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1.
Cell ; 175(2): 429-441.e16, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30245008

RESUMO

Targeting autophagy in cancer cells and in the tumor microenvironment are current goals of cancer therapy. However, components of canonical autophagy play roles in other biological processes, adding complexity to this goal. One such alternative function of autophagy proteins is LC3-associated phagocytosis (LAP), which functions in phagosome maturation and subsequent signaling events. Here, we show that impairment of LAP in the myeloid compartment, rather than canonical autophagy, induces control of tumor growth by tumor-associated macrophages (TAM) upon phagocytosis of dying tumor cells. Single-cell RNA sequencing (RNA-seq) analysis revealed that defects in LAP induce pro-inflammatory gene expression and trigger STING-mediated type I interferon responses in TAM. We found that the anti-tumor effects of LAP impairment require tumor-infiltrating T cells, dependent upon STING and the type I interferon response. Therefore, autophagy proteins in the myeloid cells of the tumor microenvironment contribute to immune suppression of T lymphocytes by effecting LAP.


Assuntos
Tolerância Imunológica/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Fagocitose/fisiologia , Animais , Autofagia/imunologia , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Tolerância Imunológica/imunologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Células Mieloides/metabolismo , Fagossomos/fisiologia , Linfócitos T/metabolismo , Microambiente Tumoral/fisiologia
2.
J Infect Dis ; 227(12): 1364-1375, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-36763010

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers activation of the NLRP3 inflammasome, which promotes inflammation and aggravates severe COVID-19. Here, we report that SARS-CoV-2 induces upregulation and activation of human caspase-4/CASP4 (mouse caspase-11/CASP11), and this process contributes to NLRP3 activation. In vivo infections performed in transgenic hACE2 humanized mice, deficient or sufficient for Casp11, indicate that hACE2 Casp11-/- mice were protected from disease development, with the increased pulmonary parenchymal area, reduced clinical score of the disease, and reduced mortality. Assessing human samples from fatal cases of COVID-19, we found that CASP4 was expressed in patient lungs and correlated with the expression of inflammasome components and inflammatory mediators, including CASP1, IL1B, IL18, and IL6. Collectively, our data establish that CASP4/11 promotes NLRP3 activation and disease pathology, revealing a possible target for therapeutic interventions for COVID-19.


Assuntos
COVID-19 , Inflamassomos , Camundongos , Animais , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Macrófagos/metabolismo , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Camundongos Transgênicos
4.
Nature ; 533(7601): 115-9, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-27096368

RESUMO

Defects in clearance of dying cells have been proposed to underlie the pathogenesis of systemic lupus erythematosus (SLE). Mice lacking molecules associated with dying cell clearance develop SLE-like disease, and phagocytes from patients with SLE often display defective clearance and increased inflammatory cytokine production when exposed to dying cells in vitro. Previously, we and others described a form of noncanonical autophagy known as LC3-associated phagocytosis (LAP), in which phagosomes containing engulfed particles, including dying cells, recruit elements of the autophagy pathway to facilitate maturation of phagosomes and digestion of their contents. Genome-wide association studies have identified polymorphisms in the Atg5 (ref. 8) and possibly Atg7 (ref. 9) genes, involved in both canonical autophagy and LAP, as markers of a predisposition for SLE. Here we describe the consequences of defective LAP in vivo. Mice lacking any of several components of the LAP pathway show increased serum levels of inflammatory cytokines and autoantibodies, glomerular immune complex deposition, and evidence of kidney damage. When dying cells are injected into LAP-deficient mice, they are engulfed but not efficiently degraded and trigger acute elevation of pro-inflammatory cytokines but not anti-inflammatory interleukin (IL)-10. Repeated injection of dying cells into LAP-deficient, but not LAP-sufficient, mice accelerated the development of SLE-like disease, including increased serum levels of autoantibodies. By contrast, mice deficient in genes required for canonical autophagy but not LAP do not display defective dying cell clearance, inflammatory cytokine production, or SLE-like disease, and, like wild-type mice, produce IL-10 in response to dying cells. Therefore, defects in LAP, rather than canonical autophagy, can cause SLE-like phenomena, and may contribute to the pathogenesis of SLE.


