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1.
Science ; 370(6514)2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060333

RESUMO

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs. LPS additionally promotes the physical and functional uncoupling of LDs from mitochondria, reducing fatty acid metabolism while increasing LD-bacterial contacts. Thus, LDs actively participate in mammalian innate immunity at two levels: They are both cell-autonomous organelles that organize and use immune proteins to kill intracellular pathogens as well as central players in the local and systemic metabolic adaptation to infection.


Assuntos
Bactérias/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Gotículas Lipídicas/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Ácidos Graxos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/imunologia
2.
Proc Natl Acad Sci U S A ; 117(37): 22984-22991, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868431

RESUMO

Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.


Assuntos
Aciltransferases/imunologia , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Lipídeo A/imunologia , Yersinia pestis/imunologia , Animais , Evolução Biológica , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único/imunologia , Células THP-1/imunologia , Células U937 , Yersinia pseudotuberculosis/imunologia
3.
Life Sci ; 261: 118429, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32931797

RESUMO

AIMS: Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been reported as the important regulators in osteoarthritis (OA). However, the detailed mechanism is implicated. The aim of this study is to reveal the functional mechanism of lncRNA ARFRP1 and miR-15a-5p in osteoarthritis. MATERIALS AND METHODS: The expression level of genes was detected by quantitative real time polymerase chain reaction (qRT-PCR) or western blot assay. Cell Counting Kit-8 (CCK-8) was used to assess cell viability. Cell apoptosis rate was analyzed by flow cytometry analysis. Furthermore, Enzyme-linked immunosorbent assay (ELISA) was performed to measure tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß contents. The interaction between miR-15a-5p and ARFRP1 or Toll-like receptor 4 (TLR4) was predicted by miRcode or PITA, and then confirmed by the dual luciferase reporter assay or pull down assay. Besides, NF-κB-driven luciferase activity was determined using NF-κB luciferase reporter assay. KEY FINDINGS: ARFRP1 and TLR4 levels were increased and miR-15a-5p level was decreased in OA cartilage tissues and lipopolysaccharides (LPS)-induced chondrocytes. ARFRP1 knockdown inhibited LPS-induced the injury of chondrocytes. Interestingly, miR-15a-5p downregulated by ARFRP1 negatively modulated TLR4 expression through interaction. ARFRP1 mediated LPS-induced the injury of chondrocytes via regulating miR-15a-5p/TLR4 axis. Furthermore, ARFRP1 exerted function by modulation of NF-κB pathway. SIGNIFICANCE: Our findings confirmed that ARFRP1 mediated LPS-induced the injury of chondrocytes through regulating NF-κB pathway by modulation of miR-15a-5p/TLR4 axis, providing theoretical basis for the treatment of OA patients.


Assuntos
Condrócitos/patologia , MicroRNAs/genética , NF-kappa B/imunologia , RNA Longo não Codificante/genética , Receptor 4 Toll-Like/genética , Adulto , Idoso , Células Cultivadas , Condrócitos/imunologia , Condrócitos/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Lipopolissacarídeos/imunologia , MicroRNAs/imunologia , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/imunologia , Osteoartrite/patologia , RNA Longo não Codificante/imunologia , Receptor 4 Toll-Like/imunologia , Regulação para Cima
4.
Science ; 369(6510)2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32943500

RESUMO

Inflammasomes are supramolecular complexes that play key roles in immune surveillance. This is accomplished by the activation of inflammatory caspases, which leads to the proteolytic maturation of interleukin 1ß (IL-1ß) and pyroptosis. Here, we show that nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3)- and pyrin-mediated inflammasome assembly, caspase activation, and IL-1ß conversion occur at the microtubule-organizing center (MTOC). Furthermore, the dynein adapter histone deacetylase 6 (HDAC6) is indispensable for the microtubule transport and assembly of these inflammasomes both in vitro and in mice. Because HDAC6 can transport ubiquitinated pathological aggregates to the MTOC for aggresome formation and autophagosomal degradation, its role in NLRP3 and pyrin inflammasome activation also provides an inherent mechanism for the down-regulation of these inflammasomes by autophagy. This work suggests an unexpected parallel between the formation of physiological and pathological aggregates.


