Mouse Tissue-Resident Peritoneal Macrophages in Homeostasis, Repair, Infection, And Tumor Metastasis Part 1

1. Introduction

The peritoneal cavity, as well as the pleural and pericardial cavities, are generated, from the embryonic coelom, a cavity resulting from the formation of the embryonic body wall, comprising the parietal plate mesoderm and the ectoderm, and the gut wall, comprising the visceral plate mesoderm and the endoderm. 

Embryo coelom is an important structure in the process of embryo formation, and its appearance marks embryonic development into the blastocyst stage. During the blastocyst stage, the embryo develops a fluid-filled sac called the coelom.

The embryonic coelom plays a very important role in embryonic development. It provides the environment for the embryo to nourish and breathe, and it can expel waste products. In addition, the embryo coelom can also provide some protection for the embryo. It can be said that without the body cavity of the embryo, the embryo cannot develop normally.

On the other hand, immunity is also one of the essential factors in human development. Immunity can protect the body from external germs and viruses, and it can also help us fight various diseases. For embryos, since the immune system is not yet fully developed, the embryo relies on the mother's immune system for protection.

Although the embryonic body cavity and immunity may seem unrelated, there is indeed a certain connection between them. Several studies have shown that the environment in the embryonic coelom plays a role in the development of immunity. For example, there are some growth factors in the body cavity of the embryo, which can promote the development of the immune system. In addition, the fluid in the embryo's body cavity also provides nutrition and protection for immune cells.

In conclusion, the relationship between embryo coelom and immunity is very close. The coelom of the embryo provides a good environment for the development of the embryo, and it also provides favorable conditions for the development of the immune system. Therefore, we should cherish the importance of the coelom of the embryo, and focus on protecting and promoting our immunity. From this point of view, we need to improve our immunity. Cistanche can significantly improve immunity, because meat ash contains a variety of biologically active components, such as polysaccharides, two mushrooms, Huang Li, etc. These components can stimulate the immune system Various types of cells in the system, increase their immune activity.

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The process by which the embryonic coelom is formed has been conserved from the primitive superphyla Protostomia and Deuterostomia, so that invertebrates of the phyla Annelida, Mollusca, Echinodermata, and Tunicata possess a coelomic cavity anatomically and developmentally equivalent to the embryonic coelom,[2] that generates the peritoneal, pleural, and pericardial cavities during the embryonic development of mammals.

The peritoneal cavity is covered by the peritoneum, the largest serous membrane of the body, with a surface area comparable to that of the skin, composed of the mesothelium, an epithelium of mesodermal origin, a basal membrane, and a submesothelial connective tissue.[3]

The parietal peritoneum lines the inner surface of the abdominal wall, whereas the visceral peritoneum integrates with the serosal layers of intra-abdominal organs. 

A double fold of the peritoneum forms the mesentery, which connects abdominal digestive organs to the abdominal wall and serves as a conduit for vessels, nerves, and lymphatics. A small volume of peritoneal fluid secreted by mesothelial cells serves as a lubricant in the peritoneal cavity and prevents mechanical friction between abdominal organs. In mice, total peritoneal fluid volume was estimated in two recent reports to be around 50–100 μL in the steady state,[4,5] and was claimed to differ between males and females (≈20 μL vs ≈100 μL) and, in the latter, to change during the estrous cycle.[6] Drainage of the peritoneal fluid into the lymphatic system allows peritoneal fluid recirculation,[7] and is achieved through openings in the mesothelium, called stomata, that are mainly located in the diaphragm and omentum.[3] 

The omentum is a visceral adipose tissue that develops by overgrowth of the mesentery and harbors a specialized vascular system and an organized lymphoid tissue, claimed to play an important role in defense against peritoneal infection.[8] Peritoneal fluid draining through the diaphragm collects into the subperitoneal lymphatic lacunae to reach the diaphragm collecting lymphatics, that drain into the mediastinal lymph nodes, whereas peritoneal fluid draining through the omentum collects in the omental lymphatics, that in turn collect into the intestinal lymphatic trunk that connects to the thoracic duct through the cisterna chyli.[3] Drainage of the peritoneal cavity allows control of peritoneal homeostasis and leukocyte recirculation but increases the risk of pathogen and metastatic tumor cell dissemination.

