The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Interleukin-22 induces interleukin-18 expression from epithelial cells during intestinal infection.

T helper 1 (Th1) cell-associated immunity exacerbates ileitis induced by oral Toxoplasma gondii infection. We show here that attenuated ileitis observed in interleukin-22 (IL-22)-deficient mice was associated with reduced production of Th1-cell-promoting IL-18. IL-22 not only augmented the expression of Il18 mRNA and inactive precursor protein (proIL-18) in intestinal epithelial cells after T. gondii or Citrobacter rodentium infection, but also maintained the homeostatic amount of proIL-18 in the ileum. IL-22, however, did not induce the processing to active IL-18, suggesting a two-step regulation of IL-18 in these cells. Although IL-18 exerted pathogenic functions during ileitis triggered by T. gondii, it was required for host defense against C. rodentium. Conversely, IL-18 was required for the expression of IL-22 in innate lymphoid cells (ILCs) upon T. gondii infection. Our results define IL-18 as an IL-22 target gene in epithelial cells and describe a complex mutual regulation of both cytokines during intestinal infection.

Succinate Produced by Intestinal Microbes Promotes Specification of Tuft Cells to Suppress Ileal Inflammation.

BACKGROUND & AIMS: Countries endemic for parasitic infestations have a lower incidence of Crohn's disease (CD) than nonendemic countries, and there have been anecdotal reports of the beneficial effects of helminths in CD patients. Tuft cells in the small intestine sense and direct the immune response against eukaryotic parasites. We investigated the activities of tuft cells in patients with CD and mouse models of intestinal inflammation. METHODS: We used microscopy to quantify tuft cells in intestinal specimens from patients with ileal CD (n = 19), healthy individuals (n = 14), and TNFΔARE/+ mice, which develop Crohn's-like ileitis. We performed single-cell RNA sequencing, mass spectrometry, and microbiome profiling of intestinal tissues from wild-type and Atoh1-knockout mice, which have expansion of tuft cells, to study interactions between microbes and tuft cell populations. We assessed microbe dependence of tuft cell populations using microbiome depletion, organoids, and microbe transplant experiments. We used multiplex imaging and cytokine assays to assess alterations in inflammatory response following expansion of tuft cells with succinate administration in TNFΔARE/+ and anti-CD3E CD mouse models. RESULTS: Inflamed ileal tissues from patients and mice had reduced numbers of tuft cells, compared with healthy individuals or wild-type mice. Expansion of tuft cells was associated with increased expression of genes that regulate the tricarboxylic acid cycle, which resulted from microbe production of the metabolite succinate. Experiments in which we manipulated the intestinal microbiota of mice revealed the existence of an ATOH1-independent population of tuft cells that was sensitive to metabolites produced by microbes. Administration of succinate to mice expanded tuft cells and reduced intestinal inflammation in TNFΔARE/+ mice and anti-CD3E-treated mice, increased GATA3+ cells and type 2 cytokines (IL22, IL25, IL13), and decreased RORGT+ cells and type 17 cytokines (IL23) in a tuft cell-dependent manner. CONCLUSIONS: We found that tuft cell expansion reduced chronic intestinal inflammation in mice. Strategies to expand tuft cells might be developed for treatment of CD.

Deoxycholic acid enhancement of lymphocyte migration through direct interaction with the intestinal vascular endothelium.

