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Sterile Injury Repair and Adhesion Formation at Serosal Surfaces

Sterile Injury Repair and Adhesion Formation at Serosal Surfaces REVIEW published: 14 May 2021 doi: 10.3389/fimmu.2021.684967 Sterile Injury Repair and Adhesion Formation at Serosal Surfaces Simone N. Zwicky, Deborah Stroka and Joel Zindel Department of Visceral Surgery and Medicine, Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland Most multicellular organisms have a major body cavity containing vital organs. This cavity is lined by a mucosa-like serosal surface and filled with serous fluid which suspends many immune cells. Injuries affecting the major body cavity are potentially life-threatening. Here we summarize evidence that unique damage detection and repair mechanisms have evolved to ensure immediate and swift repair of injuries at serosal surfaces. Furthermore, thousands of patients undergo surgery within the abdominal and thoracic cavities each day. While these surgeries are potentially lifesaving, some patients will suffer complications due to inappropriate scar formation when wound healing at serosal surfaces defects. These scars called adhesions cause profound challenges for health care systems and patients. Therefore, reviewing the mechanisms of wound repair at serosal surfaces is of Edited by: clinical importance. Serosal surfaces will be introduced with a short embryological and Javier Leceta, Complutense University of Madrid, microanatomical perspective followed by a discussion of the mechanisms of damage Spain recognition and initiation of sterile inflammation at serosal surfaces. Distinct immune cells Reviewed by: populations are free floating within the coelomic (peritoneal) cavity and contribute towards Pilar Sandoval, damage recognition and initiation of wound repair. We will highlight the emerging role of Severo Ochoa Molecular Biology Center (CSIC-UAM), Spain resident cavity GATA6+ macrophages in repairing serosal injuries and compare serosal Andrea Doni, (mesothelial) injuries with injuries to the blood vessel walls. This allows to draw some Humanitas Research Hospital, Italy parallels such as the critical role of the mesothelium in regulating fibrin deposition and how *Correspondence: Joel Zindel peritoneal macrophages can aggregate in a platelet-like fashion in response to sterile joel.zindel@dbmr.unibe.ch injury. Then, we discuss how serosal wound healing can go wrong, causing adhesions. The current pathogenetic understanding of and potential future therapeutic avenues Specialty section: against adhesions are discussed. This article was submitted to Mucosal Immunity, Keywords: peritoneal adhesions, peritoneum, sterile injury, mesothelium, post-surgical adhesions a section of the journal Frontiers in Immunology Received: 24 March 2021 Accepted: 23 April 2021 DEVELOPMENT AND MICROANATOMY OF THE COELOM Published: 14 May 2021 AND MESOTHELIUM Citation: Zwicky SN, Stroka D and Zindel J During embryology, atthe end ofthe third week, thelateralplatemesoderm isdivided intotwo layers:the (2021) Sterile Injury Repair and somatic and splanchnic mesoderm layer (1). These two layers form a cleft that becomes a cavity as the Adhesion Formation embryo undergoes a cranio-caudal and latero-lateral folding event in week four (1). This cavity is called at Serosal Surfaces. the intraembryonic coelom and contains vital organs such as the heart, the lungs, the liver, and the Front. Immunol. 12:684967. doi: 10.3389/fimmu.2021.684967 intestines. In mammals, the mesodermal lining of the coelom differentiates into a serous epithelium-like Frontiers in Immunology | www.frontiersin.org 1 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation membrane called mesothelium (2). The somatic mesoderm gives filled cavities from surrounding tissues (Figure 2). Together with rise to the parietal layer of the mesothelium which lines the body the associated sub-mesothelial connective tissue the serosa is also wall, and the splanchnic mesoderm gives rise to the visceral layer of called peritoneum, pleura, and pericardium in the peritoneal the mesothelium which lines the surfaces of organs. The intra- (abdominal), pleural and pericardial cavities, respectively. In coelomic organs stay connected to the body wall by elongations practice, the terms mesothelium, serosa, and peritoneum (or referred to as mesenteries which contain blood vessels, lymphatics, pleura or pericardium) are often used interchangeably. and nerves (1)(Figure 1). The peritoneum is less than 25 µm thick in the mouse (3) and Later, the coelomic cavity is further subdivided resulting in about 50-100 µm thick in humans (4, 5). Therefore, as we discuss three embryologically related but anatomically distinct anatomical injury at serosal surfaces, it is important to note that the compartments: the pericardial cavity, the pleural cavities, and the mesothelium will rarely be injured in an isolated fashion. In peritoneal (abdominal) cavity. All of these contain vital organs fact, serosal injuries will often compromise the tissues that are such as heart, lung, and abdominal organs (1). covered by the mesothelium as well. These underlying tissues can The serous membrane that covers the walls of all coelomic be vastly different such as: cavities as well as the borders of all organs contained within them is - smooth-muscular wall of the intestines, urinary bladder, uterus, also called the serosa and is comprised of a flat monolayer of mesothelial cells. The serosal linings ensure friction-less movement - parenchymal tissue of heart, lung, liver, gallbladder, spleen of organs and establish a water-tight barrier separating the fluid- (only mouse), ovaries, AB C FIGURE 1 | Development of the intra-embryonic coelomic cavity. (A) Schematic cross section human embryo of 3 weeks age. The mesoderm shows a somatic (dorsal) and splanchnic (ventral) aspect. (B) Cranio-caudal and latero-lateral folding in week 4. (C) After closure of the anterior abdominal wall the intra-embryonic coelomic cavity is formed. Organs (e.g. gut) are suspended by dorsal and sometimes ventral (not shown) mesenteries carrying blood vessels and nerves. FIGURE 2 | Microanatomy of mesothelial surfaces. (A, B) Cross sections of mouse abdominal wall stained with Hematoxylin & Eosin (A) and Masson’s trichrome staining (B). Scale bars: 50 µm. (C) Illustration of the structures shown in (A, B). (D) Top view on mesothelial surface stained with anti-podoplanin antibody. Frontiers in Immunology | www.frontiersin.org 2 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation - fat tissue of the omentum, macrophages (SPM) are monocyte-derived, constantly replenished and can be recruited within hours in significant amounts (14). At - striated muscle, fascia, and bone of the thoracoabdominal wall baseline, they account for about 5% of all immune cells or about and diaphragm, 10% of all macrophages (14, 18). The majority (90%) of peritoneal - and connective tissues such as that of the pericardium. macrophages belong to a distinct tissue-residential macrophage population. Since these resident cells are slightly larger than their Any experimental model system that studies serosal wound monocyte-derived sisters, they are also referred to as large peritoneal repair, may invoke some underlying tissue-specificwound repair macrophages (14). The large peritoneal macrophages (LPM) are a mechanisms. This review is targeted at serosa specific mechanisms, self-renewing population characterized by the expression of CD102 but weask thereadertobearin mindthatwe use thegeneralization (Icam2), high levels of F4/80 and the transcription factor GATA6 “at serosal surfaces” inductively; some of the mechanisms discussed (19–22). GATA6+ LPM seem to be well conserved when comparing here mayapply onlyto specific locations within the coelomic cavity. the different coelomic cavities of mice and human (23–25). Canonically, these GATA6+ cavity macrophages are thought to clear bacteria by phagocytosis (14, 26) and also by inducing intra- CELLS SUSPENDED IN abdominal formation offibrin clots that immobilize bacteria (21). In COELOMIC CAVITIES primordial species such as the purple sea urchin (Strongylocentrotus purpuratus), coelomocytes are also crucial for tissue repair, in The coelomic cavities are filled with fluid that suspend millions addition to clearing toxins and pathogens (27–30). The of cells also referred to as coelomocytes. The coelomocyte importance of GATA6+ cavity macrophages in damage composition of mice and humans has been reviewed elsewhere recognition and tissue repair will be discussed in detail. (6). Briefly, the human peritoneal cavity suspends a total of 10 leukocytes in 5-100ml of peritoneal fluid (6, 7). In mice, the number of peritoneal leukocytes varies between strains from 3 to 6 6 5x10 cells (8). The pleuropericardial cavities contain 0.3-1x10 DAMAGE RECOGNITION leukocytes per mouse (6, 9, 10). Most leukocytes in the peritoneal AND INFLAMMATION cavity are lymphocytes (10-60%) and macrophages (40-60%) (8, 11–16). In addition, the peritoneal cavity contains dendritic cells Wound repair at large starts with inflammation. Inflammation is (2 – 6%) (12, 17), mast cells, eosinophils, neutrophils (0-31%), induced when a significant deviation from homeostasis is detected. innate lymphoid cells (ILCs) including natural killer cells and According to the current paradigm, such a deviation could be the mesothelial cells (14, 16)(Figure 3). presence of microbes (infection) or damaged tissue (injury). The In terms of wound healing, the role of peritoneal macrophages is innate immune system has developed an effective arsenal of best established. Macrophages make up 40-60% of all coelomocytes surveillance cells that constantlyprobe theirmicroenvironment in both mice and humans. Two major subpopulations of peritoneal for deviations from homeostasis. On a molecular level, deviation macrophages have been described (14). The small peritoneal from homeostasis is defined by the occurrence of pre-specified AB FIGURE 3 | Cells in a mouse coelomic cavity. (A, B) Peritoneal cavity lavage of healthy C57Bl/6 mice. Dimensionality reduction dimension 1 (umap1) and 2 (umap2) of myeloid lineage markers (mass cytometry) are plotted on x- and y-axis, respectively. Dots (cells) are colored by cluster (A) or marker (B). Data with kindly permission from M. Dosch and G. Beldi. Frontiers in Immunology | www.frontiersin.org 3 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation Mesothelial Damage Is First Recognized molecular patterns. Immunostimulatory molecular patterns that by Cavity Macrophages induce inflammation in case of sterile injury, i.e., in the absence of pathogens and their products, have been termed damage associated Recent advances in intravital microscopy have allowed to characterize the sequence of cells recruited to mesothelial molecular patterns (DAMPs). DAMPs have been extensively reviewed elsewhere (31). In brief, DAMPs comprise different injuries. By using resonant-scanners, multi-photon excitation, and extremely sensitive hybrid detection systems it became molecules that are not normally present outside of cells such as double stranded DNA, nuclear proteins, mitochondrial DNA, possible to image the peritoneal cavity through the intact abdominal wall under real-life conditions (21, 44). Second, mitochondrial proteins, and molecules with high cytosolic concentrations such as ATP or K Ions.In addition,damaged multi-photon imaging allows the use of near-infrared microscopy lasers to induce focal thermal injuries during cells may induce the production and release of additional DAMPs (iDAMPs) such as heat shock proteins, defensins, galactins and intravital microscopy with high precision (44–46). By combining intravital microscopy of the abdominal cavity with peritoneal laser interleukin 1 (IL-1). Furthermore, if proteins that are constitutively present in the extracellular space such as hyaluronan, biglycan, injuries, we were able to image cellular recruitment to mesothelial injuries. Surprisingly, the first GATA6+ cavity macrophages heperansulfate and other extracellular matrix (ECM) components are modified by injuries, they can also become DAMPs. Under attached at the injuries within only a few seconds and the macrophages completely covered the lesions after 15 minutes of homeostatic conditions, the serosal surfaces are covered with glycoconjugates such as sialomucins, hyaluronic acid, and imaging (44). The recruitment of cavity macrophages to mesothelial injury was significantly faster than that of glycoproteins like fibronectin (32–35). These molecules contain large anionic sites that cover the serosal surfaces with a negatively neutrophils, which needed much longer (> 40 minutes) (44). Cavity macrophages were present in the peritoneal fluid in vast charged coat—also referred to as the glycocalyx—that may help to repulse invading microbes (32) and ensure friction-less movement numbers and traversed the peritoneal cavity in a seemingly random fashion within respiration-dependent movement of of intra-coelomic organs (35). Thelossofthisnegativelycharged coating due to serosal injury, may serve as mesothelium-specific peritoneal cavity content (44). The observations that these cells seemed to rely on passive transportation by peritoneal fluid, and DAMP or “touch me signal” (36). Molecules that allow eukaryotic cells to detect the presence of that they—upon contact with cell already adhering to the injury— were forming stable cell-cell aggregates were very reminiscent of DAMPs have been termed pattern recognition receptors (PRR). The expression of PRR such as toll-like receptors 1 through 6 the platelet aggregation that took place when a nearby blood vessel wall was damaged using laser injury. We concluded that cavity (TLR-1-6), nucleotide-binding oligomerization domain (Nod)-1 and Nod-2 and advanced glycation end product (AGE) macrophages randomly “patrol” the serosal surfaces in a platelet- like fashion and rapidly form aggregates in response to DAMPs. receptors, has been demonstrated for murine and human mesothelial cells (37). Upon activation, mesothelial cells release This is consistent with a previous electron microscopy study by Haney showing that peritoneal macrophages invariably detected cytokines and inflammatory mediators such as chemokine (C–C motif) ligand 2 (CCL2), CCL5, (C–X–C motif) ligand 8 and migrated to injuries of the peritoneal membrane (47). In addition, Wang and Kubes showed that cavity macrophages were (CXCL8), and nitric oxide (38, 39). Furthermore, mesothelial cells upregulate adhesion molecules that presumably facilitate able to detect mesothelial injuries of the liver capsule and migrated to the injured liver (36). On a molecular level, this interaction the migration of inflammatory leukocytes across and along serosal surfaces. These