The peritoneal cavity is exposed to two major pathologies, infection and tumor metastasis, generally associated with high mortality, due to the easy spreading of pathogens or tumor cells throughout intra-abdominal organs, and to the anatomical features of the peritoneal cavity that greatly hinders the development of efficient treatments against these diseases. Even though the peritoneal cavity is a confined space, not readily exposed to invading pathogens such as those penetrating the skin, the lungs, or the gut, peritoneal infections can arise due to the loss of intestinal wall integrity (caused by ulcers, strangulation of hernias, appendicitis, or tumor growth), liver cirrhosis, accidental abdominal injuries, abdominal surgery, or peritoneal dialysis. 

The peritoneal cavity is also exposed to injuries in the parietal or visceral peritoneum caused by trauma, infection, or abdominal surgery, which can lead to peritoneal adhesions. Additional pathologies of the peritoneal cavity include—peritoneal endometriosis, involving the formation of ectopic vascularized endometrial tissue in the peritoneum associated with chronic inflammation— peritoneal autoimmune serositis, chronic inflammation of the peritoneum caused by autoimmune diseases, such as Crohn’s disease and—post-surgical peritoneal adhesions.[3,9,10]

Immune defense against peritoneal infection and tumor metastasis relies on a first line of local defense supported by resident peritoneal immune cells, present in the peritoneal cavity in the steady state, with innate immunity sensing and responding properties. The second line of immune defense in the peritoneal cavity is provided by functional units of lymphoid tissue, associated with adipose tissue located in the omentum, mesentery, or gonadal fat, called fat-associated lymphoid clusters (FALCs), or milky spots for omental FALCs.[8] FALCs harbor a structural organization similar to that found in secondary lymphoid organs, including a reticular cell-based stroma, B and T cell compartments, and specialized blood and lymphatic vessels, allowing leukocyte migration to and from the peritoneal cavity.[8]

Resident peritoneal immune cells include tissue-resident peritoneal macrophages, generally named large peritoneal macrophages (LPMs) and B1 cells. Recent experimental evidence has unveiled that, apart from their primary phagocytic function, LPMs fulfill different homeostatic, repair, and immunological defense functions, that reflect a previously unexpected functional plasticity.[11] Peritoneal B1 cells are considered innate-like B cells, that constitutively produce natural IgM, providing local immune protection against a wide variety of pathogens. 

In addition, B1 cells actively produce IgM in response to viruses, bacteria, fungi, and parasites.[12] The first line of immunity in the peritoneal cavity in mammals, relying on phagocytic and antibody-mediated defense mechanisms supported by LPMs and B1 cells, is reminiscent of the primitive defense mechanisms sustained by different populations of coelomocytes present in the coelomic cavity of invertebrates.[13–15] Immune defense strategies in coelomic cavities have been therefore highly conserved throughout evolution from invertebrates to higher vertebrates.

In this review, we discuss recent evidence that has widened our knowledge of the biology of LPMs by describing the mechanisms of resident embryonic LPM replacement by resident bone marrow monocyte-derived LPMs (moLPMs), that result in phenotypic and functional LPM sexual dimorphism, and unveiling how LPMs, free in a fluidic environment in the steady state, perform repair and immune defense functions, by forming thrombus-like structures in response to peritoneal injury, and mesothelium bound dynamic LPM aggregates after bacterial infection. 

Moreover, recent experimental evidence support that peritoneal tumors can subvert LPM metabolism, leading to the acquisition of tumor-promoting functions that, nevertheless, might be reverted by experimental strategies blocking tumor-induced subversion of LPM function, that could be the basis for the development of novel immunotherapeutic approaches against peritoneal tumor metastasis based on macrophage reprogramming.

2. Large Peritoneal Macrophage Identity

LPMs are long-lived, tissue-resident macrophages formed during embryonic life, developmentally and functionally confined to the peritoneal cavity, in contrast to other peritoneal immune cell populations that are recruited to the peritoneal cavity and recirculate to other locations in the steady state, and under pathological conditions. Those include, in the steady state, B1 cells, that together with LPMs constitute the vast majority of cells harvested by peritoneal lavage, and a low percentage of monocyte-derived SPMs (for small peritoneal macrophages), B2 cells, T cells, NK cells, innate lymphoid cells, and mast cells.[11] 

As discussed in depth in this review, research performed over the last years has established that LPMs not only fulfill peritoneal homeostatic functions, but are also involved in the repair of tissue damage caused by inflammation and infection, and defense against microbial infection. Moreover, LPMs contribute to most peritoneal pathologies, particularly to peritoneal tumor metastasis, but also to peritoneal endometriosis, autoimmune serositis, and post-operative adhesions.

