Crohn's Disease-like Ileitis Research Articles

Miniaturized implantable temperature sensors for the long-term monitoring of chronic intestinal inflammation.

Diagnosing and monitoring inflammatory bowel diseases, such as Crohn's disease, involves the use of endoscopic imaging, biopsies and serology. These infrequent tests cannot, however, identify sudden onsets and severe flare-ups to facilitate early intervention. Hence, about 70% of patients with Crohn's disease require surgical intestinal resections in their lifetime. Here we report wireless, miniaturized and implantable temperature sensors for the real-time chronic monitoring of disease progression, which we tested for nearly 4 months in a mouse model of Crohn's-disease-like ileitis. Local measurements of intestinal temperature via intraperitoneally implanted sensors held in place against abdominal muscular tissue via two sutures showed the development of ultradian rhythms at approximately 5 weeks before the visual emergence of inflammatory skip lesions. The ultradian rhythms showed correlations with variations in the concentrations of stress hormones and inflammatory cytokines in blood. Decreasing average temperatures over the span of approximately 23 weeks were accompanied by an increasing percentage of inflammatory species in ileal lesions. These miniaturized temperature sensors may aid the early treatment of inflammatory bowel diseases upon the detection of episodic flare-ups.

A Novel Probiotic Combination Ameliorates Crohn's Disease-Like Ileitis by Increasing Short-Chain Fatty Acid Production and Modulating Essential Adaptive Immune Pathways.

Crohn's disease (CD) represents a significant public health challenge. We identified a combination of beneficial probiotic strains (Saccharomyces boulardii, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Bifidobacterium breve) and amylase that may antagonize elevated bacterial pathogens in the inflamed gut. Our aim was to characterize the effect(s) of this novel probiotic supplement in SAMP1/YitFc (SAMP) mice with CD-like ileitis. Three groups of 7-week-old SAMP mice were used in this study. The first experimental group was administered 1 dose of the probiotic supplement (probiotic strains + amylase) diluted in sterile phosphate-buffered saline (PBS) (0.25 mg in 100 µL of PBS) every day for 56 days through the gavage technique, the second group had a probiotic supplement (probiotic strains without amylase), and the third group was a control group in which animals were administered sterile PBS. At the end of the treatment, mice were sacrificed and ilea were collected for histological scoring of ileitis and NanoString analysis. Stool samples were evaluated by 16S ribosomal RNA and gas chromatography-mass spectrometry analyses. Histology scores showed that mice treated with probiotics + amylase had a significant decrease of ileitis severity compared with the other 2 groups. 16S ribosomal RNA and gas chromatography-mass spectrometry analysis showed that abundance of species belonging to genus Lachnoclostridium and Mucispirillum schaedleri were significantly increased compared with the other 2 groups, and this increase was associated with augmented production of short-chain fatty acids. NanoString data showed that 21 genes involved in B memory cell development and T cell infiltration were significantly upregulated in probiotic-treated mice and that 3 genes were significantly downregulated. Our data provide experimental proof for a beneficial effect of the designed probiotic formulation on the severity of CD-like ileitis in the SAMP mouse model, involving both alteration of intestinal genetic pathways and microbial rearrangements. Thus, we propose that this novel probiotic mixture should be further tested as an adjuvant therapy in the treatment of biofilm-associated disorders such as CD, in which it has been proven that polymicrobial imbalance plays a critical role in dysbiosis and gut inflammation.

Chronic stress induces colonic tertiary lymphoid organ formation and protection against secondary injury through IL-23/IL-22 signaling

