Methylation matters: binding of Ets-1 to the demethylated Foxp3 gene contributes to the stabilization of Foxp3 expression in regulatory T cells

The forkhead-box protein P3 (Foxp3) is a key transcription factor for the development and suppressive activity of regulatory T cells (Tregs), a T cell subset critically involved in the maintenance of self-tolerance and prevention of over-shooting immune responses. However, the transcriptional regulation of Foxp3 expression remains incompletely understood. We have previously shown that epigenetic modifications in the CpG-rich Treg-specific demethylated region (TSDR) in the Foxp3 locus are associated with stable Foxp3 expression. We now demonstrate that the methylation state of the CpG motifs within the TSDR controls its transcriptional activity rather than a Treg-specific transcription factor network. By systematically mutating every CpG motif within the TSDR, we could identify four CpG motifs, which are critically determining the transcriptional activity of the TSDR and which serve as binding sites for essential transcription factors, such as CREB/ATF and NF-κB, which have previously been shown to bind to this element. The transcription factor Ets-1 was here identified as an additional molecular player that specifically binds to the TSDR in a demethylation-dependent manner in vitro. Disruption of the Ets-1 binding sites within the TSDR drastically reduced its transcriptional enhancer activity. In addition, we found Ets-1 bound to the demethylated TSDR in ex vivo isolated Tregs, but not to the methylated TSDR in conventional CD4+ T cells. We therefore propose that Ets-1 is part of a larger protein complex, which binds to the TSDR only in its demethylated state, thereby restricting stable Foxp3 expression to the Treg lineage

Colitic pIV−/− K14 CIITA Tg mice lack inducible MHCII expression by colonic IECs.

<p>(A–B) Frequency of CD45.2⁻ EpCAM⁺ MHCII⁺ IECs isolated from anti-IL-10R mAb or isotype treated, <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls. Representative histograms (A) and summarized data as mean (B) from three pooled experiments (<i>n = </i>8–11 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies; IEC, intestinal epithelial cell;</p

TLR3-Mediated CD8+ Dendritic Cell Activation Is Coupled with Establishment of a Cell-Intrinsic Antiviral State

Because of their unique capacity to cross-present Ags to CD8(+) T cells, mouse lymphoid tissue-resident CD8(+) dendritic cells (DCs) and their migratory counterparts are critical for priming antiviral T cell responses. High expression of the dsRNA sensor TLR3 is a distinctive feature of these cross-presenting DC subsets. TLR3 engagement in CD8(+) DCs promotes cross-presentation and the acquisition of effector functions required for driving antiviral T cell responses. In this study, we performed a comprehensive analysis of the TLR3-induced antiviral program and cell-autonomous immunity in CD8(+) DC lines and primary CD8(+) DCs. We found that TLR3-ligand polyinosinic-polycytidylic acid and human rhinovirus infection induced a potent antiviral protection against Sendai and vesicular stomatitis virus in a TLR3 and type I IFN receptor-dependent manner. Polyinosinic-polycytidylic acid-induced antiviral genes were identified by mass spectrometry-based proteomics and transcriptomics in the CD8(+) DC line. Nanostring nCounter experiments confirmed that these antiviral genes were induced by TLR3 engagement in primary CD8(+) DCs, and indicated that many are secondary TLR3-response genes requiring autocrine IFN-β stimulation. TLR3-activation thus establishes a type I IFN-dependent antiviral program in a DC subtype playing crucial roles in priming adaptive antiviral immune responses. This mechanism is likely to shield the priming of antiviral responses against inhibition or abrogation by the viral infection. It could be particularly relevant for viruses detected mainly by TLR3, which may not trigger type I IFN production by DCs that lack TLR3, such as plasmacytoid DCs or CD8(-) DCs

Chronic <i>H. hepaticus</i> infection plus anti-IL10R mAb treatment induces colitis in pIV−/− K14 CIITA Tg mice.

<p>(A) Development of body weight during anti-IL-10R mAb or isotype treatment of <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls (<i>n = </i>9–11 per group). (B) Serum albumin concentrations in feces collected on days 26–30 (<i>n = </i>6–8 per group). Data are shown as mean and s.d. and represent two pooled experiments. (C–D) Colon histopathological analysis on day 32; (C) colitis scores, data displayed as mean, and (D) representative photomicrographs of colon sections, stained with hematoxylin and eosin. Bar, 100 µm. Data represent three pooled experiments (<i>n = </i>9–11 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies;</p

Innate effector cells and proinflammatory cytokines are elevated in colitic pIV−/− K14 CIITA Tg mice.

<p>(A–B) Frequency of Ly6G⁺ neutrophil granulocytes (A) and CD11b⁺ Ly6C⁺ inflammatory monocytes (B) isolated from the colonic intestinal epithelium (cIE, left panel) and the colonic lamina propria (cLP, right panel) of <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls. (C) <i>ccl3</i>, <i>ccl4</i>, <i>ccl5</i>, <i>il1b</i> and <i>il6</i> mRNA expression levels in colon explants. (D) IL-1β, TNF-α, IL-12p40, CXCL9 and VEGF secretion upon <i>ex vivo</i> organ culture of colon explants. All data represent three pooled experiments (<i>n = </i>9–11 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies; IL, interleukin; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor;</p

Colitic pIV−/− K14 CIITA Tg mice display elevated Th1 cells, IFN-γ, and CD4⁺ T cell: FoxP3⁺ Treg cell ratios.

<p>(A–B) Frequency of CD3⁺ CD4⁺ and CD8⁺ T cells isolated from anti-IL-10R mAb or isotype treated, <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls. Representative histograms from the colonic intestinal epithelium (cIE) (A) and summarized data (B) from cIE (left) and the colonic lamina propria (cLP) (right) as mean. (C) <i>ifng</i> and <i>tbet</i> mRNA expression levels in colon explants. (D) IFN-γ secretion upon <i>ex vivo</i> organ culture of colon explants as means and s.d. (A–D) Data represent three pooled experiments (<i>n = </i>9–11 per group). (E) Ratio of absolute numbers of CD4⁺ T cells:absolute numbers of CD25⁺ FoxP3⁺ Treg cells from cIE (left) and cLP (right) as mean from two pooled experiments (<i>n = </i>7–10 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies; FoxP3, forkhead box P3; IFN, interferon;</p

IFN-γ induces MHCII expression on IECs.

<p>(A) Frequency of CD45.2⁻ EpCAM⁺ MHCII⁺ intestinal epithelial cells (IEC) isolated from Rag1−/− or Rag1−/− IFN-γ−/− mice that were adoptively transferred with CD4⁺ CD45RB^hi T cells from WT or IFN-γ−/− mice shown as means and SEM in representative histograms (<i>n</i> = 3 mice per group). (B–C) Frequency of CD45.2⁻ EpCAM⁺ MHCII⁺ IECs (B) and CD4⁺ T cells from the colonic intestinal epithelium (C) isolated from <i>H. hepaticus</i>-infected, anti-IL-10R mAb-administered pIV−/− K14 CIITA Tg, pIV+/− K14 CIITA Tg or pIV+/− K14 CIITA Tg that were treated with neutralizing anti-IFN-γ mAb. Shown are representative FACS plots, means and SEM from two pooled experiments (<i>n</i> = 4–7 mice per group). αIFN-y, anti-interferon-γ monoclonal antibodies; FSC, forward scatter; IFN, interferon; Rag, recombination activating gene; WT, wild type;</p

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD