Interplay of gut microbiota and host epithelial mitochondrial dysfunction is necessary for the development of spontaneous intestinal inflammation in mice.

BACKGROUND: Intestinal epithelial cell (IEC) mitochondrial dysfunction involvement in inflammatory bowel diseases (IBD), including Crohn's disease affecting the small intestine, is emerging in recent studies. As the interface between the self and the gut microbiota, IECs serve as hubs of bidirectional cross-talk between host and luminal microbiota. However, the role of mitochondrial-microbiota interaction in the ileum is largely unexplored. Prohibitin 1 (PHB1), a chaperone protein of the inner mitochondrial membrane required for optimal electron transport chain function, is decreased during IBD. We previously demonstrated that mice deficient in PHB1 specifically in IECs (Phb1i∆IEC) exhibited mitochondrial impairment, Paneth cell defects, gut microbiota dysbiosis, and spontaneous inflammation in the ileum (ileitis). Mice deficient in PHB1 in Paneth cells (epithelial secretory cells of the small intestine; Phb1∆PC) also exhibited mitochondrial impairment, Paneth cell defects, and spontaneous ileitis. Here, we determined whether this phenotype is driven by Phb1 deficiency-associated ileal microbiota alterations or direct effects of loss of PHB1 in host IECs. RESULTS: Depletion of gut microbiota by broad-spectrum antibiotic treatment in Phb1∆PC or Phb1i∆IEC mice revealed a necessary role of microbiota to cause ileitis. Using germ-free mice colonized with ileal microbiota from Phb1-deficient mice, we show that this microbiota could not independently induce ileitis without host mitochondrial dysfunction. The luminal microbiota phenotype of Phb1i∆IEC mice included a loss of the short-chain fatty acid butyrate. Supplementation of butyrate in Phb1-deficient mice ameliorated Paneth cell abnormalities and ileitis. Phb1-deficient ileal enteroid models suggest deleterious epithelial-intrinsic responses to ileal microbiota that were protected by butyrate. CONCLUSIONS: These results suggest a mutual and essential reinforcing interplay of gut microbiota and host IEC, including Paneth cell, mitochondrial health in influencing ileitis. Restoration of butyrate is a potential therapeutic option in Crohn's disease patients harboring epithelial cell mitochondrial dysfunction. Video Abstract.

Distinct immune modulatory roles of regulatory T cells in gut versus joint inflammation in TNF-driven spondyloarthritis.

OBJECTIVES: Gut and joint inflammation commonly co-occur in spondyloarthritis (SpA) which strongly restricts therapeutic modalities. The immunobiology underlying differences between gut and joint immune regulation, however, is poorly understood. We therefore assessed the immunoregulatory role of CD4+FOXP3+ regulatory T (Treg) cells in a model of Crohn's-like ileitis and concomitant arthritis. METHODS: RNA-sequencing and flow cytometry was performed on inflamed gut and joint samples and tissue-derived Tregs from tumour necrosis factor (TNF)∆ARE mice. In situ hybridisation of TNF and its receptors (TNFR) was applied to human SpA gut biopsies. Soluble TNFR (sTNFR) levels were measured in serum of mice and patients with SpA and controls. Treg function was explored by in vitro cocultures and in vivo by conditional Treg depletion. RESULTS: Chronic TNF exposure induced several TNF superfamily (TNFSF) members (4-1BBL, TWEAK and TRAIL) in synovium and ileum in a site-specific manner. Elevated TNFR2 messenger RNA levels were noted in TNF∆ARE/+ mice leading to increased sTNFR2 release. Likewise, sTNFR2 levels were higher in patients with SpA with gut inflammation and distinct from inflammatory and healthy controls. Tregs accumulated at both gut and joints of TNF∆ARE mice, yet their TNFR2 expression and suppressive function was significantly lower in synovium versus ileum. In line herewith, synovial and intestinal Tregs displayed a distinct transcriptional profile with tissue-restricted TNFSF receptor and p38MAPK gene expression. CONCLUSIONS: These data point to profound differences in immune-regulation between Crohn's ileitis and peripheral arthritis. Whereas Tregs control ileitis they fail to dampen joint inflammation. Synovial resident Tregs are particularly maladapted to chronic TNF exposure.

Differently surface-labeled polystyrene nanoplastics at an environmentally relevant concentration induced Crohn's ileitis-like features via triggering intestinal epithelial cell necroptosis.