BACKGROUND AND AIM: The small intestine plays a central role in gut immunity, and enhanced lymphocyte migration is involved in the pathophysiology of various enteropathy. Bile acid (BA) is closely related to lipid metabolism and gut microbiota and essential for gut homeostasis. However, the effects of BA on gut immunity have not been studied in detail, especially on the small intestine and lymphocyte migration. Therefore, we aimed to investigate the effect of BA on small intestinal lymphocyte microcirculation. METHODS: The effect of deoxycholic acid (DCA), taurocholic acid (tCA), or cholic acid (CA) on the indomethacin (IND)-induced small intestinal enteropathy in mice was investigated. Lymphocyte movements were evaluated after exposure to BA using intravital microscopy. The effects of BA on surface expression of adhesion molecules on the vascular endothelium and lymphocytes through BA receptors were examined in vitro. RESULTS: IND-induced small intestinal enteropathy was histologically aggravated by DCA treatment alone. The expression of adhesion molecules ICAM-1 and VCAM-1 was significantly enhanced by DCA. Exposure to DCA increased lymphocyte adhesion in the microvessels of the ileum, which was partially blocked by anti-α4ß1 integrin antibody in vivo. The expression of ICAM-1 and VCAM-1 was significantly enhanced by DCA in vitro, which was partially suppressed by the sphingosine-1-phosphate receptor 2 (S1PR2) antagonist. The S1PR2 antagonist significantly ameliorated IND-induced and DCA-exaggerated small intestinal injury. CONCLUSION: DCA exacerbated IND-induced small intestinal enteropathy. DCA directly acts on the vascular endothelium and enhances the expression levels of adhesion molecules partially via S1PR2, leading to enhanced small intestinal lymphocyte migration.

Should We Divide Crohn's Disease Into Ileum-Dominant and Isolated Colonic Diseases?

Crohn's disease (CD) is an inflammatory bowel disease that can involve any region of the gastrointestinal tract. First described in 1932 as terminal ileitis or regional enteritis, it predominately involves the ileum with or without colonic involvement. Isolated colonic CD was first described in 1960 and since then the phenotypic classification of CD has evolved to stratify patients into isolated ileal, ileocolonic, or isolated colonic involvement. In the current review we evaluate the published literature regarding differences in epidemiology, natural history, pathogenesis, response to therapy, and disease monitoring, when stratified by disease location. Based on the available evidence consideration could be given to a new classification for CD, which splits it into ileum dominant (isolated ileal and ileocolonic) and isolated colonic disease. This may allow for a more optimized approach to clinical care and scientific research for CD.

Porcine lactoferrin-derived peptide LFP-20 modulates immune homoeostasis to defend lipopolysaccharide-triggered intestinal inflammation in mice.

The performance of immune system is vital for defending the body from pathogens, and it plays a crucial role in health homoeostasis. In a previous study, we have shown that LFP-20, a twenty-amino acid antimicrobial peptide in the N terminus of porcine lactoferrin, modulated inflammatory response in colitis. Here, we further investigated the effects of LFP-20 on immune homoeostasis to elucidate the mechanism of its anti-inflammation action. A lipopolysaccharide (LPS)-triggered systemic inflammatory response mice model was established. On the basis of observed mucosal lesions and apoptosis in small intestine, we found increased macrophage and neutrophil infiltration in ileum after LPS stimulation. Expectedly, LFP-20 pre-treatment attenuated the LPS-mediated immune disorders in ileum. Moreover, the flow cytometry results indicated pre-treatment with LFP-20 sustained the balance of CD3+CD8+ T cells, B cells and natural killer cells in LPS-triggered immune disturbance. Simultaneously, we demonstrated LFP-20 modulated the secretion of both activated Th1-related IL-12p70, interferon-γ, TNF-α and Th2-related IL-4, IL-5 and IL-6. Furthermore, we found LFP-20 facilitated a balanced Th1 and Th2 response, which triggered cellular defence mechanisms and induced B cells to produce opsonising antibodies belonging to certain IgG subclasses to defend against LPS stimulation. Collectively, our study indicated pre-treatment with LFP-20 could defend against LPS-triggered systemic inflammatory response in mice via modulating immune homoeostasis.

Activity of SHIP, Which Prevents Expression of Interleukin 1ß, Is Reduced in Patients With Crohn's Disease.

