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
Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.
Assuntos
Células Dendríticas Foliculares/imunologia , Fibroblastos/imunologia , Linfonodos/imunologia , Células Estromais/imunologia , Idoso , Animais , Apoptose/genética , Apoptose/imunologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Dendríticas Foliculares/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Humanos , Imunidade Celular/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfonodos/citologia , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Análise de Célula Única , Células Estromais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The DENN domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to human disease, Dennd1b(-/-) mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b(-/-) TH2, but not other TH cells, exhibit delayed receptor-induced T cell receptor (TCR) downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b(-/-) TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain-containing proteins may contribute to other human disorders.
Assuntos
Asma/imunologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Células Th2/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/imunologia , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Hipersensibilidade/imunologia , Ativação Linfocitária , Camundongos , Polimorfismo de Nucleotídeo Único , Células Th2/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.
Assuntos
Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Neoplasias/imunologia , Nucleotídeos Cíclicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , c-Mer Tirosina Quinase/imunologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Antígeno B7-H1/imunologia , Células Cultivadas , Feminino , Imunidade Inata , Imunoterapia , Interferon Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/metabolismo , Fagocitose , Receptor de Morte Celular Programada 1/imunologia , Receptores Purinérgicos P2X7/deficiência , Transdução de Sinais/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , c-Mer Tirosina Quinase/genéticaRESUMO
Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.
Assuntos
Anticorpos Monoclonais , Membrana Celular , Inflamação , Fígado , Fatores de Crescimento Neural , Traumatismo por Reperfusão , Animais , Camundongos , Alanina Transaminase , Alarminas , Anticorpos Monoclonais/imunologia , Aspartato Aminotransferases , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/imunologia , Moléculas de Adesão Celular Neuronais/ultraestrutura , Morte Celular , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Concanavalina A , Galactosamina , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Inflamação/patologia , Lactato Desidrogenases , Fígado/patologia , Microscopia Eletrônica , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/ultraestrutura , Infiltração de Neutrófilos , Traumatismo por Reperfusão/patologiaRESUMO
Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Morte Celular , Membrana Celular/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Apoptose , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Morte Celular/genética , Feminino , Humanos , Macrófagos , Masculino , Camundongos , Mutação , Necrose , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Multimerização Proteica , Piroptose/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.
Assuntos
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
Mutations in the death receptor FAS1,2 or its ligand FASL3 cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages13 was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.
Assuntos
Caspase 8/metabolismo , Citocinas/imunologia , Imunidade Inata/imunologia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Citocinas/antagonistas & inibidores , Humanos , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Type 2 innate lymphoid cells (ILC2 cells) participate in host defense against helminth parasites and in allergic inflammation. Given their functional relatedness to type 2 helper T cells (T(H)2 cells), we explored whether Gfi1 acts as a shared transcriptional determinant in ILC2 cells. Gfi1 promoted the development of ILC2 cells and controlled their responsiveness during infection with Nippostrongylus brasiliensis and protease allergen-induced lung inflammation. Gfi1 'preferentially' regulated the responsiveness of ILC2 cells to interleukin 33 (IL-33) by directly activating Il1rl1, which encodes the IL-33 receptor (ST2). Loss of Gfi1 in activated ILC2 cells resulted in impaired expression of the transcription factor GATA-3 and a dysregulated genome-wide effector state characterized by coexpression of IL-13 and IL-17. Our findings establish Gfi1 as a shared determinant that reciprocally regulates the type 2 and IL-17 effector states in cells of the innate and adaptive immune systems.
Assuntos
Proteínas de Ligação a DNA/imunologia , Imunidade Inata/imunologia , Células Th2/imunologia , Fatores de Transcrição/imunologia , Transcriptoma/imunologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/imunologia , Fator de Transcrição GATA3/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-13/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-33 , Interleucinas/farmacologia , Pulmão/imunologia , Pulmão/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Nippostrongylus/imunologia , Nippostrongylus/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia , Células Th2/metabolismo , Células Th2/parasitologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Immunoglobulin E (IgE) antibodies are pathogenic in asthma and allergic diseases, but the in vivo biology of IgE-producing (IgE(+)) cells is poorly understood. A model of the differentiation of IgE(+) B cells proposes that IgE(+) cells develop through a germinal-center IgG1(+) intermediate and that IgE memory resides in the compartment of IgG1(+) memory B cells. Here we have used a reporter mouse expressing green fluorescent protein associated with membrane IgE transcripts (IgE-GFP) to assess in vivo IgE responses. In contrast to the IgG1-centered model of IgE switching and memory, we found that IgE(+) cells developed through a germinal-center IgE(+) intermediate to form IgE(+) memory B cells and plasma cells. Our studies delineate a new model for the in vivo biology of IgE switching and memory.
