Robert D. Thurston

Dendritic Cell-Specific Disruption of TGF-β Receptor II Leads to Altered Regulatory T Cell Phenotype and Spontaneous Multiorgan Autoimmunity

In vitro data and transgenic mouse models suggest a role for TGF-β signaling in dendritic cells (DCs) to prevent autoimmunity primarily through maintenance of DCs in their immature and tolerogenic state characterized by low expression of MHC class II (MHCII) and costimulatory molecules and increased expression of IDO, among others. To test whether a complete lack of TGF-β signaling in DCs predisposes mice to spontaneous autoimmunity and to verify the mechanisms implicated previously in vitro, we generated conditional knockout (KO) mice with Cre-mediated DC-specific deletion of Tgfbr2 (DC-Tgfbr2 KO). DC-Tgfbr2 KO mice die before 15 wk of age with multiorgan autoimmune inflammation and spontaneous activation of T and B cells. Interestingly, there were no significant differences in the expression of MHCII, costimulatory molecules, or IDO in secondary lymphoid organ DCs, although Tgfbr2-deficient DCs were more proinflammatory 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 IFN-γ and were not capable of directing Ag-specific Treg conversion unless in the presence of anti–IFN-γ blocking Ab. Adoptive transfer of induced Tregs into DC-Tgfbr2 KO mice partially rescued the phenotype. Therefore, in vivo, TGF-β signaling in DCs is critical in the control of autoimmunity through both Treg-dependent and -independent mechanisms, but it does not affect MHCII and costimulatory molecule expression.

Tumor Necrosis Factor and Interferon-γ Down-regulate Klotho in Mice With Colitis

BACKGROUND & AIMS Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. METHODS We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10(-/-)), and colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-gamma on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). RESULTS Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-gamma. The combination of TNF and IFN-gamma increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-gamma was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-gamma, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. CONCLUSIONS The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.

Limited Effects of Dietary Curcumin on Th-1 Driven Colitis in IL-10 Deficient Mice Suggest an IL-10 Dependent Mechanism of Protection

Curcumin (diferulolylmethane) demonstrates profound anti-inflammatory effects in intestinal epithelial cells (IEC) and in immune cells in vitro and exhibits a protective role in rodent models of chemically induced colitis, with its presumed primary mechanism of action via inhibition of NF-kappaB. Although it has been demonstrated effective in reducing relapse rate in ulcerative colitis patients, curcumins effectiveness in Crohns disease (CD) or in Th-1/Th-17 mediated immune models of CD has not been evaluated. Therefore, we investigated the effects of dietary curcumin (0.1-1%) on the development of colitis, immune activation, and in vivo NF-kappaB activity in germ-free IL-10(-/-) or IL-10(-/-);NF-kappaB(EGFP) mice colonized with specific pathogen-free microflora. Proximal and distal colon morphology showed a mild protective effect of curcumin only at 0.1%. Colonic IFN-gamma and IL-12/23p40 mRNA expression followed similar pattern ( approximately 50% inhibition at 0.1%). Secretion of IL-12/23p40 and IFN-gamma by colonic explants and mesenteric lymph node cells was elevated in IL-10(-/-) mice and was not decreased by dietary curcumin. Surprisingly, activation of NF-kappaB in IL-10(-/-) mice (phospho-NF-kappaBp65) or in IL-10(-/-);NF-kappaB(EGFP) mice (whole organ or confocal imaging) was not noticeably inhibited by curcumin. Furthermore, we demonstrate that IL-10 and curcumin act synergistically to downregulate NF-kappaB activity in IEC and IL-12/23p40 production by splenocytes and dendritic cells. In conclusion, curcumin demonstrates limited effectiveness on Th-1 mediated colitis in IL-10(-/-) mice, with moderately improved colonic morphology, but with no significant effect on pathogenic T cell responses and in situ NF-kappaB activity. In vitro studies suggest that the protective effects of curcumin are IL-10 dependent.

Changes in mucosal homeostasis predispose NHE3 knockout mice to increased susceptibility to DSS-induced epithelial injury

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 interferon (IFN)-gamma, and distal colitis, suggesting its role in epithelial barrier homeostasis. Our aim was to investigate the role of NHE3 in maintaining mucosal integrity. METHODS Control or dextran sulfate sodium (DSS)-treated, 6- to 8-week-old 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 CD8alpha(+) T and natural killer cells in the intraepithelial lymphocytes and lamina propria lymphocytes compartments, representing the source of IFN-gamma. NHE3(-/-) mice display alterations in epithelial gene and protein expression patterns that 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-gamma and iNOS to basal levels and delay but do not prevent severe mortality in response to DSS treatment. CONCLUSIONS 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.

NHE3 modulates the severity of colitis in IL-10-deficient mice

NHE3, the major intestinal Na(+)/H(+) exchanger, was shown to be downregulated and/or inhibited in patients with inflammatory bowel disease (IBD), a phenomenon believed to contribute to inflammation-associated diarrhea. NHE3(-/-) mice spontaneously develop colitis and demonstrate high susceptibility to dextran sulfate-induced mucosal injury. We investigated the effects of NHE3 deficiency on the development of chronic colitis in an IL-10 knockout (KO) mouse model of Crohns disease. NHE3(-/-) mice were first backcrossed to 129/SvEv mice for >10 generations, with no apparent changes in their survival or phenotype. These mice were crossed with IL-10(-/-) mice on the same genetic background, and the phenotypes of 10-wk-old wild-type (WT), IL-10(-/-), NHE3(-/-), and IL-10(-/-)/NHE3(-/-) (double-KO) mice were studied. Histological and immunohistochemical examination of the colon established important architectural alterations, including increased neutrophilic and mononuclear cell infiltration in double- compared with single-KO mice. Double-KO mice demonstrated increased colonic expression of neutrophil collagenase matrix metalloproteinase-8 and the chemokines macrophage inflammatory protein-2, CXCL1, CXCL10, and CXCL11. Colonic IFNγ, IL-17, and IL-12/23 p40 protein secretion was significantly increased in double- compared with single-KO mice. IL-10(-/-)/NHE3(-/-) mouse colonic epithelium exhibited increased hallmarks of apoptosis, including a significantly increased number of cleaved caspase-3-positive surface epithelial cells. These results highlight the importance of NHE3 in the maintenance of intestinal barrier integrity and in modulating the inflammatory process in IL-10-deficient mice. Chronic NHE3 inhibition or underexpression observed in IBD may therefore contribute to the pathogenesis of IBD by influencing the extent of the epithelial barrier defect and affect the ultimate degree of inflammation.

