting evaluated. Thus, we first evaluated whether the tools we used in factors. This production makes up for the minimal access that the peritoneal cavity that regulate adhesion, including the liver–derived FV has to the peritoneum. Expression of FV al- Cre fl/fl Lyz2 ;Tln1 mouse strain and heparin, altered CFUs in the lows macrophages to generate clots that trap microorganisms spleen after i.v. delivery, as any effects in this arm of the ex- even before they have a chance to be phagocytosed. This form periment could confound interpretation of dissemination from of host defense is especially relevant in a fast-flowing envi- Cre fl/fl the peritoneum. Indeed, and surprisingly, Lyz2 ;Tln1 mice ronment of fluid that comprises a challenging space for effi- displayed fewer CFUs in the spleen after E. coli delivery i.v. cient phagocytosis. While the clots collect microorganisms in (Fig. 4 A). This protective effect of Tln1 deficiency in blood large numbers, additional macrophages adhering within the myeloid cells might be expected to offset our predicted increase omentum and upon visceral organ surfaces undoubtedly in dissemination of E. coli from the peritoneum. By contrast, promote capture of organisms that avoided entrapment in heparin administered i.p. did not show any statistically signifi- clots. These two mechanisms collectively account for the vast cant effect on CFU in the spleen after i.v. administration of E. coli majority of macrophage disappearance in the context of per- −/− (Fig. 4 B). F5 ;AlbF5Tg mice and hirudin administered i.p. also itoneal inflammation, and our data indicate that both arms of had neutral effects on E. coli CFUs in the spleen when E. coli was the MDR, adhesion and coagulation, are required for optimal administered i.v. (Fig. 4, B and C). With respect to adhesion, host defense. Finally, in many clinical scenarios, therapeutic because Tln1 deficiency was not neutral in the blood–spleen agents are used to limit coagulation to minimize its occur- compartment (Fig. 4 A), we sought to block macrophage adhe- rence in blood. The present work, however, illustrates that in sion using antibodies rather than Tln1 deficiency. Combined the particular tissue microenvironment of the peritoneum, Zhang et al. Journal of Experimental Medicine 1296 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 Figure 4. MDR restricts bacterial dissemina- tion out of the peritoneal cavity to the spleen. fl/fl Cre (A) CFUs per spleen from Tln1 mice or Lyz2 ; fl/fl Tln1 mice at 16 h after i.v. E. coli infection. (B) CFUs per spleen from mice untreated or treated with heparin, anti-integrin β1and β2 blocking antibodies, or hirudin at 16 h after i.v. E. coli infection. Differences between groups are not statistically significant. (C) CFU per spleen −/− from F5 littermates and F5 ;AlbF5Tg mice at 16 h after i.v. E. coli infection. Differences are not statistically significant. Ctrl, control. (D) CFUs per spleen from untreated and heparin-treated mice at 16 h after i.p. E. coli infection. (E) CFUs −/− per spleen from F5 littermates and F5 ;AlbF5Tg mice treated with anti-integrin β1and β2block- ing antibodies at 16 h after i.p. E. coli infection. (F) CFUs per spleen from mice treated with anti- integrin β1and β2 blocking antibodies and mice treated with hirudin and anti-integrin β1and β2 blocking antibodies at 16 h after i.p. E. coli fl/fl infection. (G) CFUs per spleen from Tln1 mice Cre fl/fl or Lyz2 ;Tln1 mice treated with hirudin at 16 h after i.p. E. coli infection. Differences are not statistically significant. Two-tailed t tests were used to test statistical significance for A and C–G. One-way ANOVA was used to test statis- tical significance for B. Symbols represent indi- vidual mice studied. Error bars represent ± SEM. All experiments were repeated at least one to three times. *, P < 0.05; **, P < 0.01; ***, P < 0.001. resident macrophage–mediated coagulation produces impor- polyclonal antibody was purchased from R&D Systems