Resident embryonic LPMs are CD11b+ F4/80hi MHC-II− cells expressing a series of markers characterizing tissue-resident macrophages, such as CD14, CD64, and MerTK.[16,17] 

Besides, tissue-resident macrophages located in the serous cavities of the body, that comprise LPMs and tissue-resident macrophages present in the pleural and pericardial cavities, appear to share the expression of the transcription factor GATA6, the scavenger receptor Tim4, and the M-CSF receptor CFSR1.[11,18] In addition, resident embryonic LPMs are characterized by the expression of several cell surface receptors reflecting LPM homeostatic, repair, regulatory, and defense functions, including molecules involved in LPM adhesion and localization, such as ICAM-2 (CD102), CD11b, CD49f, CD73, and CD62P,[19] recognition and removal dead cells, such as CD36, CD93, CD163, Tim4, MerTK, MARCO, and MSR1,[4,16,20–22] the negative regulation of macrophage activation, ensuring non-inflammatory clearance of apoptotic cells, such as V-set immunoglobulin domain containing 4 (VSIG4),[23] pathogen binding, such as CD14, CD36, and SIGN-R1 (CD209b)[11,24] and response to pathogens, such as TLR4 and TLR7.[25,26] The most representative cell surface molecules expressed by embryonic LPMs are summarized in Figure 1.

LPMs belong to the family of tissue-resident macrophages, that share the expression of core lineage-related genes determined during embryonic life, but acquire tissue-specific transcriptional and functional features established upon exposure to tissue-specific microenvironmental signals, through the expression of tissue-specific transcription factors.[16,27] In this regard, the transcription factor GATA6 is essential for LPM-specific gene expression, proliferation, and survival of LPMs.[19,28,29] Consequently, homeostatic, repair, and defense LPM functions were compromised in mice deficient in GATA6 in myeloid cells.[5,19,30] GATA6 expression is maintained in a non-cell autonomous manner[27,31] and was proposed, based on in vitro experiments, to be activated by the vitamin A metabolite retinoic acid, through retinoic acid nuclear receptors.[19] 

GATA6 expression would be thus modulated by the local availability of retinoic acid, supporting the concept that the GATA6-induced transcriptional program of LPMs is reversible,[17] which would be the basis for the functional plasticity of LPMs, which enables LPMs to switch from homeostatic to repair or immune defense functions when needed. 

In this regard, LPMs transferred into the alveolar space downregulated GATA6 and acquired an alveolar macrophage transcriptional profile.[27] Retinoic acid activating GATA6 in LPMs was claimed to be produced by omental and peritoneal stromal cells.[19] In line with these observations, expression by mesothelial and fibroblastic stromal cells of the Wilms’ tumor 1 (WT1) transcription factor, that drive the expression of two rate-limiting enzymes controlling retinol metabolism, RALDH1, and RALDH2,[32] was claimed to control GATA6 expression in LPMs and in GATA6+ resident macrophages located in the pleural and pericardial cavities, since depletion of WT1+ cells in resulted in a profound reduction in these macrophage subsets, paralleled by a concomitant diminishment of Raldh1 and Raldh2 transcripts,[18] further supporting the role of retinoic acid in sustaining GATA6 expression, that nevertheless remains to be formally demonstrated. 

The fact that in GATA6-deficient mice CD11b+ macrophages accumulated in omental milky spots, while LPMs were reduced in the peritoneal cavity,[19] supports the hypothesis that retinoid acid secreted by stromal cells in the omentum maintains the GATA6-driven transcriptional program in LPMs, and would imply that LPMs continuously recirculate through the omentum, but this remains to be formally demonstrated.

Retinoic acid is a ligand of retinoid X receptors (RXRs), which are members of the nuclear receptor superfamily of ligand-dependent transcription factors, that control lipid and glucose metabolism, and play key roles in inflammatory and autoimmune disorders.[33] Interestingly, mice deficient in RXRs 𝛼 and 𝛽 displayed a profound defect in neonatal LPM expansion, and reduced survival of adult LPMs, due to lipid accumulation resulting in apoptosis, revealing that RXRs contribute to the expansion and maintenance of LPM.[34] ATAC-seq analyses revealed that the Gata6 locus displayed reduced chromatin accessibility in RXR-deficient LPMs, which correlated with a lower Gata6 gene expression, supporting that RXRs regulate the GATA6-dependent LPM transcriptional program.