include intercellular adhesion molecule- occurred independent of integrins or selectins, instead peritoneal macrophages relied on different receptor molecules such as 1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), E-cadherin, N-cadherin, CD29 and CD44 (38, 40–42). It is macrophage receptor with collagenous structure (MARCO), Macrophage scavenger receptor 1 (MSR1), CD44, and important to note that cellular adhesion molecules expressed by mesothelial cells play a dual role in serosal wound repair. purinergic receptor P2X7. The respective DAMPs recognized by CD44 and P2X7 are hyaluronan and ATP respectively (36). The While an initial upregulation may facilitate leukocyte recruitment, these molecules, especially E-cadherin, are ligands that mediate MARCO and Msr1 dependent macrophage aggregation are yet to be identified (44). downregulated later during serosal wound repair. The latter is associated with loss of mesothelial cohesion enabling the The function of peritoneal macrophages in sterile injury is multi- facetted. Current models indicate that ligation of DAMPs to PRR on mesothelium to switch to a more mesenchymal program, a process that we will discuss in detail below. In addition, macrophages leads to their inflammatory polarization—also referred to as M1 polarization. This activation would result in the mesothelial cells modulate inflammation by synthesis and release of hyaluronan (43), which is able to sequester free production of pro-inflammatory cytokines such as tumor necrosis factor (TNF) and IL-1 (31, 48). However, peritoneal macrophages radicals and initiate tissue repair responses (38). In vivo, the initiation of inflammation at serosal surfaces does recruited to sterile liver injury were shown to skew their phenotype towards alternative or repair polarization—also referred to as M2 not rely on mesothelial cells alone but on a series of events. These comprise specialized cellular and humoral immune mechanisms macrophages—increasing their expression of CD273, CD206 and Arginase 1 (36). Interestingly, Uderhardt et al. recently investigated such as leukocyte recruitment, complement activation and production of natural antibodies. In the rest of this chapter, we the resident tissue macrophages of the muscular abdominal wall. The abdominal wall macrophages are distinct from the peritoneal will discuss these elements one by one. Frontiers in Immunology | www.frontiersin.org 4 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation cavity macrophages suspended in the peritoneal cavity. They indicate that the aggregation of cavity macrophages in response to a proposed that abdominal wall macrophages can extend their strong stimulus, such as peritonitis, causes inflammation. However, pseudopods toward local injury sites within a radius of 100- in the case of smaller insults such as focal injuries or localized 150µm. In their study, resident tissue macrophages were able to microbial challenges, MDR may compartmentalize the insult, in completely enclose lesions if their size was below a certain threshold analogy to the cloaking mechanism described for macrophages of (microlesions). This—as the authors termed it—cloaking the muscular abdominal wall (45). Along those lines, complete mechanism, was able to block scouting neutrophils from MDR could be interpreted as a threshold above which all interacting with DAMPs and thus prevented subsequent macrophages have been “used up” indicating that the attempt at neutrophil driven inflammation and tissue destruction (45). The cloaking the insult has failed, which in turn results in inflammation. cloaking mechanism was described for tissue resident macrophages Either way, it would be important to study the largely unknown in the muscular abdominal wall, i.e., on the far side of the (intracellular) changes in macrophages undergoing a disappearance mesothelium with respect to the coelomic cavity. It needs to be reaction in sterile and microbial models. determined whether scavenger receptor mediated macrophage Dendritic Cells and Mast Cells aggregation on the coelomic site of the mesothelium causes inflammation or whether aggregation of cavity macrophages The peritoneal cavity harbors CD11c dendritic cells as well as cKit mast cells both of which are canonical initiators of serves to contain injuries and is therefore—in essence—anti- inflammatory. Ultimately, aggregation of peritoneal cavity inflammation. Their role as antigen presenting cells and inducers of inflammation in response to bacterial infection is macrophages in response to mesothelial injuries was shown to improve tissue repair (36, 44, 47). well documented. In fact, CD11c+ dendritic cells are required for survival in murine polymicrobial peritoneal sepsis (66). In Cavity Macrophage addition to pathogen-derived ligands for PRR, several DAMPs have been shown to interact with dendritic cells and dramatically Disappearance Reaction affect their function (67, 68). Interestingly, the response of Aggregation of peritoneal macrophages causes their number in dendritic cells to DAMPs is not always clear-cut, with different the peritoneal lavage to drop. The decrease in their number was responses depending on dendritic cell subtypes and location (67). correlated with the injury size (44). With larger injuries of the For example, activation of dendritic cells in sterile liver injury mesothelium, such as a surgical laparotomy, the number of leads to the secretion of anti-inflammatory cytokines such as IL- GATA6+ cavity macrophages in the peritoneal lavage was 10 and TGF-b (67) while similar injury models of kidney and gut reduced to zero (44). In other words, these cells disappeared may lead to a pro-inflammatory response and secretion of IL-6, from the peritoneal fluid (lavage). However, this was not the first IL-12 and TNF-a (67, 69). So far, the response of peritoneal time, the sudden absence of macrophages was observed. In fact, dendritic cells to serosal injury is not well understood and over half a century ago, Nelson and Boyden described a sharp requires further studies. Mast cells have traditionally been decline of macrophage count in peritoneal exudates in response studied in the context off helminthic infections and Ig-E to a hypersensitivity reaction to tuberculin in Bacille Calmette- mediated reactions. It becomes clear, that mast cell Guérin (BCG)-vaccinated guinea pigs. They termed this the degranulation is also an important modulator of wound “macrophage disappearance reaction” (MDR) (49). Since then, healing of skin wounds (70) and lesions in the gastrointestinal various insults (sterile and microbial) to the peritoneal tract (71–73). Poerwosusanta et al. investigated the role of mast compartment have been found to induce the MDR (Table 1). cell degranulation in mesothelial injury. Mesothelial injury was These studies indicate that the MDR is not a specific reaction but carried out by performing laparoscopic surgeries in rats at arguably follows any inflammatory challenge to the peritoneal different intra-abdominal inflation pressures (74). They showed compartment. While some reports indicate that peritoneal that an increased intraabdominal pressure—and presumably macrophages can leave the peritoneal cavity through the draining increased stress to the mesothelium—led to an increased lymphatics (52, 60, 64), most of the more recent reports suggest that number of mast cells that infiltrated the mesothelium. This peritoneal macrophages have the tendency to adhere to each other was correlated with increased mast cell degranulation. This (aggregate) as well as to the mesothelium in response to challenge increased mast cell count is consistent with findings from skin (Table 1). Therefore, the loss of dispersion and cellular aggregation injury models and is due to chemokine-dependent mast cell are a commonality among the different models of MDR. The MDR immigration rather than local proliferation. More detailed correlates with increased inflammatory cytokine levels in the investigation, e.g. based on intravital microscopy, could help to peritoneal fluid and the influx of pro-inflammatory leukocytes elucidate whether mast cells are recruited to mesothelial injuries such as monocytes, eosinophils, and neutrophils into the by blood or directly from the peritoneal cavity. peritoneal compartment (21, 59). Cailhier et al. used CD11b driven diphtheria toxin receptor and low dose intraperitoneal Humoral Pattern Recognition Molecules injections of diphtheria toxin to selectively deplete resident and Natural Antibodies peritoneal macrophages. In an experimental peritonitis model, this resulted in a significant decrease of inflammation (infiltration The fluid of the pleural and peritoneal cavity in mice and humans not only contains cells but also large amounts of proteins of the of neutrophils) that could be restored by the adoptive transfer of resident, non-transgenic, peritoneal macrophages (65). These data coagulation system and complement system as well as large Frontiers in Immunology | www.frontiersin.org 5 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation TABLE 1 | Macrophage disappearance reaction (MDR). Studies describing MDR from 1963 until now. MDR Trigger (dose) Time between trigger and Postulated fate of disappeared Molecular mechanism Reference complete MDR macrophages Sterile Models Sterile mesothelial injury 3h Form stable cell-cell aggregates that Scavenger receptors, can be blocked with (44) (surgery, laser) cover injury and induce post-surgical Heparin and Poly-(I) adhesions Sterile Brewer’s Thioglycollate 12-72h Macrophage cell death Not demonstrated (50–52) Antigen, migration inhibitory 1 to 96h Undergo activation during MDR in MDR Inhibited by Heparin, L-Fucose, (53) factor, viruses or tumor cells delayed type hypersensitivity or acute Hyaluronidase inflammatory reaction and then reappear activated to regulate responses toward pathogens or tumor cells. Egg Antigen (10ug), purified 5h Macrophage activation Desensitization suppress MDR, in (54) protein derivate (10ug) sensitized animals normal MDR Tuberculin 2.5 - 6h Not demonstrated MDR completely inhibited by Heparin and (49, 55) Warfarin Ova peptide (50ug) into mice 5h Macrophage adhesion Suppressed in fibrinogen- (56) bearing antigen-primed T cells deficient mice, partially suppressed by thrombin antagonist Thrombin (20 Units) 1h -5h Macrophage adhesion MDR suppressed (56) in fibrinogen-deficient mice. RGES Peptide 48h Macrophage bind the mesothelium Integrin-mediated mechanisms involving (57) overlying draining lymphatics VLA-4 and VLA-5 that can be blocked by RGD (Arg-Gly-Asp peptides) and VLA-4 and VLA-5 blocking antibodies. Microbes or microbial products E. coli (5×10 UV-inactivated) 20d Do not undergo fas-mediated apoptosis No difference in fas-deficient mice. (58) S. aureus 2h (2 × 10 ) Not demonstrated Not demonstrated (59) Lipopolysaccharide 3h (10 µg) Accumulation in the omentum Macrophage interaction with mesothelial (19, 40, 5h (1 µg) cells, mainly of the omentum, was 56) proposed to be a key step in MDR. Partially inhibited by refludan. Zymosan 3-4h (1mg) Form large clots to trap MDR reversed completely with Heparin (21, 51, 4h (0.5mg) microorganisms; adherence with tissue and partially with Hirudin/loss Factor V 60–62) 3d (10 µg) and drained to lymph node Expression/loss of Integrin activation adaptor talin-1 Expression/TF deficiency INF-g (100 U/mL) + LPS 20h Binding to mesothelial cells Monocyte activated by in vitro exposure to (40) (100ng/ml) LPS and INF-Y bound with increased efficiency to mesothelial cells Synthethic Lipopetid 12h Not demonstrated Not demonstrated (59) (Pam3CSK4) Human studies Bacterial peritonitis 1 day Shedding of surface CD206 Depletion of CD206+ LPM at day 1 of SPB (63) Peritonitis with normalization to steady state after resolution of SPB Liver cirrhosis associated Not demonstrated Severity of liver disease and liver cirrhosis (25) events (Bacterial peritonitis, are correlated with reduced numbers of hi hi encephalopathy, death) CrIg macrophages. Human CrIg macrophages were transcriptionally similar to mouse F4/80hi peritoneal macrophages. amounts of natural antibodies (75, 76). In the peritoneal and correlation of the invasiveness of the procedure with the pleural cavities, the complement proteins are produced by amount of complement used, indicating that sterile injury mesothelial cells (75, 77). The complement system is an leads to complement activation in humans (84). Inversely, ancient enzymatic cascade of proteins with the main function humoral molecules that are canonically associated with innate of opsonization and lysis of bacteria (78). The alternative immunity, have been shown to mediate tissue repair and regulate pathway of the complement can be activated by injuries and in fibrosis. For example, pentraxin 3 (PTX3) was shown to reduce the last decade a role of complement activation in wound healing fibrin deposition and fibrosis in several wound models outside of (79) and regeneration (80, 81) as well as morphogenetic and the peritoneal cavity. While to our knowledge, no humoral developmental processes (82, 83) has been suggested. molecule with anti-fibrotic properties has been described in the Furthermore, some clinical studies have evaluated the role of peritoneal cavity, the discovery of such could have great blood complement during major surgery and described a therapeutic potential (85–87). Frontiers in Immunology | www.frontiersin.org 6 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation Like complement factors, natural antibodies are abundant in an abdominal adhesion, a pathology we will discuss in more the coelomic cavity fluids and are primarily thought to combat detail below. See the Perspective Box 1 speculating on parallels microbes by recognizing a wide variety of different microbial between vascular and mesothelial coagulation control. antigen patterns (88). The peritoneal natural antibodies are mainly produced by self-replenishing peritoneal B1 cells in an antigen-independent manner (89). The repertoire of natural antibodies also enables recognition of self-antigens such as RECRUITMENT OF NEUTROPHILS phosphorylcholine, phosphatidylcholine and carbohydrate AND MONOCYTES determinants. Furthermore, natural antibodies have been shown to sense apoptotic cells (88) and electronegative We discovered that the first cells recruited to mesothelial injuries are DAMPs (90). In addition, natural antibodies were able to the peritoneal cavity macrophages (44). These cells reside suspended accelerate wound healing by recruiting additional wound in the peritoneal fluid (19) and are recruited directly from their macrophages (91). Along those lines, Grönwall et al. postulate suspensive state to the mesothelium in case of injury. This that natural IgM antibodies are part of a synapse between an comprises a special