BACKGROUND & AIMS: Crohn's disease (CD) is associated with a dysregulated immune response to commensal micro-organisms in the intestine. Mice deficient in inositol polyphosphate 5'-phosphatase D (INPP5D, also known as SHIP) develop intestinal inflammation resembling that of patients with CD. SHIP is a negative regulator of PI3Kp110α activity. We investigated mechanisms of intestinal inflammation in Inpp5d(-/-) mice (SHIP-null mice), and SHIP levels and activity in intestinal tissues of subjects with CD. METHODS: We collected intestines from SHIP-null mice, as well as Inpp5d(+/+) mice (controls), and measured levels of cytokines of the interleukin 1 (IL1) family (IL1α, IL1ß, IL1ra, and IL6) by enzyme-linked immunosorbent assay. Macrophages were isolated from lamina propria cells of mice, IL1ß production was measured, and mechanisms of increased IL1ß production were investigated. Macrophages were incubated with pan-phosphatidylinositol 3-kinase inhibitors or PI3Kp110α-specific inhibitors. Some mice were given an antagonist of the IL1 receptor; macrophages were depleted from ilea of mice using clodronate-containing liposomes. We obtained ileal biopsies from sites of inflammation and peripheral blood mononuclear cells (PBMCs) from treatment-naïve subjects with CD or without CD (controls), and measured SHIP levels and activity. PBMCs were incubated with lipopolysaccharide and adenosine triphosphate, and levels of IL1ß production were measured. RESULTS: Inflamed intestinal tissues and intestinal macrophages from SHIP-null mice produced higher levels of IL1B and IL18 than intestinal tissues from control mice. We found PI3Kp110α to be required for macrophage transcription of Il1b. Macrophage depletion or injection of an IL1 receptor antagonist reduced ileal inflammation in SHIP-null mice. Inflamed ileal tissues and PBMCs from patients with CD had lower levels of SHIP protein than controls (P < .0001 and P < .0002, respectively). There was an inverse correlation between levels of SHIP activity in PBMCs and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate (R(2) = .88). CONCLUSIONS: Macrophages from SHIP-deficient mice have increased PI3Kp110α-mediated transcription of Il1b, which contributes to spontaneous ileal inflammation. SHIP levels and activity are lower in intestinal tissues and peripheral blood samples from patients with CD than controls. There is an inverse correlation between SHIP activity and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate in PBMCs. Strategies to reduce IL1B might be developed to treat patients with CD found to have low SHIP activity.

Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis.

BACKGROUND: Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. METHODS: Ileal biopsies were obtained from 50 HLA-B27+ patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. RESULTS: Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. CONCLUSIONS: Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour.

IL-33 Drives Eosinophil Infiltration and Pathogenic Type 2 Helper T-Cell Immune Responses Leading to Chronic Experimental Ileitis.

Although a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33-mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease.

SAMP1/YitFc mice develop ileitis via loss of CCL21 and defects in dendritic cell migration.

BACKGROUND & AIMS: The lymphatic chemokine CCL21 is required for dendritic cell (DC) migration from tissues to lymph nodes, which helps establish tolerance to foreign yet harmless antigens. We demonstrate that CCL21 is almost completely absent from SAMP1/YitFc (SAMP) mice, which spontaneously develop chronic ileitis that resembles Crohn's disease, and that DC migration is severely impaired in these mice compared with AKR mice (controls). Toll-like receptor agonists like the Toll-like receptor 7 agonist R848 induce DC maturation and mobilization. METHODS: We collected intestinal and other tissues and mesenteric lymph nodes (MLN) from SAMP mice. Expression of CCL21 was measured by quantitative reverse transcription polymerase chain reaction and immunofluorescence analyses; spontaneous and induced migration of DCs were assessed by flow cytometry. We analyzed production of retinoic acid by DCs and their ability to induce development of regulatory T cells. Mice were fed R848 to determine its effects on migration of DCs and development of ileitis in SAMP mice. RESULTS: SAMP mice expressed almost no CCL21 in any tissue tested. Their CD11b(+)CD103(+) DCs were defective in migration from the ileal lamina propria to the MLN. DCs from SAMP mice also had a greatly reduced ability to produce retinoic acid and induce development of regulatory T cells compared with control mice. Young SAMP mice had reduced CCL21 expression and decreased DC migration before developing ileitis. Administration of R848 to adult SAMP mice increased migration of DC to the MLN and development of regulatory T cells there, and reduced the severity of ileitis. CONCLUSIONS: Loss of CCL21 signaling and DC migration is required for development of ileitis in SAMP mice. Reagents such as R848, which activate DC migration to the MLN, may be developed as treatments for patients with Crohn's disease.

