Constitutive STAT5 activation regulates Paneth and Paneth-like cells to control Clostridium difficile colitis.

Clostridium difficile impairs Paneth cells, driving intestinal inflammation that exaggerates colitis. Besides secreting bactericidal products to restrain C. difficile, Paneth cells act as guardians that constitute a niche for intestinal epithelial stem cell (IESC) regeneration. However, how IESCs are sustained to specify Paneth-like cells as their niche remains unclear. Cytokine-JAK-STATs are required for IESC regeneration. We investigated how constitutive STAT5 activation (Ca-pYSTAT5) restricts IESC differentiation towards niche cells to restrain C. difficile infection. We generated inducible transgenic mice and organoids to determine the effects of Ca-pYSTAT5-induced IESC lineages on C. difficile colitis. We found that STAT5 absence reduced Paneth cells and predisposed mice to C. difficile ileocolitis. In contrast, Ca-pYSTAT5 enhanced Paneth cell lineage tracing and restricted Lgr5 IESC differentiation towards pYSTAT5+Lgr5-CD24+Lyso+ or cKit+ niche cells, which imprinted Lgr5hiKi67+ IESCs. Mechanistically, pYSTAT5 activated Wnt/ß-catenin signaling to determine Paneth cell fate. In conclusion, Ca-pYSTAT5 gradients control niche differentiation. Lack of pYSTAT5 reduces the niche cells to sustain IESC regeneration and induces C. difficile ileocolitis. STAT5 may be a transcription factor that regulates Paneth cells to maintain niche regeneration.

Natural compound methyl protodioscin protects against intestinal inflammation through modulation of intestinal immune responses.

Dioscoreaceae, a kind of yam plant, has been recommended for treatment of chronic inflammatory conditions. However, the mechanisms are poorly defined. Methyl protodioscin (MPD) is one of the main bioactive components in Dioscoreaceae. Here, we aim to determine the mechanisms by which MPD ameliorates intestinal inflammation. Surgical intestinal specimens were collected from inflammatory bowel diseases (IBD) patients to perform organ culture. Experimental colitis was induced in mice by dextran sulfate sodium (DSS) or Citrobacter rodentium, and was then treated with MPD. NF-κB activation, expression of mucosal pro-inflammatory cytokines, disease severity, and epithelial proliferation/apoptosis were determined. Mouse crypts and Caco-2 monolayers were cultured to observe the effect of MPD upon intestinal epithelial differentiation and barrier function. We found that MPD increased the percentage of survival from high-dose DSS-(4%) treated mice, and accelerated mucosal healing and epithelial proliferation in low-dose DSS-(2.5%) treated mice characterized by marked reduction in NF-κB activation, pro-inflammatory cytokines expression and bacterial translocation. Consistently, MPD protected colonic mucosa from C. rodentium-induced colonic inflammation and bacterial colonization. In vitro studies showed that MPD significantly increased crypt formation and restored intestinal barrier dysfunction induced by pro-inflammatory cytokines. In conclusion, MPD ameliorates the intestinal mucosal inflammation by modulating the intestinal immunity to enhance intestinal barrier differentiation. MPD could be an alternative for treating chronic intestinal inflammatory diseases.

Activated STAT5 confers resistance to intestinal injury by increasing intestinal stem cell proliferation and regeneration.

Intestinal epithelial stem cells (IESCs) control the intestinal homeostatic response to inflammation and regeneration. The underlying mechanisms are unclear. Cytokine-STAT5 signaling regulates intestinal epithelial homeostasis and responses to injury. We link STAT5 signaling to IESC replenishment upon injury by depletion or activation of Stat5 transcription factor. We found that depletion of Stat5 led to deregulation of IESC marker expression and decreased LGR5(+) IESC proliferation. STAT5-deficient mice exhibited worse intestinal histology and impaired crypt regeneration after γ-irradiation. We generated a transgenic mouse model with inducible expression of constitutively active Stat5. In contrast to Stat5 depletion, activation of STAT5 increased IESC proliferation, accelerated crypt regeneration, and conferred resistance to intestinal injury. Furthermore, ectopic activation of STAT5 in mouse or human stem cells promoted LGR5(+) IESC self-renewal. Accordingly, STAT5 promotes IESC proliferation and regeneration to mitigate intestinal inflammation. STAT5 is a functional therapeutic target to improve the IESC regenerative response to gut injury.

Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation.

