SAMP1/YitFc Mice Research Articles

P-150 CCL21 Defect in Ileitis-Prone SAMP1/YitFc Mice

The chemokine CCL21 plays an important role in homing of naive T cells and dendritic cell (DC) to lymph nodes but factors regulating its expression are not fully understood. Migratory DCs are crucial in establishing tolerance towards foreign yet harmless antigens. We discovered that CCL21 is almost completely absent in ileitis-prone SAMP1/YitFc (SAMP) mice, and DC migration is severely impaired. To study molecular basis for reduced production of CCL21 we measured gene expression in lymph node stromal cells and conducted genetic experiments to isolate region responsible for the defect. We used SAMP mice, which develop spontaneous chronic ileitis similar to Crohn's disease. We measured CCL21 expression by quantitative polymerase chain reaction and immunofluorescence, and spontaneous or induced DC migration by flow cytometry. The impact of DC trafficking on ileitis was assessed by feeding mice with the Toll-like receptor 7 ligand R848. To find genetic region conveying defective production of Ccl21 we backcrossed the offspring of SAMP mice and C57/Bl6 mice to the SAMP background and performed high-density SNP mapping. We studied gene expression by nanoString and chromatin structure by ATACseq. SAMP mice express almost no CCL21. Loss of CCL21 expression was linked to a recessive locus on chromosome 4, located adjacent to CCL21 genes. Stromal cells from MLN of SAMP mice had upregulated genes related to antigen presentation and a signature of interferon response. SAMP mice had severe defect of CD11b+CD103+ DC migration from the ileal lamina propria to the mesenteric lymph nodes (MLN), similar to the defect seen in mice lacking CCR7, the receptor for CCL21. Treatment with R848 enhanced DC migration, increased Tregs in MLN and dramatically improved disease scores. We identify the absence of CCL21 as the major pathogenic defect contributing to ileitis in SAMP mice. Identified genetic defect offers new insights into the regulation of CCL21 expression. Our data suggest that therapies aimed at improved DC trafficking might be useful in patients with Crohn's disease.

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.

Dysregulated intrahepatic CD4+ T-cell activation drives liver inflammation in ileitis-prone SAMP1/YitFc mice.

Background & AimsLiver inflammation is a common extraintestinal manifestation of inflammatory bowel disease (IBD), but whether liver involvement is a consequence of a primary intestinal defect or results from alternative pathogenic processes remains unclear. Therefore, we sought to determine the potential pathogenic mechanism(s) of concomitant liver inflammation in an established murine model of IBD.MethodsLiver inflammation and immune cell subsets were characterized in ileitis-prone SAMP1/YitFc (SAMP) and AKR/J (AKR) control mice, lymphocyte-depleted SAMP (SAMPxRag-1−/−), and immunodeficient SCID recipient mice receiving SAMP or AKR donor CD4+ T cells. Proliferation and suppressive capacity of CD4+ T-effector (Teff) and T-regulatory (Treg) cells from gut-associated lymphoid tissue (GALT) and livers of SAMP and AKR mice were measured.ResultsSurprisingly, prominent inflammation was detected in 4-week-old SAMP livers before histologic evidence of ileitis, whereas both disease phenotypes were absent in age-matched AKR mice. SAMP liver disease was characterized by abundant infiltration of lymphocytes, required for hepatic inflammation to occur, a TH1-skewed environment, and phenotypically activated CD4+ T cells. SAMP intrahepatic CD4+ T cells also had the ability to induce liver and ileal inflammation when adoptively transferred into SCID recipients, whereas GALT-derived CD4+ T cells produced milder ileitis but not liver inflammation. Interestingly, SAMP intrahepatic CD4+ Teff cells showed increased proliferation compared with both SAMP GALT- and AKR liver-derived CD4+ Teff cells, and SAMP intrahepatic Tregs were decreased among CD4+ T cells and impaired in in vitro suppressive function compared with AKR.ConclusionsActivated intrahepatic CD4+ T cells induce liver inflammation and contribute to experimental ileitis via locally impaired hepatic immunosuppressive function.

SAMP1/YitFc Mice Develop Ileitis via Loss of CCL21 and Defects in Dendritic Cell Migration

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. 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. 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. 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.

