Nitsan Maharshak

Macrophage Migration Inhibitory Factor Induces B Cell Survival by Activation of a CD74-CD44 Receptor Complex

Macrophage migration inhibitory factor (MIF) is an upstream activator of innate immunity that regulates subsequent adaptive responses. It was previously shown that in macrophages, MIF binds to a complex of CD74 and CD44, resulting in initiation of a signaling pathway. In the current study, we investigated the role of MIF in B cell survival. We show that in B lymphocytes, MIF initiates a signaling cascade that involves Syk and Akt, leading to NF-κB activation, proliferation, and survival in a CD74- and CD44-dependent manner. Thus, MIF regulates the adaptive immune response by maintaining the mature B cell population.

The Cytokine Midkine and Its Receptor RPTPζ Regulate B Cell Survival in a Pathway Induced by CD74

Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.

Increased fibrosis progression rates in hepatitis C patients carrying the prothrombin G20210A mutation

AIMnTo examine whether hepatitis C virus (HCV)-infected patients who carry hypercoagulable mutations suffer from increased rates of liver fibrosis.nnnMETHODSnWe analyzed DNA samples of 168 HCV patients for three common hypercoagulable gene mutations: prothrombin 20210 (PT20210), factor V Leiden (FV Leiden) and methylene tetrahydrofolate reductase (MTHFR). The patients were consecutively recruited as part of the prospective Fibroscore Study in France. The effect of the various mutations on the rate of fibrosis was analyzed statistically and was correlated with epidemiological, clinical and biochemical data such as grade and stage of liver biopsies, patients risk factors for liver cirrhosis, and timing of infection.nnnRESULTSnFifty two of the patients were categorized as fast fibrosers and 116 as slow fibrosers; 13% of the fast fibrosers carried the PT20210 mutation as compared with 5.5% of the slow fibrosers, with an odds ratio of 4.76 (P = 0.033; 95% CI: 1.13-19.99) for fast liver fibrosis. Carriage of MTHFR or FV Leiden mutations was not associated with enhanced liver fibrosis.nnnCONCLUSIONnCarriage of the PT20210 mutation is related to an increased rate of liver fibrosis in HCV patients.

CCL2 (pM levels) as a therapeutic agent in inflammatory bowel disease models in mice

Background: Chemokines regulate the pathways that restrict homing of specific subsets of immune cells, and thereby fine tune the immune response at specific lymphoid and peripheral tissues. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells. Previously, we demonstrated that pM levels of CCL2 dramatically inhibit migration of T cells. The aim was to test whether subphysiological doses of CCL2 can ameliorate murine colitis and inflammation‐induced colorectal cancer. Methods: TNBS (2,4,6 trinitrobenzene sulfonic acid) colitis and dextran sodium sulfate (DSS) colitis were induced in Balb/c and C57BL/6 mice, respectively. Mice were treated daily with intraperitoneal CCL2 injections. Disease activity was assessed clinically, histologically, and by measuring inflammatory cytokine levels. In addition, an inflammatory cancer model was induced by azoxymethane‐DSS (AOM‐DSS) in Balb/c mice. Mice were treated daily with CCL2 for 11 weeks and then assessed for number of tumors in the colons. Results: Daily administration of CCL2 (60–120 ng) significantly decreased the development of TNBS‐ and DSS‐induced colitis. In a DSS‐AOM model, CCL2‐treated mice developed significantly fewer tumors (P < 0.005) at 11 weeks. Chronic inflammation in the CCL2‐treated mice was significantly less pronounced as compared to phosphate‐buffered saline‐treated mice. Conclusions: Administration of pM levels of CCL2 significantly inhibits migration of T cells in amelioration of TNBS and DSS colitis and inhibits development of colorectal cancer in an AOM‐DSS colitis model in mice. Thus, pM levels of CCL2 may be clinically beneficial as an antiinflammatory agent in IBD. (Inflamm Bowel Dis 2010)

CD151 Regulates T-Cell Migration in Health and Inflammatory Bowel Disease.

