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Nontuberculous mycobacteria (NTM) infection is common in patients with structural lung damage. To address how NTM infection is established and causes lung damage, we established an NTM mouse model by intranasal inoculation of clinical isolates of M. intracellulare. During the 39-week course of infection, the bacteria persistently grew in the lung and caused progressive granulomatous and fibrotic lung damage with mortality exceeding 50%. Lung neutrophils were significantly increased at 1 week postinfection, reduced at 2 weeks postinfection and increased again at 39 weeks postinfection. IL-17A was increased in the lungs at 1-2 weeks of infection and reduced at 3 weeks postinfection. Depletion of neutrophils during early (0-2 weeks) and late (32-34 weeks) infection had no effect on mortality or lung damage in chronically infected mice. However, neutralization of IL-17A during early infection significantly reduced bacterial burden, fibrotic lung damage, and mortality in chronically infected mice. Since it is known that IL-17A regulates matrix metalloproteinases (MMPs) and that MMPs contribute to the pathogenesis of pulmonary fibrosis, we determined the levels of MMPs in the lungs of M. intracellulare-infected mice. Interestingly, MMP-3 was significantly reduced by anti-IL-17A neutralizing antibody. Moreover, in vitro data showed that exogenous IL-17A exaggerated the production of MMP-3 by lung epithelial cells upon M. intracellulare infection. Collectively, our findings suggest that early IL-17A production precedes and promotes organized pulmonary M. intracellulare infection in mice, at least in part through MMP-3 production.
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Infecção por Mycobacterium avium-intracellulare , Animais , Humanos , Interleucina-17 , Pulmão , Metaloproteinase 3 da Matriz , Camundongos , Infecção por Mycobacterium avium-intracellulare/microbiologia , Infecção por Mycobacterium avium-intracellulare/patologiaRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1008140.].
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Previously, we found that pathological immune responses enhance the mortality rate of Mycobacterium tuberculosis (Mtb)-infected mice with type 2 diabetes mellitus (T2DM). In the current study, we evaluated the role of the cytokine IL-22 (known to play a protective role in bacterial infections) and type 3 innate lymphoid cells (ILC3s) in regulating inflammation and mortality in Mtb-infected T2DM mice. IL-22 levels were significantly lower in Mtb-infected T2DM mice than in nondiabetic Mtb-infected mice. Similarly, serum IL-22 levels were significantly lower in tuberculosis (TB) patients with T2DM than in TB patients without T2DM. ILC3s were an important source of IL-22 in mice infected with Mtb, and recombinant IL-22 treatment or adoptive transfer of ILC3s prolonged the survival of Mtb-infected T2DM mice. Recombinant IL-22 treatment reduced serum insulin levels and improved lipid metabolism. Recombinant IL-22 treatment or ILC3 transfer prevented neutrophil accumulation near alveoli, inhibited neutrophil elastase 2 (ELA2) production and prevented epithelial cell damage, identifying a novel mechanism for IL-22 and ILC3-mediated inhibition of inflammation in T2DM mice infected with an intracellular pathogen. Our findings suggest that the IL-22 pathway may be a novel target for therapeutic intervention in T2DM patients with active TB disease.
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Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/microbiologia , Interleucinas/imunologia , Linfócitos/imunologia , Tuberculose/imunologia , Animais , Diabetes Mellitus Tipo 2/complicações , Humanos , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Tuberculose/complicações , Interleucina 22RESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma, which are mostly seen in immunocompromised patients, such as human immunodefeciency virus (HIV)+ individuals. Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), remains one of the deadliest infectious diseases in the world. The risk of developing TB is dramatically higher in people living with HIV than among those without HIV infection. Case reports link cutaneous or pulmonary KS in HIV+ patients with mycobacterial co-infections, however, impacts of Mtb infection or its products on KSHV-infected cells are not known. We report here that ESAT-6, a secreted Mtb virulence factor, induces viral reactivation from KSHV-infected cells. KSHV-infected pulmonary endothelial cells were resistant to ESAT-6 induced inhibition of cell growth. Our data demonstrate that Mtb virulence factors influence the biology of KSHV-infected cells, highlighting the need to study the interactions between these two pathogens commonly found in people living with HIV.