Psychological stress has been previously reported to worsen symptoms of inflammatory bowel disease (IBD). Similarly, intestinal tertiary lymphoid organs (TLOs) are associated with more severe inflammation. While there is active debate about the role of TLOs and stress in IBD pathogenesis, there are no studies investigating TLO formation in the context of psychological stress. Our mouse model of Crohn's disease-like ileitis, the SAMP1/YitFc (SAMP) mouse, was subjected to 56 consecutive days of restraint stress (RS). Stressed mice had significantly increased colonic TLO formation. However, stress did not significantly increase small or large intestinal inflammation in the SAMP mice. Additionally, 16S analysis of the stressed SAMP microbiome revealed no genus-level changes. Fecal microbiome transplantation into germ-free SAMP mice using stool from unstressed and stressed mice replicated the behavioral phenotype seen in donor mice. However, there was no difference in TLO formation between recipient mice. Stress increased the TLO formation cytokines interleukin-23 (IL-23) and IL-22 followed by up-regulation of antimicrobial peptides. SAMP × IL-23r-/- (knockout [KO]) mice subjected to chronic RS did not have increased TLO formation. Furthermore, IL-23, but not IL-22, production was increased in KO mice, and administration of recombinant IL-22 rescued TLO formation. Following secondary colonic insult with dextran sodium sulfate, stressed mice had reduced colitis on both histology and colonoscopy. Our findings demonstrate that psychological stress induces colonic TLOs through intrinsic alterations in IL-23 signaling, not through extrinsic influence from the microbiome. Furthermore, chronic stress is protective against secondary insult from colitis, suggesting that TLOs may function to improve the mucosal barrier.

Acesulfame potassium induces dysbiosis and intestinal injury with enhanced lymphocyte migration to intestinal mucosa.

The artificial sweetener acesulfame potassium (ACK) is officially approved as safe for intake and has been used in processed foods. However, ACKs have been reported to induce metabolic syndrome, along with alteration of the gut microbiota in mice. In recent years, studies have suggested that this artificial sweetener promotes myeloperoxidase reactivity in Crohn's disease-like ileitis. We aimed to investigate the effect of ACK on the intestinal mucosa and gut microbiota of normal mice. Acesulfame potassium was administered to C57BL/6J mice (8weeks old) via free drinking. Intestinal damage was evaluated histologically, and messenger RNA (mRNA) levels of TNF-α, IFN-γ, IL1-β, MAdCAM-1, GLP1R, and GLP2R were determined with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed to examine the expression of MAdCAM-1 in the small intestine. The composition of gut microbiota was assessed using high-throughput sequencing. We performed intravital microscopic observation to examine if ACK altered lymphocyte migration to the intestinal microvessels. Acesulfame potassium increased the expression of proinflammatory cytokines, decreased the expression of GLP-1R and GLP-2R, and induced small intestinal injury with an increase in intestinal permeability, and ACK treatment induced microbial changes, but the transfer of feces alone from ACK mice did not reproduce intestinal damage in recipient mice. ACK treatment significantly increased the migration of lymphocytes to intestinal microvessels. Acesulfame potassium induces dysbiosis and intestinal injury with enhanced lymphocyte migration to intestinal mucosa. Massive use of non-caloric artificial sweeteners may not be as safe as we think.

NOD2 drives early IL-33-dependent expansion of group 2 innate lymphoid cells during Crohn's disease-like ileitis.

Innate lymphoid cells (ILCs) are enriched at barrier surfaces, including the gastrointestinal tract. While most studies have focused on the balance between pathogenic group 1 ILCs (ILC1s) and protective ILC3s in maintaining gut homeostasis and during chronic intestinal inflammation, such as Crohn's disease (CD), less is known regarding ILC2s. Using an established murine model of CD-like ileitis, i.e., the SAMP1/YitFc (SAMP) mouse strain, we showed that ILC2s, compared with ILC1s and ILC3s, were increased within draining mesenteric lymph nodes and ilea of SAMP versus AKR (parental control) mice early, during the onset of disease. Gut-derived ILC2s from CD patients versus healthy controls were also increased and expanded, similarly to ILC1s, in greater proportion compared with ILC3s. Importantly, we report that the intracellular bacteria-sensing protein, nucleotide-binding oligomerization domaining-containing protein 2, encoded by Nod2, the first and strongest susceptibility gene identified for CD, promoted ILC2 expansion, which was dramatically reduced in SAMP mice lacking NOD2 and in SAMP mice raised under germ-free conditions. Furthermore, these effects occurred through a mechanism involving the IL-33/ST2 ligand-receptor pair. Collectively, our results indicate a functional link between NOD2 and ILC2s, regulated by the IL-33/ST2 axis, that mechanistically may contribute to early events leading to CD pathogenesis.