Abstract:The continuous recirculation of mature lymphocytes and their entry into the peripheral lymph nodes are crucial for the development of an immune response to foreign antigens. Occasionally, the entry and the subsequent response of T lymphocytes in these sites lead to severe inflammation and pathological conditions. Here, we characterized the tetraspanin molecule, CD151, as a regulator of T cell motility in health and in models of inflammatory bowel disease. CD151 formed a cell surface complex with VLA-4 and LFA-1 integrins, and its activation led to enhanced migration of T cells. Picomolar levels of CCL2 that were previously shown to inhibit T-cell migration to lymph nodes suppressed CD151 expression and dissociated CD151-integrin complexes in T lymphocytes, resulting in attenuated migration toward T-cell attractant chemokines. To directly inhibit CD151 function, a truncated CD151 peptide fragment mimicking of the CD151 extracellular loop was designed. CD151 extracellular loop inhibited T-cell migration in vitro and in vivo and attenuated the development of dextrane sulfate sodium-induced colitis. Thus, CD151 is a key orchestrator of T cell motility; interference with its proper function results in attenuated progression of inflammatory bowel disease.

Tu2090 Molecular Characterization of the Intestinal Microbiota Focusing on Subgroups of Patients With Irritable Bowel Syndrome.

Alterations in the intestinal microbiota have been implicated in the pathogenesis of irritable bowel syndrome (IBS). Recent studies using advanced molecular biology techniques have demonstratedmicrobiota compositional differences between IBS patients and healthy controls (HC). Many of these studies, however, are confounded by the fact that the study populations comprise a mixed group of patients with IBS or a specific subtype of IBS. Thus, there is a need for systematic comparison of microbiota composition between subjects with clinicallyrelevant subtypes of IBS. Aim: To investigate the differences in composition of the intestinal microbiota between clinically-relevant, well-defined subgroups of patients with IBS and HC using deep pyrosequencing of the 16S rRNA gene. Methods: DNA was isolated from fecal samples from 60 IBS patients (Rome III: C-IBS, D-IBS, M-IBS; n=20 per group) and 20 HC. The V1-V2 variable regions of the 16S rRNA gene were amplified using bar-coded fusion primers and pyrosequenced using Roche-454 Titanium chemistry. Sequences were processed through the Multi-CLASSIFIER algorithm for taxonomic assignment. Classified reads were assigned to a species/OTU-level status using BLAST pipeline. Unclassified readswere clustered into OTUs at 97% using CD-Hit. A Core Measurable Microbiome (CMM) was generated for OTUs detected in ≥75% of all samples. Compositional features of CMM were compared using Linear Discriminant Analysis (LDA). Quantitative differences in species/OTUs and taxonomic groups were tested by ANOVA with the Tukey correction for multiple testing. Results: Subjects with IBS differentiated from HC by LDA using continuous variation in the species/OTUs or the CMM genera as variables. When further subcategorized based on bowel characteristics, the same subjects were also well-differentiated from one another and from HC. ANOVA analysis showed quantitative species/OTUs differences between the study subgroups: I. a significant increase in members of the Actinobacteria phyla (in particular the genus Collinsiella) in the C-IBS subgroup compared to all other groups, II. a significant decrease in members of Firmicutes phyla ( Oscillibacter, Anaerovorax, Incertae sedis XIII, Streptococcus and Eubacteriaceae) in D-IBS and M-IBS compared to HC and C-IBS, and III. a significant lower levels of Lactobacillus in HC compared to C-IBS and D-IBS. Conclusion: We demonstrated clear differences in the intestinal microbiota between patients with clinically-relevant subtypes of IBS. While differences in many individual taxa were identified, there was little difference in the relative abundances of major taxonomic groups (e.g., ratio of Bacteriodetes;Firmicutes:Proteobacteria). Our results provide a rationale for further investigation of the causality and the role of these microbiota compositional alterations in the pathogenesis of IBS.

