Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis

Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 β release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD⁺:NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis.     

ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis

The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b^−/− mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b^−/− mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.     

Identification of genes involved in mucosal defense and inflammation associated with normal enteric bacteria

Normal luminal bacteria and their products play a role in experimental colitis and inflammatory bowel disease. However, what molecules from what cells are responsible for mounting and maintaining the mucosal defense against luminal flora is still uncertain. The aim of this study was to identify epithelial gene products involved in mucosal defense and inflammation associated with ubiquitous enteric bacteria. Germ-free ICR mice were given an oral bacterial suspension prepared from conventional components (bacterial reconstitution). Small intestinal and colonic epithelial cells were isolated from bacteria-reconstituted, germ-free, and specific pathogen-free mice. Differential gene expression was investigated by differential display, Northern blot, and sequence analysis. Bacterial reconstitution resulted in acute but self-limited colitis. In epithelial cells, we observed the induction of small intestine-specific genes of the cryptdin family and colon-specific expression of serum amyloid A1 gene. This novel approach allows the identification of known and novel gene products involved in mucosal defense against luminal microorganisms and the associated inflammatory response.     

Persistent epithelial dysfunction and bacterial translocation after resolution of intestinal inflammation

Epithelial secretion may play an important role in reducing bacterial colonization and translocation in intestine. If so, secretory dysfunction could result in increased susceptibility to infection and inflammation. We investigated whether long-term colonic secretory dysfunction occurs after a bout of colitis and if this is accompanied by an increase in bacterial colonization and translocation. Rats were studied 6 wk after induction of colitis with trinitrobenzene sulfonic acid when inflammation had completely resolved, and epithelial permeability was normal. Intestinal loops were stimulated with either Clostridium difficile toxin A or a phosphodiesterase inhibitor. In vitro, colonic tissue from previously sensitized rats was exposed to antigen (ovalbumin). Secretory responses to all three stimuli were suppressed in rats that had previously had colitis. These rats exhibited increased (16-fold) numbers of colonic aerobic bacteria and increased (>3-fold) bacterial translocation, similar to results in rats studied after resolution of enteritis. Postcolitis bacterial translocation was prevented by daily treatment with an inhibitor of inducible nitric oxide synthase. This study demonstrates that intestinal inflammation results in prolonged impairment of colonic epithelial secretion, which may contribute to increases in bacterial load and bacterial translocation. Epithelial dysfunction of this type could underlie an increased propensity for further bouts of inflammation, a hallmark of diseases such as inflammatory bowel disease.     

IL13 activates autophagy to regulate secretion in airway epithelial cells

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.     

Berberine promotes recovery of colitis and inhibits inflammatory responses in colonic macrophages and epithelial cells in DSS-treated mice

Inflammatory bowel disease (IBD) results from dysregulation of intestinal mucosal immune responses to microflora in genetically susceptible hosts. A major challenge for IBD research is to develop new strategies for treating this disease. Berberine, an alkaloid derived from plants, is an alternative medicine for treating bacterial diarrhea and intestinal parasite infections. Recent studies suggest that berberine exerts several other beneficial effects, including inducing anti-inflammatory responses. This study determined the effect of berberine on treating dextran sulfate sodium (DSS)-induced intestinal injury and colitis in mice. Berberine was administered through gavage to mice with established DSS-induced intestinal injury and colitis. Clinical parameters, intestinal integrity, proinflammatory cytokine production, and signaling pathways in colonic macrophages and epithelial cells were determined. Berberine ameliorated DSS-induced body weight loss, myeloperoxidase activity, shortening of the colon, injury, and inflammation scores. DSS-upregulated proinflammatory cytokine levels in the colon, including TNF, IFN-γ, KC, and IL-17 were reduced by berberine. Berberine decreased DSS-induced disruption of barrier function and apoptosis in the colon epithelium. Furthermore, berberine inhibited proinflammatory cytokine production in colonic macrophages and epithelial cells in DSS-treated mice and promoted apoptosis of colonic macrophages. Activation of signaling pathways involved in stimulation of proinflammatory cytokine production, including MAPK and NF-κB, in colonic macrophages and epithelial cells from DSS-treated mice was decreased by berberine. In summary, berberine promotes recovery of DSS-induced colitis and exerts inhibitory effects on proinflammatory responses in colonic macrophages and epithelial cells. Thus berberine may represent a new therapeutic approach for treating gastrointestinal inflammatory disorders.     

