RESUMO
BACKGROUND: The immunologic features of nontuberculous mycobacterial pulmonary disease (NTM-PD) are largely unclear. This study investigated the immunologic features of NTM-PD using digital spatial profiling techniques. METHODS: Lung tissues obtained from six patients with NTM-PD between January 1, 2006, and December 31, 2020, at Seoul National University Hospital were subjected to RNA sequencing. Cores from the peribronchial areas were stained with CD3, CD68, and DNASyto13, and gene expression at the whole-transcriptome level was quantified using PCR amplification and Illumina sequencing. Lung tissues from six patients with bronchiectasis collected during the same period were used as controls. The RNA sequencing results were validated using immunohistochemistry (IHC) in another cohort (30 patients with NTM-PD and 15 patients with bronchiectasis). RESULTS: NTM-PD exhibited distinct gene expression patterns in T cells and macrophages. Gene set enrichment analysis revealed that pathways related to antigen presentation and processing were upregulated in NTM-PD, particularly in macrophages. Macrophages were more prevalent and the expression of genes associated with the M1 phenotype (CD40 and CD80) was significantly elevated. Although macrophages were activated in the NTM-PD group T cell activity was unaltered. Notably, expression of the costimulatory molecule CD28 was decreased in NTM-PD. IHC analysis showed that T cells expressing Foxp3 or TIM-3, which facilitate the regulatory functions of T cells, were increased. CONCLUSIONS: NTM-PD exhibits distinct immunologic signatures characterized by the activation of macrophages without T cell activation.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Humanos , Masculino , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/genética , Feminino , Pessoa de Meia-Idade , Idoso , Transcriptoma , Macrófagos/imunologia , Macrófagos/metabolismo , Pulmão/microbiologia , Pulmão/imunologia , Pulmão/patologia , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/imunologia , Pneumopatias/genética , Pneumopatias/microbiologia , Pneumopatias/imunologia , Linfócitos T/imunologia , Perfilação da Expressão Gênica , Adulto , Bronquiectasia/imunologia , Bronquiectasia/genética , Bronquiectasia/microbiologiaRESUMO
BACKGROUND: The genetic signatures associated with the susceptibility to nontuberculous mycobacterial pulmonary disease (NTM-PD) are still unknown. In this study, we performed RNA sequencing to explore gene expression profiles and represent characteristic factor in NTM-PD. METHODS: Peripheral blood samples were collected from patients with NTM-PD and healthy individuals (controls). Differentially expressed genes (DEGs) were identified by RNA sequencing and subjected to functional enrichment and immune cell deconvolution analyses. RESULTS: We enrolled 48 participants, including 26 patients with NTM-PD (median age, 58.0 years; 84.6% female), and 22 healthy controls (median age, 58.5 years; 90.9% female). We identified 21 upregulated and 44 downregulated DEGs in the NTM-PD group compared to those in the control group. NTM infection did not have a significant impact on gene expression in the NTM-PD group compared to the control group, and there were no differences in the proportion of immune cells. However, through gene ontology (GO), gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) analysis, we discovered that PARK2 is a key factor associated with NTM-PD. The PARK2 gene, which is linked to the ubiquitination pathway, was downregulated in the NTM-PD group (fold change, - 1.314, P = 0.047). The expression levels of PARK2 remained unaltered after favorable treatment outcomes, suggesting that the gene is associated with host susceptibility rather than with the outcomes of infection or inflammation. The area under the receiver operating characteristic curve for the PARK2 gene diagnosing NTM-PD was 0.813 (95% confidence interval, 0.694-0.932). CONCLUSION: We identified the genetic signatures associated with NTM-PD in a cohort of Korean patients. The PARK2 gene presents as a potential susceptibility factor in NTM-PD .
