RESUMO
Mycobacterium leprae (M. leprae) infection causes nerve damage and the condition worsens often during and long after treatment. Clearance of bacterial antigens including lipoarabinomannan (LAM) during and after treatment in leprosy patients is slow. We previously demonstrated that M. leprae LAM damages peripheral nerves by in situ generation of the membrane attack complex (MAC). Investigating the role of complement activation in skin lesions of leprosy patients might provide insight into the dynamics of in situ immune reactivity and the destructive pathology of M. leprae. In this study, we analyzed in skin lesions of leprosy patients, whether M. leprae antigen LAM deposition correlates with the deposition of complement activation products MAC and C3d on nerves and cells in the surrounding tissue. Skin biopsies of paucibacillary (n = 7), multibacillary leprosy patients (n = 7), and patients with erythema nodosum leprosum (ENL) (n = 6) or reversal reaction (RR) (n = 4) and controls (n = 5) were analyzed. The percentage of C3d, MAC and LAM deposition was significantly higher in the skin biopsies of multibacillary compared to paucibacillary patients (p = <0.05, p = <0.001 and p = <0.001 respectively), with a significant association between LAM and C3d or MAC in the skin biopsies of leprosy patients (r = 0.9578, p< 0.0001 and r = 0.8585, p<0.0001 respectively). In skin lesions of multibacillary patients, MAC deposition was found on axons and co-localizing with LAM. In skin lesions of paucibacillary patients, we found C3d positive T-cells in and surrounding granulomas, but hardly any MAC deposition. In addition, MAC immunoreactivity was increased in both ENL and RR skin lesions compared to non-reactional leprosy patients (p = <0.01 and p = <0.01 respectively). The present findings demonstrate that complement is deposited in skin lesions of leprosy patients, suggesting that inflammation driven by complement activation might contribute to nerve damage in the lesions of these patients. This should be regarded as an important factor in M. leprae nerve damage pathology.
Assuntos
Ativação do Complemento/imunologia , Hanseníase/imunologia , Hanseníase/patologia , Dermatopatias/imunologia , Dermatopatias/patologia , Linfócitos T/imunologia , Adolescente , Adulto , Carga Bacteriana , Biomarcadores , Biópsia , Criança , Complemento C3d/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Feminino , Granuloma/imunologia , Granuloma/metabolismo , Granuloma/patologia , Humanos , Imuno-Histoquímica , Hanseníase/microbiologia , Lipopolissacarídeos , Masculino , Pessoa de Meia-Idade , Linfócitos T/metabolismo , Adulto JovemRESUMO
Multibacillary and paucibacillary paratuberculosis are both caused by Mycobacterium avium subspecies paratuberculosis. Multibacillary lesions are composed largely of infected epithelioid macrophages and paucibacillary lesions contain T cells but few bacteria. Multibacillary disease is similar to human lepromatous leprosy, with variable/high levels of antibody and a dysfunctional immune response. Animals with paucibacillary disease have high cell-mediated immunity and variable levels of antibody. This study aims to characterize the immunological dysfunction using TruSeq analysis of the ileocaecal lymph node that drains disease lesions. Immune dysfunction is highlighted by repression of TCR/CD3 genes, T cell co-receptors/co-stimulators, T cell activation and signal-transduction genes. Inflammation was an acute phase response and chronic inflammation, with little evidence of acute inflammation. The high levels of immunoglobulin and plasma cell transcripts is consistent with the anti-MAP antibody responses in paratuberculosis sheep. Also notable was the overwhelming reduction in mast cell transcripts, potentially affecting DC activation of the immune response. This study also shows that there were no fundamental differences in the gene expression patterns in multibacillary and paucibacillary disease, no shift in T cell genes from Th1 to Th2 pattern but rather an incremental decline into immune dysfunction leading to multibacillary pathology.
Assuntos
Imunidade Celular/genética , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/genética , Doenças dos Ovinos/genética , Transdução de Sinais/genética , Animais , Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno/imunologia , Imunidade Celular/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Mycobacterium avium subsp. paratuberculosis/fisiologia , Paratuberculose/microbiologia , Ovinos , Doenças dos Ovinos/microbiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/microbiologia , Transcriptoma/genética , Transcriptoma/imunologiaRESUMO
Mycobacterium tuberculosis secretes a number of proteins into the extracellular milieu during growth. Several of these proteins have been associated with modulation of the host immune response. Antigen 84, or Wag31, is one such protein that is conserved among all mycobacterial species and is recognized by the sera from tuberculosis and leprosy patients. Here, we examined the effect of Wag31 on the ability of activated human T cells to produce cytokines such as IL-10, IL-17 and IFN-γ in response to combined anti-CD3 and anti-CD28 stimulation. Purified recombinant Wag31 inhibited the secretion of IL-10 and IL-17, but not IFN-γ, by human T cells stimulated with plate-bound anti-CD3 and anti-CD28 monoclonal antibodies. Furthermore, the C-terminal domain, but not the N-terminal domain, inhibited the production of IL-10 and IL-17 without a significant effect on the production of IFN-γ. These data suggest that Wag31 may modulate human T cell immune responses during tuberculosis infection through its C-terminal domain.