Intestinal Stem Cell Niche Defects Result in Impaired 3D Organoid Formation in Mouse Models of Crohn's Disease-like Ileitis.

SummaryIntestinal epithelial barrier dysfunction is a risk factor in the pathogenesis of Crohn’s disease (CD); however, no corrective FDA-approved therapies exist. We used an enteroid (EnO)-based system in two murine models of experimental CD, SAMP1/YitFc (SAMP) and TNFΔARE/+ (TNF). While severely inflamed SAMP mice do not generate EnOs, “inflammation-free” SAMP mice form EnO structures with impaired morphology and reduced intestinal stem cell (ISC) and Paneth cell viability. We validated these findings in TNF mice concluding that inflammation in intestinal tissues impedes EnO generation and suppressing inflammation by steroid administration partially rescues impaired formation in SAMP mice. We generated the first high-resolution transcriptional profile of the SAMP ISC niche demonstrating that alterations in multiple key pathways contribute to niche defect and targeting them may partially rescue the phenotype. Furthermore, we correlated the defects in formation and the rescue of EnO formation to reduced viability of ISCs and Paneth cells.

Impaired estrogen signaling underlies regulatory T cell loss-of-function in the chronically inflamed intestine

Signaling of 17β-estradiol (estrogen) through its two nuclear receptors, α and β (ERα, ERβ), is an important mechanism of transcriptional regulation. Although ERs are broadly expressed by cells of the immune system, the mechanisms by which they modulate immune responses remain poorly understood. ERβ-specific signaling is reduced in patients with chronic inflammatory diseases, including systemic lupus erythematosus and inflammatory bowel disease, and our previous work suggests that dysregulation of ERβ-specific signaling contributes to enhanced intestinal inflammation in female SAMP/YitFC mice, a spontaneous model of Crohn's disease-like ileitis. The present study builds on these prior observations to identify a nonredundant, immunoprotective role for ERβ-specific signaling in TGF-β-dependent regulatory T cell (Treg) differentiation. Using a strain of congenic SAMP mice engineered to lack global expression of ERβ, we observed dramatic, female-specific exacerbation of intestinal inflammation accompanied by significant reductions in intestinal Treg frequency and function. Impaired Treg suppression in the absence of ERβ was associated with aberrant overexpression of Tsc22d3 (GILZ), a glucocorticoid-responsive transcription factor not normally expressed in mature Tregs, and ex vivo data reveal that forced overexpression of GILZ in mature Tregs inhibits their suppressive function. Collectively, our findings identify a pathway of estrogen-mediated immune regulation in the intestine, whereby homeostatic expression of ERβ normally functions to limit Treg-specific expression of GILZ, thereby maintaining effective immune suppression. Our data suggest that transcriptional cross-talk between glucocorticoid and steroid sex hormone signaling represents an important and understudied regulatory node in chronic inflammatory disease.

Ameliorative effect of a novel BET inhibitor CN210 on experimentally-induced murine Crohn's disease-like ileitis via inhibition of inflammatory cytokine expression

Ameliorative effect of a novel BET inhibitor CN210 on experimentally-induced murine Crohn's disease-like ileitis via inhibition of inflammatory cytokine expression

Death-Domain-Receptor 3 Deletion Normalizes Inflammatory Gene Expression and Prevents Ileitis in Experimental Crohn's Disease.