Macrophage colony-stimulating factor (M-CSF or CFS1) control commitment to the macrophage lineage, and therefore LPM differentiation is dependent on CFS1, as demonstrated in osteopetrosis (Csf1 op/op) mice that harbor a mutation in the Cfs1 gene, leading to a defective LPM development.[35] Moreover, based on in vitro assays, mesothelial cells were reported to secrete CSF1 that sustained LPM proliferation in mesothelial cell-LPM co-cultures; transwell assays revealed that LPM proliferation was significantly reduced when mesothelial-LPM interactions were prevented, suggesting that cell-to-cell contact contributed to LPM proliferation.[36] The concept that mesothelial-derived CSF1 is required for LPM maintenance is further supported by a recent report showing that LPMs were highly reduced in mice in which WT1+ cells were deficient in CFS1.[37] Whether mesothelial cell-derived CSF1 contributes to steady-state LPM self-renewal and/or to LPM proliferation during inflammation remains to be explored.

3. Large Peritoneal Macrophage Origin and Replacement in Homeostasis

LPMs differentiate during embryonic life and maintain themselves by in situ self-renewal during adult life. In the steady state, embryonic LPMs are gradually, yet partially, replaced from the late stages of embryonic development by resident bone marrow moLPMs that acquire a resident embryonic LPM identity, but retained some transcriptional and functional characteristics related to their origin.[38,39] The origin of embryonic LPMs remains controversial since they were reported to derive either from a dual contribution from yolk sac macrophages and fetal liver monocytes,[40] or exclusively from fetal liver monocytes.[41] An integrated model of the origin and replacement of LPMs is shown in Figure 2. 

The replacement of embryonic for bone marrow monocyte-derived tissue-resident macrophages, in the steady state, has been described for all tissue-resident macrophage populations, except microglia, Langerhans cells, and Kupffer cells, as reported by Dr. F. Ginhoux’s lab, using fate-mapping models based on the expression of the Ms4a3 gene, specifically expressed by granulocyte-monocyte progenitors.[42] The degree of replacement by bone marrow monocyte-derived macrophages appears to be essentially dictated by niche access and availability.[43] None of the tissue-resident macrophage populations exhibit a total replacement by bone marrow monocyte-derived macrophages, suggesting that an equilibrium is reached in each organ between bone marrow monocyte recruitment, and proliferation and survival of embryonic and bone marrow monocyte-derived resident macrophages.[42]

Therefore, during adult life, the resident LPM pool is maintained, in a steady state, by a combination of self-renewal of resident embryonic LPMs and differentiation and self-renewal of resident moLPMs. Consequently, in this manuscript, unless otherwise indicated, the term LPMs refers to the adult LPM population which, in the steady state, comprises resident embryonic LPMs and resident moLPMs. Interestingly, after sexual maturity, the rate of embryonic LPM replacement is higher in males, whose LPMs display a higher proliferative activity, as demonstrated by genetic fate-mapping analyses from Dr. F. Ginhoux’s and Dr. S. Jenkins’ labs, that nevertheless reported differences in the rates of replacement.[39,42] Ginhoux and colleagues[42] found a higher proportion of resident moLPMs in males at 8 weeks and 20 weeks of age (≈25% vs 10% and ≈50% vs 25%, respectively). 

In contrast, Jenkins and colleagues[39] reported that ≈30% of resident moLPMs were detected both in males and females at 4 weeks, whereas at 16 weeks, males harbored a higher proportion of resident moLPMs (≈60% vs 30%). Sexually dimorphic replacement by resident moLPMs was proposed to be controlled by changes in the peritoneal microenvironment that arise upon sexual maturation, independently of estrogen levels and peritoneal adiposity,[39] leading to divergence in the heterogeneity of the LPM population. Sex-associated divergence in the heterogeneity within the LPM population, as well as sex differences in the peritoneal microenvironment, determine significant transcriptional and functional differences between the LPM population in male and female mice, although RNA-seq analyses at the single-cell level revealed equivalent cluster identities in male and female LPMs. 

