RESUMO
The elderly population is more susceptible to pulmonary infections, including tuberculosis. In this article, we characterize the impact of aging on the phenotype of mouse alveolar macrophages (AMs) and their response to Mycobacterium tuberculosis. Uninfected AMs were isolated from bronchoalveolar lavage of young (3 mo) and old (18 mo) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-ß, IL-10, IL-12p40, TNF-α, and MIF than young mice, and old mice contained higher levels of CCL2, IL-1ß, IFN-ß, and MIF in their alveolar lining fluid. We identified two distinct AM subpopulations, a major CD11c+ CD11b- population and a minor CD11c+ CD11b+ population; the latter was significantly increased in old mice (4-fold). Expression of CD206, TLR2, CD16/CD32, MHC class II, and CD86 was higher in CD11c+ CD11b+ AMs, and these cells expressed monocytic markers Ly6C, CX3CR1, and CD115, suggesting monocytic origin. Sorted CD11c+ CD11b+ AMs from old mice expressed higher mRNA levels of CCL2, IL-1ß, and IL-6, whereas CD11c+ CD11b- AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-ß and IL-10. CD11c+ CD11b+ AMs phagocytosed significantly more M. tuberculosis, which expressed higher RNA levels of genes required for M. tuberculosis survival. Our studies identify two distinct AM populations in old mice: a resident population and an increased CD11c+ CD11b+ AM subpopulation expressing monocytic markers, a unique inflammatory signature, and enhanced M. tuberculosis phagocytosis and survival when compared with resident CD11c+ CD11b- AMs, which are more immune regulatory in nature.
Assuntos
Inflamação/imunologia , Macrófagos Alveolares/imunologia , Tuberculose/imunologia , Animais , Citocinas/imunologia , Feminino , Inflamação/patologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Tuberculose/patologiaRESUMO
As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.
Assuntos
Pulmão/imunologia , Pulmão/microbiologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Tuberculose/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Inflamação/microbiologia , Lisossomos/imunologia , Lisossomos/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Fagossomos/imunologia , Fagossomos/microbiologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína D Associada a Surfactante Pulmonar/imunologia , Tuberculose/microbiologia , Adulto JovemRESUMO
Studies have shown that exposure to psychological stressors leads to inflammation throughout the body. This has been widely studied using social disruption (SDR), a social stressor that involves repeated social defeat in subordinate mice. Exposure to SDR increases serum cytokine levels, results in accumulation of spleen CD11b+ myeloid cells, and primes macrophages for increased cytokine and microbicidal activity. Our previous studies showed that intestinal microbes are necessary for SDR-enhancement of innate immunity. In this study, we show that SDR increases spleen CD11b+Ly6CintermLy6G+ neutrophil and CD11b+Ly6ChiLy6G-monocyte numbers compared with control mice. Further, we found that neutrophils and monocytes from stressor-exposed mice expressed higher levels of IL-1ß mRNA. To determine whether bacterial translocation may contribute to these effects, bacterial 16S rRNA was quantified using quantitative real-time RT-PCR with bacterial group-specific primers. Exposure to the SDR stressor specifically increased Lactobacillus RNA in the spleen, which localized in spleen monocytes. The increased spleen levels of Lactobacillus 16S rRNA in SDR mice positively correlated with increased levels of IL-1ß and IL-23 mRNA. Our findings indicate that during stressor exposure, Lactobacillus spp. can translocate to the spleen and prime the innate immune system for enhanced reactivity.
