RESUMO
Early diagnosis of leprosy is challenging, particularly its inflammatory reactions, the major cause of irreversible neuropathy in leprosy. Current diagnostics cannot identify which patients are at risk of developing reactions. This study assessed blood RNA expression levels as potential biomarkers for leprosy. Prospective cohorts of newly diagnosed leprosy patients, including reactions, and healthy controls were recruited in Bangladesh, Brazil, Ethiopia and Nepal. RNA expression in 1,090 whole blood samples was determined for 103 target genes for innate and adaptive immune profiling by dual color Reverse-Transcription Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) followed by cluster analysis. We identified transcriptomic biomarkers associated with leprosy disease, different leprosy phenotypes as well as high exposure to Mycobacterium leprae which respectively allow improved diagnosis and classification of leprosy patients and detection of infection. Importantly, a transcriptomic signature of risk for reversal reactions consisting of five genes (CCL2, CD8A, IL2, IL15 and MARCO) was identified based on cross-sectional comparison of RNA expression. In addition, intra-individual longitudinal analyses of leprosy patients before, during and after treatment of reversal reactions, indicated that several IFN-induced genes increased significantly at onset of reaction whereas IL15 decreased. This multi-site study, situated in four leprosy endemic areas, demonstrates the potential of host transcriptomic biomarkers as correlates of risk for leprosy. Importantly, a prospective five-gene signature for reversal reactions could predict reversal reactions at least 2 weeks before onset. Thus, transcriptomic biomarkers provide promise for early detection of these acute inflammatory episodes and thereby help prevent permanent neuropathy and disability in leprosy patients.
Assuntos
Hanseníase/genética , Transcriptoma , Adolescente , Adulto , Bangladesh/epidemiologia , Biomarcadores/sangue , Brasil/epidemiologia , Etiópia/epidemiologia , Feminino , Humanos , Hanseníase/sangue , Hanseníase/epidemiologia , Masculino , Mycobacterium leprae/isolamento & purificação , Nepal/epidemiologia , Países Baixos/epidemiologia , Prognóstico , Estudos Prospectivos , Adulto JovemRESUMO
Gelatinases A and B (matrix metalloproteinase 2 [MMP-2] and MMP-9, respectively) can induce basal membrane breakdown and leukocyte migration, but their role in leprosy skin inflammation remains unclear. In this study, we analyzed clinical specimens from leprosy patients taken from stable, untreated skin lesions and during reactional episodes (reversal reaction [RR] and erythema nodosum leprosum [ENL]). The participation of MMPs in disease was suggested by (i) increased MMP mRNA expression levels in skin biopsy specimens correlating with the expression of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), (ii) the detection of the MMP protein and enzymatic activity within the inflammatory infiltrate, (iii) increased MMP levels in patient sera, and (iv) the in vitro induction of MMP-9 by Mycobacterium leprae and/or TNF-alpha. It was observed that IFN-gamma, TNF-alpha, MMP-2, and MMP-9 mRNA levels were higher in tuberculoid than lepromatous lesions. In contrast, interleukin-10 and tissue inhibitor of MMP (TIMP-1) message were not differentially modulated. These data correlated with the detection of the MMP protein evidenced by immunohistochemistry and confocal microscopy. When RR and ENL lesions were analyzed, an increase in TNF-alpha, MMP-2, and MMP-9, but not TIMP-1, mRNA levels was observed together with stronger MMP activity (zymography/in situ zymography). Moreover, following in vitro stimulation of peripheral blood cells, M. leprae induced the expression of MMP-9 (mRNA and protein) in cultured cells. Overall, the present data demonstrate an enhanced MMP/TIMP-1 ratio in the inflammatory states of leprosy and point to potential mechanisms for tissue damage. These results pave the way toward the application of new therapeutic interventions for leprosy reactions.
Assuntos
Hanseníase/imunologia , Leucócitos/imunologia , Metaloproteinases da Matriz/imunologia , Mycobacterium leprae/imunologia , Pele/imunologia , Pele/microbiologia , Adulto , Movimento Celular , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Inflamação , Mediadores da Inflamação/análise , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Pele/química , Pele/patologia , Adulto JovemRESUMO
Matrix metalloproteinases (MMPs) and tumor necrosis factor alpha (TNF-alpha) play important and related roles in the pathogenesis of nerve injury. MMP-dependent and TNF-alpha-dependent processes of neurodegeneration, such as blood-nerve breakdown and immune cell recruitment, are characteristic of leprosy nerve damage. Our work has contributed to the understanding of the role of cytokines in the process, but the role of MMPs in the pathogenesis of neuritic leprosy has not been investigated. This study analyzed the changes in mRNA expression and immunodistribution of MMP-2, MMP-9, TNF-alpha-converting enzyme (TACE), TNF-alpha in nerves of 27 pure neuritic leprosy (PNL) patients, both acid-fast bacilli positive (AFB(+)) and acid-fast bacilli negative (AFB(-)), and 8 non-leprosy patients with control peripheral neuropathic conditions. MMP-2, MMP-9, and TNF-alpha mRNA expression was significantly induced in the AFB(-) relative to the AFB(+) neuritic leprosy group and nonlepritic controls; TACE levels were also elevated in the AFB(-) group, but this change was not statistically significant. Immunoreactive profiles for TNF-alpha and MMPs demonstrated strong reactivity of myelinated axons, infiltrating macrophages, Schwann cells, endothelial cells, and perineurial cells in neuritic leprosy biopsies. This study provides the evidence of the involvement of MMPs in the pathogenesis of PNL neuropathy.