Autotaxin (ATX) inhibits autophagy leading to exaggerated disruption of intestinal epithelial barrier in colitis

Inflammatory bowel disease (IBD) is an immune-mediated disease. Autotaxin (ATX) is associated with increased inflammatory molecules, however, its effect on IBD is not well understood. Autophagy plays an important role in IBD, whether ATX and autophagy act in concert in IBD remains unknown. This study is to explore the possible mechanisms of ATX affecting autophagy leading to the disruption of intestinal epithelial barrier, thereby exacerbating colitis. The expression of ATX was upregulated in UC patients and dextran sulfate sodium (DSS)-induced colitis mice. Here, we described that providing an ATX inhibitor during DSS colitis increased autophagy and ameliorated colonic inflammation. Conversely, intrarectal administration with recombinant (r)ATX increased colitis and decreased autophagy. This pro-colitic effect was attenuated in mice treated with rapamycin, resulting in increased autophagy activity and mild colitis. Moreover, the inhibitory effect of rATX on autophagy was confirmed in vitro and was reversed by the addition of rapamycin. The damaging effects of ATX on epithelial barrier function were reversed by ATX inhibitor or rapamycin treatment. In sum, our results show that ATX can inhibit autophagy through the mTOR pathway, resulting in exaggerated damage to the intestinal epithelial barrier during colitis. These findings suggest that ATX may be a key pro-colitic factor, and represent a potential therapeutic target for treating IBD in the future.     

A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon

Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin-gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2-10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with < or = 3 copies have a significantly higher risk of developing colonic CD than did individuals with > or = 4 copies (odds ratio 3.06; 95% confidence interval 1.46-6.45). An HBD-2 gene copy number of < 4 was associated with diminished mucosal HBD-2 mRNA expression (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression.     

Toll-like receptor 2 plays a critical role in maintaining mucosal integrity during Citrobacter rodentium-induced colitis

Inflammatory bowel diseases and infectious gastroenteritis likely occur when the integrity of intestinal barriers is disrupted allowing luminal bacterial products to cross into the intestinal mucosa, stimulating immune cells and triggering inflammation. While specific Toll-like receptors (TLR) are involved in the generation of inflammatory responses against enteric bacteria, their contributions to the maintenance of intestinal mucosal integrity are less clear. These studies investigated the role of TLR2 in a model of murine colitis induced by the bacterial pathogen Citrobacter rodentium . C. rodentium supernatants specifically activated TLR2 in vitro while infected TLR2–/– mice suffered a lethal colitis coincident with colonic mucosal ulcerations, bleeding and increased cell death but not increased pathogen burden. TLR2–/– mice suffered impaired epithelial barrier function mediated via zonula occludens (ZO)-1 in naïve mice and claudin-3 in infected mice, suggesting this could underlie their susceptibility. TLR2 deficiency was also associated with impaired production of IL-6 by bone marrow-derived macrophages and infected colons cultured ex vivo . As IL-6 has antiapoptotic and epithelial repair capabilities, its reduced expression could contribute to the impaired mucosal integrity. These studies report for the first time that TLR2 plays a critical role in maintaining intestinal mucosal integrity during infection by a bacterial pathogen.     

MyD88 signalling plays a critical role in host defence by controlling pathogen burden and promoting epithelial cell homeostasis during Citrobacter rodentium-induced colitis

Myeloid differentiation factor (MyD)88, an adaptor protein shared by the Toll-interleukin 1 receptor superfamily, plays a critical role in host defence during many systemic bacterial infections by inducing protective inflammatory responses that limit bacterial growth. However, the role of innate responses during gastrointestinal (GI) infections is less clear, in part because the GI tract is tolerant to commensal antigens. The current study investigated the role of MyD88 following infection by the murine bacterial pathogen, Citrobacter rodentium . MyD88-deficient mice suffered a lethal colitis coincident with colonic mucosal ulcerations and bleeding. Their susceptibility was associated with an overwhelming bacterial burden and selectively impaired immune responses in colonic tissues, which included delayed inflammatory cell recruitment, reduced iNOS and abrogated production of TNF-α and IL-6 from MyD88-deficient macrophages and colons cultured ex vivo . Immunostaining for Ki67 and BrDU revealed that MyD88 signalling mediated epithelial hyper-proliferation in response to C. rodentium infection. Thus, MyD88-deficient mice could not promote epithelial cell turnover and repair, leading to deep bacterial invasion of colonic crypts, intestinal barrier dysfunction and, ultimately, widespread mucosal ulcerations. In conclusion, MyD88 signalling within the GI tract plays a critical role in mediating host defence against an enteric bacterial pathogen, by controlling bacterial numbers and promoting intestinal epithelial homeostasis.     