Assuntos
Autofagia , Inflamação/patologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Autoanticorpos/sangue , Autofagia/genética , Citocinas/biossíntese , Citocinas/sangue , Inflamação/sangue , Inflamação/genética , Interleucina-10/biossíntese , Rim/metabolismo , Rim/patologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Fagócitos/citologia , Fagócitos/fisiologia , Fagossomos/fisiologia
5.
PLoS Pathog ; 13(8): e1006502, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28771586

RESUMO

Legionella pneumophila is a Gram-negative, flagellated bacterium that survives in phagocytes and causes Legionnaires' disease. Upon infection of mammalian macrophages, cytosolic flagellin triggers the activation of Naip/NLRC4 inflammasome, which culminates in pyroptosis and restriction of bacterial replication. Although NLRC4 and caspase-1 participate in the same inflammasome, Nlrc4-/- mice and their macrophages are more permissive to L. pneumophila replication compared with Casp1/11-/-. This feature supports the existence of a pathway that is NLRC4-dependent and caspase-1/11-independent. Here, we demonstrate that caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in response to flagellin-positive bacteria. Accordingly, caspase-8 is activated in Casp1/11-/- macrophages in a process dependent on flagellin, Naip5, NLRC4 and ASC. Silencing caspase-8 in Casp1/11-/- cells culminated in macrophages that were as susceptible as Nlrc4-/- for the restriction of L. pneumophila replication. Accordingly, macrophages and mice deficient in Asc/Casp1/11-/- were more susceptible than Casp1/11-/- and as susceptible as Nlrc4-/- for the restriction of infection. Mechanistically, we found that caspase-8 activation triggers gasdermin-D-independent pore formation and cell death. Interestingly, caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in wild-type macrophages, but it is only activated when caspase-1 or gasdermin-D is inhibited. Our data suggest that caspase-8 activation in the Naip5/NLRC4/ASC inflammasome enable induction of cell death when caspase-1 or gasdermin-D is suppressed.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Caspase 1/imunologia , Caspase 8/imunologia , Inflamassomos/imunologia , Doença dos Legionários/imunologia , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Ligação ao Cálcio , Caspase 1/metabolismo , Caspase 8/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas de Silenciamento de Genes , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Legionella pneumophila , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Inibidora de Apoptose Neuronal , Proteínas de Ligação a Fosfato , Reação em Cadeia da Polimerase em Tempo Real
6.
J Immunol ; 195(5): 2303-11, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26232428

RESUMO

Gram-negative bacteria from the Legionella genus are intracellular pathogens that cause a severe form of pneumonia called Legionnaires' disease. The bacteria replicate intracellularly in macrophages, and the restriction of bacterial replication by these cells is critical for host resistance. The activation of the NAIP5/NLRC4 inflammasome, which is readily triggered in response to bacterial flagellin, is essential for the restriction of bacterial replication in murine macrophages. Once activated, this inflammasome induces pore formation and pyroptosis and facilitates the restriction of bacterial replication in macrophages. Because investigations related to the NLRC4-mediated restriction of Legionella replication were performed using mice double deficient for caspase-1 and caspase-11, we assessed the participation of caspase-1 and caspase-11 in the functions of the NLRC4 inflammasome and the restriction of Legionella replication in macrophages and in vivo. By using several species of Legionella and mice singly deficient for caspase-1 or caspase-11, we demonstrated that caspase-1 but not caspase-11 was required for pore formation, pyroptosis, and restriction of Legionella replication in macrophages and in vivo. By generating F1 mice in a mixed 129 × C57BL/6 background deficient (129 × Casp-11(-/-) ) or sufficient (129 × C57BL/6) for caspase-11 expression, we found that caspase-11 was dispensable for the restriction of Legionella pneumophila replication in macrophages and in vivo. Thus, although caspase-11 participates in flagellin-independent noncanonical activation of the NLRP3 inflammasome, it is dispensable for the activities of the NLRC4 inflammasome. In contrast, functional caspase-1 is necessary and sufficient to trigger flagellin/NLRC4-mediated restriction of Legionella spp. infection in macrophages and in vivo.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Caspase 1/imunologia , Caspases/imunologia , Legionella/imunologia , Doença dos Legionários/imunologia , Macrófagos/imunologia , Piroptose/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Caspases/genética , Caspases/metabolismo , Caspases Iniciadoras , Linhagem Celular , Células Cultivadas , Ativação Enzimática/imunologia , Flagelos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Legionella/classificação , Legionella/fisiologia , Legionella pneumophila/imunologia , Legionella pneumophila/fisiologia , Doença dos Legionários/genética , Doença dos Legionários/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Piroptose/genética , Especificidade da Espécie
8.
Infect Immun ; 84(9): 2439-48, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27297388