TNF-like cytokine 1A (TL1A) and its functional receptor, death-domain-receptor-3 (DR3), are multifunctional mediators of effector and regulatory immunity. We aimed to evaluate the functional role and therapeutic potential of TL1A/DR3 signaling in Crohn's disease-like ileitis. Ileitis-prone SAMP1/YitFc (SAMP) and TNFΔARE/+ mice were rendered deficient for DR3 or TL1A by microsatellite marker-assisted backcrossing. Pathological and immunological characteristics were compared between control and knockout mice, and mucosal immunophenotype was analyzed by Nanostring microarray assay. The therapeutic effect of pharmacological TL1A neutralization was also investigated. DR3 deficiency was associated with restoration of a homeostatic mucosal immunostat in SAMP mice through the regulation of several pro- and anti-inflammatory genes. This led to suppression of effector immunity, amelioration of ileitis severity, and compromised ability of either unfractionated CD4+ or CD4+CD45RBhi mucosal lymphocytes to transfer ileitis to severe combined immunodeficient mice recipients. TNF-driven ileitis was also prevented in TNFΔARE/+xDR3-/- mice, in association with decreased expression of the pro-inflammatory cytokines TNF and IFN-γ. In contrast to DR3, TL1A was dispensable for the development of ileitis although it affected the kinetics of inflammation, as TNFΔARE/+xTL1A-/- demonstrated delayed onset of inflammation, whereas administration of a neutralizing, anti-TL1A antibody ameliorated early but not late TNFΔARE/+ ileitis. We found a prominent pro-inflammatory role of DR3 in chronic ileitis, which is only partially mediated via interaction with TL1A, raising the possibility for additional DR3 ligands. Death-domain-receptor-3 appears to be a master regulator of mucosal homeostasis and inflammation and may represent a candidate therapeutic target for chronic inflammatory conditions of the bowel.

Tu1803 - The Gene Expression Landscape of Cobblestone Lesions in Crohn's Disease-Like Ileitis

Tu1803 - The Gene Expression Landscape of Cobblestone Lesions in Crohn's Disease-Like Ileitis

Tu1770 - Role of Tl1A and its Receptor Dr3 in Regulating Intestinal Th9 Cell Differentiation in Crohn's Disease-Like Ileitis

Tu1770 - Role of Tl1A and its Receptor Dr3 in Regulating Intestinal Th9 Cell Differentiation in Crohn's Disease-Like Ileitis

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

CD4+ Tcells are tightly regulated by microbiota in the intestine, but whether intestinal Tcells 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.

Tauroursodeoxycholic acid protects bile acid homeostasis under inflammatory conditions and dampens Crohn's disease-like ileitis

Bile acids regulate the expression of intestinal bile acid transporters and are natural ligands for nuclear receptors controlling inflammation. Accumulating evidence suggests that signaling through these receptors is impaired in inflammatory bowel disease. We investigated whether tauroursodeoxycholic acid (TUDCA), a secondary bile acid with cytoprotective properties, regulates ileal nuclear receptor and bile acid transporter expression and assessed its therapeutic potential in an experimental model of Crohn's disease (CD). Gene expression of the nuclear receptors farnesoid X receptor, pregnane X receptor and vitamin D receptor and the bile acid transporters apical sodium-dependent bile acid transporter and organic solute transporter α and β was analyzed in Caco-2 cell monolayers exposed to tumor necrosis factor (TNF)α, in ileal tissue of TNFΔARE/WT mice and in inflamed ileal biopsies from CD patients by quantitative real-time polymerase chain reaction. TNFΔARE/WT mice and wild-type littermates were treated with TUDCA or placebo for 11 weeks and ileal histopathology and expression of the aforementioned genes were determined. Exposing Caco-2 cell monolayers to TNFα impaired the mRNA expression of nuclear receptors and bile acid transporters, whereas co-incubation with TUDCA antagonized their downregulation. TNFΔARE/WT mice displayed altered ileal bile acid homeostasis that mimicked the situation in human CD ileitis. Administration of TUDCA attenuated ileitis and alleviated the downregulation of nuclear receptors and bile acid transporters in these mice. These results show that TUDCA protects bile acid homeostasis under inflammatory conditions and suppresses CD-like ileitis. Together with previous observations showing similar efficacy in experimental colitis, we conclude that TUDCA could be a promising therapeutic agent for inflammatory bowel disease, warranting a clinical trial.