A new role for cathelicidin in ulcerative colitis in mice

Cathelicidin, an antimicrobial peptide of the innate immune system, modulates microbial growth, wound healing, and inflammation. However, its association with inflammatory bowel diseases (IBDs) is unknown. Our objective was to determine whether cathelicidin would exert a modulatory effect on the progression of IBD and, if so, investigate the mechanism of action through which this effect occurred. We evaluated the potential for a synthetic cathelicidin, the mouse cathelin-related antimicrobial peptide (mCRAMP), to prevent the initiation and promote the healing of lesions from inflammatory colitis that was experimentally induced in mice with dextran sulfate sodium (DSS). During the experiment, mCRAMP was given: (i) as a parallel treatment starting together with 3% DSS feeding, and (ii) as a posttreatment starting 7 days after 3% DSS feeding. The body weight, fecal microflora populations, clinical symptoms, and histologic findings of colonic tissues were measured. Relative gene expression of mucins (MUC1, MUC2, MUC3, and MUC4) in colonic tissues was determined by real-time polymerase chain reaction. Intrarectal administration of mCRAMP ameliorated DSS-induced colitis with negligible effects on mucosal healing. The peptide also significantly reduced the increased number of fecal microflora in colitis animals. It reversed the decline of colonic mucus thickness during colitis through upregulation of the expression of mucin genes. Treatment with mCRAMP also prevented colitis development by suppressing the induction of apoptosis by DSS. The current study demonstrates for the first time that intrarectal administration of cathelicidin may be a novel therapeutic option for IBDs.     

Intestinal macrophage/epithelial cell-derived CCL11/eotaxin-1 mediates eosinophil recruitment and function in pediatric ulcerative colitis

Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn's disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2(-/-) and eotaxin-1/2(-/-) mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80(+)CD11b(+) macrophages in DSS-induced epithelial injury and to CD68(+) intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.     

Colonic microbiota alters host susceptibility to infectious colitis by modulating inflammation, redox status, and ion transporter gene expression

Individuals vary in their resistance to enteric infections. The role of the intestinal microbiota in altering susceptibility to enteric infection is relatively unknown. Previous studies have identified that C3H/HeOuJ mice suffer 100% mortality during Citrobacter rodentium-induced colitis, whereas C57BL/6 mice recover from infection. The basis for their differences in susceptibility is unclear and has been mainly attributed to differences in host genetics. This study investigated the role of the intestinal microbiota in altering susceptibility to C. rodentium-induced colitis. When the feces of C57BL/6 mice were gavaged into antibiotic treated C3H/HeOuJ mice, the C57BL/6 microflora led to a complete reversal in mortality patterns where 100% of the C3H/HeOuJ mice survived infection. This protection corresponded with reduced colonic pathology and less systemic pathogen load and was associated with increased inflammatory and redox responses with reduced epithelial cell death. C3H/HeOuJ mice are normally susceptible to infection-induced dehydration due to defective expression of colonic ion transporters such as Dra, CA IV, and CA I; expression of these genes was normalized when C3H/HeOuJ mice were colonized with the C57BL/6 microflora. Together, these data reveal that the colonic microbiota play a critical role in protecting against intestinal infection by inducing proinflammatory and prooxidant responses that control pathogen load as well as ion transporter gene expression previously shown to prevent fatal dehydration. Protection of mice from lethal colitis was associated with higher levels of bacteria from Bacteroidetes. This study reveals that the microbiota is sufficient to overcome inherent genetic susceptibility patterns in C3H/HeOuJ mice that cause mortality during C. rodentium infection.     

