Transcription factor RORα enforces stability of the Th17 cell effector program by binding to a Rorc cis-regulatory element.

T helper 17 (Th17) cells regulate mucosal barrier defenses but also promote multiple autoinflammatory diseases. Although many molecular determinants of Th17 cell differentiation have been elucidated, the transcriptional programs that sustain Th17 cells in vivo remain obscure. The transcription factor RORγt is critical for Th17 cell differentiation; however, it is not clear whether the closely related RORα, which is co-expressed in Th17 cells, has a distinct role. Here, we demonstrated that although dispensable for Th17 cell differentiation, RORα was necessary for optimal Th17 responses in peripheral tissues. The absence of RORα in T cells led to reductions in both RORγt expression and effector function among Th17 cells. Cooperative binding of RORα and RORγt to a previously unidentified Rorc cis-regulatory element was essential for Th17 lineage maintenance in vivo. These data point to a non-redundant role of RORα in Th17 lineage maintenance via reinforcement of the RORγt transcriptional program.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFß activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

Lung eosinophils elicited during allergic and acute aspergillosis express RORγt and IL-23R but do not require IL-23 for IL-17 production.

Exposure to the mold, Aspergillus, is ubiquitous and generally has no adverse consequences in immunocompetent persons. However, invasive and allergic aspergillosis can develop in immunocompromised and atopic individuals, respectively. Previously, we demonstrated that mouse lung eosinophils produce IL-17 in response to stimulation by live conidia and antigens of A. fumigatus. Here, we utilized murine models of allergic and acute pulmonary aspergillosis to determine the association of IL-23, IL-23R and RORγt with eosinophil IL-17 expression. Following A. fumigatus stimulation, a population of lung eosinophils expressed RORγt, the master transcription factor for IL-17 regulation. Eosinophil RORγt expression was demonstrated by flow cytometry, confocal microscopy, western blotting and an mCherry reporter mouse. Both nuclear and cytoplasmic localization of RORγt in eosinophils were observed, although the former predominated. A population of lung eosinophils also expressed IL-23R. While expression of IL-23R was positively correlated with expression of RORγt, expression of RORγt and IL-17 was similar when comparing lung eosinophils from A. fumigatus-challenged wild-type and IL-23p19-/- mice. Thus, in allergic and acute models of pulmonary aspergillosis, lung eosinophils express IL-17, RORγt and IL-23R. However, IL-23 is dispensable for production of IL-17 and RORγt.

Origins, Biology, and Diseases of Tissue Macrophages.

Tissue-resident macrophages are present in most tissues with developmental, self-renewal, or functional attributes that do not easily fit into a textbook picture of a plastic and multifunctional macrophage originating from hematopoietic stem cells; nor does it fit a pro- versus anti-inflammatory paradigm. This review presents and discusses current knowledge on the developmental biology of macrophages from an evolutionary perspective focused on the function of macrophages, which may aid in study of developmental, inflammatory, tumoral, and degenerative diseases. We also propose a framework to investigate the functions of macrophages in vivo and discuss how inherited germline and somatic mutations may contribute to the roles of macrophages in diseases.

Deciphering the regulatory landscape of fetal and adult γδ T-cell development at single-cell resolution.

γδ T cells with distinct properties develop in the embryonic and adult thymus and have been identified as critical players in a broad range of infections, antitumor surveillance, autoimmune diseases, and tissue homeostasis. Despite their potential value for immunotherapy, differentiation of γδ T cells in the thymus is incompletely understood. Here, we establish a high-resolution map of γδ T-cell differentiation from the fetal and adult thymus using single-cell RNA sequencing. We reveal novel sub-types of immature and mature γδ T cells and identify an unpolarized thymic population which is expanded in the blood and lymph nodes. Our detailed comparative analysis reveals remarkable similarities between the gene networks active during fetal and adult γδ T-cell differentiation. By performing a combined single-cell analysis of Sox13, Maf, and Rorc knockout mice, we demonstrate sequential activation of these factors during IL-17-producing γδ T-cell (γδT17) differentiation. These findings substantially expand our understanding of γδ T-cell ontogeny in fetal and adult life. Our experimental and computational strategy provides a blueprint for comparing immune cell differentiation across developmental stages.