RNA-seq analyses, at the population level, of 10- to 12-week-old male and female mice LPMs indicated that 486 mRNA transcripts were differentially expressed (>1.5-fold) between female and male LPMs. The 148 mRNA transcripts more highly expressed in female LPMs, at the population level, comprised genes associated with lipid uptake and transport, such as Apoe, Apoc1, Saa2, and Saa3, as well as, genes associated with immune defense. The latter included Timd4, Cxcl13, Tgfb2, the complement component genes C1qa, C3, and C4b, and the C-type lectin receptor genes Cd209a, Cd209b, and Clec4g. [39] In contrast, in males, the genes more highly expressed by LPMs were associated with proliferation and cell cycle-related processes, such as Cdk1, E2f2, and Mki67.

Interestingly, female mice were more resistant to acute peritonitis induced by group B streptococci[44] or by Streptococcus pneumoniae infection.[39] Since CD209 (SIGN-R1) is critical for survival after infection by S. pneumoniae infection by promoting efficient bacterial phagocytosis and clearance,[24] the sex-dependent resistance to bacterial peritonitis has been claimed to be due to the higher expression by female LPMs of CD209 and, additionally, of complement components and the B1 cell recruiting chemokine CXCL13.[39] In this regard, the higher resistance of women and infants to blood-borne infections was claimed to correlate with an enhanced CXCL13-dependent production by B1 cells of natural antibodies.

4. Large Peritoneal Macrophage Replacement Induced by Inflammation

Inflammatory reactions in the peritoneal cavity induced by sterile inflammatory stimuli,[5,39,42,45,46] abdominal surgery,[39] or bacterial infection[47] were reported to cause LPM cell death leading to a reduction in the number of resident LPMs (including resident embryonic LPMs and resident moLPMs), whose extent correlates with the severity of inflammation.[42,46] Recovery of the original LPM pool occurs by proliferation of the remaining resident LPMs[45] and replacement by LPMs derived from inflammatory monocytes (hereafter ii-moLPMs for inflammation-induced moLPMs) as demonstrated using different experimental strategies, based on fate-mapping models,[42] tissue-protected bone marrow chimeric mice and adoptive transfer experiments.[39,46]

Using an experimental model based on the induction of mild inflammation, caused by low-dose zymosan (10 μg per mouse), or severe inflammation caused by high-dose zymosan (1000 μg per mouse), and adoptive transfer experiments to track ii-moLPMs and assess how the inflammatory environment controls their differentiation, Jenkins and colleagues proposed that the degree of replacement of resident LPMs by ii-moLPMs, and the extent to which the later acquire the identity and function of resident LPMs is determined by the severity of the inflammatory process and the magnitude of LPM death[46] (Figure 2).

ii-moLPMs formed after mild inflammation co-existed long-term with remaining resident LPMs, but competition with resident LPMs and alterations in the peritoneal environment retained them in an aberrant state of activation and blocked the acquisition of a resident LPM phenotype. In contrast, severe inflammation can lead to the total ablation of resident LPMs, which are ultimately replaced by ii-moLPMs, that acquired a resident LPM identity but maintained transcriptionally and functionally divergent features, determined by their origin, peritoneal inflammation, and time-of-residency.[46] The phenotype of ii-moLPMs was proposed to comprise intrinsic markers determined by their origin, such as CD62L and Semaphorin 4a, markers whose expression is controlled by competition with resident LPMs but is reprogrammed with time, such as GATA6, MHCII, and CCR5, and markers related to time-of-residency, independent of competition with resident LPMs, such as Tim4, CD209b, and VSIG4. A significant proportion of genes differentially expressed by resident LPMs and ii-moLPMs appear to be controlled by differences in retinoic acid signaling, either directly or in a GATA6-dependent manner.[46]

ii-moLPMs exhibit higher proliferative activity than resident LPMs,[38,46] which was suggested to correlate with differences in the enhanced ability of the former to proliferate in response to CSF1 produced by mesothelial cells.[36] In addition, ii-moLPMs displayed a lower ability to phagocytose bacteria and uptake dying cells, and failed to produce CXCL13.[46] While the number of peritoneal B1 cells increases with age in homeostasis, peritoneal inflammation led to a defective accumulation of B1 cells[46] since, as pointed out above, CXCL13 production by LPMs control B1 cell homing to the peritoneal cavity.[48] Therefore, the fact that developmental and functional heterogeneity of the LPM population depends on sex and age has important implications when addressing the role of LPMs in repair, defense, and implication in peritoneal tumor metastasis, that need to be taken into account in future studies.