case of leukocyte recruitment that is unique to apoptotic cell (that binds IgM) and the phagocyte. This synapse coelomic cavities. The canonical route of leukocyte recruitment is is mediated by complement and complement receptors from the blood stream. The processes of leukocytes leaving the expressed by phagocytes (92). There is increased interest in blood stream have been referred to as leukocyte adhesion cascade studying the role of natural IgM, and IgM-dependent, and trans-endothelial migration. The underlying mechanisms have complement-mediated phagocytosis in several disease models been revisited and reviewed most comprehensively by Nourshargh (93–95). Although available data is limited, it is conceivable that et al. (97, 98). In perfused organs such as muscle or liver, neutrophils complement and natural antibodies of body cavities play an are recruited within 30 minutes to the inflammatory site from the important a role in wound repair at serosal surfaces. bloodstream (46, 99). In the mesothelium, neutrophils are the first cells that arrive after peritoneal macrophages, after about 40-60 minutes (44). In response to focal sterile injuries, neutrophils show an extremely high degree of coordination. While intravascular COAGULATION AND FIBRIN DEPOSITION chemokine gradient seems to be very important for successful directional migration over the initial distance within the vessel It is widely accepted that inflammation of the peritoneal, pleural, (31, 99), different molecules are the most potent chemotactic stimuli or pericardial compartment, is associated with fibrin exudates. once the neutrophils are close to the wound (31, 99, 100). This has After mesothelial injury, the resulting inflammation as well as the been demonstrated for molecules such as N-formyl peptides, ATP, injury itself, lead to the activation of tissue factor pathway and and leukotriene B4 (LTB4) and their respective receptors on the inhibition of the antithrombin-III (AT-III) pathway (96). neutrophils called formyl peptide receptor, P2Y2 receptor and This is followed by the spontaneous cleavage of fibrinogen and LTB4 receptor (31, 46, 99–102). These so-called gradients and cross-linking of fibrin. In addition to inflammation, injury causes autocrine feedback loops (LTB4) have been established for solid the destruction of and consequent leakage from blood vessels of organs such as the liver or muscle. Whether neutrophils rely on the abdominal wall. Extravasation of blood results in the similar mechanism to reach serosal surfaces has not been activation of the canonical coagulation cascade which further demonstrated yet but the technical advances of the last years will enhances the deposition of fibrin (96). In vivo, the deposition of now allow us to address these questions using intravital microscopy. fibrin is limited at serosal surfaces by activated plasmin which Although the canonical role of neutrophils is to clear continuously degrades fibrin. Plasmin levels are regulated by microbes, several reports suggest that they are imperative for plasminogen activators tPA and uPA and their respective timely restoration of tissue architecture after sterile injury by plasminogen activator inhibitors 1 and 2 (PAI 1, PAI 2) (96). clearing necrotic material (99) and producing growth factors Homeostatic mesothelial cells produce large amounts of Plasmin, such as transforming growth factor b (TGF-b) and vascular uPA and tPA and low amounts of PAI1 and PAI2. Therefore, the deposition of fibrin at serosal surfaces is tightly controlled. During inflammation however, the mesothelial production of BOX 1 | Perspective. PAI1 and PAI2 is significantly increased resulting in a mesothelial program switch from fibrinolytic towards anti- We find it intriguing that fibrin clots after surgery are not focally limited to sites of fibrinolytic state, resulting in the visible deposition of fibrinous injury but seem to be formed at distant sites as well while other regions of the peritoneal cavity appear to be protected. During sepsis, spontaneous exudates (96). disseminated intravascular coagulation (DIC) is a major clinical problem. The The formation of a stable fibrin clot—also referred to as fibrin distribution of fibrin clot deposits during DIC is poorly understood. Interestingly, matrix or cross-linked fibrin—serves as the scaffold for the the response of mesothelium and endothelium to inflammation share certain subsequent wound repair (granulation) tissue. This includes similarities and both lead to the spontaneous formation of disseminated fibrin clots. Studying the analogy between these two pathologies, that both involve an the infiltration of leukocytes and mesenchymal precursors and epithelial-like monolayer of mesodermal origin, may lead to the identification of results in the deposition of ECM, ingrowth of vessels and nerves common patterns and molecules governing the respective phenomena of DIC and and finally, the re-mesothelialization of the injured serosa. If peritoneal adhesions. these fibrin clots grow too large, they can be the starting point for Frontiers in Immunology | www.frontiersin.org 7 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation endothelial growth factor (VEGF) (103–105). Furthermore, once peritoneal cavity fluid. In fact, within a few hours after neutrophils have cleared all necrotic tissue, they starve each other of extensive mesothelial injury (surgery), large numbers of Gr1+ DAMPs. This leads to neutrophils becoming apoptotic and cleared cells (neutrophils) and monocyte derived macrophages were by macrophages, which is another critical step for tissue repair as it recruited into the peritoneal cavity (119). This is consistent enables macrophages within the wound to switch from an with observations in humans undergoing surgery where inflammatory to an anti-inflammatory, pro-resolution program billions of neutrophils and monocytes can be isolated from the (99, 103, 106). In this environment, pro-resolution macrophages peritoneal fluid. This recruitment was proposed to be driven by and neutrophils start to produce factors that inhibit the recruitment chemokines such as MCP-1 and CXCL1 that were released into of additional neutrophils and enhance pro-resolution properties of the peritoneal cavity by mesothelial cells in response to injury macrophages. These factors include lipoxin A4, resolvins, protectins (119, 120). It is not known what molecules and mechanisms (107), phosphatidylserine containing microvesicles shed by govern the migration of leukocytes from the interstitium, into the neutrophils, chemokine sequestration through CCR5 modulation peritoneal fluid, where these cells adopt a planktonic form once by neutrophils (108), Annexin A1 released from neutrophil granules again. Further, it would be interesting to investigate the or in microvesicles (109)and IL-10 (110). Taken together, migratory patterns of neutrophils and monocytes after they neutrophils play an important role in clearing debris and setting have reached their suspended state in the peritoneal cavity. up a pro-resolution environment. The second major leukocyte population recruited to mesothelial injuries comprises inflammatory monocytes and SEROSAL (MESOTHELIAL) REPAIR monocyte-derived macrophages. It is generally accepted that the recruitment of neutrophils precedes the recruitment of monocytes So far, we have seen how a mesothelial injury leads to in sterile injury (31, 111) but it remains controversial whether inflammation with the consecutive deposition of a fibrin neutrophils recruitment is a necessary prerequisite for subsequent matrix and infiltration of immune cells. Normal serosal repair monocyte recruitment (112, 113). Rather than being pre- is achieved when a) the underlying organ is repaired, b) the sub- determined, the fate of recruited monocytes appears to be mesothelial connective tissue layer is restored to its original largely dependent on the environment (62). In the context of composition and thickness and c) the integrity of the sterile injury repair, monocytes have been shown to differentiate mesothelial membrane is reconstituted (31, 121, 122). The into mature macrophages at the site of injury. This process has central role of the mesothelium in tissue repair and fibrosis has hi been reviewed before (114). In brief, Ly6C monocytes are been revisited and comprehensively summarized (38). recruited to the wound, where they gradually differentiate into The mesothelium is a slowly renewing tissue with less than 1% low Ly6C macrophages. One of the molecules necessary for this of cells undergoing mitosis at any time (123). After mesothelial conversion is nuclear receptor subfamily 4, group a, member 1 injury, activated macrophages induce a pronounced proliferative (Nr4a1) (115). In the peritoneal cavity, the influx of inflammatory expansion of the mesothelial compartment (124, 125). Genetic monocytes due to injury or infection, correlates with the increase lineage tracing of mesothelial cells in several injury and disease + - - of MHCII CD102 GATA6 macrophages (26). These are also models indicate that regenerating mesothelium originates from referred to as small peritoneal macrophages or bone marrow healthy mesothelium rather than submesothelial cells (126–128). derived macrophages (14, 26). In the peritoneal cavity, PAI-1 Small focal mesothelial injuries that were induced using thermal and CCL1 were shown to recruit macrophages to the wound probes on the liver capsule and abdominal wall, healed completely involving the receptor molecules CD11b and CCR8 respectively without any visible defect left after days (36, 44). Because repair of (116, 117). Depletion of macrophages using clodronate-loaded mesothelial defects is largely independent of the defect size, liposomes or genetic constructs results in decreased wound investigators have proposed that mesothelial cells not only crawl healing of sterile injuries of serosal surfaces of the liver and into the wound from the borders, but also detach from opposing abdominal wall (36, 44). However, novel experimental strategies surfaces and distant sites and migrate in a free-floating state will be necessary to experimentally isolate the role of recruited through the coelomic cavity until they settle on the wound (125, macrophages compared to that of the resident GATA6+ 129, 130). This is further supported by the fact that adoptively macrophages in mesothelial wound healing. Furthermore, transferred mesothelial cells improve mesothelial repair in the monocyte-derived macrophages have been shown to replenish recipient (130). The response of mesothelial cells to injury can be the resident GATA6+ macrophages. This process is dependent on summarized as proliferation, loss of epithelial cohesion and the transcription factor IRF4 (118). The degree of this replacement migration. These phenotypic changes are reminiscent of other strongly depends on the degree of initial macrophage serosal surfaces that undergo epithelial-to-mesenchymal transition disappearance (50, 62). Taken together, this suggests a dual role (EMT). In analogy, this reaction has been termed mesothelial-to- of infiltrating monocytes in wound repair: they act as precursors of mesenchymal transition (MMT) (131, 132). On a molecular level, bone-marrow derived macrophages that are directly needed in MMT involves the downregulation of epithelial junctional wound repair, and they can serve to replace the GATA6+ proteins such as E-cadherin and the upregulation of resident macrophages. mesenchymal marker a-smooth muscle actin (a-SMA) and Neutrophils and monocytes are not only recruited into the production of ECM (133, 134). These changes correlate with an abdominal wall (45) but they are also recruited into the upregulation of transcription factors canonically associated with Frontiers in Immunology | www.frontiersin.org 8 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation EMT such as SNAI1, SNAI2, ZEB1, ZEB2, Twist1 (38) and can be deposition led to adhesion formation in many different injury induced by exposing mesothelial cells to TGF-b1, hepatocyte models and different species (96) and became generally accepted growth factor, platelet derived growth factor and IL-1b (38). We as the “classical concept of adhesion formation” (96). We have will next discuss how mesothelial repair can become defective. seen that an exuberant inflammatory response induced by peritoneal injury or infectionpromotesanincreased procoagulatory and antifibrinolytic reaction. On a molecular level, inflammatory mediators increase the expression of tissue POST-SURGICAL ADHESIONS factor (TF) and PAI-1 and decreases the expression of tPA in Surgeries within body cavities such as the abdominal cavity are often mesothelial cells resulting in increased fibrin deposition (96). This lifesaving procedures. Following surgical trauma, mesothelial repair mechanism was confirmed in peritoneal biopsies of inflamed can lead to a restitution of serosal surfaces at integrum. However, in peritoneum of humans that underwent surgery. The reduction some patients, the healing process is disrupted, leading to a fibrotic of fibrinolytic activity during inflammation was mediated by PAI- complication called post-surgical adhesions. Adhesions are fibrous 1(96). Importantly, a prospective study in humans showed that bridges of various thickness and length containing blood vessels and PAI-1 concentrations in peritoneal fluid were correlated with the nerve tissue (135, 136). Adhesions can also be caused by infection occurrence of adhesions after 8 days (96). Strategies to prevent but today, surgeries comprise by far the most common cause of postoperative adhesion target the dysregulated fibrin clot mesothelial injury leading to adhesions (96, 135). In the peritoneal deposition by either inhibiting coagulation, increasing cavity, adhesions result in considerable morbidity as they impair the fibrinolytic activity, or reducing inflammation. free movement of organs. These problems include potentially life- Administration of fibrinolytic (tPA) or anticoagulant agents threatening intestinal occlusions, secondary infertility in women, (Heparin) significantly reduced adhesions in different animal and chronic post-operative abdominal pain (96, 137, 138). models (38, 96, 141–144). However, the only study in humans Peritoneal adhesions were described for the first time in 1836 in a that enrolled 102 patients, was unable to confirm this effect. In post-mortem examination of a patient that had died from peritoneal this study 5000 I.U. of heparin were diluted in saline and used to tuberculosis (139). It was then suggested in 1849 that these wash the peritoneal cavity. The patients in this study then abnormal structures originate from lymphatic vessels that turn underwent a second operation (laparoscopy) 12 days after the into fibrinous adhesions (135, 139, 