tant beneficial effects. (AF2065-SP). Anti-Gata6 monoclonal antibody was pur- chased from Cell Signaling Technology (5851). Anti- fibrin(ogen) polyclonal antibody was purchased from Agilent Dako (A0080). The Alexa Fluor 594–conjugated heat-killed Materials and methods bacterial particles were purchased from Thermo Fisher Sci- Mice entific (E23370). Clodronate-loaded liposome was purchased All C57BL/6J mice were purchased at the age of 8 wk from The Cre fl/fl from ClodronateLiposomes.org. Recombinant hirudin was Jackson Laboratory. Lyz2 ;Tln1 mice were a kind gift from purchased from Aniara Diagnostica (ARE120A). Zymosan was Dr. Rodger P. McEver (Oklahoma Medical Research Foundation, Cre fl/fl purchased from Sigma-Aldrich (Z4250). GFP E. coli was pur- Oklahoma City, OK). Low human TF mice, Lyz2 ;F3 mice, −/− Cre fl/fl chased from ATCC (ATCC 25922GFP). Human FV–deficient F5 ;AlbF5Tg mice, Lyz2 ;Gata6 mice (now backcrossed to Cre LSL-Tdtomato GFP plasma was purchased from Haematologic Technologies. C57BL/6 background), Lyz2 ;R26 mice, and Bhlhe40 Rabbit thromboplastin (44213), Liberase, hyaluronidase, mice were described previously (Parry et al., 1998; Sun et al., DNase I, streptokinase, and collagenase D were purchased 2003; Pawlinski et al., 2010; Gautier et al., 2014; Lin et al., 2016; MeriCreMer fl/fl from Sigma-Aldrich. Plasminogen was purchased from Lee Wang and Kubes, 2016). Csf1r ;Gata6 mice (fully Biosolutions. backcrossed to C57BL/6 background) were generated by MeriCreMer fl/fl crossing Csf1r mice with Gata6 mice. Mouse studies were approved by animal use oversight committees at Wash- Flow cytometry ington University School of Medicine and the University of Flow cytometry was performed as described previously (Gautier North Carolina (studies on TF). et al., 2014). Briefly, peritoneal exudate cells were collected by flushing the peritoneum with 6 ml HBSS with 2.5 mM EDTA and Reagents 0.2% BSA. Peritoneal clots were collected 3 h after zymosan Pac Blue anti-CD45 mAb, allophycocyanin anti-ICAM2 mAb, injection and digested using a cocktail of 1 mg/ml collagenase D, allophycocyanin/Cy7 anti-CD11b mAb, PE anti-Ly6G mAb, 50 U/ml streptokinase, 4 U/ml plasminogen, 100 µg/ml Lib- PerCP/Cy5.5 anti-Ly6C mAb, PE/Cy7 anti-F4/80 mAb, and erase, 100 µg/ml DNase I, and 0.5 mg/ml hyaluronidase in RPMI anti-ICAM2 mAb were all purchased from BioLegend. Anti-S100A9 with 1% FBS. The digestion was incubated at 37°C for 30 min and Zhang et al. Journal of Experimental Medicine 1297 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 mixed by pipetting every 5 min. It was then passed through FV activity measurement 70-µm cell strainers to collect single-cell suspension for anti- Whole blood from C57BL/6J mice was drawn from inferior vena body staining. All antibodies were incubated with cells on ice cava with one-tenth volume 3.8% sodium citrate. Plasma was and diluted 1:200. Peritoneal cells and clot digestions were in- collected by centrifuging at 2,000 g for 10 min. Peritoneal fluid cubated for 30 min with Pac Blue anti-CD45 mAb, allophyco- was centrifuged at 5,000 g for 10 min, aliquoted, and stored in cyanin anti-ICAM2 mAb, allophycocyanin/Cy7 anti-CD11b mAb, a −80°C freezer. FV activity was determined using a one-stage PE anti-Ly6G mAb, PerCP/Cy5.5 anti-Ly6C mAb, and PE/Cy7 clotting assay with human FV–deficient plasma, as previously anti-F4/80 mAb and then washed, resuspended, and analyzed on described, with a Coatron M1 machine. The mouse plasma was a BD FACSCanto II (BD Biosciences) using FlowJo software. used as standard and set as 100%. Peritonitis models Collection of peritoneal interstitial fluid Briefly, 1 mg zymosan or 2 × 10 E. coli ATCC 25922 was injected Pure peritoneal interstitial fluid was collected as described by i.p. E. coli was washed with sterile PBS before injection. Peri- Hartveit and Thunold (1966). Briefly, it was collected by as- toneal exudate cells, peritoneal fluid, blood, or spleens were pirating with