Assuntos
Predisposição Genética para Doença , Infecções por Mycobacterium não Tuberculosas , Ubiquitina-Proteína Ligases , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Predisposição Genética para Doença/genética , Ubiquitina-Proteína Ligases/genética , Idoso , Pneumopatias/genética , Pneumopatias/microbiologia , Pneumopatias/diagnósticoRESUMO
Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production, and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo, we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown/knockout exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of proinflammatory factors il-1ß and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Peixe-Zebra , Animais , Humanos , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/genética , Mycobacterium marinum , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Peixe-Zebra/microbiologia , Masculino , FemininoRESUMO
Regulation of host miRNA expression is a contested node that controls the host immune response to mycobacterial infection. The host must counter subversive efforts of pathogenic mycobacteria to launch a protective immune response. Here, we examine the role of miR-126 in the zebrafish-Mycobacterium marinum infection model and identify a protective role for infection-induced miR-126 through multiple effector pathways. We identified a putative link between miR-126 and the tsc1a and cxcl12a/ccl2/ccr2 signalling axes resulting in the suppression of non-tnfa expressing macrophage accumulation at early M. marinum granulomas. Mechanistically, we found a detrimental effect of tsc1a expression that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death via mTOR inhibition. We found that macrophage recruitment driven by the cxcl12a/ccl2/ccr2 signalling axis was at the expense of the recruitment of classically activated tnfa-expressing macrophages and increased cell death around granulomas. Together, our results delineate putative pathways by which infection-induced miR-126 may shape an effective immune response to M. marinum infection in zebrafish embryos.
Assuntos
Quimiocina CXCL12 , MicroRNAs , Infecções por Mycobacterium não Tuberculosas , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas de Peixe-Zebra , Animais , Granuloma/genética , Macrófagos , MicroRNAs/genética , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Peixe-Zebra , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Quimiocina CXCL12/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Keratinocytes are the predominant cell type in the skin epidermis, and they not only protect the skin from the influence of external physical factors but also function as an immune barrier against microbial invasion. However, little is known regarding the immune defence mechanisms of keratinocytes against mycobacteria. Here, we performed single-cell RNA sequencing (scRNA-seq) on skin biopsy samples from patients with Mycobacterium marinum infection and bulk RNA sequencing (bRNA-seq) on M. marinum-infected keratinocytes in vitro. The combined analysis of scRNA-seq and bRNA-seq data revealed that several genes were upregulated in M. marinum-infected keratinocytes. Further in vitro validation of these genes by quantitative polymerase chain reaction and western blotting assay confirmed the induction of IL-32 in the immune response of keratinocytes to M. marinum infection. Immunohistochemistry also showed the high expression of IL-32 in patients' lesions. These findings suggest that IL-32 induction is a possible mechanism through which keratinocytes defend against M. marinum infection; this could provide new targets for the immunotherapy of chronic cutaneous mycobacterial infections.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium marinum , Humanos , Mycobacterium marinum/genética , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Queratinócitos , ImunidadeRESUMO