Characterization of Transcriptional Regulatory Networks that Promote and Restrict Identities and Functions of Intestinal Innate Lymphoid Cells.

Innate lymphoid cells (ILCs) promote tissue homeostasis and immune defense but also contribute to inflammatory diseases. ILCs exhibit phenotypic and functional plasticity in response to environmental stimuli, yet the transcriptional regulatory networks (TRNs) that control ILC function are largely unknown. Here, we integrate gene expression and chromatin accessibility data to infer regulatory interactions between transcription factors (TFs) and genes within intestinal type 1, 2, and 3 ILC subsets. We predicted the "core" TFs driving ILC identities, organized TFs into cooperative modules controlling distinct gene programs, and validated roles for c-MAF and BCL6 as regulators affecting type 1 and type 3 ILC lineages. The ILC network revealed alternative-lineage-gene repression, a mechanism that may contribute to reported plasticity between ILC subsets. By connecting TFs to genes, the TRNs suggest means to selectively regulate ILC effector functions, while our network approach is broadly applicable to identifying regulators in other in vivo cell populations.

Author Correction: c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont.

In this Letter, the 'Competing interests' statement should have stated: 'D.R.L. consults for and has equity in Vedanta Biosciences.' The original Letter has not been corrected.

Leveraging chromatin accessibility for transcriptional regulatory network inference in T Helper 17 Cells.

Transcriptional regulatory networks (TRNs) provide insight into cellular behavior by describing interactions between transcription factors (TFs) and their gene targets. The assay for transposase-accessible chromatin (ATAC)-seq, coupled with TF motif analysis, provides indirect evidence of chromatin binding for hundreds of TFs genome-wide. Here, we propose methods for TRN inference in a mammalian setting, using ATAC-seq data to improve gene expression modeling. We test our methods in the context of T Helper Cell Type 17 (Th17) differentiation, generating new ATAC-seq data to complement existing Th17 genomic resources. In this resource-rich mammalian setting, our extensive benchmarking provides quantitative, genome-scale evaluation of TRN inference, combining ATAC-seq and RNA-seq data. We refine and extend our previous Th17 TRN, using our new TRN inference methods to integrate all Th17 data (gene expression, ATAC-seq, TF knockouts, and ChIP-seq). We highlight newly discovered roles for individual TFs and groups of TFs ("TF-TF modules") in Th17 gene regulation. Given the popularity of ATAC-seq, which provides high-resolution with low sample input requirements, we anticipate that our methods will improve TRN inference in new mammalian systems, especially in vivo, for cells directly from humans and animal models.

c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont.

Both microbial and host genetic factors contribute to the pathogenesis of autoimmune diseases. There is accumulating evidence that microbial species that potentiate chronic inflammation, as in inflammatory bowel disease, often also colonize healthy individuals. These microorganisms, including the Helicobacter species, can induce pathogenic T cells and are collectively referred to as pathobionts. However, how such T cells are constrained in healthy individuals is not yet understood. Here we report that host tolerance to a potentially pathogenic bacterium, Helicobacter hepaticus, is mediated by the induction of RORγt+FOXP3+ regulatory T (iTreg) cells that selectively restrain pro-inflammatory T helper 17 (TH17) cells and whose function is dependent on the transcription factor c-MAF. Whereas colonization of wild-type mice by H. hepaticus promoted differentiation of RORγt-expressing microorganism-specific iTreg cells in the large intestine, in disease-susceptible IL-10-deficient mice, there was instead expansion of colitogenic TH17 cells. Inactivation of c-MAF in the Treg cell compartment impaired differentiation and function, including IL-10 production, of bacteria-specific iTreg cells, and resulted in the accumulation of H. hepaticus-specific inflammatory TH17 cells and spontaneous colitis. By contrast, RORγt inactivation in Treg cells had only a minor effect on the bacteria-specific Treg and TH17 cell balance, and did not result in inflammation. Our results suggest that pathobiont-dependent inflammatory bowel disease is driven by microbiota-reactive T cells that have escaped this c-MAF-dependent mechanism of iTreg-TH17 homeostasis.

