RESUMO
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Asma/terapia , Mastócitos/enzimologia , Mastócitos/imunologia , Triptases/antagonistas & inibidores , Triptases/imunologia , Adolescente , Regulação Alostérica/imunologia , Animais , Linhagem Celular , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , CoelhosRESUMO
Foxp3+ regulatory T cells (Treg cells) are crucial for the maintenance of immune homeostasis both in lymphoid tissues and in non-lymphoid tissues. Here we demonstrate that the ability of intestinal Treg cells to constrain microbiota-dependent interleukin (IL)-17-producing helper T cell (TH17 cell) and immunoglobulin A responses critically required expression of the transcription factor c-Maf. The terminal differentiation and function of several intestinal Treg cell populations, including RORγt+ Treg cells and follicular regulatory T cells, were c-Maf dependent. c-Maf controlled Treg cell-derived IL-10 production and prevented excessive signaling via the kinases PI(3)K (phosphatidylinositol-3-OH kinase) and Akt and the metabolic checkpoint kinase complex mTORC1 (mammalian target of rapamycin) and expression of inflammatory cytokines in intestinal Treg cells. c-Maf deficiency in Treg cells led to profound dysbiosis of the intestinal microbiota, which when transferred to germ-free mice was sufficient to induce exacerbated intestinal TH17 responses, even in a c-Maf-competent environment. Thus, c-Maf acts to preserve the identity and function of intestinal Treg cells, which is essential for the establishment of host-microbe symbiosis.
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Imunoglobulina A/biossíntese , Intestinos/imunologia , Microbiota , Proteínas Proto-Oncogênicas c-maf/fisiologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Células Cultivadas , Colite/imunologia , Citocinas/metabolismo , Disbiose , Regulação da Expressão Gênica , Homeostase , Interleucina-10/biossíntese , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-maf/genética , Proteínas Proto-Oncogênicas c-maf/metabolismo , Linfócitos T Reguladores/enzimologiaRESUMO
Defective autophagy is linked to diseases such as rheumatoid arthritis, lupus and inflammatory bowel disease (IBD). However, the mechanisms by which autophagy limits inflammation remain poorly understood. Here we found that loss of the autophagy-related gene Atg16l1 promoted accumulation of the adaptor TRIF and downstream signaling in macrophages. Multiplex proteomic profiling identified SQSTM1 and Tax1BP1 as selective autophagy-related receptors that mediated the turnover of TRIF. Knockdown of Tax1bp1 increased production of the cytokines IFN-ß and IL-1ß. Mice lacking Atg16l1 in myeloid cells succumbed to lipopolysaccharide-mediated sepsis but enhanced their clearance of intestinal Salmonella typhimurium in an interferon receptor-dependent manner. Human macrophages with the Crohn's disease-associated Atg16l1 variant T300A exhibited more production of IFN-ß and IL-1ß. An elevated interferon-response gene signature was observed in patients with IBD who were resistant to treatment with an antibody to the cytokine TNF. These findings identify selective autophagy as a key regulator of signaling via the innate immune system.
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Proteínas Adaptadoras de Transporte Vesicular/imunologia , Autofagia/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Doença de Crohn/imunologia , Feminino , Humanos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais/imunologiaRESUMO
Microglia and other tissue-resident macrophages within the central nervous system (CNS) have essential roles in neural development, inflammation and homeostasis. However, the molecular pathways underlying their development and function remain poorly understood. Here we report that mice deficient in NRROS, a myeloid-expressed transmembrane protein in the endoplasmic reticulum, develop spontaneous neurological disorders. NRROS-deficient (Nrros-/-) mice show defects in motor functions and die before 6 months of age. Nrros-/- mice display astrogliosis and lack normal CD11bhiCD45lo microglia, but they show no detectable demyelination or neuronal loss. Instead, perivascular macrophage-like myeloid cells populate the Nrros-/- CNS. Cx3cr1-driven deletion of Nrros shows its crucial role in microglial establishment during early embryonic stages. NRROS is required for normal expression of Sall1 and other microglial genes that are important for microglial development and function. Our study reveals a NRROS-mediated pathway that controls CNS-resident macrophage development and affects neurological function.
