A distal cis-regulatory element, CNS-9, controls NFAT1 and IRF4-mediated IL-10 gene activation in T helper cells

IL-10 contributes to the maintenance of intestinal homeostasis via the regulation of inflammatory responses to enteric bacteria. Loss of IL-10 signaling results in spontaneous colitis in mice and early onset enterocolitis in humans. Nucleotide-binding oligomerization domain (NOD) 2 is an intracellular receptor of bacterial peptidoglycan products, and, although NOD2 mutations are associated with Crohn’s disease, the precise role of NOD2 in the development of intestinal inflammation remains undefined. To determine the role of NOD2 in the development of colitis on the clinically relevant genetic background of IL-10–deficient signaling, we generated mice lacking IL-10 and NOD2 (IL-10−/−NOD2−/−). Loss of NOD2 in IL-10−/− mice resulted in significant amelioration of chronic colitis, indicating that NOD2 signaling promotes the development of intestinal inflammation in IL-10−/− mice. Contrary to previous reports investigating immune function in NOD2−/− mice, T cell proliferative capacity and IL-2 production were not impaired, and immune polarization toward type 1 immunity was not affected. However, loss of NOD2 in IL-10–deficient macrophages reduced IL-6, TNF-α, and IL-12p40 production in response to bacterial stimulation. Further analysis of the intrinsic macrophage response before the onset of inflammation revealed that, in the absence of IL-10, synergistic signaling between various TLRs and NOD2 resulted in hyperresponsive, proinflammatory macrophages, thus providing the appropriate immune environment for the development of colitis. Data presented in this study demonstrate that NOD2 signaling contributes to intestinal inflammation that arises through loss of IL-10 and provides mechanistic insight into the development of colitis in inflammatory bowel disease patients with impaired IL-10 signaling.

Section: Discussionmentioning

confidence: 99%

Nucleotide-Binding Oligomerization Domain 2 Signaling Promotes Hyperresponsive Macrophages and Colitis in IL-10–Deficient Mice

Jamontt¹,

Petit

Clark

et al. 2013

“…Reporter constructs were generated by cloning the minimal promoter and conserved noncoding sequence (CNS) regions of the Il10 locus from P1 clones into the pXPG reporter vector containing the luciferase gene described in the previous report (30). Flag-Ets-1 plasmid was described previously (21).…”

Section: Plasmids and Luciferase Reporter Assaysmentioning

confidence: 99%

“…Chromatin immunoprecipitation (ChIP) assays were performed as described previously (30). In brief, cells were cross-linked with formaldehyde at a final concentration 1%, lysed, and sonicated to shear DNA.…”

Section: Chromatin Immunoprecipitation Assaysmentioning

confidence: 99%

“…We further examined the functional significance of the interaction between Ets-1 and HDAC1 in the repression of Il10 gene expression. We performed in vitro reporter assay using IL-10 reporter constructs containing a minimal IL-10 promoter and the diverse CNSs (30). Overexpression of Ets-1 alone failed to repress the IL-10 reporter activity of CNS20.12 ( Fig.…”

Section: Functional Synergism Of Ets-1 and Hdac1 To Repress Il-10 Expmentioning

confidence: 99%

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Interaction of Ets-1 with HDAC1 Represses IL-10 Expression in Th1 Cells

Lee

Kwon

Sahoo

et al. 2012

Self Cite

IL-10 is a multifunctional cytokine that plays a crucial role in immunity and tolerance. IL-10 is produced by diverse immune cell types, including B cells and subsets of T cells. Although Th1 produce IL-10, their expression levels are much lower than Th2 cells under conventional stimulation conditions. The potential role of E26 transformation-specific 1 (Ets-1) transcription factor as a negative regulator for Il10 gene expression in CD4+ T cells has been implicated previously. In this study, we investigated the underlying mechanism of Ets-1–mediated Il10 gene repression in Th1 cells. Compared with wild type Th1 cells, Ets-1 knockout Th1 cells expressed a significantly higher level of IL-10, which is comparable with that of wild type Th2 cells. Upregulation of IL-10 expression in Ets-1 knockout Th1 cells was accompanied by enhanced chromatin accessibility and increased recruitment of histone H3 acetylation at the Il10 regulatory regions. Reciprocally, Ets-1 deficiency significantly decreased histone deacetylase 1 (HDAC1) enrichment at the Il10 regulatory regions. Treatment with trichostatin A, an inhibitor of HDAC family, significantly increased Il10 gene expression by increasing histone H3 acetylation recruitment. We further demonstrated a physical interaction between Ets-1 and HDAC1. Coexpression of Ets-1 with HDAC1 synergistically repressed IL-10 transcription activity. In summary, our data suggest that an interaction of Ets-1 with HDAC1 represses the Il10 gene expression in Th1 cells.

“…A distal cis-acting element, conserved noncoding sequence (CNS)-9, showed an enhancer activity and the recruitment of NFAT1 and IFN regulatory factor (IRF)4 to the site that synergistically increased IL-10 transcription in Th2 cells (25). Based on this result, we developed a T cell-based reporter assay system to screen compounds that can modulate the regulatory activity of CNS-9.…”

mentioning

confidence: 99%

6-Methoxyflavone Inhibits NFAT Translocation into the Nucleus and Suppresses T Cell Activation

Kim

Song

et al. 2014

Self Cite

NFAT plays a crucial role in the immune system by regulating the transcription of inducible genes during immune responses. In T cells, NFAT proteins govern various cellular events related to T cell development, activation, tolerance induction, and differentiation. We previously reported the NFAT1-dependent enhancer activity of conserved noncoding sequence (CNS)-9, a distal cis-acting element, in the regulation of IL-10 transcription in T cells. In this study, we developed a T cell–based reporter system to identify compounds that modulate the regulatory activity of CNS-9. Among the identified candidates, 6-methoxyflavone (6-MF) significantly inhibited the enhancer activity of CNS-9, thereby reducing IL-10 expression in T cells without affecting cell viability. 6-MF also downregulated the transcription of NFAT1 target genes such as IL-4, IL-13, and IFN-γ. Treatment of 6-MF inhibited the translocation of NFAT1 into the nucleus, which consequently interrupted NFAT1 binding to the target loci, without affecting the expression or dephosphorylation of NFAT1. Treatment of 6-MF to CD4+ T cells or B cells isolated from mice with atopic dermatitis significantly reduced disease-associated cytokine production, as well as the levels of IgE. In addition, oral administration of 6-MF to atopic dermatitis mice ameliorated disease symptoms by reducing serum IgE levels and infiltrating lymphocytes. Conclusively, our results suggest that 6-MF can be a potential candidate for the development of an effective immunomodulator via the suppression of NFAT-mediated T cell activation.