140). Despite tremendous first, to obtain adhesion scores (145). The heparin dose that was scientific advances since 1849 including the execution of many effectively administered in these patients was not reported but it clinical and experimental studies on adhesions, understanding of must have been extremely low as most heparin containing lavage their pathogenesis has not evolved enough to develop effective solution was removed after a few minutes. Based on titration therapies. To date, few research and development resources are studies in an animal models, the threshold dose for significant dedicated towards resolving this significant health problem. The anti-adhesion effect was (without occurrence of bleeding after process of adhesion formation largely depends on the same two days) 7.5x10 U/kg/day, which is equivalent to 5250 I.U/day mechanisms as “normal” mesothelial repair: Healing is initiated for a person weighting 70kg (142). Therefore, low heparin dose by damage recognition, inflammation, and coagulation. These steps or low heparin concentration may be the reason no effect on lead to the recruitment of leukocytes and the deposition of fibrin. adhesion formation was observed in the Jansen study. Further Then, the stable fibrin matrix (fibrin clot) is infiltrated by studies would be necessary. However, whether the current myofibroblasts that start to deposit ECM proteins such as collagen. evidence justifies a human trial testing high dose heparin in The problem with adhesions is that wound healing occurs at major abdominal surgery remains to be discussed. sites it should not (96). The classical paradigm of adhesion Others have tried to use anti-inflammatory agents to restore formation states that if serosal surfaces cannot re-establish mesothelial fibrinolytic activity. Cyclo-oxygenase inhibitors and homeostatic fibrinolysis soon after injury, excessive amounts of steroids were tested in animal models (146–148)withno fibrin are deposited. We have discussed that macrophage resounding success. Experimental animal models demonstrate aggregation accompanies fibrin deposition after sterile injury at potent prevention of postoperative adhesion following serosal surfaces (3). Macrophage-fibrin deposits serve as the intraperitoneal application of HMG-CoA reductase inhibitors basic scaffold for tissue repair. Clots that span the space (statins) (149). HMG-CoA reductase inhibitors stimulate between opposing serosal surfaces are dangerous because they fibrinolytic activity in human peritoneal mesothelial cell cultures can be converted into scars that permanently link these surfaces (150) and exert anti-inflammatory functions (151). In experimental called adhesions. We will now discuss the events taking place in animal models amelioration of adhesion after administration of more detail and highlight the respective therapeutic intraperitoneal acylated ghrelin, a 28-amino acid gastric peptide considerations for each (Figure 4). with anti-fibrotic and anti-inflammatory properties, was demonstrated. The adhesion prevention by ghrelin application Macrophage Aggregation and Fibrin was modulated via blockage of the TGF-b signaling pathway (152). Clot Formation Extending on this classical paradigm we have recently The idea that mesothelial loss of baseline fibrinolytic activity after proposed yet another factor in adhesion formation: that of surgical trauma may cause adhesions has been the subject of peritoneal macrophage aggregation. We showed that in various animal models (96). Interestingly, hypofibrinolytic fibrin macrophage aggregation to focal peritoneal injuries was tightly Frontiers in Immunology | www.frontiersin.org 9 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation AB C DE F FIGURE 4 | Post-surgical adhesion formation. (A) Overview of the peritoneal cavity before surgery. (B) Non-focal mesothelial injury such as major abdominal surgery leads to the uncontrolled aggregation of peritoneal macrophages serving as the nidus for the (C) subsequent Fibrin clot deposition. Inflammation and Coagulation inter-dependently promote the deposition of fibrin (see text). (D) Overview during or after adhesion formation. The abdominal organs (e.g., intestine) are now attached to the abdominal wall at anatomic (mesentery) and non-anatomic (adhesion) locations. (E) Mesothelial to mesenchymal transition gives rise to myofibroblasts that migrate into the wound and into the fibrin clot where they start to deposit extracellular matrix (ECM) such as collagen. (F) Adhesion formation is completed when the scar tissue is covered with mesothelium. The lesion may become fully perfused and pain-sensitive by ingrowth of blood vessels and nerves. regulated with just enough cells aggregated to seal the defect. aggregation of macrophages was largely independent of fibrin However, in response to large peritoneal injuries, such as crosslinking and macrophages showed the ability to aggregate ex abdominal surgeries, the aggregation of these macrophages was vivo without addition of fibrin (44). Taken together, mesothelial dysregulated, resulting in the formation of large super aggregates inflammation, macrophage aggregation, and coagulation, can act that started to join mesothelial surfaces. We found that this cooperatively but do not necessarily depend on each other. The process was dependent on scavenger receptors MARCO and relative contribution of each of these three processes likely depends MSR1. Depleting peritoneal cavity macrophages or inhibiting on the type (sterile, microbial, combined) and strength of the insult their aggregation significantly reduced the amount and severity as well as on local shear (3), which in turn is largely dependent on of adhesions in a mouse model. We therefore propose an adaptation patient movement and post-surgical intestinal paralysis (Figure 5). of the classical paradigm to include peritoneal macrophage Fibrotic Conversion aggregation as an additional event (Figure 4). Before we discuss the later events in adhesion formation such as fibrotic conversion Alpha smooth muscle actin (a-SMA) positive myofibroblasts are considered the main collagen-producing cell in wound healing and remodeling, we would like to note that the early process of adhesion formation such as mesothelial inflammation (chapter 3), and many fibrotic diseases (155–157). The question of the origin of a-SMA positive myofibroblasts in adhesions has been a matter macrophage aggregation (chapter 3) and coagulation (chapter 4) are tightly linked. We have discussed how inflammation directly affects of debate. Myofibroblasts in adhesions were believed to be either derived from the mesothelium or alternatively derived from sub- coagulation. Inversely, coagulation provides a positive feedback to the mesothelium further increasing inflammation. For example, mesothelial cells (126). Recently, Fischer at al. used a genetic fate mapping to permanently and selectively label cells expressing activation of proteinase-activated receptor-2 (PAR2) on mesothelial CreERT tdTomato cells results in increased MIP-2 production and consecutive protein c receptor gene (Procr xRosa26 ). Tamoxifen administration in these reporter mice resulted in the neutrophil infiltration (153). Furthermore, peritoneal cavity macrophages were shown to produce coagulation factors (21, 23, selective and permanent labelling of approximately 50% of all mesothelial cells but not submesothelial cells (127). Using this 154) and Factor V produced by peritoneal macrophages was shown to be essential for the clotting of peritoneal fluid in response to approach, they were able to show that the majority of platelet derived growth factor receptor a positive (PDGFRa+) bacteria (21). Inversely, they showed that macrophage aggregation (disappearance) was partially dependent on coagulation factors. In myofibroblasts in adhesions were of mesothelial origin (127). This is in line with older studies that relied on non-genetic other models such as laser-induced sterile mesothelial injury, the Frontiers in Immunology | www.frontiersin.org 10 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation lineage tracing methods such as cell tracker dyes or lineage weeks post-surgery the nerve fibers were traversing the whole markers to infer on source of myofibroblasts in adhesions (122, adhesion from coecum to abdominal wall (161). Human 158, 159). Taken together these data suggest that mesothelial to peritoneal adhesion specimens collected during surgery from mesenchymal transition (MMT) is the major source of 25 patients contained invariably sensory nerves (162). The myofibroblasts in adhesion pathogenesis. On a molecular level, sensory innervation could partially explain the chronic pain a MMT in adhesion formation relies on the same pathways as lot of patients with adhesions experience. Animal studies mesothelial repair (159). In fact, administration of TGF-b blocking revealed blood vessels in adhesions already 6 hours after injury peptide P144 resulted in a significant reduction of adhesions in an (163). This process of remodeling from connective tissue to fully experimental mouse model (159). This was associated with a innervated and vasculated tissue might be modulated. reduced expression of MMT markers such as Snail, a-SMA and The nerve fibers in adhesions were often associated with blood Collagen I in P144 treated mice (159). In addition, the exposure of vessels indicating angiogenesis could play a key part in regulating mesothelial cells to cyclic mechanical forces was shown to increase ingrowth of nerves into adhesions (162, 164). Local production of MMT in experimental murine and human models. Biomechanical VEGF by mesothelial cells appears to play a central role in the induction of MMT cooperates with biochemical signals such as process leading to peritoneal angiogenesis (165). In different TGF-b and seemed to be regulated by caveolin-1, a plasma murine models, postsurgical adhesion formation was reduced by membrane mechanotransducer (160). Interestingly, MMT-cells inhibition of VEGF suggesting adhesion formation is in adhesions also express many markers that are found in the angiogenesis-dependent (166, 167). In a human study including mesothelium during embryonic development but not within the adhesions samples from patients years after first surgery, adult mesothelium. These markers, including Mesothelin (MSLN), adhesions expressing VEGF A and its receptor showed Uroplakin-1B and Wilms-tumor 1 (WT1), were upregulated in significantly higher numbers of immature vessels suggesting adhesions indicating that adult mesothelial cells can repurpose ongoing angiogenesis in mature adhesions (163). In addition to aspects of fetal development (158). Depletion of mesothelial cells angiogenesis, modulation of the ECM by matrix metallo- results in a complete reduction of adhesions (127). However, proteinases (MMPs) takes place. MMPs are proteolytic enzymes totally depleting the peritoneal cavity of potential myofibroblasts involved in degradation of ECM, their activity is opposed by may compromise wound healing too much for use in the clinic. tissue-derived inhibitors of MMPs (TIMPs) (168). The expression Strategies that inhibit MMT in adhesions but leave mesothelial of both VEGF and MMPs is upregulated during MMT (131). In a repair intact need to be developed. human study, peritoneal samples were collected during initial laparoscopy and during a second-look laparoscopy 48 hours later. Remodeling Patients with pelvic adhesions exhibited significantly lower After fibrotic conversion, adhesions are considered irreversible amounts of MMP-9 concentrations and significantly higher and redundant scar bands. Furthermore, animal, and human MMP-9/TIMP-1 ratios when compared with controls (169). In studies demonstrated the ingrowth of nerves and vessels into peritoneal fluid of patients with excessive adhesions, higher TIMP- adhesions. In a murine model, nerve fibers in abdominal 1 levels could be demonstrated compared with those of patients adhesions were detected already two weeks after surgery and 4 without adhesions (170). Mice treated with instillation of FIGURE 5 | Factors influencing adhesion formation. Proposed concept of early local determinants that influence the binary outcome of adhesion formation and may be exploited therapeutically. tPA, tissue plasminogen activator; uPA, urokinase-type plasminogen activator; PAI, Plasminogen activator inhibitor. Frontiers in Immunology | www.frontiersin.org 11 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation adenovirus vector encoding mutant MMP-9 gene at the time of understanding of wound healing at serosal surfaces which in peritoneal injury showed a reduced of severity of de novo turn might have great impact on the way we think of any of the adhesions (168). It remains unclear whether adhesion keep their above-mentioned pathologies. capacity for remodeling and thus have the potential to spontaneously resolve, or whether adhesions are an irreversibly fixed pathology once they have developed. AUTHOR CONTRIBUTIONS JZ and SNZ wrote the article. DS gave substantial input to the CONCLUDING REMARKS concepts underlying this review and revised and approved the article. We have discussed how mesothelial repair works for small injuries and how it can go wrong and result in peritoneal adhesions. It is important to note that mesothelial repair plays an important role in other fibrotic disorders in proximity to FUNDING serosal surfaces. These disorders include the fibrotic thickening JZ was supported by a Swiss National Science Foundation of the peritoneum (peritoneal fibrosis or encapsulating (SNSF) research fellowship (P1BEP3_181641). peritoneal sclerosis) and the pleura (pleural fibrosis). A number of studies have also demonstrated that mesothelial cells play also an important role in fibrotic diseases of the liver (128) and lung (38, 171–173). This is not surprising, since both, ACKNOWLEDGMENTS the liver and the lungs are covered with visceral mesothelium. 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Sandoval P, Jimenez-Heffernan JA, Guerra-Azcona G, Perez-Lozano ML, any commercial or financial relationships that could be construed as a potential Rynne-Vidal A, Albar-Vizcaino P, et al. Mesothelial-to-Mesenchymal conflict of interest. Transition in the Pathogenesis of Post-Surgical Peritoneal Adhesions. J Pathol (2016) 239(1):48–59. doi: 10.1002/path.4695 Copyright © 2021 Zwicky, Stroka and Zindel. This is an open-access article 160. Strippoli R, Sandoval P, Moreno-Vicente R, Rossi L, Battistelli C, Terri M, distributed under the terms of the Creative Commons Attribution License et al. Caveolin1 and YAP Drive Mechanically Induced Mesothelial to (CC BY). The use, distribution or reproduction in other forums is permitted, Mesenchymal Transition and Fibrosis. Cell Death Dis (2020) 11 provided the original author(s) and the copyright owner(s) are credited and that the (8):647fpage. doi: 10.1038/s41419-020-02822-1 original publication in this journal is cited, in accordance with accepted academic 161. Sulaiman H, Gabella G, Davis C, Mutsaers SE, Boulos P, Laurent GJ, et al. practice. No use, distribution or reproduction is permitted which does not comply Growth of Nerve Fibres Into Murine Peritoneal Adhesions. J Pathol (2000) with these terms. Frontiers in Immunology | www.frontiersin.org 16 May 2021 | Volume 12 | Article 684967 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Immunology Pubmed Central