Assuntos
Translocação Bacteriana , Imunidade Inata , Lactobacillus/fisiologia , Monócitos/imunologia , Neutrófilos/imunologia , Baço/imunologia , Estresse Psicológico/imunologia , Animais , Células Cultivadas , Exposição Ambiental/efeitos adversos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Ribossômico 16S/genética , Baço/microbiologia , Estresse Psicológico/microbiologia , Simbiose , Regulação para CimaRESUMO
IFN-γ-activated macrophages play an essential role in controlling intracellular pathogens; however, macrophages also serve as the cellular home for the intracellular pathogen Mycobacterium tuberculosis. Based on previous evidence that M. tuberculosis can modulate host microRNA (miRNA) expression, we examined the miRNA expression profile of M. tuberculosis-infected primary human macrophages. We identified 31 differentially expressed miRNAs in primary human macrophages during M. tuberculosis infection by NanoString and confirmed our findings by quantitative real-time RT-PCR. In addition, we determined a role for two miRNAs upregulated upon M. tuberculosis infection, miR-132 and miR-26a, as negative regulators of transcriptional coactivator p300, a component of the IFN-γ signaling cascade. Knockdown expression of miR-132 and miR-26a increased p300 protein levels and improved transcriptional, translational, and functional responses to IFN-γ in human macrophages. Collectively, these data validate p300 as a target of miR-132 and miR-26a, and demonstrate a mechanism by which M. tuberculosis can limit macrophage responses to IFN-γ by altering host miRNA expression.
Assuntos
Macrófagos/imunologia , Macrófagos/metabolismo , MicroRNAs/genética , Mycobacterium tuberculosis/imunologia , Tuberculose/genética , Tuberculose/imunologia , Regiões 3' não Traduzidas , Sítios de Ligação , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Interferência de RNA , RNA Mensageiro/genética , Receptores de IgG/genética , Receptores de IgG/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Exposing mice to a social stressor called social disruption (SDR) that involves repeated social defeat during intermale aggression results in increased circulating cytokines, such as interleukin-1α (IL-1α) and IL-1ß, and increased reactivity of splenic CD11b(+) macrophages to inflammatory stimuli. For example, upon lipopolysaccharide stimulation, macrophages from stressor-exposed mice produce higher levels of cytokines than do cells from nonstressed controls. Moreover, the SDR stressor enhances the ability of these macrophages to kill Escherichia coli both in vitro and in vivo, through a Toll-like receptor 4-dependent mechanism. The present study tested the hypothesis that stressor-enhanced bacterial killing is due to increases in the production of peroxynitrite. Male mice were exposed to the SDR stressor or were left undisturbed. Upon stimulation with E. coli, splenic macrophages from SDR-exposed mice expressed significantly increased levels of inducible nitric oxide synthase mRNA and produced higher levels of peroxynitrite. Blocking the production of peroxynitrite abrogated the SDR-induced increase in microbicidal activity. Studies in IL-1 receptor type 1 knockout mice indicated that the increased microbicidal activity and peroxynitrite production was dependent upon IL-1 signaling. These data confirm and extend the importance of IL-1 signaling for stressor-induced immunopotentiation; the finding that inhibiting superoxide or nitric oxide production inhibits both peroxynitrite production and killing of E. coli demonstrates that peroxynitrite mediates the stressor-induced increase in bacterial killing.
Assuntos
Escherichia coli K12/fisiologia , Macrófagos/fisiologia , Ácido Peroxinitroso/metabolismo , Baço/citologia , Agressão , Animais , Comportamento Animal , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Interleucina-1/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitrogênio/metabolismo , Ácido Peroxinitroso/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Predomínio Social , Estresse FisiológicoRESUMO
The indigenous microbiota impact mucosal, as well as systemic, immune responses, but whether the microbiota are involved in stressor-induced immunomodulation has not been thoroughly tested. A well characterized murine stressor, called social disruption (SDR), was used to study whether the microbiota are involved in stressor-induced enhancement of macrophage reactivity. Exposure to the SDR Stressor enhanced the ability of splenic macrophages to produce microbicidal mediators (e.g., inducible nitric oxide synthase (iNOS), superoxide anion, and peroxynitrite) and to kill target Escherichia coli. Exposure to the SDR Stressor also increased cytokine production by LPS-stimulated splenic macrophages. These effects, however, were impacted by the microbiota. Microbicidal activity and cytokine mRNA in splenic macrophages from Swiss Webster germfree mice that lack any commensal microbiota were not enhanced by exposure to the SDR Stressor. However, when germfree mice were conventionalized by colonizing them with microbiota from CD1 conventional donor mice, exposure to the SDR Stressor again increased microbicidal activity and cytokine mRNA. In follow-up experiments, immunocompetent conventional CD1 mice were treated with a cocktail of antibiotics to disrupt the intestinal microbiota. While exposure to the SDR Stressor-enhanced splenic macrophage microbicidal activity and cytokine production in vehicle-treated mice, treatment with antibiotics attenuated the SDR Stressor-induced increases in splenic macrophage reactivity. Treatment with antibiotics also prevented the stressor-induced increase in circulating levels of bacterial peptidoglycan, suggesting that translocation of microbiota-derived peptidoglycan into the body primes the innate immune system for enhanced activity. This study demonstrates that the microbiota play a crucial role in stressor-induced immunoenhancement.