Assuntos
Desacetilase 6 de Histona/metabolismo , Vigilância Imunológica , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pirina/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Inflamassomos/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Transporte Proteico
5.
PLoS One ; 15(8): e0237754, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804985

RESUMO

A strain of lactic acid bacteria, Lactobacillus paracasei KW3110 (KW3110), activates M2 macrophages with anti-inflammatory reactions and mitigates aging-related chronic inflammation and blue-light exposure-induced retinal inflammation in mice. However, the mechanism underlying the anti-inflammatory effects of KW3110 remains unclear. In this study, we investigated the anti-inflammatory effects of KW3110 using both mouse and human immune cells and evaluated the suppressive effect of KW3110 on the inflammatory reactions of the cells stimulated with lipopolysaccharide and adenosine 5'-triphosphate (LPS/ATP). KW3110 treatment induced anti-inflammatory cytokine interleukin (IL)-10 production in the supernatants of murine macrophage-like cells, J774A.1, and suppressed IL-1ß production in the supernatants of LPS/ATP-stimulated cells. The influence of KW3110 on the production of these cytokines was inhibited by pre-treatment with phagocytosis blocker or transfection with siRNAs for IL-10 signaling components. KW3110 treatment also suppressed activation of caspase-1, an active component of inflammasome complexes, in LPS/ATP-stimulated J774A.1 cells, and its effect was inhibited by transfection with siRNAs for IL-10 signaling components. In addition to the effects of KW3110 on J774A.1 cells, KW3110 treatment induced IL-10 production in the supernatants of human monocytes, and KW3110 or IL-10 treatment suppressed caspase-1 activation and IL-1ß production in the supernatants of LPS/ATP-stimulated cells. These results suggest that KW3110 suppresses LPS/ATP stimulation-induced caspase-1 activation and IL-1ß production by promoting IL-10 production in mouse and human immune cells. Our findings reveal a novel anti-inflammatory mechanism of LAB and the effect of KW3110 on caspase-1 activation is expected to contribute to constructing future preventive strategies for inflammation-related disorders using food ingredients.


Assuntos
Anti-Inflamatórios/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamação/terapia , Lactobacillus paracasei/imunologia , Probióticos/farmacologia , Animais , Caspase 1/metabolismo , Linhagem Celular , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Inflamação/imunologia , Interleucina-10/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
6.
Nat Commun ; 11(1): 4142, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811831

RESUMO

Glycans are involved in various life processes and represent critical targets of biomedical developments. Nevertheless, the accessibility to long glycans with precise structures remains challenging. Here we report on the synthesis of glycans consisting of [→4)-α-Rha-(1 → 3)-ß-Man-(1 → ] repeating unit, which are relevant to the O-antigen of Bacteroides vulgatus, a common component of gut microbiota. The optimal combination of assembly strategy, protecting group arrangement, and glycosylation reaction has enabled us to synthesize up to a 128-mer glycan. The synthetic glycans are accurately characterized by advanced NMR and MS approaches, the 3D structures are defined, and their potent binding activity with human DC-SIGN, a receptor associated with the gut lymphoid tissue, is disclosed.


Assuntos
Bacteroides/química , Antígenos O/química , Polissacarídeos/síntese química , Bacteroides/imunologia , Bacteroides/metabolismo , Microbioma Gastrointestinal/imunologia , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Espectroscopia de Ressonância Magnética , Antígenos O/imunologia , Antígenos O/metabolismo , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
7.
Mol Immunol ; 126: 111-119, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32818819

RESUMO

Here, we aimed to investigate the role of long noncoding RNA (lncRNA) THRIL in septic-induced acute lung injury. C57BL/6 mice were injected with Adenoviruses (Ad)-shTHRIL or negative control (NC) before caecal ligation and puncture (CLP) operation. MPVECs were transfected with Ad-shTHRIL or NC, followed by lipopolysaccharide (LPS) treatment. MiR-424 and Rho-associated kinase 2 (ROCK2) were predicted and verified as direct targets of THRIL and miR-424, respectively, by using dual-luciferase reporter assay. ROCK2 overexpression vector and shTHRIL were co-transfected into mouse pulmonary microvascular endothelial cells for 24 h before LPS treatment. Our results showed that THRIL was highly expressed in the lung of sepsis mice. CLP triggered severe lung injury and apoptosis in mice, which was abolished by THRIL knockdown. Moreover, CLP treatment visibly increased protein concentration, the number of total cell of neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF). Besides, elevated protein levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 were observed in both lung and BALF. However, inhibition of THRIL reduced the number of inflammatory cells and the production of pro-inflammatory cytokines in sepsis mouse model. The effect of THRIL on inflammatory response and apoptosis in the lung was confirmed in sepsis cell model. Moreover, mechanistic studies have shown that THRIL up-regulated ROCK2 level through sponging miR-424. Furthermore, ROCK2 overexpression reversed the inhibitory effects of THRIL knockdown on LPS-induced inflammatory response and apoptosis. Overall, in vivo and in vitro results suggested that THRIL accelerates sepsis-induced lung injury by sponging miR-424 and further restoring ROCK2.


