RESUMO
Broad-spectrum histone deacetylase inhibitors (HDACi) are used clinically as anticancer agents, and more isoform-selective HDACi have been sought to modulate other conditions, including chronic inflammatory diseases. Mouse studies suggest that HDACi downregulate immune responses and may compromise host defense. However, their effects on human macrophage antimicrobial responses are largely unknown. Here, we show that overnight pretreatment of human macrophages with HDACi prior to challenge with Salmonella enterica serovar Typhimurium or Escherichia coli results in significantly reduced intramacrophage bacterial loads, which likely reflect the fact that this treatment regime impairs phagocytosis. In contrast, cotreatment of human macrophages with HDACi at the time of bacterial challenge did not impair phagocytosis; instead, HDACi cotreatment actually promoted clearance of intracellular S. Typhimurium and E. coli. Mechanistically, treatment of human macrophages with HDACi at the time of bacterial infection enhanced mitochondrial reactive oxygen species generation by these cells. The capacity of HDACi to promote the clearance of intracellular bacteria from human macrophages was abrogated when cells were pretreated with MitoTracker Red CMXRos, which perturbs mitochondrial function. The HDAC6-selective inhibitor tubastatin A promoted bacterial clearance from human macrophages, whereas the class I HDAC inhibitor MS-275, which inhibits HDAC1 to -3, had no effect on intracellular bacterial loads. These data are consistent with HDAC6 and/or related HDACs constraining mitochondrial reactive oxygen species production from human macrophages during bacterial challenge. Our findings suggest that, whereas long-term HDACi treatment regimes may potentially compromise host defense, selective HDAC inhibitors may have applications in treating acute bacterial infections.
Assuntos
Antineoplásicos/farmacologia , Escherichia coli/imunologia , Inibidores de Histona Desacetilases/farmacologia , Macrófagos/imunologia , Mitocôndrias/imunologia , Espécies Reativas de Oxigênio/metabolismo , Salmonella typhimurium/imunologia , Carga Bacteriana/efeitos dos fármacos , Benzamidas/farmacologia , Células Cultivadas , Desacetilase 6 de Histona , Histona Desacetilases/efeitos dos fármacos , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Macrófagos/metabolismo , Compostos Orgânicos/farmacologia , Fagocitose/efeitos dos fármacos , Piridinas/farmacologiaRESUMO
Monocytes and macrophages are important innate immune cells equipped with danger-sensing receptors, including complement and Toll-like receptors. Complement protein C5a, acting via C5aR, is shown in this study to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. Whereas C5a enhanced secretion of LPS-induced IL-6 and TNF from primary human monocytes, C5a inhibited these responses while increasing IL-10 secretion in donor-matched human monocyte-derived macrophages differentiated by GM-CSF or M-CSF. Gαi/c-Raf/MEK/ERK signaling induced by C5a was amplified in macrophages but not in monocytes by LPS. Accordingly, the Gαi inhibitor pertussis toxin and MEK inhibitor U0126 blocked C5a inhibition of LPS-induced IL-6 and TNF production from macrophages. This synergy was independent of IL-10, PI3K, p38, JNK, and the differentiating agent. Furthermore, C5a did not inhibit IL-6 production from macrophages induced by other TLR agonists that are selective for Toll/IL-1R domain-containing adapter inducing IFN-ß (polyinosinic-polycytidylic acid) or MyD88 (imiquimod), demonstrating selectivity for C5a regulation of LPS responses. Finally, suppression of proinflammatory cytokines IL-6 and TNF in macrophages did not compromise antimicrobial activity; instead, C5a enhanced clearance of the Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium from macrophages. C5aR is thus a regulatory switch that modulates TLR4 signaling via the Gαi/c-Raf/MEK/ERK signaling axis in human macrophages but not monocytes. The differential effects of C5a are consistent with amplifying monocyte proinflammatory responses to systemic danger signals, but attenuating macrophage cytokine responses (without compromising microbicidal activity), thereby restraining inflammatory responses to localized infections.
Assuntos
Complemento C5a/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Receptores de Complemento/metabolismo , Aminoquinolinas , Butadienos/farmacologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Imiquimode , Inflamação/induzido quimicamente , Interleucina-10/biossíntese , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos , Sistema de Sinalização das MAP Quinases/imunologia , Fator Estimulador de Colônias de Macrófagos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Nitrilas/farmacologia , Toxina Pertussis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Poli I-C , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptor da Anafilatoxina C5a , Salmonella typhimurium/imunologia , Receptor 4 Toll-Like/metabolismo , Fatores de Necrose Tumoral/metabolismoRESUMO
Fam96a mRNA, which encodes a mammalian DUF59 protein, is enriched in macrophages. Recombinant human Fam96a forms stable monomers and dimers in solution. Crystal structures of these two forms revealed that each adopts a distinct type of domain-swapped dimer, one of which is stabilized by zinc binding. Two hinge loops control Fam96a domain swapping; both are flexible and highly conserved, suggesting that domain swapping may be a common feature of eukaryotic but not bacterial DUF59 proteins. The derived monomer fold of Fam96a diverges from that of bacterial DUF59 counterparts in that the C-terminal region of Fam96a is much longer and is positioned on the opposite side of the N-terminal core fold. The putative metal-binding site of bacterial DUF59 proteins is not conserved in Fam96a, but Fam96a interacts tightly in vitro with Ciao1, the cytosolic iron-assembly protein. Moreover, Fam96a and Ciao1 can be co-immunoprecipitated, suggesting that the interaction also occurs in vivo. Although predicted to have a signal peptide, it is shown that Fam96a is cytoplasmic. The data reveal that eukaryotic DUF59 proteins share intriguing characteristics with amyloidogenic proteins.