Sterile Injury Repair and Adhesion Formation at Serosal Surfaces

Frontiers in Immunology , Volume 12 – May 14, 2021

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Copyright © 2021 Zwicky, Stroka and Zindel
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10.3389/fimmu.2021.684967
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Abstract

REVIEW published: 14 May 2021 doi: 10.3389/fimmu.2021.684967 Sterile Injury Repair and Adhesion Formation at Serosal Surfaces Simone N. Zwicky, Deborah Stroka and Joel Zindel Department of Visceral Surgery and Medicine, Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland Most multicellular organisms have a major body cavity containing vital organs. This cavity is lined by a mucosa-like serosal surface and filled with serous fluid which suspends many immune cells. Injuries affecting the major body cavity are potentially life-threatening. Here we summarize evidence that unique damage detection and repair mechanisms have evolved to ensure immediate and swift repair of injuries at serosal surfaces. Furthermore, thousands of patients undergo surgery within the abdominal and thoracic cavities each day. While these surgeries are potentially lifesaving, some patients will suffer complications due to inappropriate scar formation when wound healing at serosal surfaces defects. These scars called adhesions cause profound challenges for health care systems and patients. Therefore, reviewing the mechanisms of wound repair at serosal surfaces is of Edited by: clinical importance. Serosal surfaces will be introduced with a short embryological and Javier Leceta, Complutense University of Madrid, microanatomical perspective followed by a discussion of the mechanisms of damage Spain recognition and initiation of sterile inflammation at serosal surfaces. Distinct immune cells Reviewed by: populations are free floating within the coelomic (peritoneal) cavity and contribute towards Pilar Sandoval, damage recognition and initiation of wound repair. We will highlight the emerging role of Severo Ochoa Molecular Biology Center (CSIC-UAM), Spain resident cavity GATA6+ macrophages in repairing serosal injuries and compare serosal Andrea Doni, (mesothelial) injuries with injuries to the blood vessel walls. This allows to draw some Humanitas Research Hospital, Italy parallels such as the critical role of the mesothelium in regulating fibrin deposition and how *Correspondence: Joel Zindel peritoneal macrophages can aggregate in a platelet-like fashion in response to sterile joel.zindel@dbmr.unibe.ch injury. Then, we discuss how serosal wound healing can go wrong, causing adhesions. The current pathogenetic understanding of and potential future therapeutic avenues Specialty section: against adhesions are discussed. This article was submitted to Mucosal Immunity, Keywords: peritoneal adhesions, peritoneum, sterile injury, mesothelium, post-surgical adhesions a section of the journal Frontiers in Immunology Received: 24 March 2021 Accepted: 23 April 2021 DEVELOPMENT AND MICROANATOMY OF THE COELOM Published: 14 May 2021 AND MESOTHELIUM Citation: Zwicky SN, Stroka D and Zindel J During embryology, atthe end ofthe third week, thelateralplatemesoderm isdivided intotwo layers:the (2021) Sterile Injury Repair and somatic and splanchnic mesoderm layer (1). These two layers form a cleft that becomes a cavity as the Adhesion Formation embryo undergoes a cranio-caudal and latero-lateral folding event in week four (1). This cavity is called at Serosal Surfaces. the intraembryonic coelom and contains vital organs such as the heart, the lungs, the liver, and the Front. Immunol. 12:684967. doi: 10.3389/fimmu.2021.684967 intestines. In mammals, the mesodermal lining of the coelom differentiates into a serous epithelium-like Frontiers in Immunology | www.frontiersin.org 1 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation membrane called mesothelium (2). The somatic mesoderm gives filled cavities from surrounding tissues (Figure 2). Together with rise to the parietal layer of the mesothelium which lines the body the associated sub-mesothelial connective tissue the serosa is also wall, and the splanchnic mesoderm gives rise to the visceral layer of called peritoneum, pleura, and pericardium in the peritoneal the mesothelium which lines the surfaces of organs. The intra- (abdominal), pleural and pericardial cavities, respectively. In coelomic organs stay connected to the body wall by elongations practice, the terms mesothelium, serosa, and peritoneum (or referred to as mesenteries which contain blood vessels, lymphatics, pleura or pericardium) are often used interchangeably. and nerves (1)(Figure 1). The peritoneum is less than 25 µm thick in the mouse (3) and Later, the coelomic cavity is further subdivided resulting in about 50-100 µm thick in humans (4, 5). Therefore, as we discuss three embryologically related but anatomically distinct anatomical injury at serosal surfaces, it is important to note that the compartments: the pericardial cavity, the pleural cavities, and the mesothelium will rarely be injured in an isolated fashion. In peritoneal (abdominal) cavity. All of these contain vital organs fact, serosal injuries will often compromise the tissues that are such as heart, lung, and abdominal organs (1). covered by the mesothelium as well. These underlying tissues can The serous membrane that covers the walls of all coelomic be vastly different such as: cavities as well as the borders of all organs contained within them is - smooth-muscular wall of the intestines, urinary bladder, uterus, also called the serosa and is comprised of a flat monolayer of mesothelial cells. The serosal linings ensure friction-less movement - parenchymal tissue of heart, lung, liver, gallbladder, spleen of organs and establish a water-tight barrier separating the fluid- (only mouse), ovaries, AB C FIGURE 1 | Development of the intra-embryonic coelomic cavity. (A) Schematic cross section human embryo of 3 weeks age. The mesoderm shows a somatic (dorsal) and splanchnic (ventral) aspect. (B) Cranio-caudal and latero-lateral folding in week 4. (C) After closure of the anterior abdominal wall the intra-embryonic coelomic cavity is formed. Organs (e.g. gut) are suspended by dorsal and sometimes ventral (not shown) mesenteries carrying blood vessels and nerves. FIGURE 2 | Microanatomy of mesothelial surfaces. (A, B) Cross sections of mouse abdominal wall stained with Hematoxylin & Eosin (A) and Masson’s trichrome staining (B). Scale bars: 50 µm. (C) Illustration of the structures shown in (A, B). (D) Top view on mesothelial surface stained with anti-podoplanin antibody. Frontiers in Immunology | www.frontiersin.org 2 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation - fat tissue of the omentum, macrophages (SPM) are monocyte-derived, constantly replenished and can be recruited within hours in significant amounts (14). At - striated muscle, fascia, and bone of the thoracoabdominal wall baseline, they account for about 5% of all immune cells or about and diaphragm, 10% of all macrophages (14, 18). The majority (90%) of peritoneal - and connective tissues such as that of the pericardium. macrophages belong to a distinct tissue-residential macrophage population. Since these resident cells are slightly larger than their Any experimental model system that studies serosal wound monocyte-derived sisters, they are also referred to as large peritoneal repair, may invoke some underlying tissue-specificwound repair macrophages (14). The large peritoneal macrophages (LPM) are a mechanisms. This review is targeted at serosa specific mechanisms, self-renewing population characterized by the expression of CD102 but weask thereadertobearin mindthatwe use thegeneralization (Icam2), high levels of F4/80 and the transcription factor GATA6 “at serosal surfaces” inductively; some of the mechanisms discussed (19–22). GATA6+ LPM seem to be well conserved when comparing here mayapply onlyto specific locations within the coelomic cavity. the different coelomic cavities of mice and human (23–25). Canonically, these GATA6+ cavity macrophages are thought to clear bacteria by phagocytosis (14, 26) and also by inducing intra- CELLS SUSPENDED IN abdominal formation offibrin clots that immobilize bacteria (21). In COELOMIC CAVITIES primordial species such as the purple sea urchin (Strongylocentrotus purpuratus), coelomocytes are also crucial for tissue repair, in The coelomic cavities are filled with fluid that suspend millions addition to clearing toxins and pathogens (27–30). The of cells also referred to as coelomocytes. The coelomocyte importance of GATA6+ cavity macrophages in damage composition of mice and humans has been reviewed elsewhere recognition and tissue repair will be discussed in detail. (6). Briefly, the human peritoneal cavity suspends a total of 10 leukocytes in 5-100ml of peritoneal fluid (6, 7). In mice, the number of peritoneal leukocytes varies between strains from 3 to 6 6 5x10 cells (8). The pleuropericardial cavities contain 0.3-1x10 DAMAGE RECOGNITION leukocytes per mouse (6, 9, 10). Most leukocytes in the peritoneal AND INFLAMMATION cavity are lymphocytes (10-60%) and macrophages (40-60%) (8, 11–16). In addition, the peritoneal cavity contains dendritic cells Wound repair at large starts with inflammation. Inflammation is (2 – 6%) (12, 17), mast cells, eosinophils, neutrophils (0-31%), induced when a significant deviation from homeostasis is detected. innate lymphoid cells (ILCs) including natural killer cells and According to the current paradigm, such a deviation could be the mesothelial cells (14, 16)(Figure 3). presence of microbes (infection) or damaged tissue (injury). The In terms of wound healing, the role of peritoneal macrophages is innate immune system has developed an effective arsenal of best established. Macrophages make up 40-60% of all coelomocytes surveillance cells that constantlyprobe theirmicroenvironment in both mice and humans. Two major subpopulations of peritoneal for deviations from homeostasis. On a molecular level, deviation macrophages have been described (14). The small peritoneal from homeostasis is defined by the occurrence of pre-specified AB FIGURE 3 | Cells in a mouse coelomic cavity. (A, B) Peritoneal cavity lavage of healthy C57Bl/6 mice. Dimensionality reduction dimension 1 (umap1) and 2 (umap2) of myeloid lineage markers (mass cytometry) are plotted on x- and y-axis, respectively. Dots (cells) are colored by cluster (A) or marker (B). Data with kindly permission from M. Dosch and G. Beldi. Frontiers in Immunology | www.frontiersin.org 3 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation Mesothelial Damage Is First Recognized molecular patterns. Immunostimulatory molecular patterns that by Cavity Macrophages induce inflammation in case of sterile injury, i.e., in the absence of pathogens and their products, have been termed damage associated Recent advances in intravital microscopy have allowed to characterize the sequence of cells recruited to mesothelial molecular patterns (DAMPs). DAMPs have been extensively reviewed elsewhere (31). In brief, DAMPs comprise different injuries. By using resonant-scanners, multi-photon excitation, and extremely sensitive hybrid detection systems it became molecules that are not normally present outside of cells such as double stranded DNA, nuclear proteins, mitochondrial DNA, possible to image the peritoneal cavity through the intact abdominal wall under real-life conditions (21, 44). Second, mitochondrial proteins, and molecules with high cytosolic concentrations such as ATP or K Ions.In addition,damaged multi-photon imaging allows the use of near-infrared microscopy lasers to induce focal thermal injuries during cells may induce the production and release of additional DAMPs (iDAMPs) such as heat shock proteins, defensins, galactins and intravital microscopy with high precision (44–46). By combining intravital microscopy of the abdominal cavity with peritoneal laser interleukin 1 (IL-1). Furthermore, if proteins that are constitutively present in the extracellular space such as hyaluronan, biglycan, injuries, we were able to image cellular recruitment to mesothelial injuries. Surprisingly, the first GATA6+ cavity macrophages heperansulfate and other extracellular matrix (ECM) components are modified by injuries, they can also become DAMPs. Under attached at the injuries within only a few seconds and the macrophages completely covered the lesions after 15 minutes of homeostatic conditions, the serosal surfaces are covered with glycoconjugates such as sialomucins, hyaluronic acid, and imaging (44). The recruitment of cavity macrophages to mesothelial injury was significantly faster than that of glycoproteins like fibronectin (32–35). These molecules contain large anionic sites that cover the serosal surfaces with a negatively neutrophils, which needed much longer (> 40 minutes) (44). Cavity macrophages were present in the peritoneal fluid in vast charged coat—also referred to as the glycocalyx—that may help to repulse invading microbes (32) and ensure friction-less movement numbers and traversed the peritoneal cavity in a seemingly random fashion within respiration-dependent movement of of intra-coelomic organs (35). Thelossofthisnegativelycharged coating due to serosal injury, may serve as mesothelium-specific peritoneal cavity content (44). The observations that these cells seemed to rely on passive transportation by peritoneal fluid, and DAMP or “touch me signal” (36). Molecules that allow eukaryotic cells to detect the presence of that they—upon contact with cell already adhering to the injury— were forming stable cell-cell aggregates were very reminiscent of DAMPs have been termed pattern recognition receptors (PRR). The expression of PRR such as toll-like receptors 1 through 6 the platelet aggregation that took place when a nearby blood vessel wall was damaged using laser injury. We concluded that cavity (TLR-1-6), nucleotide-binding oligomerization domain (Nod)-1 and Nod-2 and advanced glycation end product (AGE) macrophages randomly “patrol” the serosal surfaces in a platelet- like fashion and rapidly form aggregates in response to DAMPs. receptors, has been demonstrated