Blimp-1 and c-Maf regulate immune gene networks to protect against distinct pathways of pathobiont-induced colitis.

Intestinal immune responses to microbes are controlled by the cytokine IL-10 to avoid immune pathology. Here, we use single-cell RNA sequencing of colon lamina propria leukocytes (LPLs) along with RNA-seq and ATAC-seq of purified CD4+ T cells to show that the transcription factors Blimp-1 (encoded by Prdm1) and c-Maf co-dominantly regulate Il10 while negatively regulating proinflammatory cytokines in effector T cells. Double-deficient Prdm1fl/flMaffl/flCd4Cre mice infected with Helicobacter hepaticus developed severe colitis with an increase in TH1/NK/ILC1 effector genes in LPLs, while Prdm1fl/flCd4Cre and Maffl/flCd4Cre mice exhibited moderate pathology and a less-marked type 1 effector response. LPLs from infected Maffl/flCd4Cre mice had increased type 17 responses with increased Il17a and Il22 expression and an increase in granulocytes and myeloid cell numbers, resulting in increased T cell-myeloid-neutrophil interactions. Genes over-expressed in human inflammatory bowel disease showed differential expression in LPLs from infected mice in the absence of Prdm1 or Maf, revealing potential mechanisms of human disease.

Interferon-gamma release assay conversion after Mycobacterium tuberculosis exposure specifically associates with greater risk of progression to tuberculosis: A prospective cohort study in Leicester, UK.

OBJECTIVES: We investigated whether quantifying the serial QuantiFERON-TB Gold (QFT) response improves tuberculosis (TB) risk stratification in pulmonary TB (PTB) contacts. METHODS: A total of 297 untreated adult household PTB contacts, QFT tested at baseline and 3 months after index notification, were prospectively observed (median 1460 days). Normal variance of serial QFT responses was established in 46 extrapulmonary TB contacts. This informed categorisation of the response in QFT-positive PTB contacts as converters, persistently QFT-positive with significant increase (PPincrease), and without significant increase (PPno-increase). RESULTS: In total, eight co-prevalent TB (disease ≤3 months after index notification) and 12 incident TB (>3 months after index notification) cases were diagnosed. Genetic linkage to the index strain was confirmed in all culture-positive progressors. The cumulative 2-year incident TB risk in QFT-positive contacts was 8.4% (95% confidence interval, 3.0-13.6%); stratifying by serial QFT response, significantly higher risk was observed in QFT converters (28%), compared with PPno-increase (4.8%) and PPincrease (3.7%). Converters were characterised by exposure to index cases with a shorter interval from symptom onset to diagnosis (median reduction 50.0 days, P = 0.013). CONCLUSIONS: QFT conversion, rather than quantitative changes of a persistently positive serial QFT response, is associated with greater TB risk and exposure to rapidly progressive TB.

Blimp-1 and c-Maf regulate Il10 and negatively regulate common and unique proinflammatory gene networks in IL-12 plus IL-27-driven T helper-1 cells.

Background: CD4 + Th1 cells producing IFN-γ are required to eradicate intracellular pathogens, however if uncontrolled these cells can cause immunopathology. The cytokine IL-10 is produced by multiple immune cells including Th1 cells during infection and regulates the immune response to minimise collateral host damage. In this study we aimed to elucidate the transcriptional network of genes controlling the expression of Il10 and proinflammatory cytokines, including Ifng in Th1 cells differentiated from mouse naive CD4 + T cells. Methods: We applied computational analysis of gene regulation derived from temporal profiling of gene expression clusters obtained from bulk RNA sequencing (RNA-seq) of flow cytometry sorted naïve CD4 + T cells from mouse spleens differentiated in vitro into Th1 effector cells with IL-12 and IL-27 to produce Ifng and Il10, compared to IL-27 alone which express Il10 only , or IL-12 alone which express Ifng and no Il10, or medium control driven-CD4 + T cells which do not express effector cytokines . Data were integrated with analysis of active genomic regions from these T cells using an assay for transposase-accessible chromatin with sequencing (ATAC)-seq, integrated with literature derived-Chromatin-immunoprecipitation (ChIP)-seq data and the RNA-seq data, to elucidate the transcriptional network of genes controlling expression of Il10 and pro-inflammatory effector genes in Th1 cells. The co-dominant role for the transcription factors, Prdm1 (encoding Blimp-1) and Maf (encoding c-Maf) , in cytokine gene regulation in Th1 cells, was confirmed using T cells obtained from mice with T-cell specific deletion of these transcription factors. Results: We show that the transcription factors Blimp-1 and c-Maf each have unique and common effects on cytokine gene regulation and not only co-operate to induce Il10 gene expression in IL-12 plus IL-27 differentiated mouse Th1 cells, but additionally directly negatively regulate key proinflammatory cytokines including Ifng, thus providing mechanisms for reinforcement of regulated Th1 cell responses. Conclusions: These data show that Blimp-1 and c-Maf positively and negatively regulate a network of both unique and common anti-inflammatory and pro-inflammatory genes to reinforce a Th1 response in mice that will eradicate pathogens with minimum immunopathology.