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Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Herpesvirus Humano 8/fisiologia , Mycobacterium tuberculosis/genética , Sarcoma de Kaposi/virologia , Ativação Viral , Antígenos de Bactérias/farmacologia , Proteínas de Bactérias/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/microbiologia , Células Endoteliais/virologia , Regulação Viral da Expressão Gênica , Humanos , Pulmão/citologia , Mycobacterium tuberculosis/patogenicidade , Fatores de Virulência , Replicação ViralRESUMO
In the current study, we used a mouse model and human blood samples to determine the effects of chronic alcohol consumption on immune responses during Mycobacterium tuberculosis (Mtb) infection. Alcohol increased the mortality of young mice but not old mice with Mtb infection. CD11b+Ly6G+ cells are the major source of IFN-α in the lungs of Mtb-infected alcohol-fed young mice, and IFN-α enhances macrophage necroptosis in the lungs. Treatment with an anti-IFNAR-1 antibody enhanced the survival of Mtb-infected alcohol-fed young mice. In response to Mtb, peripheral blood mononuclear cells (PBMCs) from alcoholic young healthy individuals with latent tuberculosis infection (LTBI) produced significantly higher amounts of IFN-α than those from non-alcoholic young healthy LTBI+ individuals and alcoholic and non-alcoholic old healthy LTBI+ individuals. Our study demonstrates that alcohol enhances IFN-α production by CD11b+Ly6G+ cells in the lungs of young Mtb-infected mice, which leads to macrophage necroptosis and increased mortality. Our findings also suggest that young alcoholic LTBI+ individuals have a higher risk of developing active TB infection.
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Consumo de Bebidas Alcoólicas/imunologia , Interferon-alfa/biossíntese , Interferon-alfa/efeitos dos fármacos , Tuberculose/imunologia , Adulto , Animais , Suscetibilidade a Doenças/imunologia , Feminino , Humanos , Interferon-alfa/imunologia , Tuberculose Latente/imunologia , Masculino , Camundongos , Mycobacterium tuberculosisRESUMO
BACKGROUND/AIMS: The nontuberculous mycobacterial lung disease (NTM), caused by Mycobacterium avium complex (MAC) is an increasing health problem in the USA and worldwide. The NTM disease is prevalent in Caucasian women with a current diagnosis or history of breast cancer (BCa), posing a significant challenge towards treatment. We hypothesize that NTM affected women with considerable therapeutic resistance may harbor pathogenic microbes other than nontuberculous mycobacterium, aiding in disease progression and therapeutic resistance. METHODS: We assessed microbiome diversity in sputa from healthy women, women with nontuberculous mycobacterial lung disease (NTM) and women with both nontuberculous mycobacterial lung disease and breast cancer (NTM-BCa). First, we collected sputa and isolated DNA from sputa of these healthy women and women with NTM and NTM-BCa. We also isolated DNA from sera derived extracellular vesicles from women with NTM-BCa. To identify diverse pathogenic microbes in various groups of subjects, we then performed 16S rDNA sequencing. Data analysis was performed utilizing the analytical pipelines at the Center for Metagenomic and Microbiome Research (CMMR), Baylor College of Medicine. RESULTS: A large community of resident microbes, including bacteria, virus, Archeas and Fungi live in the human body are being increasingly recognized as the key components of human health and disease. We identified a diverse microbiome community in the sputa and the extracellular vesicles dominated by Streptococcus, Haemophillus, Veillonella, Neisseria, Prevotella, Fusobacterium, Bacteroides, Allistipes, Faecalibacterium and Staphylococcus in women with nontuberculous mycobacterial lung disease as well as women with both nontuberculous mycobacterial lung disease and breast cancer. Some of these genera, including Fusobacterium, Bacteroides, and Allistipes have estrobolome activity and associated with breast and other neoplasms. CONCLUSION: This work confirms the presence of a distinct pathogenic microbiome other than nontuberculous mycobacteria in the sputa and the circulating extracellular vesicles of these patients. This information could be useful for better therapeutic design to treat the NTM patients.