a 10-µl pipette tip without any anticoagulant, collected at different time points after injection. The cell num- centrifuged at 2,000 g for 10 min, and stored in a −80°C bers were counted using a Nexcelom cell counter. freezer. i.v. E. coli infection model Bone marrow transplantation Briefly, 2 × 10 E. coli ATCC 25922 was injected retro-orbitally. Bone marrow transplantation was performed as described pre- E. coli was washed with sterile PBS before injection. Spleens viously (Zhang et al., 2016). Briefly, bone marrow cells were −/− 6 were collected at 16 h after injection. CFUs were counted and isolated from CD45.2 or F5 ;AlbF5Tg mice. The cells (2 × 10 ) calculated as below. were injected i.v. into lethally irradiated 6-8-wk-old CD45.1/ CD45.2 mice (1,100 rad). 6 wk after transplantation, reconsti- E. coli burden measurement tution was determined by quantitating percentages of CD45.2 E. coli ATCC 25922 was cultured in LB medium with 100 µg/ml resident peritoneal macrophages with flow cytometry. ampicillin overnight. CFUs were calculated based on OD and confirmed by plating the serial dilution and culturing TAT ELISA overnightonLBplates withampicillin. 2×10 E. coli was in- TAT ELISA-paired antibodies were purchased from Cedarlane jected i.p., based on this dose as the LD from previous (CL20018K). The ELISA was performed as per the manu- studies (Xiang et al., 2013). 4 h after infection, peritoneal fluid facturer’s instructions. Mouse plasma was fully clotted by re- was collected, diluted accordingly, and cultured overnight on calcification to generate serum that has a maximal amount of LB plates with ampicillin to count and calculate CFU on the TAT. This serum was aliquoted and used as the standard. 10 µl of next day. Bar graphs were plotted as CFUs per microliter of the peritoneal fluid was diluted 1:10 into the plate. 10 µl of the peritoneal fluid, which subsequently was used to measure serum was diluted 1:10 and set as 100%. Bar graphs were plotted TAT concentration. as the percentages of the serum. Intravital two-photon microscopy Confocal microscopy GFP Bhlhe40 mice were anesthetized and placed in a supine Peritoneal clots were collected 3–5 h after zymosan injection, position. The abdominal skin was opened and separated from fixed in 4% paraformaldehyde overnight at 4°C, transferred into the muscle, leaving the peritoneal cavity intact. Some mice 20% sucrose overnight at 4°C, embedded in Tissue-Tek O.C.T. were injected with AF594-conjugated heat-killed E. coli from Compound (Triangle Biomedical Sciences), and processed into the left side of the peritoneal cavity (away from the imaging 10-µm sections. After fixation and permeabilization in 4% par- region) using a 30G needle while taking images. Images were aformaldehyde for 5 min, cryosections were rinsed with PBS, collected using a customized Leica SP8 two-photon micro- incubated in PBS with 3% BSA and 1% Triton at room temper- scope equipped with a 25×, 0.95 NA water-immersion objec- ature for 60 min, and then incubated with primary antibodies tive and a Mai Tai HP DeepSee Laser (Spectra-Physics) tuned (α-ICAM2, α-fibrin(ogen), α-Gata6, and α-S100A9) overnight at to 900 nm. Fluorescence emission was guided directly to hy- 4°C, washed with PBS, and incubated with secondary antibodies brid photodetectors. For signal separation, three dichroic (Cy2, Cy3, and Cy5) at room temperature for 1 h. After washing, beam splitters (Semrock) were used at 458, 495, and 560 nm mounting medium was added to the slides. Images were col- (FF458-Di02, FF495-Di03, and FF560-Di01). Clots from GFP lected using a Leica SP8 confocal microscope. Whole-mount E. coli–infected mice were immediately placed into an imaging imaging followed a similar protocol but extended antibody in- chamber between 50-µm mesh and a quartz coverslip to keep cubations to overnight. it at physiological temperature and was examined for up to 1 h. Most experiments focused on the 30-min time period. Gene expression analysis Clots were imaged with 20 to 35 optical sections at 2.5 µm, Gene expression analysis in macrophage herein used a previ- each taken at regular time intervals (typically 30 s), in order ously described database created by the Immunological Genome to capture cell dynamics within the clot. Project (detailed by Gautier et al., 2014). Zhang et al. Journal of Experimental Medicine 1298 Macrophage disappearance reaction https://doi.org/10.1084/jem.20182024 marrow-derived and tissue-resident macrophage lineages proliferate at Statistics key stages during inflammation. Nat. Commun. 