Introduction: Human pulmonary infection with non-tuberculous mycobacteria (NTM) such as Mycobacterium abscessus (Mabs) occurs in seemingly immunocompetent patients with underlying structural lung disease such as bronchiectasis in which normal ciliary function is perturbed. In addition to alterations in mucociliary clearance, the local immunologic milieu may be altered in patients with structural lung disease, but the nature of these changes and how they relate to NTM persistence remain unclear. Methods: We used a mouse strain containing a conditional floxed allele of the gene IFT88, which encodes for the protein Polaris. Deletion of this gene in adult mice reportedly leads to loss of cilia on lung airway epithelium and to the development of bronchiectasis. In a series of experiments, IFT88 control mice and IFT88 KO mice received different preparations of Mabs lung inocula with lung CFU assessed out to approximately 8 weeks post-infection. In addition, cytokine levels in bronchoalveolar lavage (BAL) fluid, lung T cell subset analysis, and lung histopathology and morphometry were performed at various time points. Results: Mabs embedded in agarose beads persisted in the lungs of IFT88 KO mice out to approximately 8 weeks (54 days), while Mabs agarose beads in the lungs of IFT88 control mice was cleared from the lungs of all mice at this time point. T cells subset analysis showed a decrease in the percentage of CD4+FoxP3+ T cells in the total lymphocyte population in the lungs of IFT88 KO mice relative to IFT88 control mice. Proinflammatory cytokines were elevated in the BAL fluid from infected IFT88 KO mice compared to infected IFT88 control mice, and histopathology showed an increased inflammatory response and greater numbers of granulomas in the lungs of infected IFT88 KO mice compared to the lungs of infected IFT88 control mice. Scanning lung morphometry did not show a significant difference comparing lung airway area and lung airway perimeter between IFT88 KO mice and IFT88 control mice. Discussion: Persistent lung infection in our model was established using Mabs embedded in agarose beads. The utility of using IFT88 mice is that a significant difference in Mabs lung CFU is observed comparing IFT88 KO mice to IFT88 control mice thus allowing for studies assessing the mechanism(s) of Mabs lung persistence. Our finding of minimal differences in lung airway area and lung airway diameter comparing IFT88 KO mice to IFT88 control mice suggests that the development of a proinflammatory lung phenotype in IFT88 KO mice contributes to Mabs lung persistence independent of bronchiectasis. The contribution of cilia to immune regulation is increasingly recognized, and our results suggest that ciliopathy associated with structural lung disease may play a role in NTM pulmonary infection via alteration of the local immunologic lung milieu.
Assuntos
Bronquiectasia , Pneumopatias , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Adulto , Humanos , Camundongos , Animais , Mycobacterium abscessus/genética , Tórax , Infecções por Mycobacterium não Tuberculosas/genética , Micobactérias não Tuberculosas , Citocinas , PulmãoRESUMO
PURPOSE: Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity. The most frequent genetic defects are found in IL12 or a subunit of its receptor. IL23R deficiency in MSMD has only been reported once, in two pediatric patients from the same kindred with isolated disseminated Bacille Calmette-Guérin disease. We evaluated the impact of a homozygous stop mutation in IL23R (R381X), identified by whole exome sequencing, in an adult patient with disseminated non-tuberculous mycobacterial disease. METHODS: We performed functional validation of the R381X mutation by evaluating IL23R expression and IL-23 signaling (STAT3 phosphorylation, IFN-γ production) in primary cells (PBMCs, EBV-B cells) and cell lines (HeLa) with or without back-complementation of wild-type IL23R. RESULTS: We report on a 48-year-old male with disseminated non-tuberculous mycobacterial disease. We identified and characterized a homozygous loss-of-function stop mutation underlying IL23R deficiency, resulting in near absent expression of membrane bound IL23R. IL23R deficiency was characterized by impaired IL-23-mediated IFN-γ secretion in CD4+, CD8+ T, and mucosal-associated invariant T (MAIT) cells, and low frequencies of circulating Th17 (CD3+CD45RA-CCR4+CXCR3-RORγT+), Th1* (CD45RA-CCR4-CXCR3+RORγT+), and MAIT (CD3+CD8+Vα7.2+CD161+) cells. Although the patient did not have a history of recurrent fungal infections, impaired Th17 differentiation and blunted IL-23-mediated IL-17 secretion in PBMCs were observed. CONCLUSION: We demonstrate that impaired IL-23 immunity caused by a homozygous R381X mutation in IL23R underlies MSMD, corroborating earlier findings with a homozygous p.C115Y IL23R mutation. Our report further supports a model of redundant contribution of IL-23- to IL-17-mediated anti-fungal immunity.1.