It is important to note that monocytes recruited to the peritoneal cavity during inflammatory reactions, related to noninfectious peritoneal damage, infection, or metastatic tumor growth, can potentially differentiate into monocyte-derived cells that fulfill specific repair, defense, or tumor-promoting functions, but might not acquire phenotypic or functional LPM characteristics, and thus should not be considered ii-moLPMs. However, defining the identity of cells differentiated from monocytes recruited to the inflamed peritoneum can be controversial since in most reports focusing on the functional relevance of peritoneal monocyte-derived cells, the time-of-persistency, and/or acquisition of LPMs features by these monocyte-derived cells was not addressed and, inversely, in reports on resident LPM replacement during inflammation, the function of ii-moLPMs was not explored in-depth.

In line with the hypothesis that competition for a particular physical niche, defined by cellular and molecular microenvironmental factors, determines the contribution of monocytes to tissue-resident macrophages,[43] the existence of a biochemical niche for peritoneal resident macrophages was proposed.[46] Accordingly, competition for signals and cell-to-cell interactions controlling survival, proliferation, and function of LPMs would control the balance between resident LPMs and ii-moLPMs, as well as, the acquisition of mature resident LPM identity by iimoLPMs.

5. Role of Large Peritoneal Macrophages in Peritoneal Homeostasis

LPMs fulfill an essential role in the clearance of apoptotic cells at steady state, a hallmark of tissue-resident macrophages that is crucial for the maintenance of self-tolerance,[49] through the expression of specific scavenger receptors including CD36, CD93, CD163 Tim4, and MerTK.[16,20–22] Interestingly, efficient internalization of apoptotic cells by LPMs was proposed to rely on initial binding to Tim4 of phosphatidylserine exposed by apoptotic cells, followed by MerTK-mediated engulfment.[20] LPMs are programmed by the peritoneal microenvironment to efficiently scavenge apoptotic cells, avoiding inflammation driven by TLR-mediated by self-derived nucleic acid recognition, while maintaining the ability to respond to infection.[25] 

The transcription factors Kruppel-like factors 2 and 4 were claimed to drive LPM programming for immunologically silent clearance of apoptotic cells, by controlling the expression of apoptotic cell recognition receptor genes, such as Timd4, Marco, and Olr1, and genes acting as negative regulators of TLR signaling, such as Hes1, Socs3, Pdlim2, Ptpn6, and Tnfaip3, resulting in an increased threshold of activation.[25] In line with these observations, LPMs express VSIG4, a B7 family-related receptor, reported to downregulate macrophage activation, through PDK2-mediated reprogramming of mitochondrial pyruvate oxidation and ROS production.[23]

LPMs play a pivotal role in maintaining peritoneal B1 cell homeostasis. Indeed, peritoneal B1 cells, that constitutively produce natural IgM, providing a local first line of defense against a wide variety of pathogens,[12] depend on the chemokine CXCL13, produced by LPMs and stromal cells, for their recruitment from the circulation and homing to the peritoneal cavity; CXCL13 is also required for B1 cell homing to the omentum.[48] In addition, after steady-state migration to the intestinal lamina propria, peritoneal B1 cells secrete IgA natural antibodies to provide for immune control of the intestinal microbiota.[12] Interestingly, IgA class switching in peritoneal B1 cells, and consequently, B1 cell-mediated intestinal IgA secretion, is controlled by retinoic acid/GATA6-dependent TGF-𝛽 production by LPMs.[19] In line with this observation, retinoic acid and TGF-𝛽 ad a synergistic effect on IgA class switch in peritoneal B1 cells in vitro.[50]

An additional function of LPMs linked to peritoneal homeostasis is surveillance for the detection of sterile peritoneal injury that, unless quickly repaired, could lead to the formation of peritoneal adhesions, that can turn into severe peritoneal disease, including intestinal occlusion and infertility in women.[10] Surveillance for the detection of peritoneal damage or infection is essentially fulfilled by LPMs and requires active patrolling of the peritoneal surface. In this regard, in a recent report, in which imaging of the peritoneal cavity, through the intact abdominal wall, was performed by intravital microscopy, LPMs were shown to move passively in a respiration-dependent and random manner in the steady state, with speeds of up to 800 μm s−1. [4] The role of LPMs in peritoneal tissue repair and adhesion formation is discussed in the next section.