Assuntos
Citocinas/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Metagenoma/imunologia , Estresse Psicológico/imunologia , Animais , Escherichia coli , Imunidade nas Mucosas , Imunomodulação , Camundongos , Óxido Nítrico Sintase Tipo II/imunologia , Ácido Peroxinitroso/imunologia , Baço/imunologia , Superóxidos/imunologiaRESUMO
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, debilitating, multisystem illness of unknown etiology for which no cure and no diagnostic tests are available. Despite increasing evidence implicating EBV and human herpesvirus 6A (HHV-6A) as potential causative infectious agents in a subset of patients with ME/CFS, few mechanistic studies address a causal relationship. In this study we examined a large ME/CFS cohort and controls and demonstrated a significant increase in activin A and IL-21 serum levels, which correlated with seropositivity for antibodies against the EBV and HHV-6 protein deoxyuridine triphosphate nucleotidohydrolase (dUTPases) but no increase in CXCL13. These cytokines are critical for T follicular helper (TFH) cell differentiation and for the generation of high-affinity antibodies and long-lived plasma cells. Notably, ME/CFS serum was sufficient to drive TFH cell differentiation via an activin A-dependent mechanism. The lack of simultaneous CXCL13 increase with IL-21 indicates impaired TFH function in ME/CFS. In vitro studies revealed that virus dUTPases strongly induced activin A secretion while in vivo, EBV dUTPase induced the formation of splenic marginal zone B and invariant NKTFH cells. Together, our data indicate abnormal germinal center (GC) activity in participants with ME/CFS and highlight a mechanism by which EBV and HHV6 dUTPases may alter GC and extrafollicular antibody responses.
Assuntos
Síndrome de Fadiga Crônica , Herpesvirus Humano 4 , Herpesvirus Humano 6 , Pirofosfatases , Linfócitos T Auxiliares-Indutores , Diferenciação Celular , Infecções por Vírus Epstein-Barr/enzimologia , Infecções por Vírus Epstein-Barr/virologia , Síndrome de Fadiga Crônica/diagnóstico , Síndrome de Fadiga Crônica/enzimologia , Síndrome de Fadiga Crônica/virologia , Herpesvirus Humano 4/enzimologia , Herpesvirus Humano 6/enzimologia , Humanos , Pirofosfatases/metabolismo , Infecções por Roseolovirus/enzimologia , Infecções por Roseolovirus/virologia , Linfócitos T Auxiliares-Indutores/enzimologia , Linfócitos T Auxiliares-Indutores/patologia , Linfócitos T Auxiliares-Indutores/virologiaRESUMO
Age is a major risk factor for chronic infections, including tuberculosis (TB). Elderly TB patients also suffer from elevated levels of psychological stress. It is not clear how psychological stress impacts immune response to Mycobacterium tuberculosis (M.tb). In this study, we used social disruption stress (SDR) to investigate effects of psychological stress in young and old mice. Unexpectedly, we found that SDR suppresses lung inflammation in old mice as evidenced by lower pro-inflammatory cytokine levels in bronchial lavage fluid and decreased cytokine mRNA expression by alveolar macrophages. To investigate effects of stress on M.tb infection, mice were subjected to SDR and then infected with M.tb. As previously reported, old mice were better at controlling infection at 30 days than young mice. This control was transient as CFUs at 60 days were higher in old control mice compared to young mice. Consistently, SDR significantly increased M.tb growth at 60 days in old mice compared to young mice. In addition, SDR in old mice resulted in accumulation of IL-10 mRNA and decreased IFN-γ mRNA at 60 days. Also, confocal microscopy of lung sections from old SDR mice showed increased number of CD4 T cells which express LAG3 and CD49b, markers of IL-10 secreting regulatory T cells. Further, we also demonstrated that CD4 T cells from old SDR mice express IL-10. Thus, we conclude that psychological stress in old mice prior to infection, increases differentiation of IL-10 secreting T cells, which over time results in loss of control of the infection.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Citocinas/metabolismo , Integrina alfa2 , Interleucina-10/genética , Pulmão/metabolismo , Camundongos , RNA Mensageiro , Estresse PsicológicoRESUMO