Assuntos
Linfócitos B/citologia , Diferenciação Celular/imunologia , Centro Germinativo/citologia , Imunoglobulina E/imunologia , Memória Imunológica/imunologia , Plasmócitos/imunologia , Transferência Adotiva , Animais , Linfócitos B/imunologia , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Técnicas de Introdução de Genes , Centro Germinativo/imunologia , Humanos , Switching de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Plasmócitos/citologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Genetic and environmental cues shape the evolution of the B cell Ig repertoire. Activation-induced cytidine deaminase (AID) is essential to generating Ig diversity through isotype class switching and somatic mutations, which then directly influence clonal selection. Impaired B cell development in AID-knockout mice has made it difficult to study Ig diversification in an aging repertoire. Therefore, in this report, we used a novel inducible AID-knockout mouse model and discovered that deleting AID in adult mice caused spontaneous germinal center formation. Deep sequencing of the IgH repertoire revealed that Ab diversification begins early in life and evolves over time. Our data suggest that activated B cells form germinal centers at steady state and facilitate continuous diversification of the B cell repertoire. In support, we identified shared B cell lineages that were class switched and showed age-dependent rates of mutation. Our data provide novel context to the genesis of the B cell repertoire that may benefit the understanding of autoimmunity and the strength of an immune response to infection.
Assuntos
Citidina Desaminase , Switching de Imunoglobulina , Animais , Linfócitos B , Citidina Desaminase/genética , Centro Germinativo , Switching de Imunoglobulina/genética , Camundongos , Camundongos Knockout , Hipermutação Somática de ImunoglobulinaRESUMO
OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease1,2 and embryonic lethality during mouse development3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.
Assuntos
Morte Celular , Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Inflamação/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação , Animais , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular/genética , Enzimas Desubiquitinantes/genética , Perda do Embrião/genética , Endopeptidases/genética , Inflamação/enzimologia , Inflamação/genética , Interferon Tipo I/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitinação/genética , Redução de Peso/genéticaRESUMO
BACKGROUND: Clinical studies of type 2 (T2) cytokine-related neutralizing antibodies in asthma have identified a substantial subset of patients with low levels of T2 inflammation who do not benefit from T2 cytokine neutralizing antibody treatment. Non-T2 mechanisms are poorly understood in asthma but represent a redefined unmet medical need. OBJECTIVE: We sought to gain a better understanding of genetic contributions to T2-low asthma. METHODS: We utilized an unbiased genome-wide association study of patients with moderate to severe asthma stratified by T2 serum biomarker periostin. We also performed additional expression and biological analysis for the top genetic hits. RESULTS: We identified a novel protective single nucleotide polymorphism at chr19q13.41, which is selectively associated with T2-low asthma and establishes Kallikrein-related peptidase 5 (KLK5) as the causal gene mediating this association. Heterozygous carriers of the single nucleotide polymorphisms have reduced KLK5 expression. KLK5 is secreted by human bronchial epithelial cells and elevated in asthma bronchial alveolar lavage. T2 cytokines IL-4 and IL-13 downregulate KLK5 in human bronchial epithelial cells. KLK5, dependent on its catalytic function, induces epithelial chemokine/cytokine expression. Finally, overexpression of KLK5 in airway or lack of an endogenous KLK5 inhibitor, SPINK5, leads to spontaneous airway neutrophilic inflammation. CONCLUSION: Our data identify KLK5 to be the causal gene at a novel locus at chr19q13.41 associated with T2-low asthma.
Assuntos
Asma , Estudo de Associação Genômica Ampla , Anticorpos Neutralizantes/genética , Asma/genética , Quimiocinas/genética , Citocinas/metabolismo , Humanos , Inflamação/genética , Interleucina-13/genética , Interleucina-4/genética , Calicreínas/genética , Calicreínas/metabolismoRESUMO
Both common and rare genetic variants of laccase domain-containing 1 (LACC1, previously C13orf31) are associated with inflammatory bowel disease, leprosy, Behcet disease, and systemic juvenile idiopathic arthritis. However, the functional relevance of these variants is unclear. In this study, we use LACC1-deficient mice to gain insight into the role of LACC1 in regulating inflammation. Following oral administration of Citrobacter rodentium, LACC1 knockout (KO) mice had more severe colon lesions compared with wildtype (WT) controls. Immunization with collagen II, a collagen-induced arthritis (CIA) model, resulted in an accelerated onset of arthritis and significantly worse arthritis and inflammation in LACC1 KO mice. Similar results were obtained in a mannan-induced arthritis model. Serum and local TNF in CIA paws and C. rodentium colons were significantly increased in LACC1 KO mice compared with WT controls. The percentage of IL-17A-producing CD4+ T cells was elevated in LACC1 KO mice undergoing CIA as well as aged mice compared with WT controls. Neutralization of IL-17, but not TNF, prevented enhanced mannan-induced arthritis in LACC1 KO mice. These data provide new mechanistic insight into the function of LACC1 in regulating TNF and IL-17 during inflammatory responses. We hypothesize that these effects contribute to immune-driven pathologies observed in individuals carrying LACC1 variants.