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Bactérias , Neoplasias da Mama/microbiologia , Pneumopatias/microbiologia , Microbiota , Infecções por Mycobacterium não Tuberculosas/microbiologia , Escarro/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bactérias/classificação , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Cyclic adenosine monophosphate (cAMP) is critical in immune regulation, and its role in tuberculosis infection remains unclear. We determined the levels of cAMP in peripheral blood mononuclear cells (PBMC) from tuberculosis patients and the mechanisms for cAMP suppression of IFN-γ production. PBMC from tuberculosis patients contained significantly elevated cAMP than latent tuberculosis infected subjects (LTBI), with an inverse correlation with IFN-γ production. Consistent with this, the expression of cAMP response element binding protein (CREB), activating transcription factor (ATF)-2 and c-Jun were reduced in tuberculosis patients compared with LTBI. PKA type I specific cAMP analogs inhibited Mtb-stimulated IFN-g production by PBMC through suppression of Mtb-induced IFN-γ promoter binding activities of CREB, ATF-2, and c-Jun and also miR155, the target miRNA of these transcription factors. Neutralizing both IL-10 and TGF-ß1 or supplementation of IL-12 restored cAMP-suppressed IFN-g production. We conclude that increased cAMP inhibits IFN-g production through PKA type I pathway in tuberculosis infection.
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Proteína Quinase Tipo I Dependente de AMP Cíclico/imunologia , AMP Cíclico/imunologia , Interferon gama/imunologia , Tuberculose Latente/imunologia , Mycobacterium tuberculosis/imunologia , Linfócitos T/imunologia , Fator 2 Ativador da Transcrição/imunologia , Antígenos de Bactérias/imunologia , Humanos , Interleucina-10/imunologia , Leucócitos Mononucleares/imunologia , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/imunologia , Transdução de Sinais/imunologiaRESUMO
BACKGROUND: Persistant inflammatory responses to infectious agents and other components in organic dust underlie lung injury and development of respiratory diseases. Organic dust components responsible for eliciting inflammation and the mechanisms by which they cause lung inflammation are not fully understood. We studied the mechanisms by which protease activities in poultry dust extracts and intracellular oxidant stress induce inflammatory gene expression in A549 and Beas2B lung epithelial cells. METHODS: The effects of dust extracts on inflammatory gene expression were analyzed by quantitative polymerase chain reaction (qPCR), enzyme linked immunosorbent (ELISA) and western blot assays. Oxidant stress was probed by dihydroethidium (DHE) labeling, and immunostaining for 4-hydroxynonenal (4-HNE). Effects on interleukin-8 (IL-8) promoter regulation were determined by transient transfection assay. RESULTS: Dust extracts contained trypsin and elastase activities, and activated protease activated receptor (PAR)-1 and -2. Serine protease inhibitors and PAR-1 or PAR-2 knockdown suppressed inflammatory gene induction. Dust extract induction of IL-8 gene expression was associated with increased DHE-fluorescence and 4-HNE staining, and antioxidants suppressed inflammatory gene induction. Protease inhibitors and antioxidants suppressed protein kinase C and NF-κB activation and induction of IL-8 promoter activity in cells exposed to dust extract. CONCLUSIONS: Our studies demonstrate that proteases and intracellular oxidants control organic dust induction of inflammatory gene expression in lung epithelial cells. Targeting proteases and oxidant stress may serve as novel approaches for the treatment of organic dust induced lung diseases. This is the first report on the involvement of oxidant stress in the induction of inflammatory gene expression by organic dust.
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Poeira , Células Epiteliais/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Pulmão/efeitos dos fármacos , Compostos Orgânicos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Pneumonia/induzido quimicamente , Células A549 , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Células Epiteliais/enzimologia , Regulação da Expressão Gênica , Abrigo para Animais , Humanos , Exposição por Inalação/efeitos adversos , Interleucina-8/genética , Interleucina-8/metabolismo , Pulmão/enzimologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Pneumonia/enzimologia , Pneumonia/genética , Pneumonia/prevenção & controle , Aves Domésticas , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Inibidores de Serina Proteinase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , TransfecçãoRESUMO
The regulatory mechanisms that control the ESX-1 secretion system, a key player in the pathogenesis of Mycobacterium tuberculosis, have not been fully elucidated. However, factors that regulate the ESX-1 substrate EspA usually affect ESX-1 function. Previous studies showed that espA is directly regulated by the nucleoid-associated protein EspR and the two-component system (TCS) MprAB. The PhoPR TCS also activates espA, but the direct target of PhoP was unknown. In this report, we reveal that EspR is directly regulated by MprA and PhoP-Rv, but not by PhoP-Ra. PhoP-Rv and MprA binding sites in the espR promoter were determined by gel-shift and DNase I footprinting assays, which identified a PhoP-protected region centred approximately 205 bp before the espR start codon and that encompasses MprA Region-1, one of two MprA-protected regions. MprA Region-2 is located approximately 60 bp downstream of MprA Region-1 and overlaps a known EspR binding site. Nucleotides essential for the binding of PhoP and/or MprA were identified through site-directed DNA mutagenesis. Our studies also indicate that MprA Region-2, but not MprA Region-1/PhoP region, is required for the full expression of espR. Recombinant strains carrying mutations at MprA Region-2 exhibited lower transcription levels for espR, espA and espD, and had reduced EspR and EspA levels in cell lysates. These findings indicate that EspR may mediate the regulatory effect of PhoPR and MprAB, and provide more insight into the mechanisms underlying ESX-1 control.