Assuntos
Lesão Pulmonar Aguda/imunologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Sepse/complicações , Quinases Associadas a rho/genética , Lesão Pulmonar Aguda/diagnóstico , Lesão Pulmonar Aguda/patologia , Animais , Apoptose/genética , Apoptose/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular , Modelos Animais de Doenças , Células Endoteliais , Endotélio Vascular/citologia , Técnicas de Silenciamento de Genes , Humanos , Lipopolissacarídeos/imunologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Microvasos/citologia , Sepse/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologia
8.
Nature ; 584(7820): 286-290, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760002

RESUMO

The histone deacetylases (HDACs) are a superfamily of chromatin-modifying enzymes that silence transcription through the modification of histones. Among them, HDAC3 is unique in that interaction with nuclear receptor corepressors 1 and 2 (NCoR1/2) is required to engage its catalytic activity1-3. However, global loss of HDAC3 also results in the repression of transcription, the mechanism of which is currently unclear4-8. Here we report that, during the activation of macrophages by lipopolysaccharides, HDAC3 is recruited to activating transcription factor 2 (ATF2)-bound sites without NCoR1/2 and activates the expression of inflammatory genes through a non-canonical mechanism. By contrast, the deacetylase activity of HDAC3 is selectively engaged at ATF3-bound sites that suppress Toll-like receptor signalling. Loss of HDAC3 in macrophages safeguards mice from lethal exposure to lipopolysaccharides, but this protection is not conferred upon genetic or pharmacological abolition of the catalytic activity of HDAC3. Our findings show that HDAC3 is a dichotomous transcriptional activator and repressor, with a non-canonical deacetylase-independent function that is vital for the innate immune system.


Assuntos
Histona Desacetilases/metabolismo , Inflamação/genética , Inflamação/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Animais , Biocatálise , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Masculino , Camundongos , Correpressor 1 de Receptor Nuclear , Correpressor 2 de Receptor Nuclear , Proteínas Repressoras/metabolismo , Transcrição Genética/efeitos dos fármacos
9.
Mol Immunol ; 126: 136-142, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32823238

RESUMO

Interleukin (IL)-1ß produced by macrophages plays an important role in inflammation development. However, the underlying mechanism in epigenetic regulation of IL-1ß production is not fully addressed. Though DNA methylcytosine dioxygenase ten-eleven translocation 2 (TET2) is known to be involved in the regulation of inflammatory factors by oxidizing 5-methylcytosine (5mC), the underlying molecular mechanism is largely unknown. In this study, we found that the expression of both IL-1ß and TET2 is upregulated by lipopolysaccharide (LPS)-stimulated mononuclear macrophage. We then knocked down TET2 in mouse macrophagelike cell line (J774.1) and found that LPS-induced IL-1ß is also downregulated. In addition, LPS-stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) signaling pathway and intracellular effectors of the toll-like receptor 4 (TLR4) signaling pathway were also suppressed in TET2-knockdown cells. The methylation status in the promoter regions of myeloid differentiation primary response gene (MyD)88 and TAK1 binding protein 2 (TAB2) were estimated by bisulfite polymerase chain reaction. Compared with that of the control, the 5mC level on the TAB2 promoter is downregulated in the LPS-stimulated cells which can be reversed by TET2-knockdown. These findings altogether suggest that LPS-upregulated TET2 enhances IL-1ß expression through demethylating the promoter region of TAB2, the key member of the TLR4/MAPK signaling pathway, a previously unreported molecular mechanism in TET2-regulated expression of inflammatory factors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/imunologia , Interleucina-1beta/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular , Desmetilação do DNA , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos , Camundongos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/metabolismo , Receptor 4 Toll-Like/metabolismo , Regulação para Cima/imunologia
10.
BMC Infect Dis ; 20(1): 459, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611401