Dysregulated NOD2 predisposes SAMP1/YitFc mice to chronic intestinal inflammation.

Nucleotide-binding oligomerization domain-containing 2 (NOD2) is an intracellular receptor that plays an essential role in innate immunity as a sensor of a component of the bacterial cell wall, muramyl dipeptide (MDP). Crohn's disease (CD)-associated NOD2 variants lead to defective innate immune responses, including decreased NF-κB activation and cytokine production. We report herein that SAMP1/YitFc (SAMP) mice, which develop spontaneous CD-like ileitis in the absence of NOD2 genetic mutations, fail to respond to MDP administration by displaying decreased innate cytokine production and dysregulated NOD2 signaling compared with parental AKR control mice. We show that, unlike in other mouse strains, in vivo administration of MDP does not prevent dextran sodium sulfate-induced colitis in SAMP mice and that the abnormal NOD2 response is specific to the hematopoietic cellular component. Moreover, we demonstrate that MDP fails to enhance intracellular bacterial killing in SAMP mice. These findings shed important light on the initiating molecular events underlying CD-like ileitis.

Myocyte TLR4 enhances enteric and systemic inflammation driving late murine endotoxic ileus.

Myocytes are nonhemopoietic in origin and functionally essential in generating gastrointestinal motility. In endotoxemia, a rapid-onset nonhemopoietic mechanism potently triggers early ileus in a Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88)-dependent manner. Moreover, synergistically with hemopoietic cells, nonhemopoietic cells escalate late ileus via an IL-6 receptor-dependent inflammation-driven pathway. We therefore specifically investigated the role of myocytes in TLR4-triggered inflammation and ileus. TLR4(+/+), TLR4(-/-), bmTLR4(+/+)/TLR4(-/-) chimera, SM22-Cre(-/-)TLR4(flox/flox), and selective myocyte TLR4-deficient (SM22-Cre(+/-)TLR4(flox/flox)) mice were injected intraperitoneally with purified lipopolysaccharide. SM22-driven Cre recombinase activity was selectively detected in cardiac, gastrointestinal, skeletal, and vascular myocytes, of small-sized vessels in a two-color fluorescent Cre reporter mouse. In contrast to nonhemopoietic TLR4 deficiency, deletion of myocyte TLR4 signaling prevented neither endotoxin-induced suppression of spontaneous jejunal contractility in vitro nor early ileus in vivo at 6 h. Circulating plasma colony-stimulating factor 3 was greatly elevated during endotoxemia, independent of myocyte TLR4 signaling or time. TLR4 activation of myocytes contributed significantly to an early enteric IL-6 mRNA induction and systemic IL-6 release, as well as to a late increase in circulating chemokine (C-X-C motif) ligand 1 (CXCL1) and IL-17. Consequently, inhibition of myocyte TLR4 signaling allowed functional recovery of motility by preventing inflammation-driven late ileus at 24 h. Direct TLR4 activation of myocytes is not responsible for nonhemopoietic-mediated early ileus. However, myocytes are proinflammatory cells that potently drive enteric and systemic inflammation, subsequently fueling late mediator-triggered ileus. Specifically, the myocyte TLR4-dependent inflammatory signature of elevated plasma IL-6, CXCL1, and IL-17 is strongly associated with late rodent ileus.