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Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/metabolismo , Proteínas Quinases/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Mycobacterium tuberculosis/genética , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Quinases/genética , Transcrição GênicaRESUMO
BACKGROUND: In our study, we investigated whether circulating T follicular helper (Tfh) and the related cytokines are involved in human cystic echinococcosis (CE). METHODS: A total of 64 patients with CE and 30 healthy controls were enrolled in this study. Percentages of CCR7(lo)PD-1(hi) cells within CXCR5(+) CD4(+) T cells (circulating Tfh cells) were detected by flow cytometry. Levels of IL-21 and IL-4 in peripheral blood were detected by cytometric bead array. The mRNA expression of IL-21, IL-4, Bcl-6, and Blimp-1 in peripheral blood mononuclear cells (PBMCs) were measured by real-time PCR. Levels of IgG1, IgG2, IgG3, and IgG4 in the patients' sera were measured using enzyme-linked immunosorbent assay. RESULTS: Percentages of circulating Tfh cells were significantly increased in the CE1, CE2, and CE3 groups (p < 0.05). The concentrations of IL-21 and IL-4 in the serum were significantly increased in CE1, CE2, and CE3 groups (p < 0.05). IL-21 was positively correlated with circulating Tfh cells in CE3 group (r = 0.779, p < 0.05). The mRNA levels of IL-21, IL-4, and Bcl-6 were increased in CE1, CE2, and CE3 groups. Levels of IgG1 and IgG4 in patients' sera were increased in CE1, CE2, and CE3 groups. Levels of IgG2 and IgG3 were increased in CE4-5 group. Additionally, after stimulation with hydatid fluid in vitro, the levels of circulating Tfh cells, IL-21 and IL-4 in PBMCs isolated from CE patients were significantly increased (p < 0.05). CONCLUSIONS: The levels of circulating Tfh and related cytokines were significantly increased in CE patients, suggesting that they are involved in human CE.
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Linfócitos T CD4-Positivos/imunologia , Equinococose/imunologia , Interleucinas/sangue , Receptores CXCR5/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Estudos de Casos e Controles , Citocinas/sangue , Proteínas de Ligação a DNA/sangue , Proteínas de Ligação a DNA/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Interleucina-4/sangue , Interleucina-4/genética , Interleucina-4/imunologia , Interleucinas/genética , Leucócitos Mononucleares/imunologia , Masculino , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-6 , Reação em Cadeia da Polimerase em Tempo Real , Receptores CCR7/metabolismo , Proteínas Repressoras/sangue , Proteínas Repressoras/genética , Adulto JovemRESUMO
Early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis is critical for the virulence and pathogenicity of M. tuberculosis. IL-8, a major chemotactic cytokine for neutrophils and T lymphocytes, plays important roles in the development of lung injury. To further understand the role of ESAT-6 in lung pathology associated with tuberculosis development, we studied the effects of ESAT-6 on the regulation of IL-8 expression in lung epithelial cells. ESAT-6 induced IL-8 expression by increasing IL-8 gene transcription and mRNA stability. ESAT-6 induction of IL-8 promoter activity was dependent on nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) binding and sensitive to pharmacological inhibition of PKC and ERK and p38 MAPK pathways. ESAT-6 activated ERK and p38 MAPK phosphorylation and rapidly induced reactive oxygen species (ROS) production. Dimethylthiourea but not mannitol inhibited IL-8 induction by ESAT-6, further supporting the involvement of ROS in the induction of IL-8 expression. Exposure of mice to ESAT-6 induced localized inflammatory cell aggregate formation with characteristics of early granuloma concomitant with increased keratinocyte chemoattractant CXCL1 staining in bronchiolar and alveolar type II epithelial cells and alveolar macrophages. Our studies have identified a signal transduction pathway involving ROS, PKC, ERK, and p38 MAPKs and NF-κB and AP-1 in the ESAT-6 induction of IL-8 expression in lung epithelial cells. This has important implications for the understanding of lung innate immune responses to tuberculosis and the pathogenesis of lung injury in tuberculosis.
