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In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.

The inflammatory response protects the human body against infection and injury. However, uncontrolled and unresolved inflammation can lead to tissue damage and chronic inflammatory diseases. Therefore, active resolution of inflammation is essential to restore tissue homeostasis. This review focuses on the pro-resolving molecule annexin A1 (ANXA1) and its derived peptides. Mechanisms instructed by ANXA1 are multidisciplinary and affect leukocytes as well as endothelial cells and tissue resident cells like macrophages and mast cells. ANXA1 has an outstanding role in limiting leukocyte recruitment and different aspects of ANXA1 as modulator of the leukocyte adhesion cascade are discussed here. Additionally, this review details the therapeutic relevance of ANXA1 and its derived peptides in cardiovascular diseases since atherosclerosis stands out as a chronic inflammatory disease with impaired resolution and continuous leukocyte recruitment.

Epithelial barriers play an important role in regulating mucosal homeostasis. Upon injury, the epithelium and immune cells orchestrate repair mechanisms that re-establish homeostasis. This process is highly regulated by protein and lipid mediators such as Annexin A1 (ANXA1). In this review, we focus on the pro-repair properties of ANXA1.

Neutrophil transepithelial migration (TEM) during acute inflammation is associated with mucosal injury. Using models of acute mucosal injury in vitro and in vivo, we describe a new mechanism by which neutrophils infiltrating the intestinal mucosa disrupt epithelial homeostasis. We report that junctional adhesion molecule-like protein (JAML) is cleaved from neutrophil surface by zinc metalloproteases during TEM. Neutrophil-derived soluble JAML binds to the epithelial tight junction protein coxsackie-adenovirus receptor (CAR) resulting in compromised barrier and inhibition of wound repair, through decreased epithelial proliferation. The deleterious effects of JAML on barrier and wound repair are reversed with an anti-JAML monoclonal antibody that inhibits JAML-CAR binding. JAML released from transmigrating neutrophils across inflamed epithelia may thus promote recruitment of leukocytes and aid in clearance of invading microorganisms. However, sustained release of JAML under pathologic conditions associated with persistence of large numbers of infiltrated neutrophils would compromise intestinal barrier and inhibit mucosal healing. Thus, targeting JAML-CAR interactions may improve mucosal healing responses under conditions of dysregulated neutrophil recruitment.

The mammalian gut microbiota is essential for normal intestinal development, renewal, and repair. Injury to the intestinal mucosa can occur with infection, surgical trauma, and in idiopathic inflammatory bowel disease. Repair of mucosal injury, termed restitution, as well as restoration of intestinal homeostasis involves induced and coordinated proliferation and migration of intestinal epithelial cells. N-formyl peptide receptors (FPRs) are widely expressed pattern recognition receptors that can specifically bind and induce responses to host-derived and bacterial peptides and small molecules. Here we report that specific members of the gut microbiota stimulate FPR1 on intestinal epithelial cells to generate reactive oxygen species via enterocyte NADPH oxidase 1 (NOX1), causing rapid phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase mitogen-activated protein kinase. These events stimulate migration and proliferation of enterocytes adjacent to colonic wounds. Taken together, these findings identify a novel role of FPR1 as pattern recognition receptors for perceiving the enteric microbiota that promotes repair of mucosal wounds via generation of reactive oxygen species from the enterocyte NOX1.