Host-directed immunotherapy of viral and bacterial infections: past, present and future.

The advent of COVID-19 and the persistent threat of infectious diseases such as tuberculosis, malaria, influenza and HIV/AIDS remind us of the marked impact that infections continue to have on public health. Some of the most effective protective measures are vaccines but these have been difficult to develop for some of these infectious diseases even after decades of research. The development of drugs and immunotherapies acting directly against the pathogen can be equally challenging, and such pathogen-directed therapeutics have the potential disadvantage of selecting for resistance. An alternative approach is provided by host-directed therapies, which interfere with host cellular processes required for pathogen survival or replication, or target the host immune response to infection (immunotherapies) to either augment immunity or ameliorate immunopathology. Here, we provide a historical perspective of host-directed immunotherapeutic interventions for viral and bacterial infections and then focus on SARS-CoV-2 and Mycobacterium tuberculosis, two major human pathogens of the current era, to indicate the key lessons learned and discuss candidate immunotherapeutic approaches, with a focus on drugs currently in clinical trials.

Transcriptomic Characterization of Tuberculous Sputum Reveals a Host Warburg Effect and Microbial Cholesterol Catabolism.

The crucial transmission phase of tuberculosis (TB) relies on infectious sputum and yet cannot easily be modeled. We applied one-step RNA sequencing (RNA-Seq) to sputum from infectious TB patients to investigate the host and microbial environments underlying transmission of Mycobacterium tuberculosis. In such TB sputa, compared to non-TB controls, transcriptional upregulation of inflammatory responses, including an interferon-driven proinflammatory response and a metabolic shift toward glycolysis, was observed in the host. Among all bacterial sequences in the sputum, approximately 1.5% originated from M. tuberculosis, and its transcript abundance was lower in HIV-1-coinfected patients. Commensal bacterial abundance was reduced in the presence of M. tuberculosis infection. Direct alignment to the genomes of the predominant microbiota species also reveals differential adaptation, whereby firmicutes (e.g., streptococci) displayed a nonreplicating phenotype with reduced transcription of ribosomal proteins and reduced activities of ATP synthases, while Neisseria and Prevotella spp. were less affected. The transcriptome of sputum M. tuberculosis more closely resembled aerobic replication and shared similarity in carbon metabolism to in vitro and in vivo models with significant upregulation of genes associated with cholesterol metabolism and downstream propionate detoxification pathways. In addition, and counter to previous reports on intracellular M. tuberculosis infection in vitro, M. tuberculosis in sputum was zinc, but not iron, deprived, and the phoP loci were also significantly downregulated, suggesting that the pathogen is likely extracellular in location. IMPORTANCE Although a few studies have described the microbiome composition of TB sputa based on 16S ribosomal DNA, these studies did not compare to non-TB samples and the nature of the method does not allow any functional inference. This is the first study to apply such technology using clinical specimens and obtained functional transcriptional data on all three aspects simultaneously. We anticipate that an improved understanding on the biological interactions in the respiratory tract may also allow novel interventions, such as those involving microbiome manipulation or inhibitor targeting disease-specific metabolic pathways.

Blood transcriptomics reveal the evolution and resolution of the immune response in tuberculosis.

Blood transcriptomics have revealed major characteristics of the immune response in active TB, but the signature early after infection is unknown. In a unique clinically and temporally well-defined cohort of household contacts of active TB patients that progressed to TB, we define minimal changes in gene expression in incipient TB increasing in subclinical and clinical TB. While increasing with time, changes in gene expression were highest at 30 d before diagnosis, with heterogeneity in the response in household TB contacts and in a published cohort of TB progressors as they progressed to TB, at a bulk cohort level and in individual progressors. Blood signatures from patients before and during anti-TB treatment robustly monitored the treatment response distinguishing early and late responders. Blood transcriptomics thus reveal the evolution and resolution of the immune response in TB, which may help in clinical management of the disease.

Development of a fixed module repertoire for the analysis and interpretation of blood transcriptome data.

