RESUMO
The inflammasome components NLRP3 and ASC are cytosolic proteins, which upon sensing endotoxins or danger cues, form multimeric complexes to process interleukin (IL)-1ß for secretion. Here we found that antigen (Ag)-triggered degranulation of IgE-sensitized mast cells (MCs) was mediated by NLRP3 and ASC. IgE-Ag stimulated NEK7 and Pyk2 kinases in MCs to induce the deposition of NLRP3 and ASC on granules and form a distinct protein complex (granulosome) that chaperoned the granules to the cell surface. MCs deficient in NLRP3 or ASC did not form granulosomes, degranulated poorly in vitro and did not evoke systemic anaphylaxis in mice. IgE-Ag-triggered anaphylaxis was prevented by an NLRP3 inhibitor. In endotoxin-primed MCs, pro-IL-1ß was rapidly packaged into granules after IgE-Ag stimulation and processed within granule remnants by proteases after degranulation, causing lethal anaphylaxis in mice. During IgE-Ag-mediated degranulation of endotoxin-primed MCs, granulosomes promoted degranulation, combined with exteriorization and processing of IL-1ß, resulting in severe inflammation.
Assuntos
Anafilaxia , Inflamassomos , Camundongos , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Mastócitos , Anafilaxia/metabolismo , Imunoglobulina E/metabolismo , Endotoxinas/metabolismo , Degranulação CelularRESUMO
Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage-specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1ß inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes.
Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Citoproteção , Glicólise , Macrófagos/metabolismo , Neoplasias Pancreáticas/patologia , Adenocarcinoma/imunologia , Animais , Carcinoma Ductal Pancreático/imunologia , Proliferação de Células/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transportador de Glucose Tipo 1/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Hidroxibenzoatos/farmacologia , Inflamação/patologia , Interleucina-1beta/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neoplasias Pancreáticas/imunologia , Análise de Sobrevida , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Carga Tumoral/efeitos dos fármacos , Neoplasias PancreáticasRESUMO
Calcineurin (Cn) is a protein phosphatase that regulates the activation of the nuclear factor of activated T-cells (NFAT) family of transcription factors, which are key regulators of T-cell development and function. Here, we generated a conditional Cnb1 mouse model in which Cnb1 was specifically deleted in CD4+ T cells (Cnb1CD4 mice) to delineate the role of the Cn-NFAT pathway in immune homeostasis of the intestine. The Cnb1CD4 mice developed severe, spontaneous colitis characterized at the molecular level by an increased T helper-1-cell response but an unaltered regulatory T-cell compartment. Antibiotic treatment ameliorated the intestinal inflammation observed in Cnb1CD4 mice, suggesting that the microbiota contributes to the onset of colitis. CD4+ T cells isolated from Cnb1CD4 mice produced high levels of IFNγ due to increased activation of the JAK2/STAT4 pathway induced by IL-12. Our data highlight that Cn signaling in CD4+ T cells is critical for intestinal immune homeostasis in part by inhibiting IL-12 responsiveness of CD4+ T cells.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Calcineurina/metabolismo , Colite/imunologia , Doenças Inflamatórias Intestinais/imunologia , Intestinos/imunologia , Animais , Calcineurina/genética , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Microbioma Gastrointestinal/imunologia , Homeostase , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Janus Quinase 2/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT4/metabolismo , Transdução de SinaisRESUMO
The intestinal immune system can respond to invading pathogens yet maintain immune tolerance to self-antigens and microbiota. Myeloid cells are central to these processes, but the signaling pathways that underlie tolerance versus inflammation are unclear. Here we show that mice lacking Calcineurin B in CD11chighMHCII+ cells (Cnb1 CD11c mice) spontaneously develop intestinal inflammation and are susceptible to induced colitis. In these mice, colitis is associated with expansion of T helper type 1 (Th1) and Th17 cell populations and a decrease in the number of FoxP3+ regulatory T (Treg) cells, and the pathology is linked to the inability of intestinal Cnb1-deficient CD11chighMHCII+ cells to express IL-2. Deleting IL-2 in CD11chighMHCII+ cells induces spontaneous colitis resembling human inflammatory bowel disease. Our findings identify that the calcineurin-NFAT-IL-2 pathway in myeloid cells is a critical regulator of intestinal homeostasis by influencing the balance of inflammatory and regulatory responses in the mouse intestine.