Genetic deletion of the bacterial sensor NOD2 improves murine Crohn's disease-like ileitis independent of functional dysbiosis.

Although genetic polymorphisms in NOD2 (nucleotide-binding oligomerization domain-containing 2) have been associated with the pathogenesis of Crohn’s disease (CD), little is known regarding the role of wild-type (WT) NOD2 in the gut. To date, most murine studies addressing the role of WT Nod2 have been conducted using healthy (ileitis/colitis-free) mouse strains. Here, we evaluated the effects of Nod2 deletion in a murine model of spontaneous ileitis, i.e., the SAMP1Yit/Fc (SAMP) strain, which closely resembles CD. Remarkably, Nod2 deletion improved both chronic cobblestone ileitis (by 50% assessed, as the % of abnormal mucosa at 24 wks of age), as well as acute dextran sodium sulfate (DSS) colitis. Mechanistically, Th2 cytokine production and Th2-transcription factor activation (i.e., STAT6 phosphorylation) were reduced. Microbiologically, the effects of Nod2 deletion appeared independent of fecal microbiota composition and function, assessed by 16S rRNA and metatranscriptomics. Our findings indicate that pharmacological blockade of NOD2 signaling in humans could improve health in Th2-driven chronic intestinal inflammation.

Dysbiotic gut microbiota causes transmissible Crohn's disease-like ileitis independent of failure in antimicrobial defence

ObjectivesDysbiosis of the intestinal microbiota is associated with Crohn's disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar...

266 Transmissive Crohn's Disease-Like Ileitis Is Caused by Functional Dysbiosis in the Intestinal Microbiota Independent of Inflammation-Driven Paneth Cell Failure

266 Transmissive Crohn's Disease-Like Ileitis Is Caused by Functional Dysbiosis in the Intestinal Microbiota Independent of Inflammation-Driven Paneth Cell Failure

OP001. Transmissive Crohn's disease-like ileitis is caused by functional dysbiosis in the intestinal microbiota independent of inflammation-driven Paneth cell failure

OP001. Transmissive Crohn's disease-like ileitis is caused by functional dysbiosis in the intestinal microbiota independent of inflammation-driven Paneth cell failure

Tu1760 Functional Evidence for a Causative Role of the Intestinal Microbiota in Crohn's Disease-Like Ileitis Using Antibiotic-Treated and Germ-Free TNFDeltaare/+ Mice

BACKGROUND: An increased prevalence of phylogenetic B2 group Escherichia coli (E. coli) has been associated with active disease in IBD patients (IBDEC). Many of the B2 group strains harbor virulence genes present in extraintestinal pathogenic E.coli including hemolysin (hly) and cytotoxic necrotizing factor type 1 (CNF1) the loci of which is often linked. Here we investigated the potential of B2 strains of E.coli isolated from IBD patients to damage the integrity of the intestinal epithelium and the potential contribution of Hly and CNF1. METHODS: E coli strains isolated from 35 patients with IBD were tested for virulence (Petersen et al 2009) and of those isolate C691-04A showed high cytotoxicity on dendritic cells ( submitted) and a rapidly decrease of the trans-epithelial electric resistance (TEER) of a monolayer of intestinal epithelial cell lines. Genome sequencing of C691-04A confirmed the presence of hly cluster. It is known that some E coli have two hly-clusters. In order to investigate the role of hly and CNF1 in disruption of the epithelial integrity the following mutants were created in strain C691-04A; 1: CAS965, hly cluster II upstream of CNF1; 2: CAS954, hly cluster I mutant; 3: CAS956 is a knock out of both loci of hly; 4: CAS964, CNF1 mutant; 5: CAS966, clbA mutant. Caco-2 cells were grown to confluence in 24-well trans-well devices for 14 days to form a polarized cell monolayer and TER measured constantly during exposure to E. coli strains at MOI 10. The wild type and commensal E. coli were included as controls. RESULTS: E. coli C691-04A WT as well as CNF1, clbA and single loci of hly mutants from cluster I and II were all able to damage Caco-2 cell tight junctions in less than 6 hrs co-culture (see figure). Confocal microscopy of occludin stained trans-well sections revealed loss of occludin from the tight junction within 3 hours. However, this phenotype was lost in a mutant with knock out of both hly loci (p<0.001). Commensal E. coli isolated from healthy controls also did not cause loss of TER in 6 h. CONCLUSION: IBD-associated E. coli such as C691-04A can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect is abolished in a mutant with knock out of both loci of the hly genes and diminished in one of the single knockout mutants. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.