Nanoplastics (NPs), regarded as the emerging contaminants, can enter and be mostly accumulated in the digest tract, which pose the potential threat to intestinal health. In this study, mice were orally exposed to polystyrene (PS), PS-COOH and PS-NH2 NPs with the size of ∼100 nm at a human equivalent dose for 28 consecutive days. All three kinds of PS-NPs triggered Crohn's ileitis-like features, such as ileum structure impairment, increased proinflammatory cytokines and intestinal epithelial cell (IEC) necroptosis, and PS-COOH/PS-NH2 NPs exhibited higher adverse effects on ileum tissues. Furthermore, we found PS-NPs induced necroptosis rather than apoptosis via activating RIPK3/MLKL pathway in IECs. Mechanistically, we found that PS-NPs accumulated in the mitochondria and subsequently caused mitochondrial stress, which initiated PINK1/Parkin-mediated mitophagy. However, mitophagic flux was blocked due to lysosomal deacidification caused by PS-NPs, and thus led to IEC necroptosis. We further found that mitophagic flux recovery by rapamycin can alleviate NP-induced IEC necroptosis. Our findings revealed the underlying mechanisms concerning NP-triggered Crohn's ileitis-like features and might provide new insights for the further safety assessment of NPs.

Immunoglobulin A-specific deficiency induces spontaneous inflammation specifically in the ileum.

OBJECTIVE: Although immunoglobulin A (IgA) is abundantly expressed in the gut and known to be an important component of mucosal barriers against luminal pathogens, its precise function remains unclear. Therefore, we tried to elucidate the effect of IgA on gut homeostasis maintenance and its mechanism. DESIGN: We generated various IgA mutant mouse lines using the CRISPR/Cas9 genome editing system. Then, we evaluated the effect on the small intestinal homeostasis, pathology, intestinal microbiota, cytokine production, and immune cell activation using intravital imaging. RESULTS: We obtained two lines, with one that contained a <50 base pair deletion in the cytoplasmic region of the IgA allele (IgA tail-mutant; IgAtm/tm) and the other that lacked the most constant region of the IgH α chain, which resulted in the deficiency of IgA production (IgA-/-). IgA-/- exhibited spontaneous inflammation in the ileum but not the other parts of the gastrointestinal tract. Associated with this, there were significantly increased lamina propria CD4+ T cells, elevated productions of IFN-γ and IL-17, increased ileal segmented filamentous bacteria and skewed intestinal microflora composition. Intravital imaging using Ca2+ biosensor showed that IgA-/- had elevated Ca2+ signalling in Peyer's patch B cells. On the other hand, IgAtm/tm seemed to be normal, suggesting that the IgA cytoplasmic tail is dispensable for the prevention of the intestinal disorder. CONCLUSION: IgA plays an important role in the mucosal homeostasis associated with the regulation of intestinal microbiota and protection against mucosal inflammation especially in the ileum.

Mucosal acidosis elicits a unique molecular signature in epithelia and intestinal tissue mediated by GPR31-induced CREB phosphorylation.

Metabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation. Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway. Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter Escherichia coli strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum. Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn's disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis.

Mast Cell Mediated Regulation of Small Intestinal Chloride Malabsorption in SAMP1/YitFc Mouse Model of Spontaneous Chronic Ileitis.

In Inflammatory Bowel Disease (IBD), malabsorption of electrolytes (NaCl) results in diarrhea. Inhibition of coupled NaCl absorption, mediated by the dual operation of Na:H and Cl:HCO3 exchangers on the brush border membrane (BBM) of the intestinal villus cells has been reported in IBD. In the SAMP1/YitFcs (SAMP1) mice model of spontaneous ileitis, representing Crohn's disease, DRA (Downregulated in Adenoma) mediated Cl:HCO3 exchange was shown to be inhibited secondary to diminished affinity of the exchanger for Cl. However, NHE3 mediated Na:H exchange remained unaffected. Mast cells and their secreted mediators are known to be increased in the IBD mucosa and can affect intestinal electrolyte absorption. However, how mast cell mediators may regulate Cl:HCO3 exchange in SAMP1 mice is unknown. Therefore, the aim of this study was to determine the effect of mast cell mediators on the downregulation of DRA in SAMP1 mice. Mast cell numbers and their degranulation marker enzyme (ß-hexosaminidase) levels were significantly increased in SAMP1 mice compared to control AKR mice. However, treatment of SAMP1 mice with a mast cell stabilizer, ketotifen, restored the ß-hexosaminidase enzyme levels to normal in the intestine, demonstrating stabilization of mast cells by ketotifen. Moreover, downregulation of Cl:HCO3 exchange activity was restored in ketotifen treated SAMP1 mice. Kinetic studies showed that ketotifen restored the altered affinity of Cl:HCO3 exchange in SAMP1 mice villus cells thus reinstating its activity to normal. Further, RT-qPCR, Western blot and immunofluorescence studies showed that the expression levels of DRA mRNA and BBM protein, respectively remained unaltered in all experimental conditions, supporting the kinetic data. Thus, inhibition of Cl:HCO3 exchange resulting in chloride malabsorption leading to diarrhea in IBD is likely mediated by mast cell mediators.

