Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD.
We previously reported that high levels of plasma neurotensin (NT), a gut hormone released from enteroendocrine cells of the small bowel, contribute to obesity and comorbid conditions. Gut microbiota has been implicated in the obesity development. Paneth cells are critical in maintaining gut microbiota composition and homeostasis by releasing antimicrobial proteins including α-defensins. The purpose of our current study was to determine the possible role of NT in gut microbiota composition and α-defensin gene expression associated with obesity. Here we show that the ratio of Firmicutes/Bacteroidetes (F/B ratio) and intestinal proinflammatory cytokines is significantly increased in NT+/+ mice fed with a high-fat diet (HFD) which were improved in NT-deficient mice. HFD disrupted the intestinal Mmp7/α-defensin axis, which was completely prevented in NT-/- mice. In addition, NT treatment inhibited DEFA5 expression and concurrent NF-κB activity, which was blocked by a pan PKC inhibitor (Gö6983) or an inhibitor for atypical PKCs (CRT0066854). More importantly, the shRNA-mediated knockdown of atypical PKCτ reversed NT-attenuated DEFA5 expression and increased NF-κB activity. NT contributes to the HFD-induced disruption of gut microbiota composition and α-defensin expression. PKCτ/λ plays a central role in NT-mediated α-defensin gene expression which might be mediated through the inhibition of NF-κB signaling pathways in Paneth cells.
Mucosal homeostasis and wound repair require epithelial proliferation and migration. Gut commensal microbiota are essential for normal intestinal development, renewal and repair. N‐formyl peptide receptors (FPRs) are G protein coupled receptors that function as pattern recognition receptors. FPR ligands include an array of peptides, lipids and small molecules derived from bacteria and the host. Intestinal epithelial cells (IECs) express functional FPRs in the cell surface plasma membrane. Recently we demonstrated that Annexin A1 and specific gut microbiota stimulate epithelial FPRs to generate reactive oxygen species which modulate cell signaling pathways critical for IEC proliferation and migration. However, it is incompletely understood how FPR1 maintains homeostasis of the gut symbionts important for mucosal wound healing. Here, we studied composition of murine mucosa‐associated microbiota in intact epithelia and endoscopic wound beds by pyrosequencing. We identified that specific species of essential gut bacteria is associated with mucosal wound bed. We also observed that FPR1 maintains homeostasis of mucosa‐associated microbiota characterized by their enrichment, composition, and spatial localization with respect to IECs. These changes are also associated with alterations in the colonic firm mucin layer, increased epithelial permeability and defective mucosal wound healing in FPR1 ‐/‐ mice during chronic DSS‐induced colitis. In conclusion, our study highlights a role of FPR1 in mediating important host‐microbiota interactions controlling gut mucosal homeostasis and wound repair. Grant Funding Source : CCFA and NIH
The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.
BackgroundThe microbiota of the intestinal lumen is essential for normal gut tissue development, renewal and restitution. Understanding how enteric commensal bacteria interact with intestinal epithelia via pattern recognition receptors (PRRs) is a crucial step in the delineation of mechanisms for intestinal homeostasis and reparative processes that can be harnessed for the design of novel therapeutics for inflammatory bowel disease (IBD). Pattern recognition receptors expressed in the epithelial mucosa maintain homeostatic balance with the microbiota and ensure clearance of pathogenic bacteria. N-formyl peptide receptors (FPRs) represent a family of mammalian pattern recognition receptors that can specifically bind an array of peptides and small molecules derived from bacteria and host. FPRs are seven membrane-pass G protein coupled surface receptors, which modulate multiple phagocyte immune functions. Intestinal epithelial cells also express FPRs that can stimulate migration of cultured intestinal epithelial cells in response to exogenous bacterial formylated peptides or endogenous ligands in a redox dependent manner. However, it is incompletely understood how gut symbionts function as a pro-healing and -homeostatic regulator in the intestine.
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 damaged intestinal mucosa. N‐formyl peptide receptors (FPRs) are widely expressed pattern recognition receptors that specifically respond to host‐derived and microbial peptides in a redox dependent manner. However, little is known about the host‐microbiota crosstalk mediated by FPRs during repair of gut mucosal injuries. We hypothesized that distinct members of the gut microbiota preferentially colonize the restitutive mucosa to promote wound repair processes in a FPR1‐dependent fashion. For this purpose, defined mechanical wounds were inflicted in the mouse distal colon by using an endoscope and forceps. High throughput sequencing of the V4 region of 16s rRNA gene of the bacteria harvested from mucosal wounds revealed a spatiotemporal shift in the composition and diversity of microbiota. We found several FPR1 and NADPH oxidase NOX2‐dependent microenvironmental changes in mucosal wounds, which include rapid induction of a hypoxic microenvironment, and expression of several HIF1a‐regulated genes, including the mucin muc 3. Interestingly, these events were followed by a dramatic enrichment of a distinct commensal species Akkermansia , an anaerobic, mucinophilic bacterium, which stimulates FPR1 on intestinal epithelial cells to generate reactive oxygen species (ROS) via enterocyte NOX1, resulting in enhanced migration & proliferation of enterocytes. These findings identify a novel role of FPR1 to promote microenvironmental changes that enrich a specific mucosa‐associated bacterium to enhance gut mucosal wound restitution.
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.