Colonic Expression of the Peptide Transporter PEPT1 Is Downregulated During Intestinal Inflammation and Is Not Required for NOD2-dependent Immune Activation

PEPT1 was proposed to be expressed only in inflamed colonic tissues in which it could contribute to inflammatory bowel disease (IBD) development by transporting bacterial peptides, such as muramyl dipeptide (MDP), that activate intracellular pattern recognition receptors, such as the nucleotide-binding and oligomerization domain 2. To better define the pathological relevance of this transporter, we analyzed PEPT1 expression during intestinal inflammation and studied the susceptibility of Pept1-deficient (Pept1) mice to experimental colitis. Wild-type and Pept1 mice were treated with dextran sulfate sodium and 2,4,6-trinitrobenzene sulfonic acid to induce colitis, and MDP-induced cytokine expression was studied in colonic tissue cultures. PEPT1 expression was characterized in mouse models of Crohn's disease-like ileitis (Tnf) or colitis (Il-10, Il-10XTlr2) and endoscopic tissue samples from descending colon of patients with IBD (n = 11) and controls (n = 17). Moreover, the prevalence of the PEPT1 single-nucleotide polymorphism rs2297322 was tested in German patients with IBD (n = 458) and controls (n = 452). PEPT1 expression was consistently reduced under condition of acute or chronic experimental inflammation. Wild-type and Pept1 mice revealed comparable susceptibility to dextran sulfate sodium-induced and 2,4,6-trinitrobenzene sulfonic acid-induced colitis, and MDP-induced cytokine expression was PEPT1-independent. PEPT1 expression levels were also decreased in descending colon of patients with IBD during acute inflammation, but the rs2297322 single-nucleotide polymorphism was not associated with IBD susceptibility in the German cohort. PEPT1 expression is reduced during intestinal inflammation and PEPT1 is neither required for MDP-induced immune response nor is the PEPT1 rs2297322 single-nucleotide polymorphism associated with IBD susceptibility in our German cohort. These data strongly argue against a primary role of PEPT1 in the initiation or progression of IBD.

SHIP-1 deficiency in the myeloid compartment is insufficient to induce myeloid expansion or chronic inflammation

SHIP-1 has an important role in controlling immune cell function through its ability to downmodulate PI3K signaling pathways that regulate cell survival and responses to stimulation. Mice deficient in SHIP-1 display several chronic inflammatory phenotypes including antibody-mediated autoimmune disease, Crohn's disease-like ileitis and a lung disease reminiscent of chronic obstructive pulmonary disease. The ileum and lungs of SHIP-1-deficient mice are infiltrated at an early age with abundant myeloid cells and the mice have a limited lifespan primarily thought to be due to the consolidation of lungs with spontaneously activated macrophages. To determine whether the myeloid compartment is the key initiator of inflammatory disease in SHIP-1-deficient mice, we examined two independent strains of mice harboring myeloid-restricted deletion of SHIP-1. Contrary to expectations, conditional deletion of SHIP-1 in myeloid cells did not result in consolidating pneumonia or segmental ileitis typical of germline SHIP-1 deficiency. In addition, other myeloid cell abnormalities characteristic of germline loss of SHIP-1, including flagrant splenomegaly and enhanced myelopoiesis, were absent in mice lacking SHIP-1 in myeloid cells. This study indicates that the spontaneous inflammatory disease characteristic of germline SHIP-1 deficiency is not initiated solely by LysM-positive myeloid cells but requires the simultaneous loss of SHIP-1 in other hematolymphoid lineages.