P-200 CCL21 Defect Impairs Dendritic Cell Trafficking in SAMP1/YitFc Mice

The lymphatic chemokine CCL21 is required for dendritic cell (DC) trafficking from tissues to lymph nodes. Migratory DCs are crucial in establishing tolerance towards foreign yet harmless antigens. Here, we discovered that CCL21 is almost completely absent in ileitis-prone SAMP1/YitFc (SAMP) mice, and DC migration is severely impaired. We investigated whether mobilizing DCs by oral treatment with R848 can attenuate ileitis. We used SAMP mice which develop spontaneous chronic ileitis similar to Crohn's disease. We measured CCL21 expression by quantitative polymerase chain reaction and immunofluorescence, and spontaneous or induced DC migration by flow cytometry. We analyzed DC production of retinoic acid and their ability to induce regulatory T cells (Tregs). The impact of DC trafficking on ileitis was assessed by feeding mice with the Toll-like receptor 7 ligand R848. SAMP mice express almost no CCL21 and have severe defect of CD11b+CD103+ DC migration from the ileal lamina propria to the mesenteric lymph nodes (MLN), similar to the defect seen in mice lacking CCR7, the receptor for CCL21. The ability of DCs to produce retinoic acid supporting Tregs was also drastically reduced in SAMP mice. In young mice, the defects in CCL21 expression and DC trafficking preceded the clinical manifestation of ileitis. Treatment with R848 enhanced DC migration, increased Tregs in MLN and dramatically improved disease scores. We identify the absence of CCL21 as the major pathogenic defect contributing to ileitis in SAMP mice. Our data suggest that therapies aimed at improved DC trafficking might be useful in patients with Crohn's disease.

Occurrence of spontaneous periodontal disease in the SAMP1/YitFc murine model of Crohn disease.

Oral involvement is often associated with inflammatory bowel disease (IBD). Recent evidence suggests a high incidence of periodontal disease in patients with Crohn disease (CD). To the best of the authors' knowledge, no animal model of IBD that displays associated periodontal disease was reported previously. The aim of this study is to investigate the occurrence and progression of periodontal disease in SAMP1/YitFc (SAMP) mice that spontaneously develop a CD-like ileitis. In addition, the temporal correlation between the onset and progression of periodontal disease and the onset of ileitis in SAMP mice was studied. At different time points, SAMP and parental AKR/J (AKR) control mice were sacrificed, and mandibles were prepared for stereomicroscopy and histology. Terminal ilea were collected for histologic assessment of inflammation score. Periodontal status, i.e., alveolar bone loss (ABL) and alveolar bone crest, was examined by stereomicroscopy and histomorphometry, respectively. ABL increased in both strains with age. SAMP mice showed greater ABL compared with AKR mice by 12 weeks of age, with maximal differences observed at 27 weeks of age. AKR control mice did not show the same severity of periodontal disease. Interestingly, a strong positive correlation was found between ileitis severity and ABL in SAMP mice, independent of age. The present results demonstrate the occurrence of periodontal disease in a mouse model of progressive CD-like ileitis. In addition, the severity of periodontitis strongly correlated with the severity of ileitis, independent of age, suggesting that common pathogenic mechanisms, such as abnormal immune response and dysbiosis, may be shared between these two phenotypes.

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.

Essential role for Toll-like receptor 9 in the pathogenesis of liver inflammation in a murine model of Crohn’s disease-like ileitis (P3147)

Abstract Liver inflammation, a common extraintestinal manifestation of inflammatory bowel disease (IBD), was reported to unexpectedly precede ileitis onset in SAMP1/YitFc (SAMP) mice, a spontaneous model of IBD. We hypothesized that since SAMP possess an inherent epithelial barrier defect that can facilitate bacterial translocation, ultimately into the liver, toll-like receptor (TLR) activation of resident immune cells may occur, leading to liver inflammation. To test this, SAMP X MyD88KO mice were generated and liver inflammation assessed. TLRs were measured by qPCR and TLR9+ cells identified by FACS in isolated non-parenchymal liver cells (NPLs). Cytokines were measured by ELISA from NPLs +/- a TLR9 agonist, and SAMP treated with a TLR9 agonist or antagonist evaluated. Deletion of MyD88 resulted in complete amelioration of SAMP liver inflammation as early as 4, and up to 40, wks vs age-matched WT SAMP (both p<0.001). TLR9 mRNA levels were highest for TLR expression in SAMP vs AKR (parental) control livers (↑2.4-fold, p<0.001) and TLR9 expressed in both NPL F4/80+ MΦs and CD4+ T cells, with specific TLR9 activation modulating TNF, IL-12, and IL-10 (p<0.05). Finally, administration of a TLR9 agonist exacerbated (p<0.001), while a TLR9 antagonist ameliorated, SAMP liver inflammation (vs vehicle, p<0.05). These data indicate a pivotal role for TLR9 in the pathogenesis of liver inflammation in ileitis-prone SAMP mice and suggest its importance in hepatic involvement in patients with IBD.