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Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Interleucina-8/biossíntese , Pulmão/metabolismo , Sistema de Sinalização das MAP Quinases , Mycobacterium tuberculosis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/metabolismo , Tuberculose Pulmonar/metabolismo , Animais , Antígenos de Bactérias/farmacologia , Proteínas de Bactérias/farmacologia , Linhagem Celular Tumoral , Células Epiteliais/patologia , Humanos , Interleucina-8/genética , Pulmão/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Mucosa Respiratória/patologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/genética , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/patologiaRESUMO
Early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis is a T cell Ag that is a potential vaccine candidate, but it is also a virulence factor that mediates pathogenicity. To better understand the effects of ESAT-6 on the immune response, we studied the effect of ESAT-6 on human dendritic cells (DCs). Peripheral blood monocytes were treated with GM-CSF and IL-4 to yield immature DCs, which were matured by addition of LPS and CD40 ligand (CD40L), with or without ESAT-6. ESAT-6 inhibited LPS/CD40L-induced DC expression of costimulatory molecules, reduced DC-stimulated allogeneic T cell proliferation and IL-2 and IFN-γ production, and enhanced IL-17 production. ESAT-6-treated DCs also increased IL-17 and reduced IFN-γ production by M. tuberculosis-specific autologous T cells. ESAT-6 inhibited LPS/CD40L-induced DC production of IL-12 and enhanced that of IL-23 and IL-1ß, without affecting secretion of TNF-α, IL-6, or IL-8 through specific interaction with immature DCs. The effects of ESAT-6 were not mediated through cAMP or p38 MAPK. Medium from ESAT-6-conditioned DCs increased IL-17 and reduced IFN-γ production by T cells stimulated with anti-CD3 plus anti-CD28, and ESAT-6-induced IL-17 production was blocked by neutralizing both IL-23 and IL-1ß. ESAT-6 reduced LPS/CD40L-stimulated transcription of IL-12p35 and enhanced that of IL-23p19 through inhibition of IFN regulatory factor-1 and upregulation of activating transcription factor-2 and c-Jun, transcriptional regulators of IL-12p35 and IL-23p19, respectively. We conclude that ESAT-6 increases DC production of IL-23 and IL-1ß while inhibiting that of IL-12, thus enhancing Th17 at the expense of protective Th1 responses.
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Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/fisiologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Mycobacterium tuberculosis/imunologia , Células Th1/imunologia , Células Th17/imunologia , Proteínas de Bactérias/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Regulação para Baixo/imunologia , Humanos , Células Th1/metabolismo , Células Th1/microbiologia , Células Th17/metabolismo , Células Th17/microbiologia , Regulação para Cima/imunologiaRESUMO
Immune control of Mycobacterium tuberculosis depends on interferon γ (IFN-γ)-producing CD4(+) lymphocytes. Previous studies have shown that T cells from patients with tuberculosis produce less IFN-γ, compared with healthy donors, in response to mycobacterial antigens, although IFN-γ responses to mitogens are preserved. In this work, we found that M. tuberculosis-induced IFN-γ production by human T cells correlated with phosphorylation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38. Moreover, the majority of IFN-γ-producing T cells expressed signaling lymphocyte activation molecule (SLAM), and SLAM activation further increased ERK phosphorylation. Interestingly, patients with tuberculosis had delayed activation of ERK and p38, and this was most marked in patients with the poorest IFN-γ responses (ie, low responders). Besides, SLAM signaling failed to phosphorylate ERK in low responders. Our findings suggest that activation of p38 and ERK, in part through SLAM, mediates T-cell IFN-γ production in response to M. tuberculosis, a pathway that is defective in patients with tuberculosis.
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MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interferon gama/biossíntese , Mycobacterium tuberculosis/imunologia , Linfócitos T/imunologia , Tuberculose Pulmonar/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antígenos CD/metabolismo , Ativação Enzimática , Humanos , Fosforilação , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologiaRESUMO
The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb-infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.