RESUMO

Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. Q fever is an atypical pneumonia transmitted through inhalation of contaminated aerosols. In mammalian lungs, C. burnetii infects and replicates in several cell types, including alveolar macrophages (AMs). The innate immunity and signaling pathways operating during infection are still poorly understood, in part because of the lack of relevant host cell models for infection in vitro In the study described here, we investigated and characterized the infection of primary murine AMs by C. burnetii phase II in vitro Our data reveal that AMs show a pronounced M2 polarization and are highly permissive to C. burnetii multiplication in vitro Murine AMs present an increased susceptibility to infection in comparison to primary bone marrow-derived macrophages. AMs support more than 2 logs of bacterial replication during 12 days of infection in culture, similar to highly susceptible host cells, such as Vero and THP-1 cells. As a proof of principle that AMs are useful for investigation of C. burnetii replication, we performed experiments with AMs from Nos2(-/-) or Ifng(-/-) mice. In the absence of gamma interferon and nitric oxide synthase 2 (NOS2), AMs were significantly more permissive than wild-type cells. In contrast, AMs from Il4(-/-) mice were more restrictive to C. burnetii replication, supporting the importance of M2 polarization for the permissiveness of AMs to C. burnetii replication. Collectively, our data account for understanding the high susceptibility of alveolar macrophages to bacterial replication and support the use of AMs as a relevant model of C. burnetii growth in primary macrophages.


Assuntos
Coxiella burnetii/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/microbiologia , Animais , Células Cultivadas , Imunidade Inata/imunologia , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/imunologia , Febre Q/imunologia , Febre Q/microbiologia , Transdução de Sinais/imunologia
9.
J Immunol ; 190(7): 3629-38, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23460746

RESUMO

Pathogens are detected by innate immune receptors that, upon activation, orchestrate an appropriate immune response. Recent studies revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella abortus infection. However, no report has elucidated the role of inflammasome receptors in Brucella recognition. Therefore, we decided to investigate the function of NLRC4, NLRP3, and AIM2 in sensing Brucella. In this study, we showed that NLRC4 is not required to induce caspase-1 activation and further secretion of IL-1ß by B. abortus in macrophages. In contrast, we determined that AIM2, which senses Brucella DNA, and NLRP3 are partially required for caspase-1 activation and IL-1ß secretion. Additionally, mitochondrial reactive oxygen species induced by Brucella were implicated in IL-1ß production. Furthermore, AIM2, NLRP3, ASC, and caspase-1 knockout mice were more susceptible to B. abortus infection than were wild-type animals, suggesting that multiple ASC-dependent inflammasomes contribute to host protection against infection. This protective effect is due to the inflammatory response caused by IL-1ß and IL-18 rather than pyroptosis, because we observed augmented bacterial burden in IL-1R and IL-18 knockout mice. Finally, we determined that bacterial type IV secretion system VirB and live, but not heat-killed, Brucella are required for full inflammasome activation in macrophages during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes that collectively orchestrate a robust caspase-1 activation and proinflammatory response.


