Immunoregulatory Defects In Inflammatory Bowel Disease

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Based on evidence from previous studies that activation of NOD2 by muramyl dipeptide (MDP) leads to the down-regulation of TLR2 responses and that the absence of such regulation leads to heightened Th1 responses, in initial studies we treated mice with either dextran-sulfate-induced colitis (DSS-colitis) or trinitrobenzene sulfonic acid-induced colitis (TNBS-colitis) with MDP to determine the latters effect on inflammation in vivo. Indeed, we found that MDP administration greatly decreased the inflammation in both of these models. The basis of this down-regulatory effect then became apparent in subsequent in vitro studies of dendritic cells in which we showed that pre-stimulation of the cells with MDP rendered the cells unresponsive to a wide variety of TLR stimulants in assays of a spectrum of inflammatory cytokines. Subsequent studies designed to reveal the mechanism of this effect showed that MDP stimulation of NOD2 induced the expression of IRF-4, a factor previously shown to inhibit TLR responses and that gene silencing of IRF-4 led to restoration of responses. With this insight in hand we then returned to in vivo studies of experimental colitis and showed that in vivo down-regulation of IRF-4 by administration of IRF-4-specific siRNA encapsulated in a viral envelope to facilitate cell entry abolished the effect of MDP on TNBS-colitis. In addition, it abolished the down-regulatory effect of MDP administration on TLR ligand cytokine induction. In addition, we showed that MDP administration was unable to affect DSS-colitis induced in IRF-4-deficient mice. In a final series of studies relating these findings to Crohns disease we re-constituted NOD2-deficient mice with plasmids expressing either wild type NOD2 or NOD2 bearing a frameshift mutation identical to that found in Crohns disease. We then showed that MDP administration prevented DSS-colitis in mice reconstituted with wild type NOD2 but not mice reconstituted with mutated (frameshift mutation) NOD2. Thus, the frameshift mutation conferred a phenotype characterized by an inability to acquire MDP-induced unresponsiveness.