A Multi-Omics Approach Reveals Features That Permit Robust and Widespread Regulation of IFN-Inducible Antiviral Effectors.

The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were "stronger" (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that "strong" ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.

Attenuation of TCR-induced transcription by Bach2 controls regulatory T cell differentiation and homeostasis.

Differentiation and homeostasis of Foxp3+ regulatory T (Treg) cells are strictly controlled by T-cell receptor (TCR) signals; however, molecular mechanisms that govern these processes are incompletely understood. Here we show that Bach2 is an important regulator of Treg cell differentiation and homeostasis downstream of TCR signaling. Bach2 prevents premature differentiation of fully suppressive effector Treg (eTreg) cells, limits IL-10 production and is required for the development of peripherally induced Treg (pTreg) cells in the gastrointestinal tract. Bach2 attenuates TCR signaling-induced IRF4-dependent Treg cell differentiation. Deletion of IRF4 promotes inducible Treg cell differentiation and rescues pTreg cell differentiation in the absence of Bach2. In turn, loss of Bach2 normalizes eTreg cell differentiation of IRF4-deficient Treg cells. Mechanistically, Bach2 counteracts the DNA-binding activity of IRF4 and limits chromatin accessibility, thereby attenuating IRF4-dependent transcription. Thus, Bach2 balances TCR signaling induced transcriptional activity of IRF4 to maintain homeostasis of thymically-derived and peripherally-derived Treg cells.

Absence of nonhematopoietic MHC class II expression protects mice from experimental autoimmune myocarditis.

Experimental autoimmune myocarditis (EAM) is a CD4(+) T-cell-mediated model of human inflammatory dilated cardiomyopathies. Heart-specific CD4(+) T-cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation-induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV-/- K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α-myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV-/- K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4(+) T cells and in expression of IFN-γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN-γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN-γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis.

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-ß 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.

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.

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.