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Astrócitos/metabolismo , Sistema Nervoso Central/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Microglia/metabolismo , Células Mieloides/metabolismo , Doenças do Sistema Nervoso/genética , Proteínas/genética , Animais , Astrócitos/citologia , Western Blotting , Sistema Nervoso Central/citologia , Citometria de Fluxo , Imuno-Histoquímica , Coxeadura Animal/genética , Proteínas de Ligação a TGF-beta Latente , Locomoção , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Knockout , Microglia/citologia , Células Mieloides/citologia , Postura , Fatores de Transcrição/genética , Incontinência Urinária/genética , Retenção Urinária/genéticaRESUMO
CD11b(+) dendritic cells (DCs) seem to be specialized for presenting antigens via major histocompatibility (MHC) class II complexes to stimulate helper T cells, but the genetic and regulatory basis for this is not established. Conditional deletion of Irf4 resulted in loss of CD11b(+) DCs, impaired formation of peptide-MHC class II complexes and defective priming of helper T cells but not of cytotoxic T lymphocyte (CTL) responses. Gene expression and chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analyses delineated an IRF4-dependent regulatory module that programs enhanced MHC class II antigen presentation. Expression of the transcription factor IRF4 but not of IRF8 restored the ability of IRF4-deficient DCs to efficiently process and present antigen to MHC class II-restricted T cells and promote helper T cell responses. We propose that the evolutionary divergence of IRF4 and IRF8 facilitated the specialization of DC subsets for distinct modes of antigen presentation and priming of helper T cell versus CTL responses.
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Apresentação de Antígeno/genética , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fatores Reguladores de Interferon/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Fatores Reguladores de Interferon/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/genética , Transgenes/genéticaRESUMO
Targeting interactions between α4ß7 integrin and endothelial adhesion molecule MAdCAM-1 to inhibit lymphocyte migration to the gastrointestinal tract is an effective therapy in inflammatory bowel disease (IBD). Following lymphocyte entry into the mucosa, a subset of these cells expresses αEß7 integrin, which is expressed on proinflammatory lymphocytes, to increase cell retention. The factors governing lymphocyte migration into the intestinal mucosa and αE integrin expression in healthy subjects and IBD patients remain incompletely understood. We evaluated changes in factors involved in lymphocyte migration and differentiation within tissues. Both ileal and colonic tissue from active IBD patients showed upregulation of ICAM-1, VCAM-1, and MAdCAM-1 at the gene and protein levels compared with healthy subjects and/or inactive IBD patients. ß1 and ß7 integrin expression on circulating lymphocytes was similar across groups. TGF-ß1 treatment induced expression of αE on both ß7+ and ß7- T cells, suggesting that cells entering the mucosa independently of MAdCAM-1/α4ß7 can become αEß7+ ITGAE gene polymorphisms did not alter protein induction following TGF-ß1 stimulation. Increased phospho-SMAD3, which is directly downstream of TGF-ß, and increased TGF-ß-responsive gene expression were observed in the colonic mucosa of IBD patients. Finally, in vitro stimulation experiments showed that baseline ß7 expression had little effect on cytokine, chemokine, transcription factor, and effector molecule gene expression in αE+ and αE- T cells. These findings suggest cell migration to the gut mucosa may be altered in IBD and α4ß7-, and α4ß7+ T cells may upregulate αEß7 in response to TGF-ß once within the gut mucosa.