Toll-like receptor (TLR) 2 agonists ameliorate indomethacin-induced murine ileitis by suppressing the TLR4 signaling.

BACKGROUND AND AIM: Few drugs have been found satisfactory in the treatment of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced enteropathy. Toll-like receptor (TLR) 4 and aberrant leukocyte migration to the intestinal mucosa are reported to be involved in the pathology of intestinal enteropathy and TLR2 agonists have been found to evoke hyposensitivity to TLR4 stimulation in vitro. In this study, we investigated whether and how lipoarabinomannan (LAM) or lipoteichoic acid (LTA), TLR2 agonists, attenuated indomethacin (IND)-induced intestinal damage. METHODS: LAM (0.5 mg/kg) or LTA (15 mg/kg) was administered intraperitoneally to mice before IND (10 mg/kg) administration. Disease activity was evaluated macroscopically and histologically. In the migration analysis, fluorescence-labeled leukocyte movement in the intestinal microvessels was observed by intravital microscopy. Expression of P-selectin, MAdCAM-1, TLR2, TLR4, and F4/80 was observed immunohistochemically. In the in vitro analysis, RAW264.7 macrophage cells were preincubated with LAM and stimulated with lipopolysaccharide (LPS), and the mRNA expression levels of TLR4, tumor necrosis factor-α, and interleukin-12p40 were measured. RESULTS: Pretreatment with LAM or LTA significantly decreased IND-induced injury as well as decreased leukocyte infiltration. Pretreatment with LAM decreased IND-induced TLR4 expression on F4/80(+) macrophages, the level of P-selectin expression, and leukocyte migration in the small intestinal vessels. In the in vitro study, a single administration of LAM decreased TLR4 mRNA expression and inhibited the increase in mRNA expression of inflammatory cytokines by LPS in a dose-dependent manner. CONCLUSION: TLR2 agonists attenuated IND-induced small intestinal lesions and leukocyte infiltration probably by suppressing the TLR4 signaling pathway in tissue macrophages.

Evidence that autophagy, but not the unfolded protein response, regulates the expression of IL-23 in the gut of patients with ankylosing spondylitis and subclinical gut inflammation.

OBJECTIVES: Interleukin (IL)-23 has been implicated in the pathogenesis of ankylosing spondylitis (AS). The aim of the study was to clarify the mechanisms underlying the increased IL-23 expression in the gut of AS patients. METHODS: Consecutive gut biopsies from 30 HLA-B27(+) AS patients, 15 Crohn's disease (CD) patients and 10 normal subjects were obtained. Evidence for HLA-B27 misfolding was studied. Unfolded protein response (UPR) and autophagy were assessed by RT-PCR and immunohistochemistry. The contribution of UPR and autophagy in the regulation of IL-23 expression was evaluated in in vitro experiments on isolated lamina propria mononuclear cells (LPMCs). RESULTS: Intracellular colocalisation of SYVN1 and FHCs but not a significant overexpression of UPR genes was observed in the gut of AS patients. Conversely, upregulation of the genes involved in the autophagy pathway was observed in the gut of AS and CD patients. Immunohistochemistry showed an increased expression of LC3II, ATG5 and ATG12 but not of SQSTM1 in the ileum of AS and CD patients. LC3II was expressed among infiltrating mononuclear cells and epithelial cells resembling Paneth cells (PC) and colocalised with ATG5 in AS and CD. Autophagy but not UPR was required to modulate the expression of IL-23 in isolated LPMCs of AS patients with chronic gut inflammation, CD patients and controls. CONCLUSIONS: Our data suggest that HLA-B27 misfolding occurs in the gut of AS patients and is accompanied by activation of autophagy rather than a UPR. Autophagy appears to be associated with intestinal modulation of IL-23 in AS.

Intestinal inflammation in a murine model of autism spectrum disorders.