RESUMO

BACKGROUND: Extra pulmonary manifestation of tuberculosis (TB) accounts for approximately one-half of TB cases in HIV-infected individuals with pleural TB as the second most common location. Even though mycobacteria are cleared, mycobacterial antigens may persist in infected tissues, causing sustained inflammation and chronicity of the disease. The aim of this study was to explore various mycobacterial antigens in pleural effusions, the impact of HIV infection and CD4+ T-cell depletion on the presence of antigens, and the diagnostic potential of antigens for improved and rapid diagnosis of pleural TB. METHODS: Pleural fluid specimens were collected from patients presenting with clinically suspected pleural TB, and processed routinely for culture, cytology, and adenosine deaminase activity analysis. HIV status and CD4+ T-cell counts were recorded. Pleural fluid mononuclear cells (PFMC) were isolated, and cell smears were stained with acid-fast staining and immunocytochemistry for various mycobacterial antigens. Real-time and nested-PCR were performed. Patients were categorized as pleural TB or non-TB cases using a composite reference standard. Performance of the mycobacterial antigens as diagnostic test was assessed. RESULTS: A total of 41 patients were enrolled, of which 32 were classified as pleural TB and 9 as non-TB. Thirteen patients had culture confirmed pleural TB, 26 (81%) were HIV-TB co-infected, and 64% had < 100 CD4+ T-cells/microL. Both secreted and cell-wall mycobacterial antigens were detected in PFMC. Lipoarabinomannan (LAM) was the most frequently detected antigen. There was no direct correlation between positive culture and antigens. Cases with low CD4+ T-cell counts had higher bacterial and antigen burden. By combining detection of secreted antigen or LAM, the sensitivity and specificity to diagnose pleural TB was 56 and 78%, respectively, as compared to 41 and 100% for culture, 53 and 89% for nested PCR, and 6 and 100% for real-time PCR. CONCLUSION: Mycobacterial antigens were detectable in PFMC from tuberculous pleural effusions, even in cases where viable mycobacteria or bacterial DNA were not always detected. Thus, a combination of secreted antigen and LAM detection by immunocytochemistry may be a complement to acid-fast staining and contribute to rapid and accurate diagnosis of pleural TB.


Assuntos
Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Linfócitos T CD4-Positivos/imunologia , Coinfecção/diagnóstico , Testes Diagnósticos de Rotina/métodos , Lipopolissacarídeos/genética , Lipopolissacarídeos/imunologia , Mycobacterium tuberculosis/imunologia , Derrame Pleural/microbiologia , Tuberculose Pleural/diagnóstico , Adulto , Idoso , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Contagem de Linfócito CD4 , Coinfecção/microbiologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Derrame Pleural/patologia , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Adulto Jovem
11.
Nat Commun ; 11(1): 3547, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669546

RESUMO

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81-/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.


Assuntos
Células da Medula Óssea/imunologia , Ácido Láctico/metabolismo , Neutrófilos/imunologia , Receptores Acoplados a Proteínas-G/metabolismo , Infecções por Salmonella/imunologia , Animais , Medula Óssea/irrigação sanguínea , Células da Medula Óssea/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Feminino , Humanos , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/metabolismo , Receptores Acoplados a Proteínas-G/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/imunologia , Transdução de Sinais/imunologia
12.
Proc Natl Acad Sci U S A ; 117(25): 14365-14375, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513690

RESUMO

Proper resolution of inflammation is vital for repair and restoration of homeostasis after tissue damage, and its dysregulation underlies various noncommunicable diseases, such as cardiovascular and metabolic diseases. Macrophages play diverse roles throughout initial inflammation, its resolution, and tissue repair. Differential metabolic reprogramming is reportedly required for induction and support of the various macrophage activation states. Here we show that a long noncoding RNA (lncRNA), lncFAO, contributes to inflammation resolution and tissue repair in mice by promoting fatty acid oxidation (FAO) in macrophages. lncFAO is induced late after lipopolysaccharide (LPS) stimulation of cultured macrophages and in Ly6Chi monocyte-derived macrophages in damaged tissue during the resolution and reparative phases. We found that lncFAO directly interacts with the HADHB subunit of mitochondrial trifunctional protein and activates FAO. lncFAO deletion impairs resolution of inflammation related to endotoxic shock and delays resolution of inflammation and tissue repair in a skin wound. These results demonstrate that by tuning mitochondrial metabolism, lncFAO acts as a node of immunometabolic control in macrophages during the resolution and repair phases of inflammation.