Tregs are dysfunctional in vivo in a spontaneous murine model of Crohn's disease.

Although regulatory T cells (Tregs) have been implicated in inflammatory bowel disease (IBD), Tregs from Crohn’s disease (CD) patients are increased in number and function normally in vitro. To clarify this disparity, we studied Treg function in vivo using a spontaneous model of CD-like ileitis. We first administered anti-CD25 depleting antibodies to SAMP1/YitFc (SAMP) mice to assess ileitis; mesenteric lymph node cells were then transferred into SCID recipients to induce colitis. CD25 depletion increased the severity of both spontaneous ileitis and adoptively transferred colitis. Interestingly, a second transfer of CD4+CD25+ cells from untreated AKR control mice was able to ameliorate the induced colitis, while CD4+CD25+ cells from untreated SAMP mice were not, suggesting a functional abnormality in SAMP Tregs. Anti-CD25 treatment in SAMP mice also induced proliferation of CD25−Foxp3+ Tregs, which had a proinflammatory intestinal Th1/Th2 effector phenotype. These studies demonstrate Treg dysfunction in a spontaneous model of CD-like ileitis.

Spontaneous, Immune-Mediated Gastric Inflammation in SAMP1/YitFc Mice, a Model of Crohn's-Like Gastritis

Crohn's disease (CD) can develop in any region of the gastrointestinal tract, including the stomach. The etiology and pathogenesis of Crohn's gastritis are poorly understood, treatment approaches are limited, and there are not many suitable animal models for study. We characterized the features and mechanisms of chronic gastritis in SAMP1/YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, along with possible therapeutic approaches. Stomachs from specific pathogen-free and germ-free SAMP and AKR mice (controls) were evaluated histologically; the presence of Helicobacter spp was tested in fecal pellets by polymerase chain reaction analysis. In vivo gastric permeability was quantified by fractional excretion of sucrose, and epithelial tight junction protein expression was measured by quantitative reverse-transcription polymerase chain reaction analysis. The effects of a proton pump inhibitor (PPI) or corticosteroids were measured, and the ability of pathogenic immune cells to mediate gastritis was assessed in adoptive transfer experiments. SAMP mice developed Helicobacter-negative gastritis, characterized by aggregates of mononuclear cells, diffuse accumulation of neutrophils, and disruption of epithelial architecture; SAMP mice also had increased gastric permeability compared with controls, without alterations in expression of tight junction proteins. The gastritis and associated permeability defect observed in SAMP mice were independent of bacterial colonization and reduced by administration of corticosteroids but not a PPI. CD4(+) T cells isolated from draining mesenteric lymph nodes of SAMP mice were sufficient to induce gastritis in recipient SCID mice. In SAMP mice, gastritis develops spontaneously and has many features of CD-like ileitis. These mice are a useful model to study Helicobacter-negative, immune-mediated Crohn's gastritis.

Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis

IL-33 is a novel member of the IL-1 family and ligand for the IL-1 receptor-related protein, ST2. Recent evidence suggests that the IL-33/ST2 axis plays a critical role in several autoimmune and inflammatory disorders; however, its role in inflammatory bowel disease (IBD) has not been clearly defined. We characterized IL-33 and ST2 expression and modulation after conventional anti-TNF therapy in Crohn's disease and ulcerative colitis (UC) patients and investigated the role of IL-33 in SAMP1/YitFc (SAMP) mice, a mixed Th1/Th2 model of IBD. Our results showed a specific increase of mucosal IL-33 in active UC, localized primarily to intestinal epithelial cells (IEC) and colonic inflammatory infiltrates. Importantly, increased expression of full-length IL-33, representing the most bioactive form, was detected in UC epithelium, whereas elevated levels of cleaved IL-33 were present in IBD serum. ST2 isoforms were differentially modulated in UC epithelium, and sST2, a soluble decoy receptor with anti-inflammatory properties, was also elevated in IBD serum. Infliximab (anti-TNF) treatment of UC decreased circulating IL-33 and increased sST2, whereas stimulation of HT-29 IEC confirmed IL-33 and sST2 regulation by TNF. Similarly, IL-33 significantly increased and correlated with disease severity, and potently induced IL-5, IL-6, and IL-17 from mucosal immune cells in SAMP mice. Taken together, the IL-33/ST2 system plays an important role in IBD and experimental colitis, is modulated by anti-TNF therapy, and may represent a specific biomarker for active UC.