Assuntos
Proteínas ADAM/biossíntese , Hanseníase/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Nervos Periféricos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Proteína ADAM17 , Adulto , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Hanseníase/enzimologia , Masculino , Pessoa de Meia-Idade , Nervos Periféricos/enzimologia , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The development of deformities during the course of leprosy disease is a major public health concern worldwide. It is possible that cytokine production and apoptosis of Schwann cells (SCs) directly affect nerve degeneration and regeneration leading to injury of the myelin sheath and axon. In the present study, the expression of TNFalpha, TGFbeta, and their receptors, in addition to cell death triggered by cytokines or whole Mycobacterium leprae were investigated in a human SC line. The results showed the presence of TNF-Rs and TGF-RII on the SC membrane and the shedding of TNF-Rs during the culture period. Evaluation of cell death was performed through TUNEL and flow cytometry techniques. TNFalpha/TGFbeta combination as well as M. leprae infection triggered an increase in the apoptosis rate in the cultured SC. Moreover, reverse transcriptase-polymerase chain reaction assay revealed that M. leprae upregulated the expression of such cytokines and their receptors on the SC line. Despite the detection of TNFalpha mRNA, no protein was found in the culture supernatants. The data indicate that induction of SC death after cell interaction with M. leprae may, in fact, be implicated in the pathogenesis of nerve damage, which can most likely be modulated by in vivo cytokine production.
Assuntos
Apoptose/fisiologia , Linfotoxina-alfa/metabolismo , Mycobacterium leprae/fisiologia , Células de Schwann/microbiologia , Células de Schwann/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Linhagem Celular Tumoral , Humanos , Receptores do Fator de Necrose Tumoral/metabolismo , Células de Schwann/metabolismoRESUMO
The epidermis is an important site of the immunoinflammatory response in the skin. In the present study, the expression of cytokine and ICAM-1 (intercellular adhesion molecule-1) genes was evaluated by RT-PCR in the epidermis isolated from biopsies from 25 reactional leprosy patients. TNFalpha and IL-6 mRNAs were detected in all individuals during the reactional state (reversal reaction or erythema nodosum leprosum), IL-8 message was detected in 66.6% and 62.5% of the patients, IL-12 mRNA was present in 91.6% and 62.5% and ICAM-1 in 100% and 71.4%, respectively. In addition, when skin biopsies were obtained from the same patients before and during the reactional episode, an enhancement in cytokine mRNA, but not in ICAM-1 mRNA, was observed. Seven patients were also evaluated at the onset of reaction and during antiinflammatory treatment. In contrast to a preferential decrease in the TNFalpha gene detected in the dermis, during the treatment phase, persistent/enhanced TNFalpha mRNA expression was detected in the epidermis in six out of the seven patients assessed. This peculiar pattern of expression might reflect a differential impact that in vivo antiinflammatory therapy has on the epidermis. The present findings indicate that the epidermis plays an important role in the local inflammatory response in leprosy and that the profile of response detected in the epidermis during the reactions may be regulated differently from that in the dermis.
Assuntos
Epiderme/metabolismo , Hanseníase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Anti-Inflamatórios/uso terapêutico , Citocinas/genética , Citocinas/metabolismo , Derme/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Pentoxifilina/uso terapêutico , Prednisona/uso terapêutico , RNA Mensageiro/metabolismo , Talidomida/uso terapêutico , Fator de Necrose Tumoral alfa/genéticaRESUMO
The epidermis is an important site of the immunoinflammatory response in the skin. In the present study, the expression of cytokine and ICAM-1 (intercellular adhesion molecule-1) genes was evaluated by RT-PCR in the epidermis isolated from biopsies from 25 reactional leprosy patients. TNFalpha and IL-6 mRNAs were detected in all individuals during the reactional state (reversal reaction or erythema nodosum leprosum), IL-8 message was detected in 66.6 per cent and 62.5 per cent of the patients, IL-12 mRNA was present in 91.6 per cent and 62.5 per cent and ICAM-1 in 100 per cent and 71.4 per cent, respectively. In addition, when skin biopsies were obtained from the same patients before and during the reactional episode, an enhancement in cytokine mRNA, but not in ICAM-1 mRNA, was observed. Seven patients were also evaluated at the onset of reaction and during antiinflammatory treatment. In contrast to a preferential decrease in the TNFalpha gene detected in the dermis, during the treatment phase, persistent/enhanced TNFalpha mRNA expression was detected in the epidermis in six out of the seven patients assessed. This peculiar pattern of expression might reflect a differential impact that in vivo antiinflammatory therapy has on the epidermis. The present findings indicate that the epidermis plays an important role in the local inflammatory response in leprosy and that the profile of response detected in the epidermis during the reactions may be regulated differently from that in the dermis.