The gastrointestinal epithelium functions as an important barrier that separates luminal contents from the underlying tissue compartment and is vital in maintaining mucosal homeostasis. Mucosal wounds in inflammatory disorders compromise the critical epithelial barrier. In response to injury, intestinal epithelial cells (IECs) rapidly migrate to reseal wounds. We have previously observed that a membrane-associated, actin binding protein, annexin A2 (AnxA2), is up-regulated in migrating IECs and plays an important role in promoting wound closure. To identify the mechanisms by which AnxA2 promotes IEC movement and wound closure, we used a loss of function approach. AnxA2-specific shRNA was utilized to generate IECs with stable down-regulation of AnxA2. Loss of AnxA2 inhibited IEC migration while promoting enhanced cell-matrix adhesion. These functional effects were associated with increased levels of β1 integrin protein, which is reported to play an important role in mediating the cell-matrix adhesive properties of epithelial cells. Because cell migration requires dynamic turnover of integrin-based adhesions, we tested whether AnxA2 modulates internalization of cell surface β1 integrin required for forward cell movement. Indeed, pulse-chase biotinylation experiments in IECs lacking AnxA2 demonstrated a significant increase in cell surface β1 integrin that was accompanied by decreased β1 integrin internalization and degradation. These findings support an important role of AnxA2 in controlling dynamics of β1 integrin at the cell surface that in turn is required for the active turnover of cell-matrix associations, cell migration, and wound closure.

N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1(-/-IEC) and AnxA1(-/-) mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.

Background and purpose: Over three decades of research evaluating the biology of melanocortin (MC) hormones and synthetic peptides, activation of the MC type 1 (MC1) receptor has been identified as a viable target for the development of novel anti-inflammatory therapeutic agents. Here, we have tested a recently described selective agonist of MC1 receptors, BMS-470539, on leucocyte/post-capillary venule interactions in murine microvascular beds. Experimental approach: Intravital microscopy of two murine microcirculations were utilized, applying two distinct modes of promoting inflammation. The specificity of the effects of BMS-470539 was assessed using mice bearing mutant inactive MC1 receptors (the recessive yellow e/e colony). Key results: BMS-470539, given before an ischaemia–reperfusion protocol, inhibited cell adhesion and emigration with no effect on cell rolling, as assessed 90 min into the reperfusion phase. These properties were paralleled by inhibition of tissue expression of both CXCL1 and CCL2. Confocal investigations of inflamed post-capillary venules revealed immunostaining for MC1 receptors on adherent and emigrated leucocytes. Congruently, the anti-inflammatory properties of BMS-470539 were lost in mesenteries of mice bearing the inactive mutant MC1 receptors. Therapeutic administration of BMS-470539 stopped cell emigration, but did not affect cell adhesion in the cremasteric microcirculation inflamed by superfusion with platelet-activating factor. Conclusions and implications: Activation of MC1 receptors inhibited leucocyte adhesion and emigration. Development of new chemical entities directed at MC1 receptors could be a viable approach in the development of novel anti-inflammatory therapeutic agents with potential application to post-ischaemic conditions.

The mammalian intestine houses a complex microbial community, which influences normal epithelial growth and development, and is integral to the repair of damaged intestinal mucosa(1-3). Restitution of injured mucosa involves the recruitment of immune cells, epithelial migration and proliferation(4,5). Although microenvironmental alterations have been described in wound healing(6), a role for extrinsic influences, such as members of the microbiota, has not been reported. Here, we show that a distinct subpopulation of the normal mucosal-associated gut microbiota expands and preferentially colonizes sites of damaged murine mucosa in response to local environmental cues. Our results demonstrate that formyl peptide receptor 1 (FPR1) and neutrophilic NADPH oxidase (NOX2) are required for the rapid depletion of microenvironmental oxygen and compensatory responses, resulting in a dramatic enrichment of an anaerobic bacterial consortium. Furthermore, the dominant member of this wound-mucosa-associated microbiota, Akkermansia muciniphila (an anaerobic, mucinophilic gut symbiont(7,8)), stimulated proliferation and migration of enterocytes adjacent to the colonic wounds in a process involving FPR1 and intestinal epithelial-cell-specific NOX1-dependent redox signalling. These findings thus demonstrate how wound microenvironments induce the rapid emergence of 'probiont' species that contribute to enhanced repair of mucosal wounds. Such microorganisms could be exploited as potential therapeutics.