317 Iron Supplemented Diet Protects Against Chronic Immune Mediated Colitis in IL-10 Deficient Mice

G A A b st ra ct s the waaWVL operon is not present in non pathogenic E. coli strains but is present in most AIEC from our collection and in two other sequenced AIEC strains NRG 857C and UM146. CONCLUSION: The presence of waaWVL operon is essential for AIEC bacteria to form biofilm at the surface of the intestinal mucosa. The search for the presence of the waaWVL operon could represent a useful molecular tool to identify pathogenic AIEC and targeting waaWVL operon expression could be a very potent therapeutic strategy to interfere with the ability of AIEC to form biofilm on the gut mucosa of Crohns disease patients.

Components of Enteric Dysbiosis Are Present at the Onset of Immune-mediated, Spontaneous Colitis: P-189 YI

BACKGROUND: Patients with inflammatory bowel diseases (IBD) frequently demonstrate intestinal dysbioses, but it is unknown if shifts in the microbiota drive disease or if they are a by-product of inflammation. We utilized multiple cultureindependent techniques to investigate whether enteric dysbiosis ensues prior to or subsequent to the development of inflammation in the interleukin-10 knockout (IL-10 KO) mouse model of immune-mediated, spontaneous colitis. METHODS: 8-10 week old germ-free (GF) wild-type (WT) (n 1⁄4 4) and IL-10 KO (n 1⁄4 9) 129Sv/Ev mice were colonized with identical specific pathogen-free (SPF) feces, and fecal samples were subsequently taken weekly for 4 weeks. Mice were necropsied at 2 and 4 weeks and colonic tissues were harvested. Fecal DNA was isolated and 454 pyrosequencing and qPCR of 16S rRNA genes were performed. QIIME pipeline was utilized for 454 analyses. IL-12p40 secretion from colonic tissue explants was measured by ELISA and Kruskal-Wallis one-way analysis of variance performed. RESULTS: Colonic inflammation in IL-10 KO mice as measured by pro-inflammatory IL-12p40 secretion was seen at 2 weeks after colonization (P<0.02 between groups), whereas WT mice did not develop colitis. QIIME pyrosequencing analyses revealed that microbiota a-diversity (species richness) and b-diversity (species diversity) increased from week 1 to week 2 in WT and IL-10 KO mice, then dramatically decreased at weeks 3 and 4 in IL-10 KO mice (P<0.05) despite increases in total fecal bacterial numbers (P<0.0007). Levels of abundant fecal Bacteroidetes remained unchanged in IL-10 KO mice at week 2, then decreased at weeks 3 and 4. qPCR showed that IL-10 KO mouse fecal Lactobacillus spp. levels were lesser than WT mouse levels at weeks 1 and 2, then significantly increased to exceed WT levels at weeks 3 and 4 (P<0.002). However, at 2 weeks after colonization, some obvious microbial shifts had already commenced in IL-10 KO mice. These included decreases in Firmicutes, (including several members of the Lachnospiraceae family), Verrucomicrobia, (including the abundant human intestinal colonizer Akkermansia muciniphila, which are decreased in IBD patients), and Actinobacteria. There were also dramatic increases in fecal levels of the Proteobacteria phylum in IL-10 KO mice at 2 weeks. E. coli levels, which were lower in IL-10 KO mice than WT mice at week 1, increased to approximate WT levels at 2 and 3 weeks, and then continued to increase until they exceeded WT levels at week 4 (P<0.0001). CONCLUSION(S): GF IL-10 KO mice housed in our gnotobiotic facility develop spontaneous colitis by 2 weeks after colonization with SPF microbiota. While some alterations in the enteric microbiota composition do not occur until after this time point, other changes are already observed by 2 weeks after conventionalization. Though expansion of this line of research is needed, these results suggest that it is possible that some components of intestinal dysbioses precede and drive the development of immune-mediated, spontaneous colitis in the IL-10 KO mouse, and perhaps in humans. If proven, a primary focus in preventing and/or managing IBD would involve avoidance and/or reversal of dysbioses with prebiotics, probiotics, antibiotics, fecal transplant, or anti-microbial peptide induction.