Influenza A virus (IAV) and SARS-CoV-2 virus are both acute respiratory viruses currently circulating in the human population. This study aims to determine the impact of IAV infection on SARS-CoV-2 pathogenesis and cardiomyocyte function. Infection of human bronchial epithelial cells (HBEC), A549 cells, lung fibroblasts (HLF), monocyte derived macrophages (MDMs), cardiac fibroblasts (HCF) and hiPSC-derived cardiomyocytes with IAV enhanced the expression of ACE2, the SARS-CoV-2 receptor. Similarly, IAV infection increased levels of ACE2 in the lungs of mice and humans. Of interest, we detected heavily glycosylated form of ACE2 in hiPSC-CMs and poorly glycosylated ACE2 in other cell types. Also, prior IAV infection enhances SARS-CoV-2 spike protein binding and viral entry in all cell types. However, efficient SARS-CoV-2 replication was uniquely inhibited in cardiomyocytes. Glycosylation of ACE2 correlated with enzymatic conversion of its substrate Ang II, induction of eNOS and nitric oxide production, may provide a potential mechanism for the restricted SARS-CoV-2 replication in cardiomyocytes.
RESUMO
Aging-mediated immune dysregulation affects the normal cardiac immune cell phenotypes and functions, resulting in cardiac distress. During cardiac inflammation, immune activation is critical for mounting the regenerative responses to maintain normal heart function. We investigated the impact of aging on myeloid cell phenotype and function during cardiac inflammation induced by a sub-lethal dose of LPS. Our data show that hearts of old mice contain more myeloid cells than the hearts of young mice. However, while the number of monocytic-derived suppressor cells did not differ between young and old mice, monocytic-derived suppressor cells from old mice were less able to suppress T-cell proliferation. Since cardiac resident macrophages (CRMs) are important for immune surveillance, clearance of dead cells, and tissue repair, we focused our studies on CRMs phenotype and function during steady state and LPS treatment. In the steady state, we observed significantly more MHC-IIlow and MHC-IIhigh CRMs in the hearts of old mice; however, these populations were decreased in both young and aged mice upon LPS treatment and the decrease in CRM populations correlated with defects in cardiac electrical activity. Notably, mice treated with a liver X receptor (LXR) agonist showed an increase in MerTK expression in CRMs of both young and old mice, which resulted in the reversal of cardiac electrical dysfunction caused by lipopolysaccharide (LPS). We conclude that aging alters the phenotype of CRMs, which contributes to the dysregulation of cardiac electrical dysfunction during infection in aged mice.
Assuntos
Envelhecimento/genética , Coração/fisiopatologia , Inflamação/fisiopatologia , Macrófagos/metabolismo , Animais , Humanos , Camundongos , FenótipoRESUMO
The immune system plays a pivotal role in the initiation, development and resolution of inflammation following insult or damage to organs. The heart is a vital organ which supplies nutrients and oxygen to all parts of the body. Heart failure (HF) has been conventionally described as a disease associated with cardiac tissue damage caused by systemic inflammation, arrhythmia and conduction defects. Cardiac inflammation and subsequent tissue damage is orchestrated by the infiltration and activation of various immune cells including neutrophils, monocytes, macrophages, eosinophils, mast cells, natural killer cells, and T and B cells into the myocardium. After tissue injury, monocytes and tissue-resident macrophages undergo marked phenotypic and functional changes, and function as key regulators of tissue repair, regeneration and fibrosis. Disturbance in resident macrophage functions such as uncontrolled production of inflammatory cytokines, growth factors and inefficient generation of an anti-inflammatory response or unsuccessful communication between macrophages and epithelial and endothelial cells and fibroblasts can lead to aberrant repair, persistent injury, and HF. Therefore, in this review, we discuss the role of cardiac macrophages on cardiac inflammation, tissue repair, regeneration and fibrosis.