COOPERATIVE ROLE OF NF-κB AND POLY(ADP-RIBOSE) POLYMERASE 1 (PARP-1) IN THE TNF-INDUCED INHIBITION OF PHEX EXPRESSION IN OSTEOBLASTS

Reduced bone mass is a common complication in chronic inflammatory diseases, although the mechanisms are not completely understood. The PHEX gene encodes a zinc endopeptidase expressed in osteoblasts and contributes to bone mineralization. The aim of this study was to determine the molecular mechanism involved in TNF-mediated down-regulation of Phex gene transcription. We demonstrate down-regulation of the Phex gene in two models of colitis: naive T-cell transfer and in gnotobiotic IL-10−/− mice. In vitro, TNF decreased expression of Phex in UMR106 cells and did not require de novo synthesis of a transrepressor. Transfecting UMR-106 cells with a series of deletion constructs of the proximal Phex promoter identified a region located within −74 nucleotides containing NF-κB and AP-1 binding sites. After TNF treatment, the RelA/p50 NF-κB complex interacted with two cis-elements at positions −70/−66 and −29/−25 nucleotides in the proximal Phex promoter. Inhibition of NF-κB signaling increased the basal level of Phex transcription and abrogated the effects of TNF, whereas overexpression of RelA mimicked the effect of TNF. We identified poly(ADP-ribose) polymerase 1 (PARP-1) binding immediately upstream of the NF-κB sites and showed that TNF induced poly(ADP-ribosyl)ation of RelA when bound to the Phex promoter. TNF-mediated Phex down-regulation was completely abrogated in vitro by PARP-1 inhibitor and overexpression of poly(ADP-ribose) glucohydrolase (PARG) and in vivo in PARP-1−/− mice. Our results suggest that NF-κB signaling and PARP-1 enzymatic activity cooperatively contribute to the constitutive and inducible suppression of Phex. The described phenomenon likely contributes to the loss of bone mass density in chronic inflammatory diseases, such as inflammatory bowel disease.

Mo1720 Relevance of Poly(ADP-ribose) Polymerase 1 (PARP1) in Experimental Colitis

Background: Poly(ADP-ribose) polymerases (PARPs) constitute a family of 18 cell signaling enzymes which are involved in the regulation of DNA-binding proteins and DNA repair. These enzymes catalyze the transfer of ADP-ribose units from NAD+ to a number of acceptor proteins and can also interact directly with their target proteins. Among the PARPs, PARP1 is the most abundant isoform and regulates the expression of diverse proinflammatory mediators, through its interaction with and modification of numerous transcription factors. Recent observations (including our groups) suggest that PARP1 is a cofactor for NF-κBdependent gene expression. Several studies in PARP1 KO mice have shown decreased expression of cytokines, chemokines and adhesion molecules, as well as reduced immune tissue infiltration in various models of inflammation, including streptomycin-induced diabetes and LPS-induced septic shock. Interestingly, in vivo studies have demonstrated that genetic ablation or pharmacological inhibition of PARP1 ameliorates the pathophysiological changes of experimental colitis. However, these studies failed to characterize the mechanism by which PARP1 inhibition is protective in colitis or the cell type in which Parp1 deletion has the most significant impact in IBD. Aim: Considering the essential role of PARP1 in the regulation of inflammation in IBD, we evaluated the effect of Parp1 deletion in T cellmediated colitis. Methods: The effect of Parp1 deficiency was evaluated in different models of colitis: (1) Adoptive T cell transfer: WT or Parp1-deficient CD4+CD45RBHigh T-cells were transferred into Rag2-/mice, or Rag2-/x Parp1-/double knockout (DKO) mice and (2) DSS-induced colitis in WT, Parp1-/-, Rag2-/-, and Rag2-/x Parp1-/DKO mice. In all models, we evaluated body weight loss, colonic morphology and expression of major pro-inflammatory cytokines. Results: In comparison to WT mice, Parp1-/were protected from the effects of DSS, with significantly lower weight loss, and greatly reduced proinflammatory cytokine expression. However, Parp1 deficiency in T cells was not protective in the CD4+CD45RBHigh T cell transfer model. Unexpectedly, Rag2-/x Parp1-/DKO showed only marginal improvement in both the WT CD4+CD45RBHigh T cell transfer and DSS models. Conclusions: Our preliminary data obtained with the adoptive T-cell transfer model of colitis indicate that Parp1 is dispensable in Naive and effector T-cells. While Parp1-/mice are protected from colitis resulting from DSS-induced epithelial damage, the lack of significant protection in the T cell transfer or DSS models of innate colitis in Rag2-/x Parp1-/DKO mice suggests that the pathogenic role of Parp1 in colitis may involve modulating interactions of more than one cell type, presumably innate immune cells (including colonic epithelial cells) and regulatory T cells.