for murine and human mesothelial cells (37). Upon activation, mesothelial cells release This is consistent with a previous electron microscopy study by Haney showing that peritoneal macrophages invariably detected cytokines and inflammatory mediators such as chemokine (C–C motif) ligand 2 (CCL2), CCL5, (C–X–C motif) ligand 8 and migrated to injuries of the peritoneal membrane (47). In addition, Wang and Kubes showed that cavity macrophages were (CXCL8), and nitric oxide (38, 39). Furthermore, mesothelial cells upregulate adhesion molecules that presumably facilitate able to detect mesothelial injuries of the liver capsule and migrated to the injured liver (36). On a molecular level, this interaction the migration of inflammatory leukocytes across and along serosal surfaces. These include intercellular adhesion molecule- occurred independent of integrins or selectins, instead peritoneal macrophages relied on different receptor molecules such as 1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), E-cadherin, N-cadherin, CD29 and CD44 (38, 40–42). It is macrophage receptor with collagenous structure (MARCO), Macrophage scavenger receptor 1 (MSR1), CD44, and important to note that cellular adhesion molecules expressed by mesothelial cells play a dual role in serosal wound repair. purinergic receptor P2X7. The respective DAMPs recognized by CD44 and P2X7 are hyaluronan and ATP respectively (36). The While an initial upregulation may facilitate leukocyte recruitment, these molecules, especially E-cadherin, are ligands that mediate MARCO and Msr1 dependent macrophage aggregation are yet to be identified (44). downregulated later during serosal wound repair. The latter is associated with loss of mesothelial cohesion enabling the The function of peritoneal macrophages in sterile injury is multi- facetted. Current models indicate that ligation of DAMPs to PRR on mesothelium to switch to a more mesenchymal program, a process that we will discuss in detail below. In addition, macrophages leads to their inflammatory polarization—also referred to as M1 polarization. This activation would result in the mesothelial cells modulate inflammation by synthesis and release of hyaluronan (43), which is able to sequester free production of pro-inflammatory cytokines such as tumor necrosis factor (TNF) and IL-1 (31, 48). However, peritoneal macrophages radicals and initiate tissue repair responses (38). In vivo, the initiation of inflammation at serosal surfaces does recruited to sterile liver injury were shown to skew their phenotype towards alternative or repair polarization—also referred to as M2 not rely on mesothelial cells alone but on a series of events. These comprise specialized cellular and humoral immune mechanisms macrophages—increasing their expression of CD273, CD206 and Arginase 1 (36). Interestingly, Uderhardt et al. recently investigated such as leukocyte recruitment, complement activation and production of natural antibodies. In the rest of this chapter, we the resident tissue macrophages of the muscular abdominal wall. The abdominal wall macrophages are distinct from the peritoneal will discuss these elements one by one. Frontiers in Immunology | www.frontiersin.org 4 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation cavity macrophages suspended in the peritoneal cavity. They indicate that the aggregation of cavity macrophages in response to a proposed that abdominal wall macrophages can extend their strong stimulus, such as peritonitis, causes inflammation. However, pseudopods toward local injury sites within a radius of 100- in the case of smaller insults such as focal injuries or localized 150µm. In their study, resident tissue macrophages were able to microbial challenges, MDR may compartmentalize the insult, in completely enclose lesions if their size was below a certain threshold analogy to the cloaking mechanism described for macrophages of (microlesions). This—as the authors termed it—cloaking the muscular abdominal wall (45). Along those lines, complete mechanism, was able to block scouting neutrophils from MDR could be interpreted as a threshold above which all interacting with DAMPs and thus prevented subsequent macrophages have been “used up” indicating that the attempt at neutrophil driven inflammation and tissue destruction (45). The cloaking the insult has failed, which in turn results in inflammation. cloaking mechanism was described for tissue resident macrophages Either way, it would be important to study the largely unknown in the muscular abdominal wall, i.e., on the far side of the (intracellular) changes in macrophages undergoing a disappearance mesothelium with respect to the coelomic cavity. It needs to be reaction in sterile and microbial models. determined whether scavenger receptor mediated macrophage Dendritic Cells and Mast Cells aggregation on the coelomic site of the mesothelium causes inflammation or whether aggregation of cavity macrophages The peritoneal cavity harbors CD11c dendritic cells as well as cKit mast cells both of which are canonical initiators of serves to contain injuries and is therefore—in essence—anti- inflammatory. Ultimately, aggregation of peritoneal cavity inflammation. Their role as antigen presenting cells and inducers of inflammation in response to bacterial infection is macrophages in response to mesothelial injuries was shown to improve tissue repair (36, 44, 47). well documented. In fact, CD11c+ dendritic cells are required for survival in murine polymicrobial peritoneal sepsis (66). In Cavity Macrophage addition to pathogen-derived ligands for PRR, several DAMPs have been shown to interact with dendritic cells and dramatically Disappearance Reaction affect their function (67, 68). Interestingly, the response of Aggregation of peritoneal macrophages causes their number in dendritic cells to DAMPs is not always clear-cut, with different the peritoneal lavage to drop. The decrease in their number was responses depending on dendritic cell subtypes and location (67). correlated with the injury size (44). With larger injuries of the For example, activation of dendritic cells in sterile liver injury mesothelium, such as a surgical laparotomy, the number of leads to the secretion of anti-inflammatory cytokines such as IL- GATA6+ cavity macrophages in the peritoneal lavage was 10 and TGF-b (67) while similar injury models of kidney and gut reduced to zero (44). In other words, these cells disappeared may lead to a pro-inflammatory response and secretion of IL-6, from the peritoneal fluid (lavage). However, this was not the first IL-12 and TNF-a (67, 69). So far, the response of peritoneal time, the sudden absence of macrophages was observed. In fact, dendritic cells to serosal injury is not well understood and over half a century ago, Nelson and Boyden described a sharp requires further studies. Mast cells have traditionally been decline of macrophage count in peritoneal exudates in response studied in the context off helminthic infections and Ig-E to a hypersensitivity reaction to tuberculin in Bacille Calmette- mediated reactions. It becomes clear, that mast cell Guérin (BCG)-vaccinated guinea pigs. They termed this the degranulation is also an important modulator of wound “macrophage disappearance reaction” (MDR) (49). Since then, healing of skin wounds (70) and lesions in the gastrointestinal various insults (sterile and microbial) to the peritoneal tract (71–73). Poerwosusanta et al. investigated the role of mast compartment have been found to induce the MDR (Table 1). cell degranulation in mesothelial injury. Mesothelial injury was These studies indicate that the MDR is not a specific reaction but carried out by performing laparoscopic surgeries in rats at arguably follows any inflammatory challenge to the peritoneal different intra-abdominal inflation pressures (74). They showed compartment. While some reports indicate that peritoneal that an increased intraabdominal pressure—and presumably macrophages can leave the peritoneal cavity through the draining increased stress to the mesothelium—led to an increased lymphatics (52, 60, 64), most of the more recent reports suggest that number of mast cells that infiltrated the mesothelium. This peritoneal macrophages have the tendency to adhere to each other was correlated with increased mast cell degranulation. This (aggregate) as well as to the mesothelium in response to challenge increased mast cell count is consistent with findings from skin (Table 1). Therefore, the loss of dispersion and cellular aggregation injury models and is due to chemokine-dependent mast cell are a commonality among the different models of MDR. The MDR immigration rather than local proliferation. More detailed correlates with increased inflammatory cytokine levels in the investigation, e.g. based on intravital microscopy, could help to peritoneal fluid and the influx of pro-inflammatory leukocytes elucidate whether mast cells are recruited to mesothelial injuries such as monocytes, eosinophils, and neutrophils into the by blood or directly from the peritoneal cavity. peritoneal compartment (21, 59). Cailhier et al. used CD11b driven diphtheria toxin receptor and low dose intraperitoneal Humoral Pattern Recognition Molecules injections of diphtheria toxin to selectively deplete resident and Natural Antibodies peritoneal macrophages. In an experimental peritonitis model, this resulted in a significant decrease of inflammation (infiltration The fluid of the pleural and peritoneal cavity in mice and humans not only contains cells but also large amounts of proteins of the of neutrophils) that could be restored by the adoptive transfer of resident, non-transgenic, peritoneal macrophages (65). These data coagulation system and complement system as well as large Frontiers in Immunology | www.frontiersin.org 5 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation TABLE 1 | Macrophage disappearance reaction (MDR). Studies describing MDR from 1963 until now. MDR Trigger (dose) Time between trigger and Postulated fate of disappeared Molecular mechanism Reference complete MDR macrophages Sterile Models Sterile mesothelial injury 3h Form stable cell-cell aggregates that Scavenger receptors, can be blocked with (44) (surgery, laser) cover injury and induce post-surgical Heparin and Poly-(I) adhesions Sterile Brewer’s Thioglycollate 12-72h Macrophage cell death Not demonstrated (50–52) Antigen, migration inhibitory 1 to 96h Undergo activation during MDR in MDR Inhibited by Heparin, L-Fucose, (53) factor, viruses or tumor cells delayed type hypersensitivity or acute Hyaluronidase inflammatory reaction and then reappear activated to regulate responses toward pathogens or tumor cells. Egg Antigen (10ug), purified 5h Macrophage activation Desensitization suppress MDR, in (54) protein derivate (10ug) sensitized animals normal MDR Tuberculin 2.5 - 6h Not demonstrated MDR completely inhibited by Heparin and (49, 55) Warfarin Ova peptide (50ug) into mice 5h Macrophage adhesion Suppressed in fibrinogen- (56) bearing antigen-primed T cells deficient mice, partially suppressed by thrombin antagonist Thrombin (20 Units) 1h -5h Macrophage adhesion MDR suppressed (56) in fibrinogen-deficient mice. RGES Peptide 48h Macrophage bind the mesothelium Integrin-mediated mechanisms involving (57) overlying draining lymphatics VLA-4 and VLA-5 that can be blocked by RGD (Arg-Gly-Asp peptides) and VLA-4 and VLA-5 blocking antibodies. Microbes or microbial products E. coli (5×10 UV-inactivated) 20d Do not undergo fas-mediated apoptosis No difference in fas-deficient mice. (58) S. aureus 2h (2 × 10 ) Not demonstrated Not demonstrated (59) Lipopolysaccharide 3h (10 µg) Accumulation in the omentum Macrophage interaction with mesothelial (19, 40, 5h (1 µg) cells, mainly of the omentum, was 56) proposed to be a key step in MDR. Partially inhibited by refludan. Zymosan 3-4h (1mg) Form large clots to trap MDR reversed completely with Heparin (21, 51, 4h (0.5mg) microorganisms; adherence with tissue and partially with Hirudin/loss Factor V 60–62) 3d (10 µg) and drained to lymph node Expression/loss of Integrin activation adaptor talin-1 Expression/TF deficiency INF-g (100 U/mL) + LPS 20h Binding to mesothelial cells Monocyte activated by in vitro exposure to (40) (100ng/ml) LPS and INF-Y bound with increased efficiency to mesothelial cells Synthethic Lipopetid 12h Not demonstrated Not demonstrated (59) (Pam3CSK4) Human studies Bacterial peritonitis 1 day Shedding of surface CD206 Depletion of CD206+ LPM at day 1 of SPB (63) Peritonitis with normalization to steady state after resolution of SPB Liver cirrhosis associated Not demonstrated Severity of liver disease and liver cirrhosis (25) events (Bacterial peritonitis, are correlated with reduced numbers of hi hi encephalopathy, death) CrIg macrophages. Human CrIg macrophages were transcriptionally similar to mouse F4/80hi peritoneal macrophages. amounts of natural antibodies (75, 76). In the peritoneal and correlation of the invasiveness of the procedure with the pleural cavities, the complement proteins are produced by amount of complement used, indicating that sterile injury mesothelial cells (75, 77). The complement system is an leads to complement activation in humans (84). Inversely, ancient enzymatic cascade of proteins with the main function humoral molecules that are canonically associated with innate of opsonization and lysis of bacteria (78). The alternative immunity, have been shown to mediate tissue repair and regulate pathway of the complement can be activated by injuries and in fibrosis. For example, pentraxin 3 (PTX3) was shown to reduce the last decade a role of complement activation in wound healing fibrin deposition and fibrosis in several wound models outside of (79) and regeneration (80, 81) as well as morphogenetic and the peritoneal cavity. While to our knowledge, no humoral developmental processes (82, 83) has been suggested. molecule with anti-fibrotic properties has been described in the Furthermore, some clinical studies have evaluated the role of peritoneal cavity, the discovery of such could have great blood complement during major surgery and described a therapeutic potential (85–87). Frontiers in Immunology | www.frontiersin.org 6 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation Like complement factors, natural antibodies are abundant in an abdominal adhesion, a pathology we will discuss in more the coelomic cavity fluids and are primarily thought to combat detail below. See the Perspective Box 1 speculating on parallels microbes by recognizing a wide variety of different microbial between vascular and mesothelial coagulation control. antigen patterns (88). The peritoneal natural antibodies are mainly produced by self-replenishing peritoneal B1 cells in an antigen-independent manner (89). The repertoire of natural antibodies