Assuntos
Ácidos Graxos/metabolismo , Inflamação/imunologia , Macrófagos/imunologia , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , RNA Longo não Codificante/metabolismo , Animais , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Masculino , Camundongos , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , Oxirredução , Cultura Primária de Células , RNA Longo não Codificante/genética , Pele/imunologia , Pele/lesões , Cicatrização/imunologia
13.
Proc Natl Acad Sci U S A ; 117(25): 14342-14353, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513716

RESUMO

Immature T cells undergo a process of positive selection in the thymus when their new T cell receptor (TCR) engages and signals in response to self-peptides. As the T cell matures, a slew of negative regulatory molecules, including the inhibitory surface glycoprotein CD5, are up-regulated in proportion to the strength of the self-peptide signal. Together these regulators dampen TCR-proximal signaling and help avoid any subsequent peripheral activation of T cells by self-peptides. Paradoxically, antigen-specific T cells initially expressing more CD5 (CD5hi) have been found to better persist as effector/memory cells after a peripheral challenge. The molecular mechanisms underlying such a duality in CD5 function is not clear. We found that CD5 alters the basal activity of the NF-κB signaling in resting peripheral T cells. When CD5 was conditionally ablated, T cells were unable to maintain higher expression of the cytoplasmic NF-κB inhibitor IκBα. Consistent with this, resting CD5hi T cells expressed more of the NF-κB p65 protein than CD5lo cells, without significant increases in transcript levels, in the absence of TCR signals. This posttranslationally stabilized cellular NF-κB depot potentially confers a survival advantage to CD5hi T cells over CD5lo ones. Taken together, these data suggest a two-step model whereby the strength of self-peptide-induced TCR signal lead to the up-regulation of CD5, which subsequently maintains a proportional reserve of NF-κB in peripheral T cells poised for responding to agonistic antigen-driven T cell activation.


Assuntos
Antígenos CD5/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Inibidor de NF-kappaB alfa/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Linfócitos T/imunologia , Transferência Adotiva , Animais , Apresentação do Antígeno/imunologia , Antígenos CD5/genética , Linhagem Celular Tumoral , Separação Celular , Sobrevivência Celular/imunologia , Feminino , Citometria de Fluxo , Lipopolissacarídeos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Modelos Animais , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Timo/citologia , Timo/crescimento & desenvolvimento , Timo/imunologia , Fator de Transcrição RelA/metabolismo , Regulação para Cima
14.
Nat Immunol ; 21(8): 880-891, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32541830

RESUMO

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, and GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D-dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-caspase-4 pathway in antibacterial defense.


Assuntos
Caspases Iniciadoras/imunologia , Proteínas de Ligação ao GTP/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Inflamassomos/imunologia , Transdução de Sinais/imunologia , Animais , Bactérias Gram-Negativas/imunologia , Células HeLa , Humanos , Lipopolissacarídeos/imunologia , Camundongos , Piroptose/imunologia
15.
Mol Immunol ; 124: 18-24, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32485435

RESUMO

Autophagy has been identified as an important immune regulatory mechanism. Recent studies have linked macrophage autophagy with innate immune responses against Mycobacterium tuberculosis (M. tuberculosis), which can survive within macrophages by blocking fusion of the phagosome with lysosomes. These findings suggest that autophagy is a regulatable cellular mechanism of M. tuberculosis defense in macrophages. Transcriptomic profiles in human blood in TB patients suggest that M. tuberculosis affects autophagy related pathways. In order to better understand the role of macrophage autophagy in enhancing protective immunity against M. tuberculosis, in this study, we investigate the effects of the autophagy activators rapamycin and LPS in macrophage autophagy and immunity against M. tuberculosis. We confirm that rapamycin and LPS induce autophagy in M. tuberculosis infected THP-1-derived macrophages or PMA primed THP-1 macrophages [THP-1(A)]. LPS restores M. tuberculosis-inhibited IL-12 synthesis and secretion in THP-1(A) cells via autophagy. Similarly, autophagy activators increase IL-12 synthesis and secretion in THP-1(A) cells. These studies demonstrate the importance of autophagy in M. tuberculosis elimination in macrophages and may lead to novel therapies for tuberculosis and other bacterial infections.


