RESUMO
Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.
Assuntos
Quimiocinas/metabolismo , Hanseníase/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Transcriptoma , Células Cultivadas , Quimiocinas/genética , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno , Humanos , Hanseníase/imunologia , Hanseníase/microbiologia , Masculino , Mitocôndrias/microbiologia , Mycobacterium bovis/imunologia , Mycobacterium leprae/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Nervos Periféricos/metabolismo , Células de Schwann/imunologia , Células de Schwann/metabolismo , Células de Schwann/microbiologiaRESUMO
Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.
Assuntos
Hanseníase/genética , Mycobacterium leprae/imunologia , Polimorfismo de Nucleotídeo Único/genética , Receptor 1 Toll-Like/genética , Adulto , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Frequência do Gene/genética , Genótipo , Haplótipos , Heterozigoto , Humanos , Imunidade/genética , Hanseníase/imunologia , Leucócitos Mononucleares/imunologia , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/fisiologia , Fatores de Risco , Receptor 1 Toll-Like/fisiologiaRESUMO
Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.
Assuntos
Humanos , Feminino , Adulto , Pessoa de Meia-Idade , Haplótipos , Ensaio de Imunoadsorção Enzimática , Leucócitos Mononucleares/imunologia , Estudos de Casos e Controles , Fatores de Risco , Polimorfismo de Nucleotídeo Único/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Receptor 1 Toll-Like/fisiologia , Receptor 1 Toll-Like/genética , Citometria de Fluxo , Frequência do Gene/genética , Genótipo , Heterozigoto , Imunidade/genética , Hanseníase/genética , Hanseníase/imunologia , Mycobacterium leprae/imunologiaRESUMO
Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.