Assuntos
Infecções por Mycobacterium não Tuberculosas , Infecções por Mycobacterium , Masculino , Adulto , Humanos , Criança , Pessoa de Meia-Idade , Interleucina-17/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Infecções por Mycobacterium/etiologia , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/complicações , Mutação/genética , Interleucina-23 , Predisposição Genética para Doença , Receptores de Interleucina/genéticaAssuntos
Infecções por Mycobacterium não Tuberculosas , Micobactérias não Tuberculosas , Biomarcadores , Humanos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/genéticaRESUMO
Galanin is a peptide that is conserved among different species and plays various roles in an organism, although its entire role is not completely understood. For many years, galanin has been linked mainly with the neurotransmission in the nervous system; however, recent reports underline its role in immunity. Zebrafish (Danio rerio) is an intensively developing animal model to study infectious diseases. In this study, we used larval zebrafish to determine the role of galanin in bacterial infection. We showed that knockout of galanin in zebrafish leads to a higher bacterial burden and mortality during Mycobacterium marinum and Staphylococcus aureus infection, whereas administration of a galanin analogue, NAX 5055, improves the ability of fish to control the infection caused by both pathogens. Moreover, the transcriptomics data revealed that a lower number of genes were regulated in response to mycobacterial infection in gal-/- mutants compared with their gal+/+ wild-type counterparts. We also found that galanin deficiency led to significant changes in immune-related pathways, mostly connected with cytokine and chemokine functions. The results show that galanin acts not only as a neurotransmitter but is also involved in immune response to bacterial infections, demonstrating the complexity of the neuroendocrine system and its possible connection with immunity.
Assuntos
Galanina/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/patogenicidade , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Galanina/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno , Mediadores da Inflamação/metabolismo , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/metabolismo , Mycobacterium marinum/imunologia , Transdução de Sinais , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/imunologia , Transcriptoma , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Hemophagocytic lymphohistiocytosis (HLH) is a disorder of uncontrolled immune activation with distinct clinical features including fever, cytopenia, splenomegaly, and sepsis-like symptoms. In a young adolescent patient a novel germline GATA2 variant (NM_032638.5 (GATA2): c.177C>G, p.Tyr59Ter) was discovered and had resulted in non-tuberculous mycobacterial (NTM) infection and aggressive HLH. Strikingly, impaired degranulation of cytotoxic T-lymphocytes (CTL) and natural killer (NK)-cells was detected in CD107a-analyses. The affected patient was treated with HLA-matched unrelated alloHSCT, and subsequently all hematologic and infectious abnormalities including HLH and NTM resolved. This case supports early alloHSCT in GATA2 deficiencies as curative approach regardless of active NTM infection. Future studies on GATA2 c.177C>G, p.Tyr59*Ter might unravel its potential role in cytotoxic effector cell function and its contribution to HLH pathogenesis.
Assuntos
Fator de Transcrição GATA2/genética , Predisposição Genética para Doença , Variação Genética , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/genética , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/genética , Biomarcadores , Gerenciamento Clínico , Feminino , Estudos de Associação Genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunofenotipagem , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfo-Histiocitose Hemofagocítica/terapia , Masculino , Infecções por Mycobacterium não Tuberculosas/terapia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Resultado do TratamentoRESUMO
Inherited disorders of interferon gamma (IFN) γ, also known as Mendelian Susceptibility to Mycobacterial Diseases (MSMD), have been classified as Primary Immuno Deficiency 6, ie, defect in intrinsic and innate immunity. As IFN-γ plays an important role in conferring immunity to mycobacterial infections, its disorders have been increasingly reported in association with disseminated BCG/Non Tubercular Mycobacterial infections. So far germline mutations in 16 genes have been reported, most common being IL12RB1 followed by IFNGR1 and IFNGR2. There is limited published data on MSMD from India and here we report 4 unrelated children with proven mutations in IL12RB1 in 2 children and IFNGR1 and IFNGR2 in one each with disseminated opportunistic mycobacterial infections from a tertiary care centre in India.