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Mycobacterium tuberculosis , Terapia por Fagos , Tuberculose , Animais , Camundongos , Humanos , Tuberculose/terapia , Tuberculose/microbiologia , Macrófagos/microbiologiaRESUMO
The ESX-1 secretion system exports the immunomodulatory protein ESAT-6 and other proteins important in the pathogenesis of Mycobacterium tuberculosis. Components and substrates of ESX-1 are encoded at several loci, but the regulation of the encoding genes is only partially understood. In this study, we investigated the role of the MprAB two-component system in the regulation of ESX-1 activity. We determined that MprAB directly regulates the espA gene cluster, a locus necessary for ESX-1 function. Transcript mapping determined that the five genes in the cluster form an operon with two transcriptional start points, and several MprA binding sites were detected in the espA promoter. Expression analyses and promoter constructs indicated that MprAB represses the espA operon. However, the MprAB mutant Rv-D981 secreted lower levels of EspA, ESAT-6, and the ESX-1 substrate EspB than control strains. Secretion of CFP10, which is normally cosecreted with ESAT-6, was similar in Rv-D981 and control strains, further demonstrating aberrant ESX-1 activity in the mutant. ESAT-6 induces proinflammatory cytokines, and macrophages infected with Rv-D981 elicited lower levels of interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α), consistent with the reduced levels of ESAT-6. These findings indicate that MprAB modulates ESX-1 function and reveal a new role for MprAB in host-pathogen interactions.
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Proteínas de Bactérias/metabolismo , Citocinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Quinases/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Células Cultivadas , Citocinas/genética , Proteínas de Homeodomínio/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Macrófagos/metabolismo , Família Multigênica/fisiologia , Mutação , Mycobacterium tuberculosis/genética , Óperon/fisiologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Quinases/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we tested three bacteriophage strains for their Mtb-killing activities and found that two of them efficiently lysed Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently killed H37Rv in liquid culture and in Mtb-infected human primary macrophages. In subsequent experiments, we infected humanized mice with aerosolized H37Rv, then treated these mice with DS6A intravenously to test its in vivo efficacy. We found that DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduced Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrated the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.
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Background: In patients with severe acute respiratory distress syndrome (ARDS) associated with sepsis, lung recovery is considerably delayed, and mortality is much high. More insight into the process of lung regeneration in ARDS patients is needed. Exosomes are important cargos for intercellular communication by serving as autocrine and/or paracrine. Cutting-edge exomics (exosomal proteomics) makes it possible to study the mechanisms of re-alveolarization in ARDS lungs. Aims: This study aimed to identify potential regenerative niches by characterizing differentially expressed proteins in the exosomes of bronchioalveolar lavage (BAL) in ARDS patients. Methods: We purified exosomes from BAL samples collected from ARDS patients by NIH-supported ALTA and SPIROMICS trials. The abundance of exosomal proteins/peptides was quantified using liquid chromatography-mass spectrometry (LC-MS). Differentially expressed exosomal proteins between healthy controls and ARDS patients were profiled for functional annotations, cell origins, signaling pathways, networks, and clinical correlations. Results: Our results show that more exosomal proteins were identified in the lungs of late-stage ARDS patients. Immune cells and lung epithelial stem cells were major contributors to BAL exosomes in addition to those from other organs. We enriched a wide range of functions, stem cell signals, growth factors, and immune niches in both mild and severe patients. The differentially expressed proteins that we identified were associated with key clinical variables. The severity-associated differences in protein-protein interaction, RNA crosstalk, and epigenetic network were observed between mild and severe groups. Moreover, alveolar type 2 epithelial cells could serve as both exosome donors and recipients via autocrine and paracrine mechanisms. Conclusions: This study identifies novel exosomal proteins associated with diverse functions, signaling pathways, and cell origins in ARDS lavage samples. These differentiated proteins may serve as regenerative niches for re-alveolarization in injured lungs.