Assuntos
Proteínas de Bactérias/farmacologia , Citocinas/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Linfócitos T/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Células Cultivadas , Citocinas/imunologia , Relação Dose-Resposta a Droga , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Antigen presenting cells (APC) are critical components of innate immunity and consequently shape the adaptive response. Leukocyte Ig Like Receptors (LILR) are innate immune receptors predominantly expressed on myeloid cells. LILR can influence the antigen presenting phenotype of monocytic cells to determine the nature of T cell responses in infections including Mycobaterium leprae. We therefore investigated the relevance of LILR in the context of Mycobacterium tuberculosis. Real-time PCR studies indicated that the transcriptional profile of the orphan receptor LILRB5 was significantly up-regulated following exposure to mycobacteria. Furthermore, LILRA1 and LILRB5 were able to trigger signalling through direct engagement of mycobacteria using tranfectant cells incorporating a reporter system. We describe for the first time the expression of this receptor on T cells, and highlight the potential relevance to mycobacterial recognition. Furthermore, we demonstrate that crosslinking of this receptor on T cells increases proliferation of cytotoxic, but not helper, T cells.
Assuntos
Antígenos CD/metabolismo , Receptores Imunológicos/metabolismo , Antígenos CD/genética , Vacina BCG/imunologia , Proliferação de Células , Células Cultivadas , Células Dendríticas/metabolismo , Expressão Gênica , Humanos , Imunidade Inata , Mycobacterium tuberculosis/imunologia , Receptores Imunológicos/genética , Linfócitos T/metabolismo , VacinaçãoRESUMO
The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742-752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs.
Assuntos
Proteínas de Ligação a DNA/imunologia , Eritema Nodoso/imunologia , Hanseníase Virchowiana/imunologia , Mycobacterium leprae/imunologia , Linfócitos T/imunologia , Adulto , Antígenos de Bactérias/imunologia , Proliferação de Células , Feminino , Antígenos HLA-A/imunologia , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Linfócitos T/metabolismo , Adulto JovemRESUMO
Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.
Assuntos
Antígenos CD1/metabolismo , Lipopolissacarídeos/metabolismo , Manose/metabolismo , Mycobacterium tuberculosis/metabolismo , Linfócitos T/metabolismo , Tuberculose/metabolismo , Antígenos CD1/imunologia , Proliferação de Células , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Interferon gama/metabolismo , Ponto Isoelétrico , Hanseníase/imunologia , Hanseníase/metabolismo , Lipopolissacarídeos/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Fosfatos/metabolismo , Isoformas de Proteínas , Succinatos/metabolismo , Linfócitos T/imunologia , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
Sequence variants at or near the leucine-rich repeat kinase 2 (LRRK2) locus have been associated with susceptibility to three human conditions: Parkinson's disease (PD), Crohn's disease and leprosy. As all three disorders represent complex diseases with evidence of inflammation, we hypothesized a role for LRRK2 in immune cell functions. Here, we report that full-length Lrrk2 is a relatively common constituent of human peripheral blood mononuclear cells (PBMC) including affinity isolated, CD14(+) monocytes, CD19(+) B cells, and CD4(+) as well as CD8(+) T cells. Up to 26% of PBMC from healthy donors and up to 43% of CD14(+) monocytes were stained by anti-Lrrk2 antibodies using cell sorting. PBMC lysates contained full-length (>260 kDa) and higher molecular weight Lrrk2 species. The expression of LRRK2 in circulating leukocytes was confirmed by microscopy of human blood smears and in sections from normal midbrain and distal ileum. Lrrk2 reactivity was also detected in mesenteric lymph nodes and spleen (including in dendritic cells), but was absent in splenic mononuclear cells from lrrk2-null mice, as expected. In cultured bone marrow-derived macrophages from mice we made three observations: (i) a predominance of higher molecular weight lrrk2; (ii) the reduction of autophagy marker LC3-II in (R1441C)lrrk2-mutant cells (<31%); and (iii) a significant up-regulation of lrrk2 mRNA (>fourfold) and protein after exposure to several microbial structures including bacterial lipopolysaccharide and lentiviral particles. We conclude that Lrrk2 is a constituent of many cell types in the immune system. Following the recognition of microbial structures, stimulated macrophages respond with altered lrrk2 gene expression. In the same cells, lrrk2 appears to co-regulate autophagy. A pattern recognition receptor-type function for LRRK2 could explain its locus' association with Crohn's disease and leprosy risk. We speculate that the role of Lrrk2 in immune cells may also be relevant to the susceptibility of developing PD or its progression.