6. Role of Large Peritoneal Macrophages in Repair of Peritoneal Injury

Damage of the abdominal or visceral peritoneum can be caused by sterile injury resulting from accidental trauma or abdominal surgery or can result from peritoneal pathologies, such as infection, liver or intestinal diseases, or metastatic tumor growth. Recent studies, discussed below, have shed light on the mechanisms involved in the repair of peritoneal sterile injury, and on the role of LPMs in this process.[10] In contrast, how the peritoneum damaged by peritoneal infection or tumor metastasis is subsequently restored, remains to be explored in depth.

Sensing of damage in the peritoneal lining is achieved through the recognition of danger-associated molecular patterns (DAMPs), released by damaged cells, including mesothelial cells, cells located in the submesothelial connective tissue, and potentially those forming the underlying tissues. DAMPs include constitutively-expressed DAMPs, such as nuclear and mitochondrial DNA, nuclear and mitochondrial proteins (HMGB1, histones, cytochrome c), ATP, K+ ions, or S100 calcium-binding proteins, inducible DAMPs, such as heat shock proteins, defensins, galectins, and IL-1𝛼, and extracellular DAMPs, such as hyaluronan or heparan sulfate.[51] DAMP-activated mesothelial cells trigger peritoneal inflammation through the release of proinflammatory cytokines and chemokines, that promote leukocyte recruitment to the damaged areas and complement activation, resulting in additional inflammation. This inflammatory reaction triggers tissue factor-dependent fibrin polymerization as a result of an imbalance between fibrinogenesis and fibrinolysis, leading to the formation of a fibrin matrix, serving as the scaffold for wound repair. 

The latter involves the recruitment to the submesothelial compartment of leukocytes fulfilling a repair function, including LPMs, neutrophils, monocytes, and monocyte-derived macrophages, recruitment of mesenchymal precursors, deposition of extracellular matrix, ingrowth of nerves and blood vessels, and re-epithelialization of the damaged peritoneum.[10] Persistence of peritoneal inflammation can lead to excessive fibrin deposition and, ultimately, to the formation of fibrous bridges between opposing peritoneal surfaces, containing nerves and blood vessels, called abdominal adhesions.[52] Adhesions predominantly result from abdominal surgery, but can also be caused by infection, endometriosis, radiotherapy, or peritoneal dialysis, and are associated with considerable morbidity that can involve life-threatening complications.[52]

The analysis by electron microscopy of peritoneal healing after experimental surgical injury, revealing that macrophages adhered to the damaged tissue 24 h after injury was caused, and subsequently migrated into the wound,[53] provided the first evidence of the possible role of macrophages in peritoneal injury repair. This hypothesis was further supported by intravital microscopy studies from Dr. P. Kubes’ lab demonstrating that, after laser-induced injury of the liver capsule, F4/80high GATA6+ LPMs were recruited to the damaged areas, and migrated across the mesothelium into the liver injuries, within 1 h after laser-induced injury.[30] 

LPMs sensed damaged tissue through the recognition of ATP released by liver necrotic cells through the DAMP receptor PX27 and infiltrated the liver parenchyma through CD44- mediating binding to hyaluronan present in the damaged areas. Interestingly, recruitment to the injured tissue triggered LPM proliferation and upregulation of molecules associated with an alternative activated /repair phenotype, such as CD206, CD273, and arginase 1. Accordingly, recruited LPMs actively contributed to necrotic cell removal, which was claimed to be critical for revascularization and tissue repair, as supported by experiments showing that healing of injured areas was delayed in clodronate-loaded liposome-mediated LPM-depleted or GATA6-deficient mice.[30] 

Similar ATP-induced recruitment and CD44-dependent migration to the damaged area of F4/80 high GATA6+ LPMs were described in a model of intestinal thermal injury.[54] Clodronateloaded liposome-mediated LPM depletion experiments also supported the concept that LPMs contributed to injured intestinal repair in this experimental setting. However, whether, as described in this report, LPMs are recruited to the intestinal serosa after damage of the intestinal epithelium, would require further investigation, since it remains possible that this phenomenon was artefactual if, in these experiments, damage was not just limited to the intestinal luminal surface, but affected the intestinal mucosa and submucosa, taking into account the experimental strategy employed to address this issue in this study. 