RESUMO
Protein tyrosine phosphorylation is a fundamental mechanism for diverse physiological processes, which is regulated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs). In this study, we searched for protein substrates of PTP-MEG2 (also called PTPN9), a nonreceptor PTP, and investigated its function in endothelial cells (ECs). By using a PTP-MEG2 substrate-trapping DA mutant, we found that a couple of tyrosine-phosphorylated proteins were associated with the DA mutant but not wild-type PTP-MEG2 and that the association was enhanced by vascular endothelial growth factor (VEGF) in ECs. We further found that VEGF receptor 2 (VEGFR2) was coimmunopricipitated with the DA mutant but not wild-type PTP-MEG2. The VEGF-induced phosphorylation of VEGFR2 on Tyr1175, a critical autophosphorylation site for VEGFR2 signaling, was inhibited 70% by overexpression of wild-type PTP-MEG2 but was enhanced (2.2-fold) by the DA mutant of PTP-MEG2. We also found that PTP-MEG2 DA mutant preferentially associated with Janus kinase 1 (JAK1) but not with other JAK kinases (Tyk2 and JAK2) present in ECs and regulated JAK1 tyrosine phosphorylation. Lastly, the VEGF-induced signal transduction and the production of interleukin (IL)-6 were significantly enhanced by PTP-MEG2 knockdown in ECs, whereas the VEGF-induced IL-6 production was inhibited 50% by PTP-MEG2 overexpression. Thus we have indentified VEGFR2 as a PTP-MEG2 substrate, and our findings indicate that PTP-MEG2 is a negative regulator of VEGFR2 signaling and function in ECs.
Assuntos
Células Endoteliais/fisiologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Humanos , Proteínas Tirosina Fosfatases não Receptoras/genética , Interferência de RNA , Proteínas Recombinantes , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
We reported previously that the early secreted antigenic target of 6 kDa (ESAT-6) from Mycobacterium tuberculosis directly inhibits human T cell IFN-γ production and proliferation in response to stimulation with anti-CD3 and anti-CD28. To determine the mechanism of this effect, we treated T cells with kinase inhibitors before stimulation with ESAT-6. Only the p38 MAPK inhibitor, SB203580, abrogated ESAT-6-mediated inhibition of IFN-γ production in a dose-dependent manner. SB203580 did not reverse ESAT-6-mediated inhibition of IL-17 and IL-10 production, suggesting a specific effect of SB203580 on IFN-γ production. SB203580 did not act through inhibition of AKT (PKB) as an AKT inhibitor did not affect ESAT-6 inhibition of T cell IFN-γ production and proliferation. ESAT-6 did not reduce IFN-γ production by expanding FoxP3(+) T regulatory cells. Incubation of T cells with ESAT-6 induced phosphorylation and increased functional p38 MAPK activity, but not activation of ERK or JNK. Incubation of peripheral blood mononuclear cells with ESAT-6 induced activation of p38 MAPK, and inhibition of p38 MAPK with SB203580 reversed ESAT-6 inhibition of M. tuberculosis-stimulated IFN-γ production by peripheral blood mononuclear cells from subjects with latent tuberculosis infection. Silencing of p38α MAPK with siRNA rendered T cells resistant to ESAT-6 inhibition of IFN-γ production. Taken together, our results demonstrate that ESAT-6 inhibits T cell IFN-γ production in a p38 MAPK-dependent manner.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Interferon gama/biossíntese , Tuberculose Latente/metabolismo , Mycobacterium tuberculosis/metabolismo , Linfócitos T/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Humanos , Imidazóis/farmacologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Tuberculose Latente/genética , Tuberculose Latente/imunologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , MAP Quinase Quinase 4/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Linfócitos T/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Histone deacetylases (HDACs) are critical immune regulators. However, their roles in interleukin-1ß (IL-1ß) production remain unclear. By screening 11 zinc-dependent HDACs with chemical inhibitors, we found that HDAC1 inhibitor, 4-(dimethylamino)-N-[6-(hydroxyamino)-6-oxohexyl]-benzamide (DHOB), enhanced IL-1ß production by macrophage and dendritic cells upon TLR4 stimulation or Mycobacterium tuberculosis infection through IL-1ß maturation via elevated NLRP3 expression, increased cleaved caspase-1, and enhanced ASC oligomerization. DHOB rescued defective IL-1ß production by dendritic cells infected with M. tuberculosis with ESAT-6 deletion, a virulence factor shown to activate NLRP3 inflammasome. DHOB increased IL-1ß production and NLRP3 expression in a tuberculosis mouse model. Although DHOB inhibited HDAC activities of both HDAC1 and HDAC2 by direct binding, knockdown of HDAC2, but not HDAC1, increased IL-1ß production and NLRP3 expression in M. tuberculosis-infected macrophages. These data suggest that HDAC2, but not HDAC1, controls IL-1ß production through NLRP3 inflammasome activation, a mechanism with a significance in chronic inflammatory diseases including tuberculosis.