Assuntos
Autofagia/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Autofagia/efeitos dos fármacos , Humanos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia
16.
PLoS One ; 15(5): e0232432, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365067

RESUMO

CR3 and CR4, the leukocyte specific ß2-integrins, involved in cellular adherence, migration and phagocytosis, are often assumed to have similar functions. Previously however, we proved that under physiological conditions CR4 is dominant in the adhesion to fibrinogen of human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Here, using inflammatory conditions, we provide further evidence that the expression and function of CR3 and CR4 are not identical in these cell types. We found that LPS treatment changes their expression differently on MDMs and MDDCs, suggesting a cell type specific regulation. Using mAb24, specific for the high affinity conformation of CD18, we proved that the activation and recycling of ß2-integrins is significantly enhanced upon LPS treatment. Adherence to fibrinogen was assessed by two fundamentally different approaches: a classical adhesion assay and a computer-controlled micropipette, capable of measuring adhesion strength. While both receptors participated in adhesion, we demonstrated that CR4 exerts a dominant role in the strong attachment of MDDCs. Studying the formation of podosomes we found that MDMs retain podosome formation after LPS activation, whereas MDDCs lose this ability, resulting in a significantly reduced adhesion force and an altered cellular distribution of CR3 and CR4. Our results suggest that inflammatory conditions reshape differentially the expression and role of CR3 and CR4 in macrophages and dendritic cells.


Assuntos
Células Dendríticas/imunologia , Inflamação/imunologia , Integrina alfaXbeta2/imunologia , Antígeno de Macrófago 1/imunologia , Macrófagos/imunologia , Podossomos/imunologia , Anticorpos Bloqueadores/imunologia , Antígenos CD18/imunologia , Adesão Celular/imunologia , Adesão Celular/fisiologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Movimento Celular/fisiologia , Células Dendríticas/patologia , Células Dendríticas/fisiologia , Fibrinogênio/imunologia , Humanos , Técnicas In Vitro , Inflamação/patologia , Lipopolissacarídeos/imunologia , Macrófagos/patologia , Macrófagos/fisiologia , Fagocitose/imunologia , Fagocitose/fisiologia , Podossomos/patologia
17.
Nat Immunol ; 21(7): 736-745, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32367036

RESUMO

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.


Assuntos
Dissulfiram/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Ligação a Fosfato/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Sepse/tratamento farmacológico , Animais , Caspase 1/genética , Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Linhagem Celular Tumoral , Dissulfiram/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Lipossomos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sepse/imunologia , Células Sf9 , Spodoptera
18.
PLoS One ; 15(5): e0226233, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379832

RESUMO

Allergic asthma is the most common phenotype of the pathology, having an early-onset in childhood and producing a Th2-driven airways remodeling process that leads to symptoms and pathophysiological changes. The avoidance of aeroallergen exposure in early life has been shown to prevent asthma, but without repeated success and with the underlying preventive mechanisms at the beginning of asthma far to be fully recognized. In the present study, we aimed to evaluate if neonatal LPS-induced boost in epithelial host defenses contribute to prevent OVA-induced asthma in adult mice. To this, we focused on the response of bronchiolar club cells (CC), which are highly specialized in maintaining the epithelial homeostasis in the lung. In these cells, neonatal LPS administration increased the expression of TLR4 and TNFα, as well as the immunodulatory/antiallergic proteins: club cell secretory protein (CCSP) and surfactant protein D (SP-D). LPS also prevented mucous metaplasia of club cells and reduced the epidermal growth factor receptor (EGFR)-dependent mucin overproduction, with mice displaying normal breathing patterns after OVA challenge. Furthermore, the overexpression of the epithelial Th2-related molecule TSLP was blunted, and normal TSLP and IL-4 levels were found in the bronchoalveolar lavage. A lower eosinophilia was detected in LPS-pretreated mice, along with an increase in phagocytes and regulatory cells (CD4+CD25+FOXP3+ and CD4+IL-10+), together with higher levels of IL-12 and TNFα. In conclusion, our study demonstrates stable asthma-preventive epithelial effects promoted by neonatal LPS stimulation, leading to the presence of regulatory cells in the lung. These anti-allergic dynamic mechanisms would be overlaid in the epithelium, favored by an adequate epidemiological environment, during the development of asthma.