Assuntos
Predisposição Genética para Doença , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Diagnóstico Diferencial , Feminino , Humanos , Índia , Lactente , Masculino , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/genética , Receptores de Interferon/genética , Receptores de Interleucina-12/genética , Centros de Atenção Terciária , Receptor de Interferon gamaRESUMO
Human inborn errors of IFN-γ underlie mycobacterial disease, due to insufficient IFN-γ production by lymphoid cells, impaired myeloid cell responses to this cytokine, or both. We report four patients from two unrelated kindreds with intermittent monocytosis and mycobacterial disease, including bacillus Calmette-Guérin-osis and disseminated tuberculosis, and without any known inborn error of IFN-γ. The patients are homozygous for ZNFX1 variants (p.S959* and p.E1606Rfs*10) predicted to be loss of function (pLOF). There are no subjects homozygous for pLOF variants in public databases. ZNFX1 is a conserved and broadly expressed helicase, but its biology remains largely unknown. It is thought to act as a viral double-stranded RNA sensor in mice, but these patients do not suffer from severe viral illnesses. We analyze its subcellular localization upon overexpression in A549 and HeLa cell lines and upon stimulation of THP1 and fibroblastic cell lines. We find that this cytoplasmic protein can be recruited to or even induce stress granules. The endogenous ZNFX1 protein in cell lines of the patient homozygous for the p.E1606Rfs*10 variant is truncated, whereas ZNFX1 expression is abolished in cell lines from the patients with the p.S959* variant. Lymphocyte subsets are present at normal frequencies in these patients and produce IFN-γ normally. The hematopoietic and nonhematopoietic cells of the patients tested respond normally to IFN-γ. Our results indicate that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria.
Assuntos
Antígenos de Neoplasias/genética , Leucocitose/genética , Infecções por Mycobacterium não Tuberculosas/genética , Células A549 , Adolescente , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Criança , Grânulos Citoplasmáticos/metabolismo , Feminino , Células HEK293 , Células HeLa , Homozigoto , Humanos , Lactente , Interferon gama/metabolismo , Leucocitose/patologia , Masculino , Mutação , Infecções por Mycobacterium não Tuberculosas/patologia , Linhagem , Células THP-1 , Adulto JovemRESUMO
BACKGROUND: Nontuberculous mycobacteria (NTM) infection is similar to Mycobacterium tuberculosis (MTB) infection. Early clinical identification and differentiation of NTM and MTB infections continues to be a major challenge. Nucleic acid amplification tests (NAATs) have the ability to efficiently and rapidly detect pathogens and are widely used in mycobacterial infections. The objective of this study is to estimate the diagnostic accuracy of NAATs for NTM. METHODS: We will search candidate studies that assessing the accuracy of NAATs for diagnosis of NTM through PubMed, Embase and the Cochrane Library until May 2021. Studies with full text that meet the inclusion criteria will be included. Following a revised tool for Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2), two researchers will independently evaluate the study quality. The STATA software (version 15.0) will be used to carry out meta-analyses. When heterogeneity is observed, subgroup analyses and meta-regression analyses will be used to explore sources of heterogeneity. Sensitivity analyses will be used to check the robustness of analyses. CONCLUSION: We hope that this study will provide meaningful evidence for the early and rapid diagnosis of NAATs for NTM, which will help to guide the treatment of NTM and improve the prognosis of patients.
Assuntos
DNA Bacteriano/isolamento & purificação , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA Bacteriano/genética , Testes Diagnósticos de Rotina , Humanos , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/patogenicidadeRESUMO
Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.