also enables recognition of self-antigens such as RECRUITMENT OF NEUTROPHILS phosphorylcholine, phosphatidylcholine and carbohydrate AND MONOCYTES determinants. Furthermore, natural antibodies have been shown to sense apoptotic cells (88) and electronegative We discovered that the first cells recruited to mesothelial injuries are DAMPs (90). In addition, natural antibodies were able to the peritoneal cavity macrophages (44). These cells reside suspended accelerate wound healing by recruiting additional wound in the peritoneal fluid (19) and are recruited directly from their macrophages (91). Along those lines, Grönwall et al. postulate suspensive state to the mesothelium in case of injury. This that natural IgM antibodies are part of a synapse between an comprises a special case of leukocyte recruitment that is unique to apoptotic cell (that binds IgM) and the phagocyte. This synapse coelomic cavities. The canonical route of leukocyte recruitment is is mediated by complement and complement receptors from the blood stream. The processes of leukocytes leaving the expressed by phagocytes (92). There is increased interest in blood stream have been referred to as leukocyte adhesion cascade studying the role of natural IgM, and IgM-dependent, and trans-endothelial migration. The underlying mechanisms have complement-mediated phagocytosis in several disease models been revisited and reviewed most comprehensively by Nourshargh (93–95). Although available data is limited, it is conceivable that et al. (97, 98). In perfused organs such as muscle or liver, neutrophils complement and natural antibodies of body cavities play an are recruited within 30 minutes to the inflammatory site from the important a role in wound repair at serosal surfaces. bloodstream (46, 99). In the mesothelium, neutrophils are the first cells that arrive after peritoneal macrophages, after about 40-60 minutes (44). In response to focal sterile injuries, neutrophils show an extremely high degree of coordination. While intravascular COAGULATION AND FIBRIN DEPOSITION chemokine gradient seems to be very important for successful directional migration over the initial distance within the vessel It is widely accepted that inflammation of the peritoneal, pleural, (31, 99), different molecules are the most potent chemotactic stimuli or pericardial compartment, is associated with fibrin exudates. once the neutrophils are close to the wound (31, 99, 100). This has After mesothelial injury, the resulting inflammation as well as the been demonstrated for molecules such as N-formyl peptides, ATP, injury itself, lead to the activation of tissue factor pathway and and leukotriene B4 (LTB4) and their respective receptors on the inhibition of the antithrombin-III (AT-III) pathway (96). neutrophils called formyl peptide receptor, P2Y2 receptor and This is followed by the spontaneous cleavage of fibrinogen and LTB4 receptor (31, 46, 99–102). These so-called gradients and cross-linking of fibrin. In addition to inflammation, injury causes autocrine feedback loops (LTB4) have been established for solid the destruction of and consequent leakage from blood vessels of organs such as the liver or muscle. Whether neutrophils rely on the abdominal wall. Extravasation of blood results in the similar mechanism to reach serosal surfaces has not been activation of the canonical coagulation cascade which further demonstrated yet but the technical advances of the last years will enhances the deposition of fibrin (96). In vivo, the deposition of now allow us to address these questions using intravital microscopy. fibrin is limited at serosal surfaces by activated plasmin which Although the canonical role of neutrophils is to clear continuously degrades fibrin. Plasmin levels are regulated by microbes, several reports suggest that they are imperative for plasminogen activators tPA and uPA and their respective timely restoration of tissue architecture after sterile injury by plasminogen activator inhibitors 1 and 2 (PAI 1, PAI 2) (96). clearing necrotic material (99) and producing growth factors Homeostatic mesothelial cells produce large amounts of Plasmin, such as transforming growth factor b (TGF-b) and vascular uPA and tPA and low amounts of PAI1 and PAI2. Therefore, the deposition of fibrin at serosal surfaces is tightly controlled. During inflammation however, the mesothelial production of BOX 1 | Perspective. PAI1 and PAI2 is significantly increased resulting in a mesothelial program switch from fibrinolytic towards anti- We find it intriguing that fibrin clots after surgery are not focally limited to sites of fibrinolytic state, resulting in the visible deposition of fibrinous injury but seem to be formed at distant sites as well while other regions of the peritoneal cavity appear to be protected. During sepsis, spontaneous exudates (96). disseminated intravascular coagulation (DIC) is a major clinical problem. The The formation of a stable fibrin clot—also referred to as fibrin distribution of fibrin clot deposits during DIC is poorly understood. Interestingly, matrix or cross-linked fibrin—serves as the scaffold for the the response of mesothelium and endothelium to inflammation share certain subsequent wound repair (granulation) tissue. This includes similarities and both lead to the spontaneous formation of disseminated fibrin clots. Studying the analogy between these two pathologies, that both involve an the infiltration of leukocytes and mesenchymal precursors and epithelial-like monolayer of mesodermal origin, may lead to the identification of results in the deposition of ECM, ingrowth of vessels and nerves common patterns and molecules governing the respective phenomena of DIC and and finally, the re-mesothelialization of the injured serosa. If peritoneal adhesions. these fibrin clots grow too large, they can be the starting point for Frontiers in Immunology | www.frontiersin.org 7 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation endothelial growth factor (VEGF) (103–105). Furthermore, once peritoneal cavity fluid. In fact, within a few hours after neutrophils have cleared all necrotic tissue, they starve each other of extensive mesothelial injury (surgery), large numbers of Gr1+ DAMPs. This leads to neutrophils becoming apoptotic and cleared cells (neutrophils) and monocyte derived macrophages were by macrophages, which is another critical step for tissue repair as it recruited into the peritoneal cavity (119). This is consistent enables macrophages within the wound to switch from an with observations in humans undergoing surgery where inflammatory to an anti-inflammatory, pro-resolution program billions of neutrophils and monocytes can be isolated from the (99, 103, 106). In this environment, pro-resolution macrophages peritoneal fluid. This recruitment was proposed to be driven by and neutrophils start to produce factors that inhibit the recruitment chemokines such as MCP-1 and CXCL1 that were released into of additional neutrophils and enhance pro-resolution properties of the peritoneal cavity by mesothelial cells in response to injury macrophages. These factors include lipoxin A4, resolvins, protectins (119, 120). It is not known what molecules and mechanisms (107), phosphatidylserine containing microvesicles shed by govern the migration of leukocytes from the interstitium, into the neutrophils, chemokine sequestration through CCR5 modulation peritoneal fluid, where these cells adopt a planktonic form once by neutrophils (108), Annexin A1 released from neutrophil granules again. Further, it would be interesting to investigate the or in microvesicles (109)and IL-10 (110). Taken together, migratory patterns of neutrophils and monocytes after they neutrophils play an important role in clearing debris and setting have reached their suspended state in the peritoneal cavity. up a pro-resolution environment. The second major leukocyte population recruited to mesothelial injuries comprises inflammatory monocytes and SEROSAL (MESOTHELIAL) REPAIR monocyte-derived macrophages. It is generally accepted that the recruitment of neutrophils precedes the recruitment of monocytes So far, we have seen how a mesothelial injury leads to in sterile injury (31, 111) but it remains controversial whether inflammation with the consecutive deposition of a fibrin neutrophils recruitment is a necessary prerequisite for subsequent matrix and infiltration of immune cells. Normal serosal repair monocyte recruitment (112, 113). Rather than being pre- is achieved when a) the underlying organ is repaired, b) the sub- determined, the fate of recruited monocytes appears to be mesothelial connective tissue layer is restored to its original largely dependent on the environment (62). In the context of composition and thickness and c) the integrity of the sterile injury repair, monocytes have been shown to differentiate mesothelial membrane is reconstituted (31, 121, 122). The into mature macrophages at the site of injury. This process has central role of the mesothelium in tissue repair and fibrosis has hi been reviewed before (114). In brief, Ly6C monocytes are been revisited and comprehensively summarized (38). recruited to the wound, where they gradually differentiate into The mesothelium is a slowly renewing tissue with less than 1% low Ly6C macrophages. One of the molecules necessary for this of cells undergoing mitosis at any time (123). After mesothelial conversion is nuclear receptor subfamily 4, group a, member 1 injury, activated macrophages induce a pronounced proliferative (Nr4a1) (115). In the peritoneal cavity, the influx of inflammatory expansion of the mesothelial compartment (124, 125). Genetic monocytes due to injury or infection, correlates with the increase lineage tracing of mesothelial cells in several injury and disease + - - of MHCII CD102 GATA6 macrophages (26). These are also models indicate that regenerating mesothelium originates from referred to as small peritoneal macrophages or bone marrow healthy mesothelium rather than submesothelial cells (126–128). derived macrophages (14, 26). In the peritoneal cavity, PAI-1 Small focal mesothelial injuries that were induced using thermal and CCL1 were shown to recruit macrophages to the wound probes on the liver capsule and abdominal wall, healed completely involving the receptor molecules CD11b and CCR8 respectively without any visible defect left after days (36, 44). Because repair of (116, 117). Depletion of macrophages using clodronate-loaded mesothelial defects is largely independent of the defect size, liposomes or genetic constructs results in decreased wound investigators have proposed that mesothelial cells not only crawl healing of sterile injuries of serosal surfaces of the liver and into the wound from the borders, but also detach from opposing abdominal wall (36, 44). However, novel experimental strategies surfaces and distant sites and migrate in a free-floating state will be necessary to experimentally isolate the role of recruited through the coelomic cavity until they settle on the wound (125, macrophages compared to that of the resident GATA6+ 129, 130). This is further supported by the fact that adoptively macrophages in mesothelial wound healing. Furthermore, transferred mesothelial cells improve mesothelial repair in the monocyte-derived macrophages have been shown to replenish recipient (130). The response of mesothelial cells to injury can be the resident GATA6+ macrophages. This process is dependent on summarized as proliferation, loss of epithelial cohesion and the transcription factor IRF4 (118). The degree of this replacement migration. These phenotypic changes are reminiscent of other strongly depends on the degree of initial macrophage serosal surfaces that undergo epithelial-to-mesenchymal transition disappearance (50, 62). Taken together, this suggests a dual role (EMT). In analogy, this reaction has been termed mesothelial-to- of infiltrating monocytes in wound repair: they act as precursors of mesenchymal transition (MMT) (131, 132). On a molecular level, bone-marrow derived macrophages that are directly needed in MMT involves the downregulation of epithelial junctional wound repair, and they can serve to replace the GATA6+ proteins such as E-cadherin and the upregulation of resident macrophages. mesenchymal marker a-smooth muscle actin (a-SMA) and Neutrophils and monocytes are not only recruited into the production of ECM (133, 134). These changes correlate with an abdominal wall (45) but they are also recruited into the upregulation of transcription factors canonically associated with Frontiers in Immunology | www.frontiersin.org 8 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation EMT such as SNAI1, SNAI2, ZEB1, ZEB2, Twist1 (38) and can be deposition led to adhesion formation in many different injury induced by exposing mesothelial cells to TGF-b1, hepatocyte models and different species (96) and became generally accepted growth factor, platelet derived growth factor and IL-1b (38). We as the “classical concept of adhesion formation” (96). We have will next discuss how mesothelial repair can become defective. seen that an exuberant inflammatory response induced by peritoneal injury or infectionpromotesanincreased procoagulatory and antifibrinolytic reaction. On a molecular level, inflammatory mediators increase the expression of tissue POST-SURGICAL ADHESIONS factor (TF) and PAI-1 and decreases the expression of tPA in Surgeries within body cavities such as the abdominal cavity are often mesothelial cells resulting in increased fibrin deposition (96). This lifesaving procedures. Following surgical trauma, mesothelial repair mechanism was confirmed in peritoneal biopsies of inflamed can lead to a restitution of serosal surfaces at integrum. However, in peritoneum of humans that underwent surgery. The reduction some patients, the healing process is disrupted, leading to a fibrotic of fibrinolytic activity during inflammation was mediated by PAI- complication called post-surgical adhesions. Adhesions are fibrous 1(96). Importantly, a prospective study in humans showed that bridges of various thickness and length containing blood vessels and PAI-1 concentrations in peritoneal fluid were correlated with the nerve tissue (135, 136). Adhesions can also be caused by infection occurrence of adhesions after 8 days (96). Strategies to prevent but today, surgeries comprise by far the most common cause of postoperative adhesion target the dysregulated fibrin clot mesothelial injury leading to adhesions (96, 135). In the peritoneal deposition by either inhibiting coagulation, increasing cavity, adhesions result in considerable morbidity as they impair the fibrinolytic activity, or reducing inflammation. free movement of organs. These problems include potentially life- Administration of fibrinolytic (tPA) or anticoagulant agents threatening