CAR directs T cell adaptation to bile acids in the small intestine.

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.

Unique Regulation of Intestinal Villus Epithelial Cl-/HCO3- Exchange by Cyclooxygenase Pathway Metabolites of Arachidonic Acid in a Mouse Model of Spontaneous Ileitis.

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na+/H+ and Cl-/HCO3- exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl-/HCO3- exchange, measured as DIDS-sensitive 36Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered Km with no effects on Vmax. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl-/HCO3- exchangers, were unaltered. Thus, inhibition of villus cell Cl-/HCO3- exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl-, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.

A myeloid-stromal niche and gp130 rescue in NOD2-driven Crohn's disease.

Crohn's disease is a chronic inflammatory intestinal disease that is frequently accompanied by aberrant healing and stricturing complications. Crosstalk between activated myeloid and stromal cells is critical in the pathogenicity of Crohn's disease1,2, and increases in intravasating monocytes are correlated with a lack of response to anti-TNF treatment3. The risk alleles with the highest effect on Crohn's disease are loss-of-function mutations in NOD24,5, which increase the risk of stricturing6. However, the mechanisms that underlie pathogenicity driven by NOD2 mutations and the pathways that might rescue a lack of response to anti-TNF treatment remain largely uncharacterized. Here we use direct ex vivo analyses of patients who carry risk alleles of NOD2 to show that loss of NOD2 leads to dysregulated homeostasis of activated fibroblasts and macrophages. CD14+ peripheral blood mononuclear cells from carriers of NOD2 risk alleles produce cells that express high levels of collagen, and elevation of conserved signatures is observed in nod2-deficient zebrafish models of intestinal injury. The enrichment of STAT3 regulation and gp130 ligands in activated fibroblasts and macrophages suggested that gp130 blockade might rescue the activated program in NOD2-deficient cells. We show that post-treatment induction of the STAT3 pathway is correlated with a lack of response to anti-TNF treatment in patients, and demonstrate in vivo in zebrafish the amelioration of the activated myeloid-stromal niche using the specific gp130 inhibitor bazedoxifene. Our results provide insights into NOD2-driven fibrosis in Crohn's disease, and suggest that gp130 blockade may benefit some patients with Crohn's disease-potentially as a complement to anti-TNF therapy.

Inflammasome Activation in Ankylosing Spondylitis Is Associated With Gut Dysbiosis.