Assuntos
Quimiocina CXCL12/metabolismo , MicroRNAs/genética , Infiltração de Neutrófilos/imunologia , Receptores CXCR4/metabolismo , Animais , Quimiocina CXCL12/imunologia , Técnicas de Silenciamento de Genes/métodos , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/metabolismo , Receptores CXCR4/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Peixe-Zebra/imunologiaRESUMO
Infectious diseases caused by nontuberculous mycobacteria (NTM) are increasingly becoming a major global problem. Additionally, Mycobacteroides abscessus subsp. abscessus (MAB) infections are refractory to macrolides. This study was conducted to investigate the epidemiology of rapidly growing mycobacteria (RGM) species isolated from clinical specimens in Japan and assess differences in the regional distribution of lower respiratory specimens (LRS)- and non-lower respiratory specimens (NLRS)-derived species. 532 strains (427 LRS, 92 NLRS and 15 unknown specimens) were isolated in nine areas of Japan. We collected 418 specimens from Bio Medical Laboratories (BML), Inc., and 114 specimens from 45 hospitals in Japan. Their epidemiological differences were examined according to the specimen type, region, and climate. Fifteen species were identified. The proportion of M. abscessus group (MAG) strains was significantly lower in NLRS than in LRS (35.9% vs. 68.4%). The proportion of MAG strains was higher in northern Japan than in other regions (83.7% vs. 60.5%). Variations in strain abundance among RGM species was evident in regions with a mean annual temperature below 15 °C. We conclude that the proportions of MAG strains differed between NLRS and LRS in Japan. In addition, the mean annual temperature likely influenced the distribution of RGM species.
Assuntos
Clima , Variação Genética , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Humanos , Japão/epidemiologia , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Infecções por Mycobacterium não Tuberculosas/genética , Mycobacterium abscessus/genética , Mycobacterium abscessus/isolamento & purificaçãoRESUMO
RATIONALE: Nontuberculous mycobacteria (NTM) are environmental mycobacteria that can cause a chronic progressive lung disease. Although epidemiological data indicate potential genetic predisposition, its nature remains unclear. OBJECTIVES: We aimed to identify host susceptibility loci for Mycobacterium avium complex (MAC), the most common NTM pathogen. METHODS: This genome-wide association study (GWAS) was conducted in Japanese patients with pulmonary MAC and healthy controls, followed by genotyping of candidate single-nucleotide polymorphisms (SNPs) in another Japanese cohort. For verification by Korean and European ancestry, we performed SNP genotyping. RESULTS: The GWAS discovery set included 475 pulmonary MAC cases and 417 controls. Both GWAS and replication analysis of 591 pulmonary MAC cases and 718 controls revealed the strongest association with chromosome 16p21, particularly with rs109592 (p=1.64×10-13, OR 0.54), which is in an intronic region of the calcineurin-like EF-hand protein 2 (CHP2). Expression quantitative trait loci analysis demonstrated an association with lung CHP2 expression. CHP2 was expressed in the lung tissue in pulmonary MAC disease. This SNP was associated with the nodular bronchiectasis subtype. Additionally, this SNP was significantly associated with the disease in patients of Korean (p=2.18×10-12, OR 0.54) and European (p=5.12×10-03, OR 0.63) ancestry. CONCLUSIONS: We identified rs109592 in the CHP2 locus as a susceptibility marker for pulmonary MAC disease.
Assuntos
Pneumopatias , Infecções por Mycobacterium não Tuberculosas , Infecção por Mycobacterium avium-intracellulare , Estudo de Associação Genômica Ampla , Humanos , Infecções por Mycobacterium não Tuberculosas/genética , Complexo Mycobacterium avium , Micobactérias não TuberculosasRESUMO
Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells.
