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. Herein we demonstrate a novel approach of comprehensive serum multiplex cytokine profiling 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. 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 TNFα, IL6, IL-17 and KC) to a predominant Th2-mediated inflammatory response (increase in IL-4 and IL-10 and concomitant decrease in TNFα, IL6, IL-17 and KC) in the chronic state. Profiles of multiple cytokines seen systemically were also validated locally in colonic mucosa. Moreover, advanced multivariate analyses 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. Our studies provide insight into disease immunopathogenesis and illustrate the significant potential of utilizing multiplex cytokine profiles and bioinformatics as diagnostic tools in IBD.

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The intestinal and renal proximal tubule brush border (BB) Na + -H + exchanger NHE3 binds to members of the NHERF (Na + -H + exchanger regulatory factor) family. These are four proteins (current most used names include NHERF1, NHERF2, PDZK1 and IKEPP) which are related to each other, are present in locations in or close to the BB, and scaffold a variable series of proteins in NHE3-containing complexes in a dynamic manner that is altered by changes in signal transduction which affects NHE3 activity. The specific roles of these proteins in terms of NHE3 regulation as well as interactions with each other and with their many other substrates are only now being defined. Specificity for only one member of the NHERF family in one example of NHE3 regulation, inhibition by elevation in cGMP, is used to describe how NHERF family proteins are involved in NHE3 complex formation and its regulation. In this case, NHERF2 directly binds cGKII in the brush border to form an NHE3 complex, with cGKII also associating with the BB via its myristoylation.

Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (TH1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring TH17 and TH2 responses for clearance (bacterial Citrobacter rodentium and helminth Trichuris muris infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell–mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use.

NHE3 is the brush-border (BB) Na(+)/H(+) exchanger of small intestine, colon, and renal proximal tubule which is involved in large amounts of neutral Na(+) absorption. NHE3 is a highly regulated transporter, being both stimulated and inhibited by signaling that mimics the postprandial state. It also undergoes downregulation in diarrheal diseases as well as changes in renal disorders. For this regulation, NHE3 exists in large, multiprotein complexes in which it associates with at least nine other proteins. This review deals with short-term regulation of NHE3 and the identity and function of its recognized interacting partners and the multiprotein complexes in which NHE3 functions.

+ -H+ exchange activity.3. Disrupting rafts by removal of cholesterol with methyl-b-cyclodextrin (MbCD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibodycoated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and MbCD treatment.4. We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF-and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3.

Background & Aims One of the most common symptoms among patients with inflammatory bowel diseases (IBD) is diarrhea, which is thought to be contributed by changes in electrolyte transport associated with intestinal inflammation. This study is designed to test the hypothesis that intestinal Na+-related transporters/channels and their regulatory proteins may be down-regulated as a potential contributor to IBD-associated diarrhea. Methods SDS-PAGE and Western blotting and/or confocal immunomicroscopy were used to examine the expression of Na+/H+-exchangers 1-3 (NHE1-3), epithelial Na+ channel (ENaC), Na+/K+-ATPase, the intracellular Cl- channel 5 (ClC-5), and NHE3 regulatory factors (NHERF1 & 2), in ileal and colonic pinch biopsies from IBD patients and noninflammatory controls, as well as from colonic mucosa of DSS- and TNBS-induced acute murine IBD models. Results NHE1&3 (but not NHE2), β-ENaC, Na+/K+-ATPase-α, ClC-5 and NHERF1 were all down-regulated in sigmoid mucosal biopsies from most cases of active UC and/or CD, compared to controls. NHE3 was also decreased in ileal mucosal biopsies of active CD, as well as in ~50% of sigmoid biopsies from inactive UC or CD. Importantly, similar down-regulation of NHE1&3, β-ENaC, and NHERF1&2 was also observed in the mouse colon (but not ileum) of DSS- and TNBS-induced colitis. Conclusions IBD-associated diarrhea may be due to a coordinated down regulation of multiple Na+ transporter and related regulatory proteins, including NHE1&3, Na+/K+-ATPase, and ENaC, as well as NHERF1 & 2, and ClC-5, all of which are involved directly or indirectly in intestinal Na+ absorption.

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