Autism spectrum disorder (ASD) is a cluster of neurodevelopmental disorders characterized by impairments in communication, social interest and stereotypical behaviour. Dysfunction of the intestinal tract is reported in patients with ASD and implicated in the development and severity of ASD symptoms. However, more research is required to investigate the association of intestinal problems with ASD and the potential underlying mechanisms. The purpose of this study was to investigate comorbid symptoms of intestinal inflammation in a murine model of ASD induced by prenatal exposure to valproic acid (VPA). Pregnant BALB/c females were treated subcutaneously with 600 mg/kg VPA or phosphate buffered saline on gestational day 11. Offspring were housed with their mother until weaning on postnatal day 21 (P21). All pups were exposed to a social behaviour test on P28. Inflammatory correlates and activity of the serotonergic system were measured in brain and intestinal tissue. Here we demonstrate, in addition to reduced social behaviour and increased expression of neuroinflammatory markers in the brain, that VPA in utero- exposed male offspring showed epithelial cell loss and neutrophil infiltration in the intestinal tract. Furthermore, reduced levels of serotonin were not only observed the prefrontal cortex and amygdala of VPA in utero- exposed males, but also in the small intestine. Overall, we demonstrate that gender-specific inflammatory conditions are present in the small intestines of VPA in utero- exposed mice and are accompanied by a disturbed serotonergic system in the brain as well as in the intestinal tract.

Endoplasmic reticulum stress response promotes cytotoxic phenotype of CD8αß+ intraepithelial lymphocytes in a mouse model for Crohn's disease-like ileitis.

Endoplasmic reticulum (ER) unfolded protein responses (UPR) are implicated in the pathogenesis of inflammatory bowel disease. Cytotoxic CD8αß(+) intraepithelial lymphocytes (IEL) contribute to the development of Crohn's disease-like ileitis in TNF(ΔARE/+) mice. In this study, we characterized the role of ER-UPR mechanisms in contributing to the disease-associated phenotype of cytotoxic IEL under conditions of chronic inflammation. Inflamed TNF(ΔARE/+) mice exhibited increased expression of Grp78, ATF6, ATF4, and spliced XBP1 in CD8αß(+) IEL but not in CD8αα(+) IEL or in lamina propria lymphocytes. Chromatin immunoprecipitation analysis in CD8αß(+) T cells showed selective recruitment of ER-UPR transducers to the granzyme B gene promoter. Heterozygous Grp78(-/+) mice exhibited an attenuated granzyme B-dependent cytotoxicity of CD8αß(+) T cells against intestinal epithelial cells, suggesting a critical activity of this ER-associated chaperone in maintaining a cytotoxic T cell phenotype. Granzyme B-deficient CD8αß(+) T cells showed a defect in IL-2-mediated proliferation in Grp78(-/+) mice. Adoptively transferred Grp78(-/+) CD8αß(+) T cells had a decreased frequency of accumulation in the intestine of RAG2(-/-) recipient mice. The tissue pathology in TNF(ΔARE/+) × Grp78(-/+) mice was similar to TNF(ΔARE/+) mice, even though the cytotoxic effector functions of CD8αß(+) T cells were significantly reduced. In conclusion, ER stress-associated UPR mechanisms promote the development and maintenance of the pathogenic cytotoxic CD8αß(+) IEL phenotype in the mouse model of Crohn's disease-like ileitis.

Ectopic lymphoid tissue alters the chemokine gradient, increases lymphocyte retention and exacerbates murine ileitis.