Assuntos
Artrite Experimental/imunologia , Artrite Juvenil/imunologia , Citrobacter rodentium/fisiologia , Infecções por Enterobacteriaceae/imunologia , Doenças Inflamatórias Intestinais/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxirredutases/metabolismo , Células Th17/imunologia , Alelos , Animais , Artrite Experimental/microbiologia , Artrite Juvenil/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Doenças Inflamatórias Intestinais/genética , Interleucina-17/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases/genética , Polimorfismo Genético , Fatores de Necrose Tumoral/metabolismoRESUMO
Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1ß processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1ß maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1ß secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Inflamassomos/metabolismo , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Caspases Iniciadoras , Linhagem Celular , Feminino , Bactérias Gram-Negativas/imunologia , Humanos , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Mutação/genética , Necrose , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Fosfato , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Sepse/microbiologia , Transdução de Sinais/genética , Análise de SobrevidaRESUMO
Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.
Assuntos
Anticorpos/uso terapêutico , Transdiferenciação Celular , Pulmão/citologia , Pulmão/metabolismo , Receptores Notch/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Asma/tratamento farmacológico , Asma/metabolismo , Asma/patologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Rastreamento de Células , Transdiferenciação Celular/efeitos dos fármacos , Cílios/metabolismo , Modelos Animais de Doenças , Feminino , Células Caliciformes/citologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Homeostase/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteína Jagged-2 , Ligantes , Pulmão/efeitos dos fármacos , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Serrate-Jagged , Transdução de Sinais/efeitos dos fármacosRESUMO
Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.
Assuntos
Cisteína Endopeptidases/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Animais , Morte Celular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Feminino , Inflamação/genética , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Poliubiquitina/química , Poliubiquitina/metabolismo , Ligação Proteica , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/metabolismo , UbiquitinaçãoAssuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunoglobulina E/imunologia , Memória Imunológica/imunologia , Plasmócitos/imunologia , Transferência Adotiva , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Citometria de Fluxo , Centro Germinativo/citologia , Centro Germinativo/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Parasita/imunologia , Humanos , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Nippostrongylus/imunologia , Nippostrongylus/fisiologia , Plasmócitos/citologia , Plasmócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologiaRESUMO
The connection between an altered gut microbiota and metabolic disorders such as obesity, diabetes, and cardiovascular disease is well established. Defects in preserving the integrity of the mucosal barriers can result in systemic endotoxaemia that contributes to chronic low-grade inflammation, which further promotes the development of metabolic syndrome. Interleukin (IL)-22 exerts essential roles in eliciting antimicrobial immunity and maintaining mucosal barrier integrity within the intestine. Here we investigate the connection between IL-22 and metabolic disorders. We find that the induction of IL-22 from innate lymphoid cells and CD4(+) T cells is impaired in obese mice under various immune challenges, especially in the colon during infection with Citrobacter rodentium. While innate lymphoid cell populations are largely intact in obese mice, the upregulation of IL-23, a cytokine upstream of IL-22, is compromised during the infection. Consequently, these mice are susceptible to C. rodentium infection, and both exogenous IL-22 and IL-23 are able to restore the mucosal host defence. Importantly, we further unveil unexpected functions of IL-22 in regulating metabolism. Mice deficient in IL-22 receptor and fed with high-fat diet are prone to developing metabolic disorders. Strikingly, administration of exogenous IL-22 in genetically obese leptin-receptor-deficient (db/db) mice and mice fed with high-fat diet reverses many of the metabolic symptoms, including hyperglycaemia and insulin resistance. IL-22 shows diverse metabolic benefits, as it improves insulin sensitivity, preserves gut mucosal barrier and endocrine functions, decreases endotoxaemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues. In summary, we identify the IL-22 pathway as a novel target for therapeutic intervention in metabolic diseases.