Assuntos
Antígeno CD11c/imunologia , Calcineurina/imunologia , Colite/imunologia , Interleucina-2/imunologia , Intestinos/imunologia , Células Mieloides/imunologia , Animais , Antígeno CD11c/genética , Calcineurina/genética , Colite/genética , Feminino , Genes MHC da Classe II , Homeostase , Humanos , Interleucina-2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th1/imunologia , Células Th17/imunologiaRESUMO
Acinetobacter baumannii (A. baumannii) is a significant cause of severe nosocomial pneumonia in immunocompromised individuals world-wide. With limited treatment options available, a better understanding of host immnity to A. baumannii infection is critical to devise alternative control strategies. Our previous study has identified that intracellular Nod1/Nod2 signaling pathway is required for the immune control of A. baumannii in airway epithelial cells in vitro. In the current study, using Nod2-/- mice and an in vivo sublethal model of pulmonary infection, we show that Nod2 contributes to the early lung defense against A. baumannii infection through reactive oxygen species (ROS)/reactive nitrogen species (RNS) production as Nod2-/- mice showed significantly reduced production of ROS/RNS in the lungs following A. baumannii infection. Consistent with the higher bacterial load, A. baumannii-induced neutrophil recruitment, cytokine/chemokine response and lung pathology was also exacerbated in Nod2-/- mice at early time points post-infection. Finally, we show that administration of Nod2 ligand muramyl dipeptide (MDP) prior to infection protected the wild- type mice from A. baumannii pulmonary challenge. Collectively, Nod2 is an important player in the early lung immunity against A. baumannii and modulating Nod2 pathway could be considered as a viable therapeutic strategy to control A. baumannii pulmonary infection.
Assuntos
Infecções por Acinetobacter/imunologia , Acinetobacter baumannii/imunologia , Imunidade Inata/fisiologia , Pulmão/imunologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/patologia , Animais , Anti-Infecciosos/farmacologia , Feminino , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD2/genética , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
NLRP10 is a nucleotide-binding oligomerization domain-like receptor that functions as an intracellular pattern recognition receptor for microbial products. Here, we generated a Nlrp10-/- mouse to delineate the role of NLRP10 in the host immune response and found that Nlrp10-/- dendritic cells (DCs) elicited sub-optimal IFNγ production by antigen-specific CD4+ T cells compared to wild-type (WT) DCs. In response to T-cell encounter, CD40 ligation or Toll-like receptor 9 stimulation, Nlrp10-/- DCs produced low levels of IL-12, due to a substantial decrease in NF-κB activation. Defective IL-12 production was also evident in vivo and affected IFNγ production by CD4+ T cells. Upon Mycobacterium tuberculosis (Mtb) infection, Nlrp10-/- mice displayed diminished T helper 1-cell responses and increased bacterial growth compared to WT mice. These data indicate that NLRP10-mediated IL-12 production by DCs is critical for IFNγ induction in T cells and contributes to promote the host defense against Mtb.
RESUMO
This corrects the article DOI: 10.1038/ncomms15865.
RESUMO
Optimal regulation of the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is essential for controlling bacterial infections and inflammatory disorders. Chronic NOD2 stimulation induces non-responsiveness to restimulation, termed NOD2-induced tolerance. Although the levels of the NOD2 adaptor, RIP2, are reported to regulate both acute and chronic NOD2 signalling, how RIP2 levels are modulated is unclear. Here we show that ZNRF4 induces K48-linked ubiquitination of RIP2 and promotes RIP2 degradation. A fraction of RIP2 localizes to the endoplasmic reticulum (ER), where it interacts with ZNRF4 under either 55 unstimulated and muramyl dipeptide-stimulated conditions. Znrf4 knockdown monocytes have sustained nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, and Znrf4 knockdown mice have reduced NOD2-induced tolerance and more effective control of Listeria monocytogenes infection. Our results thus demonstrate E3-ubiquitin ligase ZNRF4-mediated RIP2 degradation as a negative regulatory mechanism of NOD2-induced NF-κB, cytokine and anti-bacterial responses in vitro and in vivo, and identify a ZNRF4-RIP2 axis of fine-tuning NOD2 signalling to promote protective host immunity.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Tolerância Imunológica , Proteína Adaptadora de Sinalização NOD2/metabolismo , Acetilmuramil-Alanil-Isoglutamina/imunologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Células HEK293 , Humanos , Tolerância Imunológica/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Listeriose/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/efeitos dos fármacosRESUMO
Despite a long history and extensive usage of insoluble aluminum salts (alum) as vaccine adjuvants, the molecular mechanisms underpinning Ag-specific immunity upon vaccination remain unclear. Dendritic cells (DCs) are crucial initiators of immune responses, but little is known about the molecular pathways used by DCs to sense alum and, in turn, activate T and B cells. In this article, we show that alum adjuvanticity requires IL-2 specifically released by DCs, even when T cell secretion of IL-2 is intact. We demonstrate that alum, as well as other sterile particulates, such as uric acid crystals, induces DCs to produce IL-2 following initiation of actin-mediated phagocytosis that leads to Src and Syk kinase activation, Ca2+ mobilization, and calcineurin-dependent activation of NFAT, the master transcription factor regulating IL-2 expression. Using chimeric mice, we show that DC-derived IL-2 is required for maximal Ag-specific proliferation of CD4+ T cells and optimal humoral responses following alum-adjuvanted immunization. These data identify DC-derived IL-2 as a key mediator of alum adjuvanticity in vivo and the Src-Syk pathway as a potential leverage point in the rational design of novel adjuvants.
Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen/farmacologia , Células Dendríticas/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Interleucina-2/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fatores de Transcrição NFATC/imunologia , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/imunologiaRESUMO
Inflammasomes are cytosolic multi-protein complexes that regulate the secretion of the proinflammatory cytokines, IL-1ß and IL-18, and induce pyroptosis, an inflammatory form of cell death. The NLRP3 inflammasome is the most well-characterized member of this family and functions by sensing intracellular pathogen- and damage-associated molecular patterns and activating caspase-1, which processes the biologically inactive IL-1ß and IL-18 precursors into active cytokines. Recent studies have identified an alternative mechanism of inflammasome activation, termed the non-canonical inflammasome, which is triggered by cytosolic sensing of lipopolysaccharide (LPS) derived from bacteria that have escaped phagolysosomes. This pathway is independent of Toll-like receptor 4 (TLR4), the well-known extracellular receptor for LPS, but instead depends on the inflammatory protease, caspase-11. Although our understanding of caspase-11 activation is still in its infancy, it appears to be an essential mediator of septic shock and attenuates intestinal inflammation. In this review, we bring together the latest data on the roles of caspase-11 and the mechanisms underlying caspase-11-mediated activation of the non-canonical inflammasome, and consider the implications of this pathway on TLR4-independent immune responses to LPS.
RESUMO
The NLRC4 inflammasome mediates the rapid release of proinflammatory cytokines in response to various microbial stimuli, but its role in the pathology of human diseases remains unknown. Two new studies now report gain-of-function mutations in the NLRC4 gene that cosegregate with distinct autoinflammatory syndromes in affected families.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Ligação ao Cálcio/genética , Enterocolite/genética , Inflamassomos/genética , Inflamação/genética , Síndrome de Ativação Macrofágica/genética , Mutação de Sentido Incorreto , Feminino , Humanos , MasculinoRESUMO
GM-CSF is mostly known for its capacity to promote bone marrow progenitor differentiation, to mobilize and mature myeloid cells as well as to enhance host immune responses. However the molecular actions of GM-CSF are still poorly characterized. Here we describe a new surprising facet of this "old" growth factor as a key regulator involved in IL-1ß secretion. We found that IL-1ß release, a pivotal component of the triggered innate system, is heavily dependent on the signaling induced by GM-CSF in such an extent that in its absence IL-1ß is only weakly secreted. GM-CSF synergizes with LPS for IL-1ß secretion mainly at the level of pro-IL-1ß production via strengthening the NF-κB signaling. In addition, we show that expression of Rab39a, a GTPase required for caspase-1 dependent IL-1ß secretion is greatly augmented by LPS and GM-CSF co-stimulation suggesting a potential GM-CSF contribution in enhancing IL-1ß exocytosis. The role of GM-CSF in regulating IL-1ß secretion is extended also in vivo, since GM-CSF R-/- mice are more resistant to LPS-mediated septic shock. These results identify GM-CSF as a key regulator of IL-1ß production and indicate GM-CSF as a previously underestimated target for therapeutic intervention.
Assuntos
Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-1beta/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/fisiologia , Animais , Western Blotting , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Interleucina-1beta/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Choque Séptico/etiologia , Choque Séptico/metabolismo , Choque Séptico/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Extensive amplification of hematopoietic stem cells (HSCs) and their multipotent primitive progenitors (MPPs) in culture would greatly benefit not only clinical transplantation but also provide a potential tool to manipulate all cellular lineages derived from these cells for gene therapy and experimental purposes. Here, we demonstrate that mouse bone marrow cultures containing cells engineered to over-express NUP98-HOXB4 fusion protein support self-renewal of physiologically normal HSC and MPP for several weeks leading practically to their unlimited expansion. This allows time consuming and cumulative in vitro experimental manipulations without sacrificing their ability to differentiate in vivo or in vitro to any hematopoietic lineage.