Chronic Psychological Stress Disrupted the Composition of the Murine Colonic Microbiota and Accelerated a Murine Model of Inflammatory Bowel Disease

The effect of psychological stress on the gastrointestinal microbiota is widely recognized. Chronic psychological stress may be associated with increased disease activity in inflammatory bowel disease, but the relationships among psychological stress, the gastrointestinal microbiota, and the severity of colitis is not yet fully understood. Here, we examined the impact of 12-week repeated water-avoidance stress on the microbiota of two inbred strains of T cell receptor alpha chain gene knockout mouse (background, BALB/c and C57BL/6) by means of next-generation sequencing of bacterial 16S rRNA genes. In both mouse strains, knockout of the T cell receptor alpha chain gene caused a loss of gastrointestinal microbial diversity and stability. Chronic exposure to repeated water-avoidance stress markedly altered the composition of the colonic microbiota of C57BL/6 mice, but not of BALB/c mice. In C57BL/6 mice, the relative abundance of genus Clostridium, some members of which produce the toxin phospholipase C, was increased, which was weakly positively associated with colitis severity, suggesting that expansion of specific populations of indigenous pathogens may be involved in the exacerbation of colitis. However, we also found that colitis was not exacerbated in mice with a relatively diverse microbiota even if their colonic microbiota contained an expanded phospholipase C-producing Clostridium population. Exposure to chronic stress also altered the concentration of free immunoglobulin A in colonic contents, which may be related to both the loss of bacterial diversity in the colonic microbiota and the severity of the colitis exacerbation. Together, these results suggest that long-term exposure to psychological stress induces dysbiosis in the immunodeficient mouse in a strain-specific manner and also that alteration of microbial diversity, which may be related to an altered pattern of immunoglobulin secretion in the gastrointestinal tract, might play a crucial role in the development of chronic stress-induced colitis.     

Saccharomyces boulardii ameliorates Citrobacter rodentium-induced colitis through actions on bacterial virulence factors

Saccharomyces boulardii has received increasing attention as a probiotic effective in the prevention and treatment of infectious and inflammatory bowel diseases. The aim of this study was to examine the ameliorating effects of S. boulardii on Citrobacter rodentium colitis in vivo and identify potential mechanisms of action. C57BL/6 mice received 2.5 x 10(8) C. rodentium by gavage on day 0, followed by S. boulardii (25 mg; 5 x 10(8) live cells) gavaged twice daily from day 2 to day 9. Animal weights were monitored until death on day 10. Colons were removed and assessed for epithelial barrier function, histology, and myeloperoxidase activity. Bacterial epithelial attachment and type III secreted proteins translocated intimin receptor Tir (the receptor for bacterial intimin) and EspB (a translocation apparatus protein) required for bacterial virulence were assayed. In infected mice, S. boulardii treatment significantly attenuated weight loss, ameliorated crypt hyperplasia (234.7 +/- 7.2 vs. 297.8 +/- 17.6 microm) and histological damage score (0.67 +/- 0.67 vs. 4.75 +/- 0.75), reduced myeloperoxidase activity (2.1 +/- 0.4 vs. 4.7 +/- 0.9 U/mg), and attenuated increased mannitol flux (17.2 +/- 5.0 vs. 31.2 +/- 8.2 nm.cm(-2).h(-1)). The ameliorating effects of S. boulardii were associated with significantly reduced numbers of mucosal adherent C. rodentium, a marked reduction in Tir protein secretion and translocation into mouse colonocytes, and a striking reduction in EspB expression and secretion. We conclude that S. boulardii maintained colonic epithelial barrier integrity and ameliorated inflammatory sequelae associated with C. rodentium infection by attenuating C. rodentium adherence to host epithelial cells through putative actions on the type III secretion system.     

Increased colonic apelin production in rodents with experimental colitis and in humans with IBD