OBJECTIVE: We undertook this study to evaluate the activation and functional relevance of inflammasome pathways in ankylosing spondylitis (AS) patients and rodent models and their relationship to dysbiosis. METHODS: An inflammasome pathway was evaluated in the gut and peripheral blood from 40 AS patients using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), flow cytometry, and confocal microscopy, and was compared to that of 20 healthy controls and 10 patients with Crohn's disease. Bacteria was visualized using silver stain in human samples, and antibiotics were administered to HLA-B27-transgenic rats. The NLRP3 inhibitor MCC950 was administered to SKG mice, and ileal and joint tissues were assessed by IHC analysis and real-time qRT-PCR. The role of inflammasome in modulating the interleukin-23 (IL-23)/IL-17 axis was studied ex vivo. RESULTS: Expression levels of Nlrp3, Nlrc4, and Aim2 were increased in the gut of HLA-B27-transgenic rats and reduced by antibiotic treatment (P < 0.05). In curdlan-treated SKG mice, NLRP3 blockade prevented ileitis and delayed arthritis onset (P < 0.05). Compared to healthy controls, AS patients demonstrated overexpression of NLRP3 (fold induction 2.33 versus 22.2; P < 0.001), NLRC4 (fold induction 1.90 versus 6.47; P < 0.001), AIM2 (fold induction 2.40 versus 20.8; P < 0.001), CASP1 (fold induction 2.53 versus 24.8; P < 0.001), IL1B (fold induction 1.07 versus 10.93; P < 0.001), and IL18 (fold induction 2.56 versus 15.67; P < 0.001) in the ileum, and caspase 1 activity was increased (P < 0.01). The score of adherent and invasive mucosa-associated bacteria was higher in AS (P < 0.01) and correlated with the expression of inflammasome components in peripheral blood mononuclear cells (P < 0.001). NLRP3 expression was associated with disease activity (the Ankylosing Spondylitis Disease Activity Score using the C-reactive protein level) (r2 = 0.28, P < 0.01) and with IL23A expression (r2 = 0.34, P < 0.001). In vitro, inflammasome activation in AS monocytes was paralleled by increased serum levels of IL-1ß and IL-18. Induction of IL23A, IL17A, and IL22 was IL-1ß-dependent. CONCLUSION: Inflammasome activation occurs in rodent models of AS and in AS patients, is associated with dysbiosis, and is involved in triggering ileitis in SKG mice. Inflammasomes drive type III cytokine production with an IL-1ß-dependent mechanism in AS patients.

STAT3 phosphorylation inhibition for treating inflammation and new bone formation in ankylosing spondylitis.

OBJECTIVE: AS is a rheumatic disease characterized by chronic inflammation and bony ankylosis. This study was to evaluate whether a signal transducer and activator of transcription 3 phosphorylation inhibitor (stat3-p Inh) could treat both chronic inflammation and bone formation in AS. METHODS: Primary AS osteoprogenitor cells and spinal entheseal cells were examined for osteogenic differentiation. SF mononuclear cells (SFMCs) and lamina propria mononuclear cells (LPMCs) were obtained from AS patients. Inflammatory cytokine-producing cells were analysed using flow cytometry and ELISA. Female SKG mice were treated with stat3-p Inh, IL-17A blocker or vehicle. Inflammation and new bone formation were evaluated using immunohistochemistry, PET and micro-CT. RESULTS: In the SKG mouse model, stat3-p Inh significantly suppressed arthritis, enthesitis, spondylitis and ileitis. In experiments culturing SFMCs and LPMCs, the frequencies of IFN-γ-, IL-17A- and TNF-α-producing cells were significantly decreased after stat3-p Inh treatment. When comparing current treatments for AS, stat3-p Inh showed a comparable suppression effect on osteogenesis to Janus kinase inhibitor or IL-17A blocker in AS-osteoprogenitor cells. Stat3-p Inh suppressed differentiation and mineralization of AS-osteoprogenitor cells and entheseal cells toward osteoblasts. Micro-CT analysis of hind paws revealed less new bone formation in stat3-p Inh-treated mice than vehicle-treated mice (P = 0.005). Hind paw and spinal new bone formation were similar between stat3-p Inh- and anti-IL-17A-treated SKG mice (P = 0.874 and P = 0.117, respectively). CONCLUSION: Stat-3p inhibition is a promising treatment for both inflammation and new bone formation in AS.

Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn's disease model mice.

Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.

Hepatocyte nuclear factor-4α regulates expression of the serotonin transporter in intestinal epithelial cells.

The serotonin transporter (SERT) functions to regulate the availability of serotonin (5-HT) in the brain and intestine. An intestine-specific mRNA variant arising from a unique transcription start site and alternative promoter in the SERT gene has been identified (iSERT; spanning exon 1C). A decrease in SERT is implicated in several gut disorders, including inflammatory bowel diseases (IBD). However, little is known about mechanisms regulating the iSERT variant, and a clearer understanding is warranted for targeting SERT for the treatment of gut disorders. The current studies examined the expression of iSERT across different human intestinal regions and investigated its regulation by HNF4α (hepatic nuclear factor-4α), a transcription factor important for diverse cellular functions. iSERT mRNA abundance was highest in the human ileum and Caco-2 cell line. iSERT mRNA expression was downregulated by loss of HNF4α (but not HNF1α, HNF1ß, or FOXA1) in Caco-2 cells. Overexpression of HNF4α increased iSERT mRNA concomitant with an increase in SERT protein. Progressive promoter deletion and site-directed mutagenesis revealed that the HNF4α response element spans nucleotides -1,163 to -1150 relative to the translation start site. SERT mRNA levels in the intestine were drastically reduced in the intestine-specific HNF4α-knockout mice relative to HNF4αFL/FL mice. Both HNF4α and SERT mRNA levels were also downregulated in mouse model of ileitis (SAMP) compared with AKR control mice. These results establish the transcriptional regulation of iSERT at the gut-specific internal promoter (hSERTp2) and have identified HNF4α as a critical modulator of basal SERT expression in the intestine.