Epithelial myosin light chain kinase (MLCK)-dependent barrier dysfunction contributes to the pathogenesis of inflammatory bowel diseases (IBD). We reported that epithelial GM-CSF-STAT5 signalling is essential for intestinal homeostatic response to gut injury. However, mechanism, redundancy by STAT5 or cell types involved remained foggy. We here generated intestinal epithelial cell (IEC)-specific STAT5 knockout mice, these mice exhibited a delayed mucosal wound healing and dysfunctional intestinal barrier characterized by elevated levels of NF-κB activation and MLCK, and a reduction of zonula occludens expression in IECs. Deletion of MLCK restored intestinal barrier function in STAT5 knockout mice, and facilitated mucosal wound healing. Consistently, knockdown of stat5 in IEC monolayers led to increased NF-κB DNA binding to MLCK promoter, myosin light chain phosphorylation and tight junction (TJ) permeability, which were potentiated by administration of tumour necrosis factor-α (TNF-α), and prevented by concurrent NF-κB knockdown. Collectively, enterocyte STAT5 signalling protects against TJ barrier dysfunction and promotes intestinal mucosal wound healing via an interaction with NF-κB to suppress MLCK. Targeting IEC STAT5 signalling may be a novel therapeutic approach for treating intestinal barrier dysfunction in IBD.

Loss of guanylyl cyclase C (GCC) signaling leads to dysfunctional intestinal barrier.

BACKGROUND: Guanylyl Cyclase C (GCC) signaling via uroguanylin (UGN) and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function. METHODOLOGY/PRINCIPAL FINDINGS: Paracellular permeability of intestinal segments was assessed in wild type (WT) and GCC deficient (GCC-/-) mice with and without lipopolysaccharide (LPS) challenge, as well as in UGN deficient (UGN-/-) mice. IFNγ and myosin light chain kinase (MLCK) levels were determined by real time PCR. Expression of tight junction proteins (TJPs), phosphorylation of myosin II regulatory light chain (MLC), and STAT1 activation were examined in intestinal epithelial cells (IECs) and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi). We found that intestinal permeability was increased in GCC-/- and UGN-/- mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC-/- mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC-/- and UGN-/- mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability. CONCLUSIONS/SIGNIFICANCE: GCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury.

Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury.

BACKGROUND: Administration of granulocyte-macrophage colony stimulating factor (GM-CSF) relieves symptoms in Crohn's disease (CD). It has been reported that reduced GM-CSF bioactivity is associated with more aggressive ileal behaviour and that GM-CSF-null mice exhibit ileal barrier dysfunction and develop a transmural ileitis following exposure to non-steroidal anti-inflammatory drugs (NSAIDs). STAT5 signalling is central to GM-CSF action. It was therefore hypothesised that GM-CSF signalling in non-haematopoietic cells is required for ileal homeostasis. METHODS: Bone marrow (BM) chimeras were generated by reconstituting irradiated GM-CSF receptor (gm-csfr) beta chain or GM-CSF (gm-csf) deficient mice with wild type BM (WTBM-->GMRKO and WTBM-->GMKO). Intestinal barrier function and the response to NSAID-induced ileal injury were examined. Expression of gm-csf, gm-csfr or stat5 in Caco-2 and HT-29 intestinal epithelial cell (IEC) lines was knocked down and the effect of GM-CSF signalling on IEC survival and proliferation was determined. RESULTS: Elevated levels of GM-CSF autoantibodies in ileal CD were found to be associated with dysregulation of IEC survival and proliferation. GM-CSF receptor-deficient mice and WTBM-->GMRKO chimeras exhibited ileal hyperpermeability. NSAID exposure induced a transmural ileitis in GM-CSF receptor-deficient mice and WTBM-->GMRKO chimeras. Transplantation of wild type BM into GM-CSF-deficient mice prevented NSAID ileal injury and restored ileal barrier function. Ileal crypt IEC proliferation was reduced in WTBM-->GMRKO chimeras, while STAT5 activation in ileal IEC following NSAID exposure was abrogated in WTBM-->GMRKO chimeras. Following knock down of gm-csf, gm-csfr alpha or beta chain or stat5a/b expression in Caco-2 cells, basal proliferation was suppressed. GM-CSF normalised proliferation of Caco-2 cells exposed to NSAID, which was blocked by stat5a/b RNA interference. CONCLUSIONS: Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury; furthermore, GM-CSF signalling in non-haematopoietic cells regulates ileal epithelial homeostasis via the STAT5 pathway. The therapeutic use of GM-CSF may therefore be beneficial in chronic ileitis associated with CD.