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Antígenos CD/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Cadeias alfa de Integrinas/metabolismo , Cadeias beta de Integrinas/metabolismo , Mucosa Intestinal/imunologia , Receptores de Retorno de Linfócitos/metabolismo , Linfócitos T/imunologia , Adulto , Idoso , Movimento Celular , Feminino , Humanos , Cadeias beta de Integrinas/genética , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
OBJECTIVE: To establish whether synovial pathobiology improves current clinical classification and prognostic algorithms in early inflammatory arthritis and identify predictors of subsequent biological therapy requirement. METHODS: 200 treatment-naïve patients with early arthritis were classified as fulfilling RA1987 American College of Rheumatology (ACR) criteria (RA1987) or as undifferentiated arthritis (UA) and patients with UA further classified into those fulfilling RA2010 ACR/European League Against Rheumatism (EULAR) criteria. Treatment requirements at 12 months (Conventional Synthetic Disease Modifying Antirheumatic Drugs (csDMARDs) vs biologics vs no-csDMARDs treatment) were determined. Synovial tissue was retrieved by minimally invasive, ultrasound-guided biopsy and underwent processing for immunohistochemical (IHC) and molecular characterisation. Samples were analysed for macrophage, plasma-cell and B-cells and T-cells markers, pathotype classification (lympho-myeloid, diffuse-myeloid or pauci-immune) by IHC and gene expression profiling by Nanostring. RESULTS: 128/200 patients were classified as RA1987, 25 as RA2010 and 47 as UA. Patients classified as RA1987 criteria had significantly higher levels of disease activity, histological synovitis, degree of immune cell infiltration and differential upregulation of genes involved in B and T cell activation/function compared with RA2010 or UA, which shared similar clinical and pathobiological features. At 12-month follow-up, a significantly higher proportion of patients classified as lympho-myeloid pathotype required biological therapy. Performance of a clinical prediction model for biological therapy requirement was improved by the integration of synovial pathobiological markers from 78.8% to 89%-90%. CONCLUSION: The capacity to refine early clinical classification criteria through synovial pathobiological markers offers the potential to predict disease outcome and stratify therapeutic intervention to patients most in need.
Assuntos
Algoritmos , Artrite Reumatoide/terapia , Terapia Biológica/métodos , Membrana Sinovial/diagnóstico por imagem , Antirreumáticos/uso terapêutico , Artrite Reumatoide/classificação , Artrite Reumatoide/diagnóstico , Progressão da Doença , Feminino , Humanos , Biópsia Guiada por Imagem , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Índice de Gravidade de Doença , Membrana Sinovial/metabolismo , UltrassonografiaRESUMO
OBJECTIVES: To unravel the hierarchy of cellular/molecular pathways in the disease tissue of early, treatment-naïve rheumatoid arthritis (RA) patients and determine their relationship with clinical phenotypes and treatment response/outcomes longitudinally. METHODS: 144 consecutive treatment-naïve early RA patients (<12 months symptoms duration) underwent ultrasound-guided synovial biopsy before and 6 months after disease-modifying antirheumatic drug (DMARD) initiation. Synovial biopsies were analysed for cellular (immunohistology) and molecular (NanoString) characteristics and results compared with clinical and imaging outcomes. Differential gene expression analysis and logistic regression were applied to define variables correlating with treatment response and predicting radiographic progression. RESULTS: Cellular and molecular analyses of synovial tissue demonstrated for the first time in early RA the presence of three pathology groups: (1) lympho-myeloid dominated by the presence of B cells in addition to myeloid cells; (2) diffuse-myeloid with myeloid lineage predominance but poor in B cells nd (3) pauci-immune characterised by scanty immune cells and prevalent stromal cells. Longitudinal correlation of molecular signatures demonstrated that elevation of myeloid- and lymphoid-associated gene expression strongly correlated with disease activity, acute phase reactants and DMARD response at 6 months. Furthermore, elevation of synovial lymphoid-associated genes correlated with autoantibody positivity and elevation of osteoclast-targeting genes predicting radiographic joint damage progression at 12 months. Patients with predominant pauci-immune pathology showed less severe disease activity and radiographic progression. CONCLUSIONS: We demonstrate at disease presentation, prior to pathology modulation by therapy, the presence of specific cellular/molecular synovial signatures that delineate disease severity/progression and therapeutic response and may pave the way to more precise definition of RA taxonomy, therapeutic targeting and improved outcomes.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Membrana Sinovial/patologia , Adulto , Idoso , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Biomarcadores/sangue , Biópsia , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fenótipo , Prognóstico , Radiografia , Índice de Gravidade de Doença , Membrana Sinovial/metabolismo , Membrana Sinovial/fisiopatologia , Transcriptoma , Ultrassonografia de Intervenção/métodosRESUMO