Sphingosine-1-Phosphate Lyase Inhibition Alters the S1P Gradient and Ameliorates Crohn's-Like Ileitis by Suppressing Thymocyte Maturation.

BACKGROUND: Lymphocytes recirculate from tissues to blood following the sphingosine-1-phosphate (S1P) gradient (low in tissues, high in blood), maintained by synthetic and degradative enzymes, among which the S1P lyase (SPL) irreversibly degrades S1P. The role of SPL in the intestine, both during homeostasis and IBD, is poorly understood. We hypothesized that modulation of tissue S1P levels might be advantageous over S1P receptor (S1PR) agonists (eg, fingolimod, ozanimod, etrasimod), as without S1PR engagement there might be less likelihood of potential off-target effects. METHODS: First we examined SPL mRNA transcripts and SPL localization in tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The in vivo effects of the SPL inhibitors 4-deoxypyridoxine hydrochloride (30 mg/L) and 2-acetyl-4 (tetrahydroxybutyl)imidazole (50 mg/L) were assessed through their oral administration to adult TNF∆ARE mice, which spontaneously develop Crohn's-like chronic ileitis. The effect of SPL inhibition on circulating and tissue lymphocytes, transcriptional regulation of proinflammatory cytokines, and on the histological severity of ileitis was additionally examined. Tissue S1P levels were determined by liquid chromatography-mass spectrometry. Mechanistically, the potential effects of high S1P tissue levels on intestinal leukocyte apoptosis were assessed via terminal deoxynucleotidyl transferase dUTP nick end-labeling assay and annexin 5 staining. Finally, we examined the ability of T cells to home to the intestine, along with the effects of SPL inhibition on cellular subsets within immune compartments via flow and mass cytometry. RESULTS: S1P lyase was ubiquitously expressed. In the gut, immunohistochemistry predominantly localized it to small intestinal epithelia, although the lamina propria leukocyte fraction had higher mRNA transcripts. Inhibition of SPL markedly increased local intestinal S1P levels, induced peripheral lymphopenia, downregulated proinflammatory cytokines, and attenuated chronic ileitis in mice. SPL inhibition reduced T and myeloid cells in secondary lymphoid tissues and the intestine and decreased naïve T-cell recruitment. The anti-inflammatory activity of SPL inhibition was not mediated by leukocyte apoptosis, nor by interference with the homing of lymphocytes to the intestine, and was independent of its peripheral lymphopenic effect. However, SPL inhibition promoted thymic atrophy and depleted late immature T cells (CD4+CD8+ double positive), with accumulation of mature CD4+CD8- and CD4-CD8+ single-positive cells. CONCLUSIONS: Inhibition of the S1P lyase alters the S1P gradient and attenuates chronic ileitis via central immunosuppression. SPL inhibition could represent a potential way to tame an overactive immune response during IBD and other T-cell-mediated chronic inflammatory diseases.

Nod2 Protects the Gut From Experimental Colitis Spreading to Small Intestine.

BACKGROUND AND AIMS: Nucleotide oligomerization domain 2 [NOD2] mutations are key risk factors for Crohn's disease [CD]. NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the exact roles of NOD2 in CD and other NOD2-associated disorders remain poorly known. METHODS: We initially observed that NOD2 expression was increased in epithelial cells away from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation, and cytokines expression were examined in the small bowel of wild-type [WT], Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulphonic acid [TNBS]. RESULTS: In WT mice, Nod2 upregulation upstream to rectal injury was associated with pro-inflammatory cytokine expression but no overt histological inflammatory lesions. Conversely, in Nod2-deficient mice the inflammation spread from colitis to ileum and duodenum. CONCLUSIONS: Nod2 protects the gut from colitis spreading to small intestine.