Critical role of IRF1 and BATF in forming chromatin landscape during type 1 regulatory cell differentiation.

Type 1 regulatory T cells (Tr1 cells) are induced by interleukin-27 (IL-27) and have critical roles in the control of autoimmunity and resolution of inflammation. We found that the transcription factors IRF1 and BATF were induced early on after treatment with IL-27 and were required for the differentiation and function of Tr1 cells in vitro and in vivo. Epigenetic and transcriptional analyses revealed that both transcription factors influenced chromatin accessibility and expression of the genes required for Tr1 cell function. IRF1 and BATF deficiencies uniquely altered the chromatin landscape, suggesting that these factors serve a pioneering function during Tr1 cell differentiation.

A small-molecule inhibitor of hepatitis C virus infectivity.

One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.

An adaptive mutation in NS2 is essential for efficient production of infectious 1b/2a chimeric hepatitis C virus in cell culture.

The development of JFH1 based intergenotypic recombinants which exploit the unique replication characteristics of JFH1 has made it possible to study infectious HCV encoding the structural genes of additional HCV genotypes including genotype 1b. Although, intergenotypic 1b/2a chimeric genomes replicate efficiently in transfected cells they produce very low viral titers, limiting the utility of this system. Here, intergenotypic 1b/2a variants were generated by serially passaging the virus in a novel highly permissive Huh-7 cell clone. The adapted virus was 1000-fold more infectious than the parental unadapted virus and six adapted mutations were identified throughout the genome. Of the mutations identified, L839S in the NS2 gene was the most critical for the adapted phenotype by enhancing the infectivity of assembled viral particles. Overall, the efficient production of infectious 1b/2a virus particles will facilitate the discovery and characterization of inhibitors targeting steps that involve the structural genes of genotype 1b HCV.

Cardiac myosin activation: a potential therapeutic approach for systolic heart failure.

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Selection of clinically relevant protease inhibitor-resistant viruses using the genotype 2a hepatitis C virus infection system.

Treatment of patients infected with hepatitis C virus (HCV) with direct acting antivirals can lead to the emergence of drug-resistant variants that may pose a long-term threat to viral eradication. HCV replicons have been used to select resistance mutations; however, genotype 2a JFH-1-based viruses provide the opportunity to perform resistance selection in a bona fide infection system. In this study, we used a tissue culture-adapted J6/JFH-1 virus to select resistance to the NS3 protease inhibitors BILN-2061 and VX-950. Lunet-CD81 cells were infected with J6/JFH-1 virus and maintained in the presence of inhibitors until high-titer viral supernatant was produced. Viral supernatants were passaged over naive cells at escalating drug concentrations, and the resulting viruses were then characterized. Three NS3 resistance mutations were identified in BILN-2061-resistant viruses: A156G, D168A, and D168V. Interestingly, D168A, D168V, and A156T/V, but not A156G, were selected in parallel using a genotype 2a replicon. For VX-950, the T54A and A156S NS3 resistance mutations were identified in the virus selections, whereas only A156T/V emerged in genotype 2a replicon selections. Of note, VX-950 resistance mutations selected using the 2a virus (T54A and A156S) were also observed during VX-950 clinical studies in genotype 2 patients. We also performed viral fitness evaluations and determined that the mutations selected in the viral system did not confer marked reductions in virus production kinetics or peak titers. Overall, the HCV infection system is an efficient tool for drug resistance selections and has advantages for the rapid identification and characterization of clinically relevant resistance mutations.

Novel mutations in a tissue culture-adapted hepatitis C virus strain improve infectious-virus stability and markedly enhance infection kinetics.

Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 × 10(8) 50% tissue culture infective doses (TCID(50))/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.