BACKGROUND & AIMS: Etrolizumab is a humanized monoclonal antibody against the ß7 integrin subunit that has shown efficacy vs placebo in patients with moderate to severely active ulcerative colitis (UC). Patients with colon tissues that expressed high levels of the integrin αE gene (ITGAE) appeared to have the best response. We compared differences in colonic expression of ITGAE and other genes between patients who achieved clinical remission with etrolizumab vs those who did. METHODS: We performed a retrospective analysis of data collected from 110 patients with UC who participated in a phase 2 placebo-controlled trial of etrolizumab, as well as from 21 patients with UC or without inflammatory bowel disease (controls) enrolled in an observational study at a separate site. Colon biopsies were collected from patients in both studies and analyzed by immunohistochemistry and gene expression profiling. Mononuclear cells were isolated and analyzed by flow cytometry. We identified biomarkers associated with response to etrolizumab. In the placebo-controlled trial, clinical remission was defined as total Mayo Clinic Score ≤2, with no individual subscore >1, and mucosal healing was defined as endoscopic score ≤1. RESULTS: Colon tissues collected at baseline from patients who had a clinical response to etrolizumab expressed higher levels of T-cell-associated genes than patients who did not respond (P < .05). Colonic CD4(+) integrin αE(+) cells from patients with UC expressed higher levels of granzyme A messenger RNA (GZMA mRNA) than CD4(+) αE(-) cells (P < .0001); granzyme A and integrin αE protein were detected in the same cells. Of patients receiving 100 mg etrolizumab, a higher proportion of those with high levels of GZMA mRNA (41%) or ITGAE mRNA (38%) than those with low levels of GZMA (6%) or ITGAE mRNA (13%) achieved clinical remission (P < .05) and mucosal healing (41% GZMA(high) vs 19% GZMA(low) and 44% ITGAE(high) vs 19% ITGAE(low)). Compared with ITGAE(low) and GZMA(low) patients, patients with ITGAE(high) and GZMA(high) had higher baseline numbers of epithelial crypt-associated integrin αE(+) cells (P < .01 for both), but a smaller number of crypt-associated integrin αE(+) cells after etrolizumab treatment (P < .05 for both). After 10 weeks of etrolizumab treatment, expression of genes associated with T-cell activation and genes encoding inflammatory cytokines decreased by 40%-80% from baseline (P < .05) in patients with colon tissues expressing high levels of GZMA at baseline. CONCLUSIONS: Levels of GZMA and ITGAE mRNAs in colon tissues can identify patients with UC who are most likely to benefit from etrolizumab; expression levels decrease with etrolizumab administration in biomarker(high) patients. Larger, prospective studies of markers are needed to assess their clinical value.
Assuntos
Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD/metabolismo , Colite Ulcerativa/tratamento farmacológico , Colo/efeitos dos fármacos , Fármacos Gastrointestinais/uso terapêutico , Granzimas/metabolismo , Cadeias alfa de Integrinas/metabolismo , Antígenos CD/genética , Biópsia , Ensaios Clínicos Fase II como Assunto , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/enzimologia , Colite Ulcerativa/genética , Colo/enzimologia , Colo/patologia , Perfilação da Expressão Gênica/métodos , Granzimas/genética , Humanos , Imuno-Histoquímica , Cadeias alfa de Integrinas/genética , Valor Preditivo dos Testes , RNA Mensageiro/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto , Indução de Remissão , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento , Cicatrização/efeitos dos fármacosRESUMO
UNLABELLED: It is common for computational analyses to generate large amounts of complex data that are difficult to process and share with collaborators. Standard methods are needed to transform such data into a more useful and intuitive format. We present ReportingTools, a Bioconductor package, that automatically recognizes and transforms the output of many common Bioconductor packages into rich, interactive, HTML-based reports. Reports are not generic, but have been individually designed to reflect content specific to the result type detected. Tabular output included in reports is sortable, filterable and searchable and contains context-relevant hyperlinks to external databases. Additionally, in-line graphics have been developed for specific analysis types and are embedded by default within table rows, providing a useful visual summary of underlying raw data. ReportingTools is highly flexible and reports can be easily customized for specific applications using the well-defined API. AVAILABILITY: The ReportingTools package is implemented in R and available from Bioconductor (version ≥ 2.11) at the URL: http://bioconductor.org/packages/release/bioc/html/ReportingTools.html. Installation instructions and usage documentation can also be found at the above URL.