Assuntos
Proteínas de Transporte/química , Multimerização Proteica , Animais , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Células HeLa , Humanos , Metalochaperonas/metabolismo , Metaloproteínas , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Homologia Estrutural de ProteínaRESUMO
Differences in human and mouse immune responses may partly reflect species-specific adaptations and can provide important insights into human immunity. In this study, we show that RNF144B, which encodes an E3 ubiquitin ligase, was lipopolysaccharide-inducible in primary human macrophages and in human macrophage-like THP-1 cells. In contrast, Rnf144b was not lipopolysaccharide-inducible in several mouse cell populations, including primary macrophages from C57BL/6 and BALB/c mice and RAW264.7 macrophages. Similarly, Rnf144b was not up-regulated by infection of C57BL/6 mice with Escherichia coli Although the human and mouse RNF144B genes have conserved transcription start sites, cap analysis of gene expression data confirmed that the RNF144B promoter directs transcription in human but not mouse macrophages. The human and mouse RNF144B genes are controlled by highly conserved TATA-containing promoters, but subtle differences in transcription factor binding sites may account for differential regulation. Using gene silencing, we showed that RNF144B is necessary for priming of inflammasome responses in primary human macrophages. Specifically, RNF144B promotes lipopolysaccharide-inducible IL-1b mRNA expression but does not regulate expression of several other lipopolysaccharide-inducible cytokines (e.g., interleukin-10, interferon-γ) or affect expression of inflammasome components or substrates (e.g., procaspase-1, pro-interleukin-18). Our findings thus revealed a species-specific regulatory mechanism for selective inflammasome priming in human macrophages.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamassomos/metabolismo , Interleucina-1beta/genética , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Humanos , Interleucina-1beta/metabolismo , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
Infections are an important cause of morbidity and mortality in patients with decompensated cirrhosis and ascites. Hypothesizing that innate immune dysfunction contributes to susceptibility to infection, we assessed ascitic fluid macrophage phenotype and function. The expression of complement receptor of the immunoglobulin superfamily (CRIg) and CCR2 defined two phenotypically and functionally distinct peritoneal macrophage subpopulations. The proportion of CRIghi macrophages differed between patients and in the same patient over time, and a high proportion of CRIghi macrophages was associated with reduced disease severity (model for end-stage liver disease) score. As compared with CRIglo macrophages, CRIghi macrophages were highly phagocytic and displayed enhanced antimicrobial effector activity. Transcriptional profiling by RNA sequencing and comparison with human macrophage and murine peritoneal macrophage expression signatures highlighted similarities among CRIghi cells, human macrophages, and mouse F4/80hi resident peritoneal macrophages and among CRIglo macrophages, human monocytes, and mouse F4/80lo monocyte-derived peritoneal macrophages. These data suggest that CRIghi and CRIglo macrophages may represent a tissue-resident population and a monocyte-derived population, respectively. In conclusion, ascites fluid macrophage subset distribution and phagocytic capacity is highly variable among patients with chronic liver disease. Regulating the numbers and/or functions of these macrophage populations could provide therapeutic opportunities in cirrhotic patients.
Assuntos
Ascite/fisiopatologia , Cirrose Hepática/fisiopatologia , Macrófagos Peritoneais/citologia , Receptores de Complemento 3b/metabolismo , Idoso , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptores de ComplementoRESUMO
Ilya Metchnikoff's use of starfish larvae to discover phagocytosis, and Bruno Lemaitre's and Jules Hoffmann's identification of host defence functions for Drosophila Toll provide compelling examples of the utility of model organisms for discovery of human innate immune pathways. Bruce Beutler's mapping of lipopolysaccharide non-responsiveness in C3H/HeJ mice to the Toll-like Receptor 4 gene similarly highlights the power of the mouse as a model. Models have limitations however, and characterising the functional relevance of human innate immune responses not conserved in the mouse presents both a challenge and an opportunity. Here we review differences between human and mouse Toll-like Receptors and inflammasome-forming Nod-like Receptors in repertoire, regulation and function, highlighting the significance of these differences for human innate immunity.