BACKGROUND: The earliest endoscopically-evident lesion in Crohn's disease is the aphthous ulcer, which develops over ectopic lymphoid tissues (ie, inducible lymphoid follicles (ILF), tertiary lymphoid tissue (TLT)) in the chronically inflamed intestine. ILF/TLT are induced within effector sites by homeostatic lymphoid chemokines, but their role in the development of intestinal ILF/TLT and in the pathogenesis of Crohn's disease is poorly understood. DESIGN: Using a mouse model of Crohn's-like ileitis (TNFARE) which develops florid induction of ILF/TLT within its terminal ileum, the contribution of the CCR7/CCL19/CCL21 chemokine axis during the development of TLT and its role in disease pathogenesis were assessed. RESULTS: Both CCL19 and CCL21 were increased within the inflamed ileum of TNFARE mice, which resulted in CCR7 internalisation and impaired T cell chemotaxis. ILF/TLT were a major source of CCL19 and CCL21 and increased local synthesis, augmented recruitment/retention of effector, naïve and central memory T cell subsets within the inflamed ileum. Immunoblockade of CCR7 resulted in further effector T cell retention and exacerbation of ileitis. CONCLUSIONS: Induction of ILF/TLT in the chronically inflamed intestine alters the homeostatic CCL19-CCL21 lymphoid-chemokine gradient and increases recruitment/retention of effector CCR7+ T cell subsets within the terminal ileum, contributing to the perpetuation of chronic inflammation. Thus, blockade of CCR7 or its ligands might result in deleterious consequences for subjects with chronic inflammatory diseases.

ß-glucan triggers spondylarthritis and Crohn's disease-like ileitis in SKG mice.

OBJECTIVE: The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 ß-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-ß-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. METHODS: SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. RESULTS: After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. CONCLUSION: Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic ß-glucan or mannan exposure.

Probiotics promote gut health through stimulation of epithelial innate immunity.

Probiotic formulations are widely available and have a variety of proposed beneficial effects, including promotion of gut health. The mechanisms of action of probiotic bacteria in the intestine are still unclear but are generally attributed to an antiinflammatory effect. Here, we demonstrate that the multiple probiotic formulation VSL#3 prevents the onset of intestinal inflammation by local stimulation of epithelial innate immune responses (i.e., increased production of epithelial-derived TNF-alpha and restoration of epithelial barrier function in vivo). We also demonstrate that probiotic bacteria stimulate epithelial production of TNF-alpha and activate NF-kappaB in vitro. Our results support the hypothesis that probiotics promote gut health through stimulation, rather than suppression, of the innate immune system. Furthermore, our findings provide the perspective that defects in innate immunity may play a critical role in the pathogenesis and progression of intestinal disorders, such as inflammatory bowel disease.

Flt3 ligand expands CD103⁺ dendritic cells and FoxP3⁺ T regulatory cells, and attenuates Crohn's-like murine ileitis.

UNLABELLED: BACKGROUND; Imprinting an effector or regulatory phenotype on naïve T cells requires education at induction sites by dendritic cells (DC). Objectives To analyse the effect of inflammation on the frequency of mononuclear phagocytes (MP) and the effect of altering their frequency by administration of Flt3-L in chronic ileitis. METHODS: Using a tumour necrosis factor (TNF) driven model of ileitis (ie, TNFΔARE) that recapitulates many features of Crohn's disease (CD), dynamic changes in the frequency and functional state of MP within the inflamed ileum were assessed by flow cytometry, immunofluorescence and real-time reverse-transcription PCR and by generating CX(3)CR1 GFP-reporter TNFΔARE mice. The effect of Flt3-L supplementation on the severity of ileitis, and the frequency of CD103(+) DC and of FoxP3(+) regulatory T cells was also studied in TNFΔARE mice. RESULTS: CD11c(Hi)/MHCII(+) MP accumulated in inflamed ilea, predominantly mediated by expansion of the CX(3)CR1(+) MP subpopulation. This coincided with a decreased pro-regulatory CD103(+) DC. The phenotype of these MP was that of activated cells, as they expressed increased CD80 and CD86 on their surface. Flt3-ligand administration resulted in a preferential expansion of CD103(+) DC that attenuated the severity of ileitis in 20-week-old TNFΔARE mice, mediated by increased CD4(+)/CD25(+)/FoxP3(+) regulatory T cells. CONCLUSIONS: Results support a role for Flt3-L as a potential therapeutic agent in Crohn's-like ileitis.