Assuntos
Fibrose/metabolismo , Traumatismos Cardíacos/metabolismo , Macrófagos/metabolismo , Regeneração , Animais , Arritmias Cardíacas/imunologia , Arritmias Cardíacas/metabolismo , Citocinas/metabolismo , Cardiomiopatias Diabéticas/imunologia , Cardiomiopatias Diabéticas/metabolismo , Fibrose/imunologia , Coração/fisiopatologia , Traumatismos Cardíacos/imunologia , Homeostase , Humanos , Hipertensão/imunologia , Hipertensão/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Macrófagos/citologia , Mitocôndrias/imunologia , Miocárdio/citologia , Miocárdio/imunologiaRESUMO
Intracellular pathogens, including Mycobacterium tuberculosis, obtain iron from the host for their survival. Ferroportin 1 (FPN1; SLC40A1) is the sole iron exporter from mammalian cells and is expressed in the duodenum and macrophages. In the present study, we show that FPN1 mRNA levels in the mouse macrophage cell line RAW264.7 are synergistically induced by treatment with live or gamma-irradiated M. tuberculosis and IFN-gamma. FPN1 mRNA levels were also induced by Mycobacterium avium and IFN-gamma in RAW264.7 cells and the mouse alveolar macrophage cell line AMJ2-C8. Treatment of mouse resident peritoneal macrophages with M. tuberculosis and IFN-gamma resulted in a sixfold increase in FPN1 mRNA expression. In contrast, M. tuberculosis and IFN-gamma inhibited FPN1 mRNA expression in bone marrow-derived macrophages and lung macrophages, which have high basal levels of FPN1 mRNA expression. Using confocal microscopy, FPN1 protein localized rapidly to M. tuberculosis phagosomes after infection in RAW264.7 macrophages. In RAW264.7 cells expressing wild-type natural resistance-associated macrophage protein 1 (Nramp1(Gly169)), FPN1 and Nramp1 partially colocalized in late endosomes/lysosomes prior to infection. After 2 h of infection, Nramp1 and FPN1 were present in M. tuberculosis phagosomes. Our studies provide evidence for transcriptional regulation of FPN1 by pathogenic mycobacteria and IFN-gamma, which is dependent on the macrophage type. The trafficking of FPN1 to the M. tuberculosis phagosome suggests that it is involved in regulating iron availability to the mycobacteria in this locale.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Peritoneais/metabolismo , Mycobacterium avium/fisiologia , Mycobacterium tuberculosis/imunologia , Fagossomos/metabolismo , Tuberculose/metabolismo , Animais , Western Blotting , Medula Óssea/imunologia , Medula Óssea/microbiologia , Medula Óssea/patologia , Proteínas de Transporte de Cátions/genética , Células Cultivadas , Endossomos/imunologia , Endossomos/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Imunidade Inata , Interferon gama/farmacologia , Ferro/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Lisossomos/imunologia , Lisossomos/metabolismo , Macrófagos Alveolares/microbiologia , Macrófagos Peritoneais/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/metabolismo , Fagossomos/imunologia , Fagossomos/microbiologia , Tuberculose/imunologia , Tuberculose/patologiaRESUMO
Hepcidin is an antimicrobial peptide produced by the liver in response to inflammatory stimuli and iron overload. Hepcidin regulates iron homeostasis by mediating the degradation of the iron export protein ferroportin 1, thereby inhibiting iron absorption from the small intestine and release of iron from macrophages. Here, we examined the expression of hepcidin in macrophages infected with the intracellular pathogens Mycobacterium avium and Mycobacterium tuberculosis. Stimulation of the mouse RAW264.7 macrophage cell line and mouse bone marrow-derived macrophages with mycobacteria and IFN-gamma synergistically induced high levels of hepcidin mRNA and protein. Similar results were obtained using the human THP-1 monocytic cell line. Stimulation of macrophages with the inflammatory cytokines IL-6 and IL-beta did not induce hepcidin mRNA expression. Iron loading inhibited hepcidin mRNA expression induced by IFN-gamma and M. avium, and iron chelation increased hepcidin mRNA expression. Intracellular protein levels and secretion of hepcidin were determined by a competitive chemiluminescence ELISA. Stimulation of RAW264.7 cells with IFN-gamma and M. tuberculosis induced intracellular expression and secretion of hepcidin. Furthermore, confocal microscopy analyses showed that hepcidin localized to the mycobacteria-containing phagosomes. As hepcidin has been shown to possess direct antimicrobial activity, we investigated its activity against M. tuberculosis. We found that hepcidin inhibited M. tuberculosis growth in vitro and caused structural damage to the mycobacteria. In summary, our data show for the first time that hepcidin localizes to the phagosome of infected, IFN-gamma-activated cells and has antimycobacterial activity.
Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Imunidade Inata , Mycobacterium avium/imunologia , Mycobacterium tuberculosis/imunologia , Fagossomos/imunologia , Tuberculose/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/farmacologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Hepcidinas , Homeostase/efeitos dos fármacos , Homeostase/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon gama/farmacologia , Absorção Intestinal/imunologia , Ferro/imunologia , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Sobrecarga de Ferro/imunologia , Sobrecarga de Ferro/metabolismo , Fígado/imunologia , Fígado/metabolismo , Camundongos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fagossomos/metabolismo , Fagossomos/microbiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/imunologia , Tuberculose/metabolismoRESUMO
Despite its prominent role as a C-type lectin (CTL) pattern recognition receptor, mannose receptor (MR, CD206)-specific signaling molecules and pathways are unknown. The MR is highly expressed on human macrophages, regulating endocytosis, phagocytosis, and immune responses and mediating Mycobacterium tuberculosis (M.tb) phagocytosis by human macrophages, thereby limiting phagosome-lysosome (P-L) fusion. We identified human MR-associated proteins using phosphorylated and non-phosphorylated MR cytoplasmic tail peptides. We found that MR binds FcRγ-chain, which is required for MR plasma membrane localization and M.tb cell association. Additionally, we discovered that MR-mediated M.tb association triggers immediate MR tyrosine residue phosphorylation and Grb2 recruitment, activating the Rac/Pak/Cdc-42 signaling cascade important for M.tb uptake. MR activation subsequently recruits SHP-1 to the M.tb-containing phagosome, where its activity limits PI(3)P generation at the phagosome and M.tb P-L fusion and promotes M.tb growth. In sum, we identify human MR signaling pathways that temporally regulate phagocytosis and P-L fusion during M.tb infection.
Assuntos
Proteína Adaptadora GRB2/genética , Interações Hospedeiro-Patógeno , Lectinas Tipo C/genética , Macrófagos/metabolismo , Lectinas de Ligação a Manose/genética , Mycobacterium tuberculosis/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Receptores de Superfície Celular/genética , Receptores de IgG/genética , Proteína Adaptadora GRB2/metabolismo , Regulação da Expressão Gênica , Humanos , Lectinas Tipo C/metabolismo , Lisossomos/metabolismo , Lisossomos/microbiologia , Macrófagos/microbiologia , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Fusão de Membrana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fagocitose/genética , Fagossomos/metabolismo , Fagossomos/microbiologia , Fosforilação , Cultura Primária de Células , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de IgG/metabolismo , Transdução de Sinais , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/microbiologia , Tirosina/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Mycobacteria-infected macrophages are poor responders to interferon-gamma (IFN-gamma), resulting in decreased expression of IFN-gamma-induced genes. In the present study, we examined the inhibition of IFN-gamma-induced gene expression by Mycobacterium tuberculosis and four different Mycobacterium avium strains in mouse RAW264.7 macrophages. Gamma-irradiated M. tuberculosis inhibited mRNA expression of a panel of six different IFN- gamma-induced genes. All four of the M. avium strains completely inhibited IFN-gamma-induced expression of MHC class II Aalpha and Ebeta mRNA. However, the Mac101 strain, which is serovar 1, inhibited IFN-gamma induction of IFN regulatory factor-1 (IRF-1) and guanylate-binding protein-1 (GBP-1) mRNA to a greater extent than the other M. avium strains, which are serovar 2. In this study, we also show that mycobacteria inhibit gene expression by both toll-like receptor 2 (TLR2)-dependent and independent pathways. The inhibition of IFN-gamma-induced gene expression by M. avium was reduced but not completely blocked in macrophages from TLR2(/) mice. IFN-gamma-induced gene expression was also inhibited by mycobacteria in RAW264.7 cells expressing dominantnegative TLR2 or myeloid differentiation factor 88 (MyD88), further indicating the existence of a pathway independent of TLR2 and MyD88. These data suggest that mycobacteria inhibit IFN-gamma-induced gene expression by multiple pathways involving both TLR2 and non-TLR receptors.