Blockade of IL-33R/ST2 Signaling Attenuates Toxoplasma gondii Ileitis Depending on IL-22 Expression.

Oral T. gondii infection (30 cysts of 76K strain) induces acute lethal ileitis in sensitive C57BL/6 (B6) mice with increased expression of IL-33 and its receptor ST2 in the ileum. Here we show that IL-33 is involved in ileitis, since absence of IL-33R/ST2 attenuated neutrophilic inflammation and Th1 cytokines upon T. gondii infection with enhanced survival. Blockade of ST2 by neutralizing ST2 antibody in B6 mice conferred partial protection, while rmIL-33 aggravated ileitis. Since IL-22 expression further increased in absence of ST2, we blocked IL-22 by neutralizing antibody, which abrogated protection from acute ileitis in ST2 deficient mice. In conclusion, severe lethal ileitis induced by oral T. gondii infection is attenuated by blockade of ST2 signaling and may be mediated in part by endogenous IL-22.

Faecal Calprotectin and Magnetic Resonance Enterography in Ileal Crohn's Disease: Correlations Between Disease Activity and Long-Term Follow-Up.

BACKGROUND AND AIMS: Magnetic resonance enterography [MRE] is the gold standard for assessing ileal inflammation in Crohn's disease [CD]. The aim of the present study was to correlate faecal calprotectin [FC] to MRE via a simple score in an exclusive ileal cohort with long-term follow-up for association with time to surgery or biologic therapy. METHODS: In total, 150 MRE studies with matched FC [±30 days] were identified from the Edinburgh FC Register [2008-12; n = 18138]. Scans were re-read blinded to clinical data, independently, by two expert gastrointestinal radiologists, to generate a simple MRE score [range 0-10] from assessment of the worst intestinal segment plus total disease extent. RESULTS: In total, 119 MRE scans were evaluated from 104 patients with ileal CD [L1 or L3 with panproctocolectomy]. Receiver operating characteristic analysis showed an area under the curve of 0.77 [0.67-0.87, p < 0.0001] for FC and MRE score >1, with an optimal cut-off of 145 µg/g for severe inflammation on MRE with 69.3% [57.6-79.5] sensitivity and 71.4% [53.7-85.4] specificity. Long-term follow-up over a median [interquartile range] of 2086 days [1786-2353] revealed FC ≥ 145 µg/g was associated with reduced biologic-free survival until 3 years following MRE, whereas MRE score [severe vs absent] was associated with reduced surgery- and biologic-free survival throughout follow-up. Backwards stepwise logistic regression revealed that length of ileal disease (odds ratio [OR] 3.8, 1.1-13.2, p = 0.034) and increased bowel wall thickness at MRE [OR 4.2, 1.6-10.7, p < 0.0001] or female sex [OR 5.2, 1.5-18.7, p = 0.011] increased the risk of biologic use or surgery, respectively. CONCLUSIONS: FC correlates well with MRE assessment of ileal CD with MRE parameters associated with long-term biologic- and surgery-free remission.

Inflammation-independent TL1A-mediated intestinal fibrosis is dependent on the gut microbiome.

Tumor necrosis factor-like cytokine 1A (TL1A, TNFSF15) is implicated in inflammatory bowel disease (IBD), modulating the location and severity of intestinal inflammation and fibrosis. TL1A expression is increased in inflamed gut mucosa and associated with fibrostenosing Crohn's disease. Tl1a-overexpression in mice lead to spontaneous ileitis, and exacerbated induced proximal colitis and fibrosis. IBD is associated with shifts in the gut microbiome, but the effect of differing microbial populations and their interaction with TL1A on fibrosis has not been investigated. We demonstrate that the pro-fibrotic and inflammatory phenotype resulting from Tl1a-overexpression is abrogated in the absence of resident microbiota. To evaluate if this is due to the absence of a unique bacterial population, as opposed to any bacteria per se, we gavaged germ-free (GF) wild-type and Tl1a-transgenic (Tl1a-Tg) mice with stool from specific pathogen free (SPF) mice and a healthy human donor (Hu). Reconstitution with SPF, but not Hu microbiota, resulted in increased intestinal collagen deposition and fibroblast activation in Tl1a-Tg mice. Notably, there was reduced fibroblast migration and activation under GF conditions compared to native conditions. We then identified several candidate organisms that correlated directly with increased fibrosis in reconstituted mice and showed that these organisms directly impact fibroblast function in vitro. Thus, Tl1a-mediated intestinal fibrosis and fibroblast activation are dependent on specific microbial populations.