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Biologia Computacional , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Software , Algoritmos , Bases de Dados FactuaisRESUMO
Tryptase, the most abundant mast cell granule protein, is elevated in severe asthma patients independent of type 2 inflammation status. Higher active ß tryptase allele counts are associated with higher levels of peripheral tryptase and lower clinical benefit from anti-IgE therapies. Tryptase is a therapeutic target of interest in severe asthma and chronic spontaneous urticaria. Active and inactive allele counts may enable stratification to assess response to therapies in asthmatic patient subpopulations. Tryptase gene loci TPSAB1 and TPSB2 have high levels of sequence identity, which makes genotyping a challenging task. Here, we report a targeted next-generation sequencing (NGS) assay and downstream bioinformatics analysis for determining polymorphisms at tryptase TPSAB1 and TPSB2 loci. Machine learning modeling using multiple polymorphisms in the tryptase loci was used to improve the accuracy of genotyping calls. The assay was tested and qualified on DNA extracted from whole blood of healthy donors and asthma patients, achieving accuracy of 96%, 96% and 94% for estimation of inactive α and ßΙΙΙFS tryptase alleles and α duplication on TPSAB1, respectively. The reported NGS assay is a cost-effective method that is more efficient than Sanger sequencing and provides coverage to evaluate known as well as unreported tryptase polymorphisms.
Assuntos
Asma , Mastócitos , Humanos , Triptases/genética , Triptases/metabolismo , Mastócitos/metabolismo , Genótipo , Asma/tratamento farmacológico , Asma/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
PURPOSE: Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is characterized by chronic gastrointestinal inflammation. A high unmet need exists for noninvasive biomarkers in IBD to monitor changes in disease activity and guide treatment decisions. Stool is an easily accessed, disease proximal matrix in IBD, however the composition of the IBD fecal proteome remains poorly characterized. EXPERIMENTAL DESIGN: A data-independent acquisition LC-MS/MS approach was used to profile the human fecal proteome in two independent cohorts (Cohort 1: healthy n = 5, UC n = 5, CD n = 5, Cohort 2: healthy n = 20, UC n = 10, and CD n = 10) to identify noninvasive biomarkers reflective of disease activity. RESULTS: 688 human proteins were quantified, with 523 measured in both cohorts. In UC stool 96 proteins were differentially abundant and in CD stool 126 proteins were differentially abundant compared to healthy stool (absolute log2 fold change > 1, p-value < 0.05). Many of these fecal proteins are associated with infiltrating immune cells and ulceration/rectal bleeding, which are hallmarks of IBD pathobiology. Mapping the identified fecal proteins to a whole blood single-cell RNA sequencing data set revealed the involvement of various immune cell subsets to the IBD fecal proteome. CONCLUSIONS AND CLINICAL RELEVANCE: Findings from this study not only confirmed the presence of established fecal biomarkers for IBD, such as calprotectin and lactoferrin, but also revealed new fecal proteins from multiple pathways known to be dysregulated in IBD. These novel proteins could serve as potential noninvasive biomarkers to monitor specific aspects of IBD disease activity which could expedite clinical development of novel therapeutic targets.
RESUMO
High interleukin (IL)-6 levels are associated with greater COVID-19 severity. IL-6 receptor blockade by tocilizumab (anti-IL6R; Actemra) is used globally for the treatment of severe COVID-19, yet a molecular understanding of the therapeutic benefit remains unclear. We characterized the immune profile and identified cellular and molecular pathways modified by tocilizumab in peripheral blood samples from patients enrolled in the COVACTA study, a phase 3, randomized, double-blind, placebo-controlled trial of the efficacy and safety of tocilizumab in hospitalized patients with severe COVID-19. We identified markers of inflammation, lymphopenia, myeloid dysregulation, and organ injury that predict disease severity and clinical outcomes. Proteomic analysis confirmed a pharmacodynamic effect for tocilizumab and identified novel pharmacodynamic biomarkers. Transcriptomic analysis revealed that tocilizumab treatment leads to faster resolution of lymphopenia and myeloid dysregulation associated with severe COVID-19, indicating greater anti-inflammatory activity relative to placebo and potentially leading to faster recovery in patients hospitalized with COVID-19.