Assuntos
Asma/imunologia , Bronquíolos/efeitos dos fármacos , Bronquíolos/imunologia , Citocinas/metabolismo , Epitélio/imunologia , Imunidade Inata , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Alérgenos/imunologia , Animais , Animais Recém-Nascidos , Asma/prevenção & controle , Modelos Animais de Doenças , Epitélio/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Ovalbumina/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia
19.
J Surg Res ; 252: 231-239, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32299011

RESUMO

BACKGROUND: Standard treatment for diffuse peritonitis due to colorectal perforation may be insufficient to suppress inflammatory reaction in sepsis. Thus, developing new treatments is important. This study aimed to examine whether intraperitoneal irradiation by artificial sunlight suppresses inflammatory reaction in a lipopolysaccharide (LPS)-induced peritonitis model after surgical treatments. MATERIALS AND METHODS: Mice were divided into naive, nontreatment (NT), and phototherapy (PT) groups. In the latter two groups, LPS was intraperitoneally administered to induce peritonitis and removed by intraperitoneal lavage after laparotomy. The PT group was irradiated with artificial sunlight intraperitoneally. We evaluated the local and systemic inflammatory reactions. Murine macrophages were irradiated with artificial sunlight after stimulation by LPS, and cell viability and expression of tumor necrotizing factor-α (TNF-α) were evaluated. RESULTS: As a local inflammatory reaction, the whole cell count, the expression of interleukin-6 and TNF-α in the intra-abdominal fluid, and the peritoneal thickness were significantly lower in the PT group than in the NT group. As a systematic inflammatory reaction, the expression of serum TNF-α, granulocyte macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, and MIP-1ß were significantly lower in the PT group than in the NT group. Irradiation by artificial sunlight suppressed the expression of TNF-α in murine macrophages without affecting cell viability. CONCLUSIONS: Intraperitoneal irradiation by artificial sunlight could suppress local and systemic inflammatory reactions in the LPS-induced peritonitis murine model. These effects may be associated with macrophage immune responses.


Assuntos
Perfuração Intestinal/complicações , Peritônio/efeitos da radiação , Peritonite/terapia , Fototerapia/métodos , Luz Solar , Animais , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Perfuração Intestinal/imunologia , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/efeitos da radiação , Masculino , Camundongos , Peritônio/imunologia , Peritonite/imunologia , Células RAW 264.7
20.
Iran J Immunol ; 17(1): 75-86, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32224543

RESUMO

BACKGROUND: Pseudomonas aeruginosa has an important role in nosocomial infections. OBJECTIVE: To evaluate biological activity of the detoxified LPS (D-LPS) entrapped into Poly lactic-co-glycolic acid (PLGA) nanoparticles. MATERIALS: LPS was extracted and detoxified from the P. aeruginosa strain PAO1. The D-LPS, conjugated to the PLGA nanoparticles with 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDAC) and N-hydroxy-succinimide (NHS). The connection was evaluated by FTIR (Fourier transform infrared), Zetasizer, and Atomic Force Microscope (AFM). The BALB/c mice injected intramuscularly with the D-LPS-PLGA with two-week intervals and then challenged two weeks after the last immunization. The bioactivity of the induced specific antisera and cytokines responses against D-LPS-PLGA antigen was assessed by ELISA. RESULTS: D-LPS-PLGA conjugation was confirmed by FTIR, Zetasizer, and AFM. The ELISA results showed that D-LPS was successful in the stimulation of the humoral immune response. The immune responses raised against the D-LPS-PLGA, significantly decreased bacterial titer in the spleen of the immunized mice after challenge with PAO1 strain in comparison with the control groups. CONCLUSION: The conjugation of the bacterial LPS to the PLGA nanoparticle increased their functional activity by decrease in bacterial dissemination and increase the killing of opsonized bacteria.


Assuntos
Antígenos de Bactérias , Lipopolissacarídeos , Nanopartículas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Vacinas contra Pseudomonas , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/farmacologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa
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