The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn's Disease-Like Ileitis.

Background: Epidemiological studies indicate that the use of artificial sweeteners doubles the risk for Crohn's disease (CD). Herein, we experimentally quantified the impact of 6-week supplementation with a commercial sweetener (Splenda; ingredients sucralose maltodextrin, 1:99, w/w) on both the severity of CD-like ileitis and the intestinal microbiome alterations using SAMP1/YitFc (SAMP) mice. Methods: Metagenomic shotgun DNA sequencing was first used to characterize the microbiome of ileitis-prone SAMP mice. Then, 16S rRNA microbiome sequencing, quantitative polymerase chain reaction, fluorescent in situ hybridization (FISH), bacterial culture, stereomicroscopy, histology, and myeloperoxidase (MPO) activity analyses were then implemented to compare the microbiome and ileitis phenotype in SAMP with that of control ileitis-free AKR/J mice after Splenda supplementation. Results: Metagenomics indicated that SAMP mice have a gut microbial phenotype rich in Bacteroidetes, and experiments showed that Helicobacteraceae did not have an exacerbating effect on ileitis. Splenda did not increase the severity of (stereomicroscopic/histological) ileitis; however, biochemically, ileal MPO activity was increased in SAMP treated with Splenda compared with nonsupplemented mice (P < 0.022) and healthy AKR mice. Splenda promoted dysbiosis with expansion of Proteobacteria in all mice, and E. coli overgrowth with increased bacterial infiltration into the ileal lamina propria of SAMP mice. FISH showed increase malX gene-carrying bacterial clusters in the ilea of supplemented SAMP (but not AKR) mice. Conclusions: Splenda promoted gut Proteobacteria, dysbiosis, and biochemical MPO reactivity in a spontaneous model of (Bacteroidetes-rich) ileal CD. Our results indicate that although Splenda may promote parallel microbiome alterations in CD-prone and healthy hosts, this did not result in elevated MPO levels in healthy mice, only CD-prone mice. The consumption of sucralose/maltodextrin-containing foods might exacerbate MPO intestinal reactivity only in individuals with a pro-inflammatory predisposition, such as CD.

Acute small intestinal inflammation results in persistent lymphatic alterations.

Inflammatory bowel disease (IBD) has a complex pathophysiology with limited treatments. Structural and functional changes in the intestinal lymphatic system have been associated with the disease, with increased risk of IBD occurrence linked to a history of acute intestinal injury. To examine the potential role of the lymphatic system in inflammation recurrence, we evaluated morphological and functional changes in mouse mucosal and mesenteric lymphatic vessels, and within the mesenteric lymph nodes during acute ileitis caused by a 7-day treatment with dextran sodium sulfate (DSS). We monitored whether the changes persisted during a 14-day recovery period and determined their potential consequences on dendritic cell (DC) trafficking between the mucosa and lymphoid tissues. DSS administration was associated with marked lymphatic abnormalities and dysfunctions exemplified by lymphangiectasia and lymphangiogenesis in the ileal mucosa and mesentery, increased mesenteric lymphatic vessel leakage, and lymphadenopathy. Lymphangiogenesis and lymphadenopathy were still evident after recovery from intestinal inflammation and correlated with higher numbers of DCs in mucosal and lymphatic tissues. Specifically, a deficit in CD103+ DCs observed during acute DSS in the lamina propria was reversed and further enhanced during recovery. We concluded that an acute intestinal insult caused alterations of the mesenteric lymphatic system, including lymphangiogenesis, which persisted after resolution of inflammation. These morphological and functional changes could compromise DC function and movement, increasing susceptibility to further gastrointestinal disease. Elucidation of the changes in mesenteric and intestinal lymphatic function should offer key insights for new therapeutic strategies in gastrointestinal disorders such as IBD. NEW & NOTEWORTHY Lymphatic integrity plays a critical role in small intestinal homeostasis. Acute intestinal insult in a mouse model of acute ileitis causes morphological and functional changes in mesenteric and intestinal lymphatic vessels. While some of the changes significantly regressed during inflammation resolution, others persisted, including lymphangiogenesis and altered dendritic cell function and movement, potentially increasing susceptibility to the recurrence of gastrointestinal inflammation.