Clcn5 knockout mice exhibit novel immunomodulatory effects and are more susceptible to dextran sulfate sodium-induced colitis.

Although the intracellular Cl(-)/H(+) exchanger Clc-5 is expressed in apical intestinal endocytic compartments, its pathophysiological role in the gastrointestinal tract is unknown. In light of recent findings that CLC-5 is downregulated in active ulcerative colitis (UC), we tested the hypothesis that loss of CLC-5 modulates the immune response, thereby inducing susceptibility to UC. Acute dextran sulfate sodium (DSS) colitis was induced in Clcn5 knockout (KO) and wild-type (WT) mice. Colitis, monitored by disease activity index, histological activity index, and myeloperoxidase activity were significantly elevated in DSS-induced Clcn5 KO mice compared with those in WT mice. Comprehensive serum multiplex cytokine profiling demonstrated a heightened Th1-Th17 profile (increased TNF-alpha, IL-6, and IL-17) in DSS-induced Clcn5 KO mice compared with that in WT DSS colitis mice. Interestingly, Clcn5 KO mice maintained on a high vitamin D diet attenuated DSS-induced colitis. Immunofluorescence and Western blot analyses of colonic mucosa validated the systemic cytokine patterns and further revealed enhanced activation of the NF-kappaB pathway in DSS-induced Clcn5 KO mice compared with those in WT mice. Intriguingly, high baseline levels of IL-6 and phospho-IkappaB were observed in Clcn5 KO mice, suggesting a novel immunopathogenic role for the functional defects that result from the loss of Clc-5. Our studies demonstrate that the loss of Clc-5 1) exhibits IL-6-mediated immunopathogenesis, 2) significantly exacerbated DSS-induced colitis, which is influenced by dietary factors, including vitamin D, and 3) portrays distinct NF-kappaB-modulated Th1-Th17 immune dysregulation, implying a role for CLC-5 in the immunopathogenesis of UC.

Distinct cytokine patterns identified from multiplex profiles of murine DSS and TNBS-induced colitis.

BACKGROUND: The cytokine network in inflammatory bowel disease (IBD) is a complex, dynamic system that plays an important role in regulating mucosal innate and adaptive immune responses. While several studies have been done to evaluate immunomodulatory profiles in murine IBD, they have been limited to a relatively small number of cytokines that do not take into account its dependency of the interplay of multiple factors, and therefore the diagnostic potential of their cytokine profiles have been inconclusive. METHODS: A novel approach of comprehensive serum multiplex cytokine profiling with biometric immunosandwich ELISA's was used to describe the modulation of 16 Th1, Th2, Th17 cytokines and chemokines in both acute and chronic murine models of DSS and TNBS-induced colitis. Advanced multivariate discriminant functional analyses (DFA) was used to identify statistically interrelated sets of variables with the most significant power to discriminate among the groups. Profiles of multiple cytokines seen systemically were also validated locally in colonic mucosa using Western blot analysis and fluorescent immunohistochemistry. RESULTS: Distinctive disease-specific cytokine profiles were identified with significant correlations to disease activity and duration of disease. TNBS colitis exhibits heightened Th1-Th17 response (increased IL-12 and IL-17) as the disease becomes chronic. In contrast, DSS colitis switches from a Th1-Th17-mediated acute inflammation (increased TNF-alpha, IL6, IL-17, and KC) to a predominant Th2-mediated inflammatory response (increase in IL-4 and IL-10 and concomitant decrease in TNF-alpha, IL6, IL-17, and KC) in the chronic state. Moreover, DFA identified discriminatory cytokine profiles that can be sufficiently used to distinguish unaffected controls from diseases, and one disease type from another. IL-6 and IL-12 stratified gender-associated disease activity in chronic colitis. CONCLUSIONS: Our studies provide insight into disease immunopathogenesis and illustrate the significant potential of utilizing multiplex cytokine profiles and bioinformatics as diagnostic tools in IBD.

Multiplex cytokine profiling of initial therapeutic response in patients with chronic hepatitis C virus infection.

Currently available prognostic tools are inadequate to discern the molecular basis of the heterogenic response in hepatitis C virus (HCV)-infected patients treated with the current standard of therapy. The expression and biological function of immune mediators have been shown to be critical in all phases of the immune response to HCV infection and likely therefore influence host response. Herein, a biometric multiplex serum cytokine assay was utilized to characterize the immunomodulatory effects of host response in 10 HCV patients. Serum levels of 17 cytokines were compared before and after 1 month of treatment and against controls. Overall serum cytokine levels were significantly higher in patients (P < 0.05) than controls. Additionally, viral titers decreased in all patients after 1 month of therapy, as did overall serum cytokine levels in the cohort (P < 0.05). To assess relationships between changes in cytokine levels and changes in viral titer, the cohort was divided into three statistically distinct subgroups based on changes in viral titers. Specific sets of cytokines decreased in each group: decreases in CCL4, interleukin (IL)-2, CXCL8, and IL-1beta correlated with the greatest drops in viral titer, decreases in IL-5, granulocyte colony stimulating factor (G-CSF), and CCL4 correlated with moderate drops in viral titer, and only CCL2 correlated with the lowest drops in viral titer. Interestingly, decreases in CCL4 levels correlated with decreases in viral titers in all patients. CCL4 controls leukocyte influx and thus propagates inflammation. In conclusion, these data raise the possibility that characteristic changes in host response modulate the therapeutic response, demonstrating the prognostic power of serum cytokine profiling in chronic HCV.