On the other hand, regarding the experiments of LPM depletion by treatment with clodronate-loaded liposomes, carried out to address the role of LPMs in liver or intestinal serosal repair,[30,54] whether the delayed wound healing observed in clodronate-treated mice was due, at least in part, to the depletion of peritoneal monocyte-derived macrophages and tissue-resident macrophage populations present in the omentum, peritoneal membrane, or liver capsule, cannot be excluded. Indeed, monocytes have been demonstrated to be recruited to peritoneal injured areas, where they differentiate into monocyte-derived macrophages that can promote tissue repair.[55]

In line with these observations, the concept that after LPMs attach to damaged mesothelium, they migrate into serosal injuries and fulfill a critical repair function, has been challenged by a recent report in which genetic fate-mapping allowed to trace resident LPMs after liver sterile injury.[56] These studies revealed that GATA6+ resident LPMs accumulated on the injured surface of the liver, but minimally invaded the necrotic liver parenchyma. Moreover, by using the diphtheria toxin dependent G6Mø-CreER; R26-tdTomato/iDTR mouse line, which allowed the genetic ablation of most GATA6+ resident LPMs, the authors concluded that the absence of GATA6+ resident LPMs did not significantly impact on liver wound healing, and thus that GATA6+ resident LPMs were not critical for damaged serosal tissue regeneration. Therefore, additional research has to be conducted to establish whether, and eventually how, LPMs contribute to peritoneal healing.

Interestingly, a recent report by Dr. P. Kubes’ lab, based on the imaging of the peritoneal cavity after laser-induced focal thermal peritoneal injury, by intravital microscopy through the intact abdominal wall, supports a direct role of LPMs in serosal repair.[4] Indeed, resident GATA6+ LPMs were the first cells recruited to mesothelial injuries, a process that required peritoneal fluid shear flow. Recruited LPMs attached to the damaged peritoneum and completely covered the lesions 15 min after injury was caused, forming thrombus-like structures, in a process that mirrored platelet aggregation in response to blood vessel injury. LPM aggregation was not dependent on canonical adhesion molecules or fibrin polymerization but on scavenger receptors containing scavenger receptor cysteine-rich (SRCR) domains, such as MARCO or MSR1, that bind to a high number of polyanionic ligands, and that are highly conserved throughout evolution from invertebrates. Indeed, in echinoderms, such as the sea urchin, injury in the coelomic cavity led to the aggregation of coelomocytes, expressing SRCR-containing homologs, that sealed the damaged areas.[57,58] LPMs were claimed to contribute to the repair of focal peritoneal lesions, by achieving a physical sealing of peritoneal injuries, since blockade of macrophage aggregation led to a delayed healing of injured parietal peritoneum.[4]

In contrast, using an experimental model of peritoneal adhesion formation induced by surgical sterile injury, involving the formation of a peritoneal button by suturing a portion of the peritoneal wall, high numbers of LPMs were shown to be recruited to the buttons within 3 h after surgery.[4] In this iatrogenic setting, macrophages formed extensive aggregates that promoted the deposition of fibrin and the growth of scar tissue, leading to the formation of peritoneal adhesions within 7 days after surgery. 

Interestingly, the number and development of peritoneal adhesions were markedly reduced in mice in which LPMs were depleted by 24 h before surgery, supporting that LPMs contributed to peritoneal adhesion formation. Interestingly, by using a similar model of experimental adhesion formation, LPMs were shown to form a cell barrier over the fibrin clots formed in damaged mesothelial areas, a process leading to adhesion formation if the macrophage barrier was insufficient to cover the fibrin clot, but that precluded adhesion formation if the macrophage barrier completely shielded the fibrin clots.[59] Indeed, IL-4-mediated reinforcement of the macrophage barrier prevented adhesion formation and could be the basis for the development of innovative treatments to prevent post-operative adhesions. Therefore, although initial macrophage recruitment and aggregation, together with fibrin deposition, appears to be required for a correct serosal repair, it can also cause pathogenic scarring leading to adhesion formation, a situation that has been correlated with a low mesothelial fibrinolytic activity.[52]

In conclusion, the role of LPMs in sterile peritoneal damage repair is to a large extent dictated by the severity of the injury. LPMs promote adhesion formation after large peritoneal injury but fulfill an essential function of rapid repair of focal mesothelial injuries, reminiscent of primitive repair mechanisms conserved throughout evolution (Figure 3). On the other hand, the potential of LPMs to invade deep damaged submesothelial tissue, and contribute to its restoration together with monocyte-derived macrophages and neutrophils, is still controversial and thus requires to be further investigated. In this regard, whether, as described for other macrophage populations, LPMs produce profound healing mediators, such as platelet-derived growth factor, insulin-like growth factor 1, TGF-𝛽1, or VEGF-𝛼, [60] remains to be explored.

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