Assuntos
Sistemas de Secreção Bacterianos , Brucella abortus/imunologia , Brucella abortus/metabolismo , Brucelose/imunologia , Brucelose/metabolismo , Caspase 1/metabolismo , Proteínas do Citoesqueleto/metabolismo , Inflamassomos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 1/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA , Ativação Enzimática , Predisposição Genética para Doença , Granuloma/imunologia , Granuloma/metabolismo , Granuloma/microbiologia , Imunidade Inata , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Fígado/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo
10.
Arthritis Rheum ; 64(2): 474-84, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21952942

RESUMO

OBJECTIVE: Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)-derived leukotriene B(4) (LTB(4) ) in driving tissue inflammation and hypernociception in a murine model of gout. METHODS: Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1ß (IL-1ß), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB(4) activity, cytokine (IL-1ß, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. RESULTS: Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophil-dependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1ß/MyD88-dependent manner. LTB(4) was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1ß production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB(4) after MSU crystal injection, and LTB(4) was relevant in the MSU crystal-induced maturation of IL-1ß. Mechanistically, LTB(4) drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. CONCLUSION: These results reveal the role of the NLRP3 inflammasome in mediating MSU crystal-induced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB(4) in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.


Assuntos
Proteínas de Transporte/metabolismo , Gota/metabolismo , Hiperalgesia/metabolismo , Inflamassomos/metabolismo , Leucotrieno B4/metabolismo , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo , Animais , Caspase 1/metabolismo , Citocinas/metabolismo , Gota/induzido quimicamente , Gota/imunologia , Hiperalgesia/imunologia , Inflamassomos/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Leucotrieno B4/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/metabolismo , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Ácido Úrico/farmacologia
11.
Bio Protoc ; 13(24): e4903, 2023 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-38156029

RESUMO

Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

12.
J Clin Invest ; 133(12)2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37104043

RESUMO

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions and plays immunopathological roles in inflammatory diseases, we investigated whether the C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill patients with COVID-19 compared with patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular traps-dependent (NETs-dependent) immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonists of C5aR1 could be useful for COVID-19 treatment.


Assuntos
COVID-19 , Armadilhas Extracelulares , Humanos , Animais , Camundongos , COVID-19/genética , COVID-19/patologia , Armadilhas Extracelulares/metabolismo , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/metabolismo , Pulmão/patologia , Complemento C5a/genética , Complemento C5a/metabolismo
13.
J Immunol ; 184(3): 1148-52, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20042586

RESUMO

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)-like receptor proteins in host response to T. cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappaB-dependent products in response to infection and failed to restrict T. cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T. cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T. cruzi infection by mechanisms independent of cytokine production.


Assuntos
Doença de Chagas/imunologia , Imunidade Inata , Proteína Adaptadora de Sinalização NOD1/fisiologia , Trypanosoma cruzi/imunologia , Animais , Doença de Chagas/genética , Doença de Chagas/metabolismo , Predisposição Genética para Doença , Imunidade Inata/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/fisiologia , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/fisiologia
14.
Sci Adv ; 8(37): eabo5400, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36103544

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces mild or asymptomatic COVID-19 in most cases, but some patients develop an excessive inflammatory process that can be fatal. As the NLRP3 inflammasome and additional inflammasomes are implicated in disease aggravation, drug repositioning to target inflammasomes emerges as a strategy to treat COVID-19. Here, we performed a high-throughput screening using a 2560 small-molecule compound library and identified FDA-approved drugs that function as pan-inflammasome inhibitors. Our best hit, niclosamide (NIC), effectively inhibits both inflammasome activation and SARS-CoV-2 replication. Mechanistically, induction of autophagy by NIC partially accounts for inhibition of NLRP3 and AIM2 inflammasomes, but NIC-mediated inhibition of NAIP/NLRC4 inflammasome are autophagy independent. NIC potently inhibited inflammasome activation in human monocytes infected in vitro, in PBMCs from patients with COVID-19, and in vivo in a mouse model of SARS-CoV-2 infection. This study provides relevant information regarding the immunomodulatory functions of this promising drug for COVID-19 treatment.