intestinal occlusions, secondary infertility in women, (Heparin) significantly reduced adhesions in different animal and chronic post-operative abdominal pain (96, 137, 138). models (38, 96, 141–144). However, the only study in humans Peritoneal adhesions were described for the first time in 1836 in a that enrolled 102 patients, was unable to confirm this effect. In post-mortem examination of a patient that had died from peritoneal this study 5000 I.U. of heparin were diluted in saline and used to tuberculosis (139). It was then suggested in 1849 that these wash the peritoneal cavity. The patients in this study then abnormal structures originate from lymphatic vessels that turn underwent a second operation (laparoscopy) 12 days after the into fibrinous adhesions (135, 139, 140). Despite tremendous first, to obtain adhesion scores (145). The heparin dose that was scientific advances since 1849 including the execution of many effectively administered in these patients was not reported but it clinical and experimental studies on adhesions, understanding of must have been extremely low as most heparin containing lavage their pathogenesis has not evolved enough to develop effective solution was removed after a few minutes. Based on titration therapies. To date, few research and development resources are studies in an animal models, the threshold dose for significant dedicated towards resolving this significant health problem. The anti-adhesion effect was (without occurrence of bleeding after process of adhesion formation largely depends on the same two days) 7.5x10 U/kg/day, which is equivalent to 5250 I.U/day mechanisms as “normal” mesothelial repair: Healing is initiated for a person weighting 70kg (142). Therefore, low heparin dose by damage recognition, inflammation, and coagulation. These steps or low heparin concentration may be the reason no effect on lead to the recruitment of leukocytes and the deposition of fibrin. adhesion formation was observed in the Jansen study. Further Then, the stable fibrin matrix (fibrin clot) is infiltrated by studies would be necessary. However, whether the current myofibroblasts that start to deposit ECM proteins such as collagen. evidence justifies a human trial testing high dose heparin in The problem with adhesions is that wound healing occurs at major abdominal surgery remains to be discussed. sites it should not (96). The classical paradigm of adhesion Others have tried to use anti-inflammatory agents to restore formation states that if serosal surfaces cannot re-establish mesothelial fibrinolytic activity. Cyclo-oxygenase inhibitors and homeostatic fibrinolysis soon after injury, excessive amounts of steroids were tested in animal models (146–148)withno fibrin are deposited. We have discussed that macrophage resounding success. Experimental animal models demonstrate aggregation accompanies fibrin deposition after sterile injury at potent prevention of postoperative adhesion following serosal surfaces (3). Macrophage-fibrin deposits serve as the intraperitoneal application of HMG-CoA reductase inhibitors basic scaffold for tissue repair. Clots that span the space (statins) (149). HMG-CoA reductase inhibitors stimulate between opposing serosal surfaces are dangerous because they fibrinolytic activity in human peritoneal mesothelial cell cultures can be converted into scars that permanently link these surfaces (150) and exert anti-inflammatory functions (151). In experimental called adhesions. We will now discuss the events taking place in animal models amelioration of adhesion after administration of more detail and highlight the respective therapeutic intraperitoneal acylated ghrelin, a 28-amino acid gastric peptide considerations for each (Figure 4). with anti-fibrotic and anti-inflammatory properties, was demonstrated. The adhesion prevention by ghrelin application Macrophage Aggregation and Fibrin was modulated via blockage of the TGF-b signaling pathway (152). Clot Formation Extending on this classical paradigm we have recently The idea that mesothelial loss of baseline fibrinolytic activity after proposed yet another factor in adhesion formation: that of surgical trauma may cause adhesions has been the subject of peritoneal macrophage aggregation. We showed that in various animal models (96). Interestingly, hypofibrinolytic fibrin macrophage aggregation to focal peritoneal injuries was tightly Frontiers in Immunology | www.frontiersin.org 9 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation AB C DE F FIGURE 4 | Post-surgical adhesion formation. (A) Overview of the peritoneal cavity before surgery. (B) Non-focal mesothelial injury such as major abdominal surgery leads to the uncontrolled aggregation of peritoneal macrophages serving as the nidus for the (C) subsequent Fibrin clot deposition. Inflammation and Coagulation inter-dependently promote the deposition of fibrin (see text). (D) Overview during or after adhesion formation. The abdominal organs (e.g., intestine) are now attached to the abdominal wall at anatomic (mesentery) and non-anatomic (adhesion) locations. (E) Mesothelial to mesenchymal transition gives rise to myofibroblasts that migrate into the wound and into the fibrin clot where they start to deposit extracellular matrix (ECM) such as collagen. (F) Adhesion formation is completed when the scar tissue is covered with mesothelium. The lesion may become fully perfused and pain-sensitive by ingrowth of blood vessels and nerves. regulated with just enough cells aggregated to seal the defect. aggregation of macrophages was largely independent of fibrin However, in response to large peritoneal injuries, such as crosslinking and macrophages showed the ability to aggregate ex abdominal surgeries, the aggregation of these macrophages was vivo without addition of fibrin (44). Taken together, mesothelial dysregulated, resulting in the formation of large super aggregates inflammation, macrophage aggregation, and coagulation, can act that started to join mesothelial surfaces. We found that this cooperatively but do not necessarily depend on each other. The process was dependent on scavenger receptors MARCO and relative contribution of each of these three processes likely depends MSR1. Depleting peritoneal cavity macrophages or inhibiting on the type (sterile, microbial, combined) and strength of the insult their aggregation significantly reduced the amount and severity as well as on local shear (3), which in turn is largely dependent on of adhesions in a mouse model. We therefore propose an adaptation patient movement and post-surgical intestinal paralysis (Figure 5). of the classical paradigm to include peritoneal macrophage Fibrotic Conversion aggregation as an additional event (Figure 4). Before we discuss the later events in adhesion formation such as fibrotic conversion Alpha smooth muscle actin (a-SMA) positive myofibroblasts are considered the main collagen-producing cell in wound healing and remodeling, we would like to note that the early process of adhesion formation such as mesothelial inflammation (chapter 3), and many fibrotic diseases (155–157). The question of the origin of a-SMA positive myofibroblasts in adhesions has been a matter macrophage aggregation (chapter 3) and coagulation (chapter 4) are tightly linked. We have discussed how inflammation directly affects of debate. Myofibroblasts in adhesions were believed to be either derived from the mesothelium or alternatively derived from sub- coagulation. Inversely, coagulation provides a positive feedback to the mesothelium further increasing inflammation. For example, mesothelial cells (126). Recently, Fischer at al. used a genetic fate mapping to permanently and selectively label cells expressing activation of proteinase-activated receptor-2 (PAR2) on mesothelial CreERT tdTomato cells results in increased MIP-2 production and consecutive protein c receptor gene (Procr xRosa26 ). Tamoxifen administration in these reporter mice resulted in the neutrophil infiltration (153). Furthermore, peritoneal cavity macrophages were shown to produce coagulation factors (21, 23, selective and permanent labelling of approximately 50% of all mesothelial cells but not submesothelial cells (127). Using this 154) and Factor V produced by peritoneal macrophages was shown to be essential for the clotting of peritoneal fluid in response to approach, they were able to show that the majority of platelet derived growth factor receptor a positive (PDGFRa+) bacteria (21). Inversely, they showed that macrophage aggregation (disappearance) was partially dependent on coagulation factors. In myofibroblasts in adhesions were of mesothelial origin (127). This is in line with older studies that relied on non-genetic other models such as laser-induced sterile mesothelial injury, the Frontiers in Immunology | www.frontiersin.org 10 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation lineage tracing methods such as cell tracker dyes or lineage weeks post-surgery the nerve fibers were traversing the whole markers to infer on source of myofibroblasts in adhesions (122, adhesion from coecum to abdominal wall (161). Human 158, 159). Taken together these data suggest that mesothelial to peritoneal adhesion specimens collected during surgery from mesenchymal transition (MMT) is the major source of 25 patients contained invariably sensory nerves (162). The myofibroblasts in adhesion pathogenesis. On a molecular level, sensory innervation could partially explain the chronic pain a MMT in adhesion formation relies on the same pathways as lot of patients with adhesions experience. Animal studies mesothelial repair (159). In fact, administration of TGF-b blocking revealed blood vessels in adhesions already 6 hours after injury peptide P144 resulted in a significant reduction of adhesions in an (163). This process of remodeling from connective tissue to fully experimental mouse model (159). This was associated with a innervated and vasculated tissue might be modulated. reduced expression of MMT markers such as Snail, a-SMA and The nerve fibers in adhesions were often associated with blood Collagen I in P144 treated mice (159). In addition, the exposure of vessels indicating angiogenesis could play a key part in regulating mesothelial cells to cyclic mechanical forces was shown to increase ingrowth of nerves into adhesions (162, 164). Local production of MMT in experimental murine and human models. Biomechanical VEGF by mesothelial cells appears to play a central role in the induction of MMT cooperates with biochemical signals such as process leading to peritoneal angiogenesis (165). In different TGF-b and seemed to be regulated by caveolin-1, a plasma murine models, postsurgical adhesion formation was reduced by membrane mechanotransducer (160). Interestingly, MMT-cells inhibition of VEGF suggesting adhesion formation is in adhesions also express many markers that are found in the angiogenesis-dependent (166, 167). In a human study including mesothelium during embryonic development but not within the adhesions samples from patients years after first surgery, adult mesothelium. These markers, including Mesothelin (MSLN), adhesions expressing VEGF A and its receptor showed Uroplakin-1B and Wilms-tumor 1 (WT1), were upregulated in significantly higher numbers of immature vessels suggesting adhesions indicating that adult mesothelial cells can repurpose ongoing angiogenesis in mature adhesions (163). In addition to aspects of fetal development (158). Depletion of mesothelial cells angiogenesis, modulation of the ECM by matrix metallo- results in a complete reduction of adhesions (127). However, proteinases (MMPs) takes place. MMPs are proteolytic enzymes totally depleting the peritoneal cavity of potential myofibroblasts involved in degradation of ECM, their activity is opposed by may compromise wound healing too much for use in the clinic. tissue-derived inhibitors of MMPs (TIMPs) (168). The expression Strategies that inhibit MMT in adhesions but leave mesothelial of both VEGF and MMPs is upregulated during MMT (131). In a repair intact need to be developed. human study, peritoneal samples were collected during initial laparoscopy and during a second-look laparoscopy 48 hours later. Remodeling Patients with pelvic adhesions exhibited significantly lower After fibrotic conversion, adhesions are considered irreversible amounts of MMP-9 concentrations and significantly higher and redundant scar bands. Furthermore, animal, and human MMP-9/TIMP-1 ratios when compared with controls (169). In studies demonstrated the ingrowth of nerves and vessels into peritoneal fluid of patients with excessive adhesions, higher TIMP- adhesions. In a murine model, nerve fibers in abdominal 1 levels could be demonstrated compared with those of patients adhesions were detected already two weeks after surgery and 4 without adhesions (170). Mice treated with instillation of FIGURE 5 | Factors influencing adhesion formation. Proposed concept of early local determinants that influence the binary outcome of adhesion formation and may be exploited therapeutically. tPA, tissue plasminogen activator; uPA, urokinase-type plasminogen activator; PAI, Plasminogen activator inhibitor. Frontiers in Immunology | www.frontiersin.org 11 May 2021 | Volume 12 | Article 684967 Zwicky et al. Serosal Repair and Adhesion Formation adenovirus vector encoding mutant MMP-9 gene at the time of understanding of wound healing at serosal surfaces which in peritoneal injury showed a reduced of severity of de novo turn might have great impact on the way we think of any of the adhesions (168). It remains unclear whether adhesion keep their above-mentioned pathologies. capacity for remodeling and thus have the potential to spontaneously resolve, or whether adhesions are an irreversibly fixed pathology once they have developed. AUTHOR CONTRIBUTIONS JZ and SNZ wrote the article. DS gave substantial input to the CONCLUDING REMARKS concepts underlying this review and revised and approved the article. We have discussed how mesothelial repair works for small injuries and how it can go wrong and result in peritoneal adhesions. It is important to note that mesothelial repair plays an important role in other fibrotic disorders in proximity to FUNDING serosal surfaces. These disorders include the fibrotic thickening JZ was supported by a Swiss National Science Foundation of the peritoneum (peritoneal fibrosis or encapsulating (SNSF) research fellowship (P1BEP3_181641). peritoneal sclerosis) and the pleura (pleural fibrosis). A number of studies have also demonstrated that mesothelial cells play also an important role in fibrotic diseases of the liver (128) and lung (38, 171–173). This is not surprising, since both, ACKNOWLEDGMENTS the liver and the lungs are covered with visceral mesothelium. 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