As the capacity for generating large-scale molecular profiling data continues to grow, the ability to extract meaningful biological knowledge from it remains a limitation. Here, we describe the development of a new fixed repertoire of transcriptional modules, BloodGen3, that is designed to serve as a stable reusable framework for the analysis and interpretation of blood transcriptome data. The construction of this repertoire is based on co-clustering patterns observed across sixteen immunological and physiological states encompassing 985 blood transcriptome profiles. Interpretation is supported by customized resources, including module-level analysis workflows, fingerprint grid plot visualizations, interactive web applications and an extensive annotation framework comprising functional profiling reports and reference transcriptional profiles. Taken together, this well-characterized and well-supported transcriptional module repertoire can be employed for the interpretation and benchmarking of blood transcriptome profiles within and across patient cohorts. Blood transcriptome fingerprints for the 16 reference cohorts can be accessed interactively via:  https://drinchai.shinyapps.io/BloodGen3Module/ .

Airway macrophage-intrinsic TGF-ß1 regulates pulmonary immunity during early-life allergen exposure.

BACKGROUND: Early life represents a major risk window for asthma development. However, the mechanisms controlling the threshold for establishment of allergic airway inflammation in early life are incompletely understood. Airway macrophages (AMs) regulate pulmonary allergic responses and undergo TGF-ß-dependent postnatal development, but the role of AM maturation factors such as TGF-ß in controlling the threshold for pathogenic immune responses to inhaled allergens remains unclear. OBJECTIVE: Our aim was to test the hypothesis that AM-derived TGF-ß1 regulates pathogenic immunity to inhaled allergen in early life. METHODS: Conditional knockout (Tgfb1ΔCD11c) mice, with TGF-ß1 deficiency in AMs and other CD11c+ cells, were analyzed throughout early life and following neonatal house dust mite (HDM) inhalation. The roles of specific chemokine receptors were determined by using in vivo blocking antibodies. RESULTS: AM-intrinsic TGF-ß1 was redundant for initial population of the neonatal lung with AMs, but AMs from Tgfb1ΔCD11c mice failed to adopt a mature homeostatic AM phenotype in the first weeks of life. Evidence of constitutive TGF-ß1 signaling was also observed in pediatric human AMs. TGF-ß1-deficient AMs expressed enhanced levels of monocyte-attractant chemokines, and accordingly, Tgfb1ΔCD11c mice exposed to HDM throughout early life accumulated CCR2-dependent inflammatory CD11c+ mononuclear phagocytes into the airway niche that expressed the proallergic chemokine CCL8. Tgfb1ΔCD11c mice displayed augmented TH2, group 2 innate lymphoid cell, and airway remodeling responses to HDM, which were ameliorated by blockade of the CCL8 receptor CCR8. CONCLUSION: Our results highlight a causal relationship between AM maturity, chemokines, and pathogenic immunity to environmental stimuli in early life and identify TGF-ß1 as a key regulator of this.

Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis.

Tuberculosis (TB) is a leading cause of mortality due to infectious disease, but the factors determining disease progression are unclear. Transcriptional signatures associated with type I IFN signalling and neutrophilic inflammation were shown to correlate with disease severity in mouse models of TB. Here we show that similar transcriptional signatures correlate with increased bacterial loads and exacerbate pathology during Mycobacterium tuberculosis infection upon GM-CSF blockade. Loss of GM-CSF signalling or genetic susceptibility to TB (C3HeB/FeJ mice) result in type I IFN-induced neutrophil extracellular trap (NET) formation that promotes bacterial growth and promotes disease severity. Consistently, NETs are present in necrotic lung lesions of TB patients responding poorly to antibiotic therapy, supporting the role of NETs in a late stage of TB pathogenesis. Our findings reveal an important cytokine-based innate immune effector network with a central role in determining the outcome of M. tuberculosis infection.

JEM women in STEM: Unique journeys with a common purpose.

Before one can think of the challenges that face women in science and the hurdles that impair their development into leadership positions, it is worth considering the diversity within the collective of women scientists at the level of culture and past experience and life events.

Transcriptomic analysis reveals diverse gene expression changes in airway macrophages during experimental allergic airway disease.