Assuntos
Tratamento Farmacológico da COVID-19 , Inflamassomos , Animais , Humanos , Agentes de Imunomodulação , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , SARS-CoV-2
15.
Elife ; 112022 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-35666101

RESUMO

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV-2-infected apoptotic cells exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence supporting that lung monocytes and macrophages from severe COVID-19 patients have compromised efferocytic capacity. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppresses macrophage anti-inflammation and efficient tissue repair programs and provides mechanistic insights for the excessive production of pro-inflammatory cytokines and accumulation of tissue damage associated with COVID-19 immunopathogenesis.


Assuntos
COVID-19 , SARS-CoV-2 , Anti-Inflamatórios/farmacologia , Apoptose , Humanos , Macrófagos/metabolismo , Fagocitose
16.
J Biol Chem ; 285(42): 32087-95, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20702413

RESUMO

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1ß/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1ß and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Inflamação/imunologia , Complexos Multiproteicos/imunologia , Proteína Inibidora de Apoptose Neuronal/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Ativação Enzimática , Feminino , Flagelina/imunologia , Flagelina/farmacologia , Imunidade Inata/imunologia , Interleucina-18/imunologia , Interleucina-1beta/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Proteína Inibidora de Apoptose Neuronal/genética , Transdução de Sinais/fisiologia , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/metabolismo
17.
Cell Host Microbe ; 29(8): 1216-1217, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34384524

RESUMO

Engagement of LC3-associated phagocytosis (LAP) in response to the uptake of certain particles modulates innate immune responses. Now in Cell Host and Microbe, Akoumianaki et al. (2021) show how a regulatory role of IL-6 on LAP may be at the core of susceptibility to secondary infection during severe sepsis.


Assuntos
Autofagia , Fagossomos , Humanos , Imunidade Inata , Proteínas Associadas aos Microtúbulos , Fagocitose
18.
RMD Open ; 7(1)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33542047

RESUMO

OBJECTIVE: To evaluate whether the addition of colchicine to standard treatment for COVID-19 results in better outcomes. DESIGN: We present the results of a randomised, double-blinded, placebo-controlled clinical trial of colchicine for the treatment of moderate to severe COVID-19, with 75 patients allocated 1:1 from 11 April to 30 August 2020. Colchicine regimen was 0.5 mg thrice daily for 5 days, then 0.5 mg twice daily for 5 days. The primary endpoints were the need for supplemental oxygen, time of hospitalisation, need for admission and length of stay in intensive care unit and death rate. RESULTS: Seventy-two patients (36 for placebo and 36 for colchicine) completed the study. Median (and IQR) time of need for supplemental oxygen was 4.0 (2.0-6.0) days for the colchicine group and 6.5 (4.0-9.0) days for the placebo group (p<0.001). Median (IQR) time of hospitalisation was 7.0 (5.0-9.0) days for the colchicine group and 9.0 (7.0-12.0) days for the placebo group (p=0.003). At day 2, 67% versus 86% of patients maintained the need for supplemental oxygen, while at day 7, the values were 9% versus 42%, in the colchicine and the placebo groups, respectively (log rank; p=0.001). Two patients died, both in placebo group. Diarrhoea was more frequent in the colchicine group (p=0.26). CONCLUSION: Colchicine reduced the length of both, supplemental oxygen therapy and hospitalisation. The drug was safe and well tolerated. Once death was an uncommon event, it is not possible to ensure that colchicine reduced mortality of COVID-19. TRIAL REGISTRATION NUMBER: RBR-8jyhxh.


Assuntos
Tratamento Farmacológico da COVID-19 , Colchicina/administração & dosagem , Tempo de Internação , Oxigenoterapia , SARS-CoV-2/genética , Índice de Gravidade de Doença , Adulto , Idoso , COVID-19/mortalidade , COVID-19/virologia , Colchicina/efeitos adversos , Diarreia/induzido quimicamente , Método Duplo-Cego , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Resultado do Tratamento
19.
J Exp Med ; 218(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33231615

RESUMO

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1ß, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.


Assuntos
COVID-19/patologia , COVID-19/virologia , Inflamassomos/metabolismo , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Apoptose , Comorbidade , Citocinas/biossíntese , Humanos , Pulmão/patologia , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Mudanças Depois da Morte , Resultado do Tratamento
20.
Sci Adv ; 8(43): eadf0585, 2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36288307
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