Mechanisms of Tight Junction Dysregulation in the SAMP1/YitFc Model of Crohn's Disease–like Ileitis

To date, the precise etiology of inflammatory bowel disease (IBD) remains largely unknown; however, it is well accepted that IBD results from a dysregulated mucosal immune response to environmental factors in genetically susceptible hosts. The primary defect, in at least a subpopulation of IBD patients, may be due to abnormal intestinal epithelial barrier function. The SAMP1/YitFc (SAMP) mouse strain is a spontaneous model of IBD, closely resembling Crohn's disease for its histologic features and localization to the terminal ileum. Dysregulated epithelial barrier function that precedes histologic evidence of ileitis has been reported to be the primary defect in SAMP mice. Data suggest that barrier dysfunction occurs in the absence of commensal bacteria and is accompanied by aberrant expression of the tight junction proteins claudin-2 and occludin. Further investigation is needed to define the precise role of the intestinal epithelium, as well as the apical junctional complex and its associated proteins, in the pathogenesis of IBD in order to determine the etiology and aid in the development of novel treatment modalities for these devastating diseases.

The primary defect in experimental ileitis originates from a nonhematopoietic source

The initiating etiologic factor in Crohn's disease (CD) remains unclear. SAMP1/YitFc (SAMP) mice develop chronic ileitis similar to human CD. We used bone marrow chimeras to determine if SAMP ileitis results from a primary immunological defect or from dysregulated mucosal immunity secondary to intrinsic, nonhematopoietic (e.g., epithelial) dysfunction. SAMP mice receiving wild-type (AKR) BM developed severe ileitis, whereas SAMP BM did not confer ileitis to WT recipients. WT lymphocytes from reconstituted SAMP mice resembled native SAMP populations in regard to surface phenotype and cytokine production. Ilea from native SAMP mice and SAMP recipients of wild-type BM displayed decreased epithelial barrier resistance ex vivo and increased epithelial permeability in vivo compared to native WT mice and AKR recipients of SAMP BM. This permeability defect preceded the development of ileal inflammation, was present in the absence of commensal bacteria, and was accompanied by altered ileal mRNA expression of the tight junction proteins claudin-2 and occludin. Our results provide evidence that the primary defect conferring ileitis in SAMP mice originates from a nonhematopoietic source. Generation of pathogenic lymphocytes is a consequence of this defect and does not reflect intrinsic proinflammatory leukocyte properties. Decreased barrier function suggests that defects in the epithelium may represent the primary source of SAMP ileitis susceptibility.

Targeting Mucosal Addressin Cellular Adhesion Molecule (MAdCAM)-1 to Noninvasively Image Experimental Crohn’s Disease

Inflammatory bowel disease (IBD) is the second most common chronic inflammatory disorder worldwide; however, a noninvasive means of accurately assessing the severity and extent of intestinal inflammation is currently not available. The aim of the present study was to develop a noninvasive imaging modality to detect and evaluate ileitis in SAMP1/YitFc (SAMP) mice. An image-enhancing ultrasound (US) contrast agent, consisting of encapsulated gaseous microbubbles (MB), was developed specifically to bind mucosal addressin cellular adhesion molecule-1 (MAdCAM-1), a mucosal-restricted addressin up-regulated during gut inflammation. MAdCAM-1-targeted MB (MB(M)) were tested for binding specificity on MAdCAM-1 protein and tumor necrosis factor (TNF)-stimulated SVEC4-10 endothelial cells using an in vitro flow chamber assay and for their ability to detect and quantify ileitis by intravital microscopy and transabdominal US. Under in vitro flow conditions, a 100-fold increase in MB(M) binding was observed on MAdCAM-1 protein compared with nonspecific MB (P < .001). TNF-stimulated endothelial cells bound significantly more MB(M) vs nonspecific MB (P < .001), which was abrogated after preincubation with anti-MAdCAM-1 antibodies (P < .001). In vivo, MB(M) specifically accumulated in focal areas of ileal inflammation and produced stronger acoustic echoes, measured by average video intensity, in SAMP vs uninflamed AKR mice (P < .001) or SAMP given nonspecific MB (P < .001). MB(M)-specific video intensity showed a strong positive correlation with total ileal inflammatory scores (R2 = 0.92). We have developed a novel intravascular US contrast agent targeting MAdCAM-1 that specifically detects and quantifies intestinal inflammation in experimental ileitis, providing the potential for a reliable, noninvasive means to diagnose and monitor disease in patients with IBD.