Apelin and its receptor, the APJ receptor, are expressed in the gastrointestinal tract. The aims of this study were to examine the effects of sodium dextran sulfate (DSS)-induced experimental colitis in rats and mice and inflammatory bowel disease (IBD) in humans on intestinal apelin production, and the influence of exogenous apelin on colonic epithelial cell proliferation in mice. In rodents with experimental colitis, colonic apelin mRNA levels were elevated during the inflammatory reaction as well as during the tissue repair phase that ensues after DSS withdrawal. Fluctuations in colonic apelin expression were paralleled by similar changes in apelin immunostaining. Apelin immunostaining was increased in the surface epithelium, in epithelial cells along the length of the tubular gland and in the stem cell region at the gland base. In ulcerative colitis (UC) and Crohn's disease patients, apelin immunostaining revealed a pattern of increased intestinal apelin content similar to that observed in rodents with experimental colitis. Administration of synthetic apelin to mice during the recovery phase of DSS-induced colitis stimulated colonic epithelial cell proliferation significantly. Our observations that colonic apelin production is increased during and after DSS exposure indicate that apelin plays multiple roles during the different stages of colitis. Additionally, the stimulatory action of exogenous apelin on colonic epithelial proliferation suggests that the increased apelin production during intestinal recovery stage may contribute to the repair of the intestinal epithelium in experimental rodent models of colitis and in IBD patients.     

Relative contributions of NOS isoforms during experimental colitis: endothelial-derived NOS maintains mucosal integrity

The role of nitric oxide (NO) in inflammatory bowel diseases has traditionally focused on the inducible form of NO synthase (iNOS). However, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms may also impact on colitis, either by contributing to the inflammation or by regulating mucosal integrity in response to noxious stimuli. To date, studies examining the roles of the NOS isoforms in experimental colitis have been conflicting, and the mechanisms by which these enzymes exert their effects remain unclear. To investigate and clarify the roles of the NOS isoforms in gut inflammation, we induced trinitrobenzenesulfonic acid colitis in eNOS, nNOS, and iNOS knockout (KO) mice, assessing the course of colitis at early and late times. Both eNOS and iNOS KO mice developed a more severe colitis compared with wild-type mice. During colitis, iNOS expression dramatically increased on epithelial and lamina propria mononuclear cells, whereas eNOS expression remained localized to endothelial cells. Electron and fluorescence microscopy identified bacteria in the ulcerated colonic mucosa of eNOS KO mice, but not in wild-type, iNOS, or nNOS KO mice. Furthermore, eNOS KO mice had fewer colonic goblet cells, impaired mucin production, and exhibited increased susceptibility to an inflammatory stimulus that was subthreshold to other mice. This susceptibility was reversible, because the NO donor isosorbide dinitrate normalized goblet cell numbers and ameliorated subsequent colitis in eNOS KO mice. These results identify a protective role for both iNOS and eNOS during colitis, with eNOS deficiency resulting in impaired intestinal defense against lumenal bacteria and increased susceptibility to colitis.     

Vasoactive Intestinal Polypeptide Promotes Intestinal Barrier Homeostasis and Protection Against Colitis in Mice

Inflammatory bowel disease is a chronic gastrointestinal inflammatory disorder associated with changes in neuropeptide expression and function, including vasoactive intestinal peptide (VIP). VIP regulates intestinal vasomotor and secretomotor function and motility; however, VIP’s role in development and maintenance of colonic epithelial barrier homeostasis is unclear. Using VIP deficient (VIPKO) mice, we investigated VIP’s role in epithelial barrier homeostasis, and susceptibility to colitis. Colonic crypt morphology and epithelial barrier homeostasis were assessed in wildtype (WT) and VIPKO mice, at baseline. Colitic responses were evaluated following dinitrobenzene sulfonic acid (DNBS) or dextran-sodium sulfate (DSS) exposure. Mice were also treated with exogenous VIP. At baseline, VIPKO mice exhibited distorted colonic crypts, defects in epithelial cell proliferation and migration, increased apoptosis, and altered permeability. VIPKO mice also displayed reduced goblet cell numbers, and reduced expression of secreted goblet cell factors mucin 2 and trefoil factor 3. These changes were associated with reduced expression of caudal type homeobox 2 (Cdx2), a master regulator of intestinal function and homeostasis. DNBS and DSS-induced colitis were more severe in VIPKO than WT mice. VIP treatment rescued the phenotype, protecting VIPKO mice against DSS colitis, with results comparable to WT mice. In conclusion, VIP plays a crucial role in the development and maintenance of colonic epithelial barrier integrity under physiological conditions and promotes epithelial repair and homeostasis during colitis.     

Prohibitin 1 modulates mitochondrial stress-related autophagy in human colonic epithelial cells

Introduction

Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn''s disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.

Methods

We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A₁ or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.

Results

TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.

Conclusions

Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease.