Background & Aims Klotho deficiency in hypomorphic KL mice leads to premature senescence and phenotype consistent with impaired mineral homeostasis. Klotho has anti-inflammatory properties protecting from NO-induced endothelial dysfunction, reduces the expression of endothelial adhesion molecules, and may contribute to T-cell dysfunction. Since defective Ca2+/Pi homeostasis leading to osteopenia/osteoporosis is frequently associated with human IBD, we investigated the changes in Klotho gene expression as a consequence of experimental colitis. Methods We utilized three murine IBD models: TNBS colitis, microflora-induced colitis in gnotobiotic IL-10−/− mice, and adoptive CD4+CD45RBhigh T-cell transfer colitis. These studies were followed by in vitro approaches using renal epithelial cells (mpkDCT4 and mIMCD3), and the cloned murine KL gene promoter. Results Renal expression of Klotho mRNA and protein was significantly inhibited in all three models of human IBD. This degree of inhibition was correlated with the severity of colitis, and was reversed by neutralizing anti-TNF antibodies. In vitro, TNF resulted in a significant inhibition of KL expression and was further potentiated by IFN-γ. TNF/IFN-γ combination resulted in increased iNOS expression and significantly elevated the concentration of NO in medium. The effect of IFN-γ could be reproduced by cell exposure to SNAP (NO donor), and reversed by iNOS inhibitor, L-NIL. The cytokine effects were transcriptionally mediated since Klotho mRNA stability remained unaffected, while reporter constructs with the mKL gene promoter displayed significant downregulation in transiently transfected renal epithelial cells. Conclusions These novel findings could help explain several extraintestinal complications including abnormalities in bone homeostasis in patients with chronic colitis.

Na+/H+ exchanger 3 (NHE3) provides a major route for intestinal Na+ absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3(-/-) mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2'-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer276-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF-alpha (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3(-/-) mice. NHE3(-/-) mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.

In vitrodata and transgenic mouse models suggest a role for TGFβ signaling in dendritic cells (DC) to prevent autoimmunity primarily through maintenance of DCs in their immature and tolerogenic state characterized by low expression of MHCII and co-stimulatory molecules, and increased expression of indoleamine 2,3-dioxygenase (IDO), among others. To test whether a complete lack of TGFβ signaling in DCs predisposes mice to spontaneous autoimmunity, and to verify the mechanisms implicated previously in vitro, we generated conditional knock-out mice with Cre-mediated DC-specific deletion of Tgfbr2 (DC-Tgfbr2 KO). DC-Tgfbr2 KO mice die before 15 weeks of age with multi-organ autoimmune inflammation and spontaneous activation of T and B cells. Interestingly, there were no significant differences in the expression of MHCII, co-stimulatory molecules, or IDO in secondary lymphoid organ DCs, although Tgfbr2-deficient DCs were more pro-inflammatory in vitro and in vivo. DC-Tgfbr2 KO showed attenuated FoxP3 expression in regulatory T cells (Tregs) and abnormal expansion of CD25−FoxP3+ Tregs in vivo. Tgfbr2-deficient DCs secreted elevated levels of IFNγ and were not capable of directing antigen-specific Treg conversion unless in the presence of anti-IFNγ blocking antibody. Adoptive transfer of iTregs into DC-Tgfbr2 KO mice partially rescued the phenotype. Therefore, in vivo, TGFβ signaling in DCs is critical in the control of autoimmunity through both Treg dependent and independent mechanisms, but it does not affect MHCII and co-stimulatory molecule expression.

Background & Aims NHE3 is a target of inhibition by proinflammatory cytokines and pathogenic bacteria, an event contributing to diarrhea in infectious and idiopathic colitis. In mice, NHE3 deficiency leads to mild diarrhea, increased intestinal expression of IFN-γ, and distal colitis, suggesting its role in epithelial barrier homeostasis. Aim To investigate the role of NHE3 in maintaining mucosal integrity. Methods Control or DSS-treated, 6–8 wk wild-type (WT) and NHE3−/− mice were used for the experiments. Small intestines were dissected for further analysis. Results NHE3−/− mice have elevated numbers of CD8α+ T and NK cells in the IEL and LPL compartments, representing the source of IFN-γ. NHE3−/− mice display alterations in epithelial gene and protein expression patterns which predispose them to a high susceptibility to DSS, with accelerated mortality resulting from intestinal bleeding, hypovolemic shock, and sepsis, even at a very low DSS concentration. Microarray analysis and intestinal hemorrhage indicate that NHE3 deficiency predisposes mice to DSS-induced small intestinal injury, a segment never reported as affected by DSS, and demonstrate major differences in the colonic response to DSS challenge in WT and NHE3−/− mice. In NHE3−/− mice, broad spectrum oral antibiotics or anti-asialo GM1 antibodies reduce the expression of IFN-γ and iNOS to basal levels and delay, but do not prevent, severe mortality in response to DSS treatment. Conclusion These results suggest that NHE3 participates in mucosal responses to epithelial damage acting as a modifier gene determining the extent of the gut inflammatory responses in the face of intestinal injury.

Larmonier CB, Uno JK, Lee KM, Karrasch T, Laubitz D, Thurston R, Midura-Kiela MT, Ghishan FK, Sartor RB, Jobin C, Kiela PR. Limited effects of dietary curcumin on Th-1 driven colitis in IL-10 deficient mice suggest an IL-10-dependent mechanism of protection.