4:1886. https://doi.org/ Statistical analysis was performed using the Student’s t test for 10.1038/ncomms2877 unpaired samples or one-way ANOVA with a post-hoc Tukey’s Echtenacher, B., K. Weigl, N. Lehn, and D.N. Mannel. 2001. Tumor necrosis multiple comparisons test. Results were considered significant factor-dependent adhesions as a major protective mechanism early in septic peritonitis in mice. Infect. Immun. 69:3550–3555. https://doi.org/ at P < 0.05. Results display all replicated experiments, and val- 10.1128/IAI.69.6.3550-3555.2001 ues are mean ± SEM. Faust, N., F. Varas, L.M. Kelly, S. Heck, and T. Graf. 2000. Insertion of en- hanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood. 96: Online supplemental material 719–726. Fig. S1 shows coagulation and adhesion additively cooperate to Gautier, E.L., T. Shay, J. Miller, M. Greter, C. Jakubzick, S. Ivanov, J. Helft, A. account for the MDR in response to inflammation. Video 1 shows Chow, K.G. Elpek, S. Gordonov, et al; Immunological Genome Consor- tium. 2012. Gene-expression profiles and transcriptional regulatory intravital imaging of peritoneal macrophages through the intact pathways that underlie the identity and diversity of mouse tissue abdominal wall. Video 2 shows intravital imaging of a clot after macrophages. Nat. Immunol. 13:1118–1128. https://doi.org/10.1038/ni removal from a mouse 3 h after injection of GFP–E. coli. .2419 Gautier, E.L., S. Ivanov, P. Lesnik, and G.J. Randolph. 2013. Local apoptosis mediates clearance of macrophages from resolving inflammation in mice. Blood. 122:2714–2722. https://doi.org/10.1182/blood-2013-01 Acknowledgments -478206 Cre fl/fl Gautier, E.L., S. Ivanov, J.W. Williams, S.C. Huang, G. Marcelin, K. Fairfax, We thank R.P. McEver for providing Lyz2 ;Tln1 mice, M. P.L. Wang, J.S. Francis, P. Leone, D.B. Wilson, et al 2014. Gata6 regulates Wohltmann for assistance with mouse breeding and care, and aspartoacylase expression in resident peritoneal macrophages and T.J. Girard for technical assistance. controls their survival. J. Exp. Med. 211:1525–1531. https://doi.org/10 .1084/jem.20140570 This research was supported in large part by a National In- Ginhoux, F., and M. Guilliams. 2016. Tissue-Resident Macrophage Ontogeny stitutes of Heath grant (5R37AI049653 to G.J. Randolph) and a and Homeostasis. Immunity. 44:439–449. https://doi.org/10.1016/j National Institute of Diabetes and Digestive and Kidney Diseases .immuni.2016.02.024 Guilliams, M., and C.L. Scott. 2017. Does niche competition determine the pilot and feasibility grant (P30 DK052574 to B.H. Zinselmeyer), origin of tissue-resident macrophages? Nat. Rev. Immunol. 17:451–460. with additional support from the National Institutes of Heath https://doi.org/10.1038/nri.2017.42 (grants DP1DK109668 and R01HL118206 to G.J. Randolph), the Hartveit, F., and S. Thunold. 1966. Peritoneal fluid volume and the oestrus cycle in mice. Nature. 210:1123–1125. https://doi.org/10.1038/2101123a0 American Heart Association (grant 16SDG30480008 to B.H. Lim, T.J.F., and I.H. Su. 2018. Talin1 Methylation Is Required for Neutrophil Zinselmeyer), National Institutes of Heath (grant R01AI113118 to Infiltration and Lipopolysaccharide-Induced Lethality. J. Immunol. 201: B.T. Edelson), and a John C. 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Published: May 2, 2019

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