Assuntos
Interferon gama/antagonistas & inibidores , Macrófagos/microbiologia , Mycobacterium avium/fisiologia , Mycobacterium tuberculosis/fisiologia , Receptor 2 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide , Fator de Transcrição STAT1/biossíntese , Transdução de Sinais , Receptor 2 Toll-Like/genéticaRESUMO
Previous studies have demonstrated that Leishmania donovani attenuates STAT1-mediated signaling in macrophages; however it is not clear whether other species of Leishmania, which cause cutaneous disease, also interfere with macrophage IFN-gamma signaling. Therefore, we determined the effect of Leishmania major and Leishmania mexicana infection on STAT1-mediated IFN-gamma signaling pathway in J774A.1 and RAW264.7 macrophages. We found that both L. major and L. mexicana suppressed IFNgammaRalpha (alpha subunit of interferon gamma receptor) and IFN-gammaRbeta (beta subunit of interferon gamma receptor) expression, reduced levels of total Jak1 and Jak2, and down-regulated IFN-gamma-induced Jak1, Jak2 and STAT1 activation. The effect of L. mexicana infection on Jak1, Jak2 and STAT1 activation was more profound when compared with L. major. Although tyrosine phosphorylation of STAT1alpha was decreased in IFN-gamma stimulated macrophages infected with L. major or L. mexicana, those infected with L. mexicana showed a significant increase in phosphorylation of the dominant negative STAT1beta. These findings indicate that L. major and L. mexicana attenuate STAT1-mediated IFN-gamma signaling in macrophages. Furthermore, they also demonstrate that L. mexicana preferentially enhances tyrosine phosphorylation of dominant negative STAT1beta, which may be one of the several survival mechanisms used by this parasite to evade the host defense mechanisms.
Assuntos
Proteínas de Ligação a DNA/imunologia , Interferon gama/biossíntese , Leishmania major/imunologia , Leishmania mexicana/imunologia , Macrófagos/imunologia , Transativadores/imunologia , Tirosina/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Humanos , Janus Quinase 1 , Janus Quinase 2 , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT1 , Transdução de Sinais/imunologia , Transativadores/metabolismo , Regulação para Cima/imunologiaRESUMO
Interferon-gamma (IFN-gamma) is a key cytokine in the immune defense against mycobacteria. IFN-gamma activates macrophages to resist the growth of mycobacteria and induces expression of MHC class II molecules required for antigen presentation. Macrophages infected with mycobacteria or stimulated by the interaction of mycobacterial products with toll-like receptor 2 (TLR2) have reduced responses to IFN-gamma. Previous research has shown that infection of mouse macrophages with Mycobacterium avium causes decreased expression of the IFN-gamma receptor (IFNGR). In the present study, we show that TLR2 stimulation of RAW264.7 macrophages with a synthetic lipoprotein, Pam3CSK4, also causes rapid decrease in expression of IFNGR-1 protein, with little change in IFNGR-2 protein levels. The decrease in IFNGR-2 expression in TLR2-stimulated cells required receptor internalization and proteasomal degradation. The level of IFNGR-1 mRNA also decreased in TLR2-stimulated RAW264.7 cells and M. avium-infected cells. The decrease in IFNGR-1 mRNA was shown to be due to decreased transcription. In spite of the decrease in IFNGR-2 receptor expression, activation of Stat1 activation by an optimal dose of IFN-gamma was identical between control and TLR2-stimulated RAW264.7 cells. However, at low suboptimal doses of IFN-gamma, Stat1 activation was decreased in TLR2-stimulated cells.