Assuntos
Granuloma/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/crescimento & desenvolvimento , Fator 88 de Diferenciação Mieloide/metabolismo , Tuberculose/microbiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/microbiologia , Animais , Animais Geneticamente Modificados , Carga Bacteriana , Modelos Animais de Doenças , Granuloma/genética , Granuloma/metabolismo , Granuloma/patologia , Concentração de Íons de Hidrogênio , Leucócitos/metabolismo , Leucócitos/microbiologia , Leucócitos/ultraestrutura , Lisossomos/metabolismo , Lisossomos/microbiologia , Lisossomos/ultraestrutura , Microscopia Eletrônica de Transmissão , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/metabolismo , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum/ultraestrutura , Fator 88 de Diferenciação Mieloide/genética , Transdução de Sinais , Tuberculose/genética , Tuberculose/metabolismo , Tuberculose/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Two unrelated cystic fibrosis patients were co-infected with Mycobacterium abscessus smooth and rough phenotypes. Smooth M. abscessus is proposed as the infecting form, and the subsequent loss of glycopeptidolipids in the host leads to a rough phenotype. Whole-genome sequencing (WGS) diagnosed two different M. abscessus strains in patient N°1 but only one strain in patient N°2. In patient N°1, rough isolate had novel mutations potentially involved in smooth-to-rough morphology changes. In patient N°2, four genes were present in only the smooth isolate. In addition, we obtained different susceptibility profiles in the four clinical isolates. We revealed a new paradigm describing a cystic fibrosis patient infected with two different clones, including a rough isolate, and identifying a rough M. abscessus clone that did not lose glycopeptidolipids. We propose WGS for the identification of heterogenic isolates and genetic determinants of antimicrobial resistance, which we believe will positively influence treatment prognosis.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/genética , Mycobacterium abscessus/genética , Polimorfismo de Nucleotídeo Único , Adulto , Fibrose Cística/microbiologia , Genótipo , Humanos , FenótipoRESUMO
Infection with rapidly growing nontuberculous mycobacteria is emerging as a global health issue; however, key host factors remain elusive. Here, we investigated the characteristic immune profiles of peripheral blood mononuclear cells (PBMCs) from patients infected with Mycobacteroides abscessus subsp. abscessus (Mabc) and M. abscessus subsp. massiliense (Mmass). Using an integrated analysis of global mRNA and microRNA expression profiles, we found that several inflammatory cytokines/chemokines [interleukin (IL)-1ß, IL-6, C-X-C motif chemokine ligand 2, and C-C motif chemokine ligand 2] and miR-144-3p were significantly upregulated in PBMCs from patients compared with those from healthy controls (HCs). Notably, there was a strong correlation between the expression levels of miR-144-3p and proinflammatory cytokines/chemokines. Similarly, upregulated expression of miR-144-3p and proinflammatory cytokines/chemokines was found in macrophages and lungs from mice after infection with Mabc and Mmass. We showed that the expression of negative regulators of inflammation (SARM1 and TNIP3) was significantly downregulated in PBMCs from the patients, although they were not putative targets of miR-144-3p. Furthermore, overexpression of miR-144-3p led to a marked increase in proinflammatory cytokines/chemokines and promoted bacterial growth in macrophages. Together, our results highlight the importance of miR-144-3p linking to pathological inflammation during M. abscessus infection.
Assuntos
MicroRNAs , Infecções por Mycobacterium não Tuberculosas , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Células Cultivadas , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Mycobacterium abscessus/patogenicidade , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologiaRESUMO
Mycobacterium abscessus (Mab) is an emerging, nontuberculosis mycobacterium (NTM) that infects humans. Mab has two morphotypes, smooth (S) and rough (R), related to the production of glycopeptidolipid (GPL), that differ in pathogenesis. To further understand the pathogenicity of these morphotypes in vivo, the amphibian Xenopus laevis was used as an alternative animal model. Mab infections have been previously modeled in zebrafish embryos and mice, but Mab are cleared early from immunocompetent mice, preventing the study of chronic infection, and the zebrafish model cannot be used to model a pulmonary infection and T cell involvement. Here, we show that X. laevis tadpoles, which have lungs and T cells, can be used as a complementary model for persistent Mab infection and pathogenesis. Intraperitoneal (IP) inoculation of S and R Mab morphotypes disseminated to tadpole tissues including liver and lungs, persisting for up to 40 days without significant mortality. Furthermore, the R morphotype was more persistent, maintaining a higher bacterial load at 40 days postinoculation. In contrast, the intracardiac (IC) inoculation with S Mab induced significantly greater mortality than inoculation with the R Mab form. These data suggest that X. laevis tadpoles can serve as a useful comparative experimental organism to investigate pathogenesis and host resistance to M. abscessus.