Background: Airway macrophages (AMs) are the most abundant leukocytes in the healthy airway lumen and have a highly specialised but plastic phenotype that is governed by signals in the local microenvironment. AMs are thought to maintain immunological homeostasis in the steady state, but have also been implicated in the pathogenesis of allergic airway disease (AAD). In this study, we aimed to better understand these potentially contrasting AM functions using transcriptomic analysis. Methods: Bulk RNA sequencing was performed on AMs (CD11c + Siglec F + CD64 + CD45 + SSC hi) flow cytometry sorted from C57BL/6 mice during experimental AAD driven by repeated house dust mite inhalation (AMs HDM), compared to control AMs from non-allergic mice. Differentially expressed genes were further analysed by hierarchical clustering and biological pathway analysis. Results: AMs HDM showed increased expression of genes associated with antigen presentation, inflammatory cell recruitment and tissue repair, including several chemokine and matrix metalloproteinase genes. This was accompanied by increased expression of mitochondrial electron transport chain subunit genes and the retinoic acid biosynthetic enzyme gene Raldh2. Conversely, AMs HDM displayed decreased expression of a number of cell cycle genes, genes related to cytoskeletal functions and a subset of genes implicated in antimicrobial innate immunity, such as Tlr5, Il18 and Tnf. Differential gene expression in AMs HDM was consistent with upstream effects of the cytokines IL-4 and IFN-γ, both of which were present at increased concentrations in lung tissue after HDM treatment. Conclusions: These data highlight diverse gene expression changes in the total AM population in a clinically relevant mouse model of AAD, collectively suggestive of contributions to inflammation and tissue repair/remodelling, but with decreases in certain steady state cellular and immunological functions.

Mouse transcriptome reveals potential signatures of protection and pathogenesis in human tuberculosis.

Although mouse infection models have been extensively used to study the host response to Mycobacterium tuberculosis, their validity in revealing determinants of human tuberculosis (TB) resistance and disease progression has been heavily debated. Here, we show that the modular transcriptional signature in the blood of susceptible mice infected with a clinical isolate of M. tuberculosis resembles that of active human TB disease, with dominance of a type I interferon response and neutrophil activation and recruitment, together with a loss in B lymphocyte, natural killer and T cell effector responses. In addition, resistant but not susceptible strains of mice show increased lung B cell, natural killer and T cell effector responses in the lung upon infection. Notably, the blood signature of active disease shared by mice and humans is also evident in latent TB progressors before diagnosis, suggesting that these responses both predict and contribute to the pathogenesis of progressive M. tuberculosis infection.

A T cell-myeloid IL-10 axis regulates pathogenic IFN-γ-dependent immunity in a mouse model of type 2-low asthma.

BACKGROUND: Although originally defined as a type 2 (T2) immune-mediated condition, non-T2 cytokines, such as IFN-γ and IL-17A, have been implicated in asthma pathogenesis, particularly in patients with severe disease. IL-10 regulates TH cell phenotypes and can dampen T2 immunity to allergens, but its functions in controlling non-T2 cytokine responses in asthmatic patients are unclear. OBJECTIVE: We sought to determine how IL-10 regulates the balance of TH cell responses to inhaled allergen. METHODS: Allergic airway disease was induced in wild-type, IL-10 reporter, and conditional IL-10 or IL-10 receptor α (IL-10Rα) knockout mice by means of repeated intranasal administration of house dust mite (HDM). IL-10 and IFN-γ signaling were disrupted by using blocking antibodies. RESULTS: Repeated HDM inhalation induced a mixed IL-13/IL-17A response and accumulation of IL-10-producing forkhead box P3-negative effector CD4+ T cells in the lungs. Ablation of T cell-derived IL-10 increased the IFN-γ and IL-17A response to HDM, reducing IL-13 levels and airway eosinophilia without affecting IgE levels or airway hyperresponsiveness. The increased IFN-γ response could be recapitulated by IL-10Rα deletion in CD11c+ myeloid cells or local IL-10Rα blockade. Disruption of the T cell-myeloid IL-10 axis resulted in increased pulmonary monocyte-derived dendritic cell numbers and increased IFN-γ-dependent expression of CXCR3 ligands by airway macrophages, which is suggestive of a feedforward loop of TH1 cell recruitment. Augmented IFN-γ responses in the HDM allergic airway disease model were accompanied by increased disruption of airway epithelium, which was reversed by therapeutic blockade of IFN-γ. CONCLUSIONS: IL-10 from effector T cells signals to CD11c+ myeloid cells to suppress an atypical and pathogenic IFN-γ response to inhaled HDM.

Biology and therapeutic potential of interleukin-10.

The cytokine IL-10 is a key anti-inflammatory mediator ensuring protection of a host from over-exuberant responses to pathogens and microbiota, while playing important roles in other settings as sterile wound healing, autoimmunity, cancer, and homeostasis. Here we discuss our current understanding of the regulation of IL-10 production and of the molecular pathways associated with IL-10 responses. In addition to IL-10's classic inhibitory effects on myeloid cells, we also describe the nonclassic roles attributed to this pleiotropic cytokine, including how IL-10 regulates basic processes of neural and adipose cells and how it promotes CD8 T cell activation, as well as epithelial repair. We further discuss its therapeutic potential in the context of different diseases and the outstanding questions that may help develop an effective application of IL-10 in diverse clinical settings.