The epithelium serves as a highly selective barrier at mucosal surfaces. Upon injury, epithelial wound closure is orchestrated by a series of events that emanate from the epithelium itself as well as by the temporal recruitment of immune cells into the wound bed. Epithelial cells adjoining the wound flatten out, migrate, and proliferate to rapidly cover denuded surfaces and re-establish mucosal homeostasis. This process is highly regulated by proteins and lipids, proresolving mediators such as Annexin A1 protein and resolvins released into the epithelial milieu by the epithelium itself and infiltrating innate immune cells including neutrophils and macrophages. Failure to achieve these finely tuned processes is observed in chronic inflammatory diseases that are associated with non-healing wounds. An improved understanding of mechanisms that mediate repair is important in the development of therapeutics aimed to promote mucosal wound repair.

Restitution of mucosal injury involves induced and coordinated proliferation and migration of intestinal epithelial cells. The commensal microbiota of the intestine is integral to the repair of dam...

In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, a-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.

Junctional adhesion molecule-A (JAM-A) is a tight junction protein that plays a key role in regulation of intestinal mucosal homeostasis. Previous studies have shown that loss of JAM-A decreases be...

Intestinal barrier function is regulated by epithelial tight junctions, structures that control paracellular permeability. JAM-A regulates epithelial permeability through association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton., Intestinal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional adhesion molecule-A (JAM-A) is a TJ-associated protein that regulates barrier; however, mechanisms linking JAM-A to epithelial permeability are poorly understood. Here we report that JAM-A associates directly with ZO-2 and indirectly with afadin, and this complex, along with PDZ-GEF1, activates the small GTPase Rap2c. Supporting a functional link, small interfering RNA–mediated down-regulation of the foregoing regulatory proteins results in enhanced permeability similar to that observed after JAM-A loss. JAM-A–deficient mice and cultured epithelial cells demonstrate enhanced paracellular permeability to large molecules, revealing a potential role of JAM-A in controlling perijunctional actin cytoskeleton in addition to its previously reported role in regulating claudin proteins and small-molecule permeability. Further experiments suggest that JAM-A does not regulate actin turnover but modulates activity of RhoA and phosphorylation of nonmuscle myosin, both implicated in actomyosin contraction. These results suggest that JAM-A regulates epithelial permeability via association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton.

Actin dynamics are necessary at multiple steps in the formation of multinucleated muscle cells. BAR domain proteins can regulate actin dynamics in several cell types, but have been little studied in skeletal muscle. Here, we identify novel functions for the N-BAR domain protein, Bridging integrator 3 (Bin3), during myogenesis in mice. Bin3 plays an important role in regulating myofiber size in vitro and in vivo. During early myogenesis, Bin3 promotes migration of differentiated muscle cells, where it colocalizes with F-actin in lamellipodia. In addition, Bin3 forms a complex with Rac1 and Cdc42, Rho GTPases involved in actin polymerization, which are known to be essential for myotube formation. Importantly, a Bin3-dependent pathway is a major regulator of Rac1 and Cdc42 activity in differentiated muscle cells. Overall, these data classify N-BAR domain proteins as novel regulators of actin-dependent processes in myogenesis, and further implicate BAR domain proteins in muscle growth and repair.

Maintenance of the epithelial barrier in the intestinal tract is necessary to protect the host from the hostile luminal environment. Phospholipase C-β (PLC-β) has been implicated to control myriad signaling cascades. However, the biological effects of selective PLC-β isozymes are poorly understood. We describe novel findings that lysophosphatidic acid (LPA) regulates PLC-β1 and PLC-β2 via two distinct pathways to enhance intestinal epithelial cell (IEC) proliferation and migration that facilitate wound closure and recovery of the intestinal epithelial barrier. LPA acting on the LPA1 receptor promotes IEC migration by facilitating the interaction of Gαq with PLC-β2. LPA-induced cell proliferation is PLC-β1 dependent and involves translocation of Gαq to the nucleus, where it interacts with PLC-β1 to induce cell cycle progression. An in vivo study using LPA1-deficient mice (Lpar1(-/-)) shows a decreased number of proliferating IECs and migration along the crypt-luminal axis. Additionally, LPA enhances migration and proliferation of IECs in an LPA1-dependent manner, and Lpar1(-/-) mice display defective mucosal wound repair that requires cell proliferation and migration. These findings delineate novel LPA1-dependent lipid signaling that facilitates mucosal wound repair via spatial targeting of distinct PLC-βs within the cell.