RESUMO
Argonaute (AGO) proteins execute microRNA (miRNA)-mediated gene silencing. However, it is unclear whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3, and AGO4) are required for miRNA activity. We generate Ago1, Ago3, and Ago4-deficient mice (Ago134Δ) and find AGO1/3/4 to be redundant for miRNA biogenesis, homeostasis, or function, a role that is carried out by AGO2. Instead, AGO1/3/4 regulate the expansion of type 2 immunity via precursor mRNA splicing in CD4+ T helper (Th) lymphocytes. Gain- and loss-of-function experiments demonstrate that nuclear AGO3 interacts directly with SF3B3, a component of the U2 spliceosome complex, to aid global mRNA splicing, and in particular the isoforms of the gene Nisch, resulting in a dysregulated Nisch isoform ratio. This work uncouples AGO1, AGO3, and AGO4 from miRNA-mediated RNA interference, identifies an AGO3:SF3B3 complex in the nucleus, and reveals a mechanism by which AGO proteins regulate inflammatory diseases.
Assuntos
MicroRNAs , Precursores de RNA , Animais , Camundongos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Receptores de Imidazolinas/genética , Receptores de Imidazolinas/metabolismo , Mamíferos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The ability of stem cells to rapidly proliferate and differentiate is integral to the steady-state maintenance of tissues with high turnover such as the blood and intestine. Mutations that alter these processes can cause primary immunodeficiencies, malignancies and defects in barrier function. The Rho-kinases, Rock1 and Rock2, regulate cell shape and cytoskeletal rearrangement, activities essential to mitosis. Here, we use inducible gene targeting to ablate Rock1 and Rock2 in adult mice, and identify an obligate requirement for these enzymes in the preservation of the hematopoietic and gastrointestinal systems. Hematopoietic cell progenitors devoid of Rho-kinases display cell cycle arrest, blocking the differentiation to mature blood lineages. Similarly, these mice exhibit impaired epithelial cell renewal in the small intestine, which is ultimately fatal. Our data reveal a novel role for these kinases in the proliferation and viability of stem cells and their progenitors, which is vital to maintaining the steady-state integrity of these organ systems.
RESUMO
Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.
Assuntos
Colite , Neoplasias , Humanos , Fagocitose , Neoplasias/tratamento farmacológico , Neutrófilos/metabolismo , Inflamação/patologia , Colite/metabolismoRESUMO
Ulcerative colitis (UC) is an idiopathic chronic inflammatory disease of the colon with sharply rising global prevalence. Dysfunctional epithelial compartment (EC) dynamics are implicated in UC pathogenesis although EC-specific studies are sparse. Applying orthogonal high-dimensional EC profiling to a Primary Cohort (PC; n=222), we detail major epithelial and immune cell perturbations in active UC. Prominently, reduced frequencies of mature BEST4+OTOP2+ absorptive and BEST2+WFDC2+ secretory epithelial enterocytes were associated with the replacement of homeostatic, resident TRDC+KLRD1+HOPX+ γδ+ T cells with RORA+CCL20+S100A4+ TH17 cells and the influx of inflammatory myeloid cells. The EC transcriptome (exemplified by S100A8, HIF1A, TREM1, CXCR1) correlated with clinical, endoscopic, and histological severity of UC in an independent validation cohort (n=649). Furthermore, therapeutic relevance of the observed cellular and transcriptomic changes was investigated in 3 additional published UC cohorts (n=23, 48 and 204 respectively) to reveal that non-response to anti-Tumor Necrosis Factor (anti-TNF) therapy was associated with EC related myeloid cell perturbations. Altogether, these data provide high resolution mapping of the EC to facilitate therapeutic decision-making and personalization of therapy in patients with UC.
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Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.