Transcriptional profiling unveils type I and II interferon networks in blood and tissues across diseases.

Understanding how immune challenges elicit different responses is critical for diagnosing and deciphering immune regulation. Using a modular strategy to interpret the complex transcriptional host response in mouse models of infection and inflammation, we show a breadth of immune responses in the lung. Lung immune signatures are dominated by either IFN-γ and IFN-inducible, IL-17-induced neutrophil- or allergy-associated gene expression. Type I IFN and IFN-γ-inducible, but not IL-17- or allergy-associated signatures, are preserved in the blood. While IL-17-associated genes identified in lung are detected in blood, the allergy signature is only detectable in blood CD4+ effector cells. Type I IFN-inducible genes are abrogated in the absence of IFN-γ signaling and decrease in the absence of IFNAR signaling, both independently contributing to the regulation of granulocyte responses and pathology during Toxoplasma gondii infection. Our framework provides an ideal tool for comparative analyses of transcriptional signatures contributing to protection or pathogenesis in disease.

IL-10 Family Cytokines IL-10 and IL-22: from Basic Science to Clinical Translation.

Cytokines are among the most important effector and messenger molecules in the immune system. They profoundly participate in immune responses during infection and inflammation, protecting against or contributing to diseases such as allergy, autoimmunity, and cancer. Manipulating cytokine pathways, therefore, is one of the most effective strategies to treat various diseases. IL-10 family cytokines exert essential functions to maintain tissue homeostasis during infection and inflammation through restriction of excessive inflammatory responses, upregulation of innate immunity, and promotion of tissue repairing mechanisms. Their important functions in diseases are supported by data from many preclinical models, human genetic studies, and clinical interventions. Despite significant efforts, however, there is still no clinically approved therapy through manipulating IL-10 family cytokines. Here, we summarize the recent progress in understanding the biology of this family of cytokines, suggesting more specific strategies to maneuver these cytokines for the effective treatment of inflammatory diseases and cancers.

Author Correction: Progression of whole-blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in severe influenza.

In the version of this article initially published, a source of funding was not included in the Acknowledgements section. That section should include the following: P.J.M.O. was supported by EU FP7 PREPARE project 602525. The error has been corrected in the HTML and PDF version of the article.

Publisher Correction: c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells.

In the version of this article initially published, the Supplementary Data file was an incorrect version. The correct version is now provided. The error has been corrected in the HTML and PDF version of the article.

High-Dose IL-2 Skews a Glucocorticoid-Driven IL-17+IL-10+ Memory CD4+ T Cell Response towards a Single IL-10-Producing Phenotype.

Glucocorticoids are known to increase production of the anti-inflammatory cytokine IL-10, and this action is associated with their clinical efficacy in asthmatics. However, glucocorticoids also enhance the synthesis of IL-17A by PBMCs, which, in excess, is associated with increased asthma severity and glucocorticoid-refractory disease. In this study, we show that the glucocorticoid dexamethasone significantly increased IL-10 production by human memory CD4+ T cells from healthy donors, as assessed by intracellular cytokine staining. In addition, dexamethasone increased production of IL-17A, IL-17F, and IL-22, with the most striking enhancement in cells coproducing Th17-associated cytokines together with IL-10. Of note, an increase in IFN-γ+IL-10+ cells was also observed despite overall downregulation of IFN-γ production. These dexamethasone-driven IL-10+ cells, and predominantly the IL-17+IL-10+ double-producing cells, were markedly refractory to the inhibitory effect of dexamethasone on proliferation and IL-2Rα expression, which facilitated their preferential IL-2-dependent expansion. Although lower concentrations of exogenous IL-2 promoted IL-10+ cells coproducing proinflammatory cytokines, higher IL-2 doses, both alone and in combination with dexamethasone, increased the proportion of single IL-10+ T cells. Thus, glucocorticoid-induced IL-10 is only accompanied by an increase of IL-17 in a low IL-2 setting, which is, nevertheless, likely to be protective owing to the induction of regulatory IL-17+IL-10+-coproducing cells. These findings open new avenues of investigation with respect to the role of IL-2 in glucocorticoid responsiveness that have potential implications for optimizing the benefit/risk ratio of glucocorticoids in the clinic.