Ulcerative colitis and Crohn's disease frequently cause epithelial damage in the intestine, leading to gut inflammation accompanied by areas of ulceration. The regeneration of damaged mucosa as well as the restoration of intestinal homeostasis involve induced and coordinated proliferation and migration of intestinal epithelial cells. Commensal bacterial colonization of the intestine is essential for normal intestinal development, renewal, and repair. N-formyl peptide receptors (FPRs) are widely expressed pattern recognition receptors that can specifically bind and induce responses to host-derived and bacterial peptides and small molecules during repair of mucosal injury in a redox dependent manner. However, little is known about the host-microbiota crosstalk mediated by FPRs during repair of gut mucosal injuries. Herein, purpose of this study is to exploit the mechanism of mucosal healing promoted by a consortium of gut microbiota that preferentially colonizes ulcerated mucosa undergoing resealing. The regeneration of injured epithelial was studied using defined mechanical wounds inflicted in the mouse distal colon by employing a miniature endoscope and forceps. Microbiota studies were performed by high throughput sequencing of the V4 region of 16s rRNA gene of the bacteria harvested from mucosal wounds undergoing different stages of regenerative events. The high throughput sequencing analysis of bacterial 16s rDNA determined rapid, reversible spatiotemporal alterations in the composition and diversity of microbiota in the wound microenvironment. Our data demonstrated that these ecological changes are dependent on FPR1/NOX2-mediated local tissue hypoxia, depletion of muc2 mucin, and compensatory effect of the over expression of HIF1-regulated MUC3 mucin. Our data show that these events of mucosal regeneration enrich a dominant member of this wound-associated consortium, Akkermansia muciniphila, which is associated with the pro-restitutive function. A. muciniphila, an anaerobic, mucinophilic commensal bacterium, enhanced proliferation and migration of enterocytes adjacent to the colonic wound beds in a process involving FPR1/NOX1 dependent redox signaling. These findings highlight a novel role of FPR1 to promote changes in the wound microenvironment to such extent that enriches a specific mucosa-associated bacterium to enhance enterocyte migration and proliferation in an FPR1-mediated and redox-dependent fashion.

Stimulus-specific role for α5β1 in the process of leukocyte recruitment; fibronectin levels are increased in the inflamed tissue so that interaction with α5β1 becomes functional. The potential role of α5β1 (VLA-5) in leukocyte trafficking in zymosan-induced acute peritonitis was determined. In naïve mice, ∼98% of Gr1high cells (PMN) in bone marrow and circulation were α5β1-negative; these profiles were modestly affected by peritoneal injection of zymosan. In contrast, ∼30% of Gr1high cells recruited by zymosan (24 h) to the peritoneal cavity expressed α5β1. With respect to F4/80+ cells, ∼60% of bone marrow and peripheral blood populations expressed α5β1, with ∼90% positivity in resident cells of noninflamed peritoneum. Analysis of α5β1 expression revealed inflammation-dependent increased expression on Gr1high and F4/80+ cells in bone marrow, blood, and peritoneal cavity. Blockade of α5β1, by an anti-α5 mAb, attenuated zymosan-induced 24 h recruitment of Gr1high and F4/80+ cells. At least one underlying mechanism of this action was reduction of cell adhesion and transmigration across microvascular vessels, as revealed by intravital microscopy. Confocal analyses indicated that deposition of fibronectin, the principal ligand for α5β1, was up-regulated significantly on and around the inflamed mesenteric microvasculature. These data suggest that the effects of α5-blockade may be a result of inhibition of α5β1-dependent leukocyte adhesion to and migration along the fibronectin matrix. This is the first report that identifies a functional role for α5β1 in leukocyte trafficking during acute inflammation.