Assuntos
Macrófagos/imunologia , Glicoproteínas de Membrana/fisiologia , Mycobacterium avium/imunologia , Receptores de Superfície Celular/fisiologia , Receptores de Interferon/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Endocitose/efeitos dos fármacos , Expressão Gênica , Interferon gama/farmacologia , Lipoproteínas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Glicoproteínas de Membrana/agonistas , Camundongos , Fosforilação , Regiões Promotoras Genéticas/genética , Inibidores de Proteassoma , Estabilidade de RNA , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/agonistas , Receptores de Interferon/genética , Fator de Transcrição STAT1 , Receptor 2 Toll-Like , Receptores Toll-Like , Transativadores/metabolismo , Transativadores/farmacologia , Transativadores/fisiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Receptor de Interferon gamaRESUMO
Systemic inflammation that occurs with increasing age (inflammaging) is thought to contribute to the increased susceptibility of the elderly to several disease states. The elderly are at significant risk for developing pulmonary disorders and infectious diseases, but the contribution of inflammation in the pulmonary environment has received little attention. In this study, we demonstrate that the lungs of old mice have elevated levels of proinflammatory cytokines and a resident population of highly activated pulmonary macrophages that are refractory to further activation by IFN-γ. The impact of this inflammatory state on macrophage function was determined in vitro in response to infection with M.tb. Macrophages from the lungs of old mice secreted more proinflammatory cytokines in response to M.tb infection than similar cells from young mice and also demonstrated enhanced M.tb uptake and P-L fusion. Supplementation of mouse chow with the NSAID ibuprofen led to a reversal of lung and macrophage inflammatory signatures. These data indicate that the pulmonary environment becomes inflammatory with increasing age and that this inflammatory environment can be reversed with ibuprofen.
Assuntos
Envelhecimento/imunologia , Citocinas/biossíntese , Inflamação/imunologia , Pulmão/patologia , Macrófagos Alveolares/imunologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Genes Reporter , Ibuprofeno/farmacologia , Ibuprofeno/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/patologia , Interferon gama/farmacologia , Pulmão/crescimento & desenvolvimento , Pulmão/imunologia , Ativação de Macrófagos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Fusão de Membrana , Camundongos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Fagossomos/fisiologia , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/farmacologia , Organismos Livres de Patógenos EspecíficosRESUMO
As we age, there is an increased risk for the development of pulmonary diseases, including infections, but few studies have considered changes in lung surfactant and components of the innate immune system as contributing factors to the increased susceptibility of the elderly to succumb to infections. We and others have demonstrated that human alveolar lining fluid (ALF) components, such as surfactant protein (SP)-A, SP-D, complement protein C3, and alveolar hydrolases, play a significant innate immune role in controlling microbial infections. However, there is a lack of information regarding the effect of increasing age on the level and function of ALF components in the lung. Here we addressed this gap in knowledge by determining the levels of ALF components in the aging lung that are important in controlling infection. Our findings demonstrate that pro-inflammatory cytokines, surfactant proteins and lipids, and complement components are significantly altered in the aged lung in both mice and humans. Further, we show that the aging lung is a relatively oxidized environment. Our study provides new information on how the pulmonary environment in old age can potentially modify mucosal immune responses, thereby impacting pulmonary infections and other pulmonary diseases in the elderly population.
Assuntos
Envelhecimento/metabolismo , Pulmão/metabolismo , Estresse Oxidativo , Alvéolos Pulmonares/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Adulto , Animais , Feminino , Humanos , Hidrolases/metabolismo , Masculino , CamundongosRESUMO
Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, ß-globin, ALAS2) were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR) and transcription factors (GATA1, GATA2, FOG1) were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL), was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by cytokine-mediated apoptosis of erythroblasts.