Assuntos
Predisposição Genética para Doença/genética , Leucócitos/metabolismo , Macrófagos/metabolismo , Doença de Parkinson , Proteínas Serina-Treonina Quinases/genética , Regulação para Cima/fisiologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Autofagia/genética , Linfócitos B/metabolismo , Células Cultivadas , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Transgênicos , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/imunologia , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Linfócitos T/metabolismoRESUMO
To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.
Assuntos
Proteínas de Choque Térmico HSP70/imunologia , Ativação Linfocitária/imunologia , Proteínas de Membrana/imunologia , Mycobacterium bovis/imunologia , Linfócitos T/imunologia , Urease/deficiência , Animais , Apresentação de Antígeno/imunologia , Vacinas Bacterianas/imunologia , Western Blotting , Separação Celular , Citocinas/biossíntese , Citocinas/imunologia , Células Dendríticas/imunologia , Citometria de Fluxo , Humanos , Hanseníase/imunologia , Hanseníase/prevenção & controle , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/metabolismoAssuntos
Antígenos de Bactérias/imunologia , Antígenos de Bactérias/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologia , Antígenos de Bactérias/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Humanos , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/metabolismoRESUMO
This study reports three cases of an unusual leprotic reaction characterized by superficial bullous ulcerative cutaneous lesions associated with high fever, malaise and oedema in patients with leprosy. Two patients responded to thalidomide treatment, with regression of the symptoms and skin ulcers. The third patient responded to thalidomide plus prednisone. Analysis of the ulcerated skin lesions showed dermal oedema with mononuclear cell infiltrate enriched for gammadelta-positive T lymphocytes and an increased number of Mycobaterium leprae bacilli within capillary endothelium. In contrast, gammadelta+ cells were decreased in or absent from the blood. Tumour necrosis factor-alpha and interleukin-6 were raised in the serum of the patients at the onset of the reaction. After the episode, cytokine levels and the percentage of gammadelta+ cells in the blood returned to normal. These cases characterize an uncommon leprotic reaction with clinical similarities to type II reaction and may indicate a significant role for gammadelta+ T cells in its pathogenesis.
Assuntos
Eritema Nodoso/patologia , Hanseníase Virchowiana/patologia , Idoso , Antivirais/uso terapêutico , Eritema Nodoso/tratamento farmacológico , Eritema Nodoso/metabolismo , Humanos , Interferon gama/uso terapêutico , Hanseníase Virchowiana/tratamento farmacológico , Hanseníase Virchowiana/imunologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae , Prednisona/uso terapêutico , Linfócitos T/metabolismo , Talidomida/uso terapêutico , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVES: Development of a user-friendly test alternative to ELISA-based assays to detect IFN-gamma by in vitro cultured peripheral blood mononuclear cells (PBMC) stimulated with pathogen-derived antigens. DESIGN AND METHODS: The molecular components of an operational IFN-gamma ELISA-based test were applied in a lateral flow (LF) immuno-sandwich assay using up-converting phosphor (UCP) reporter particles. The analytical sensitivity of the UCP-LF IFN-gamma assay (ULIGA) was determined and the assay was qualitatively validated with a selection of 60 supernatants derived from PBMC cultures stimulated with M. leprae derived antigens, mitogen or medium alone. RESULTS: ULIGA indicated an analytical sensitivity better than 2 pg/mL, and demonstrated four orders of magnitude dynamic range. The assay correlated well with the IFN-gamma ELISA. CONCLUSIONS: ULIGA allows detection well below the cutoff value (100 pg/mL) used to define positive responses in the IFN-gamma ELISA. The test procedure is less demanding in respect to equipment and labor, and is suited for testing single samples.
Assuntos
Bioensaio/métodos , Interferon gama/metabolismo , Linfócitos T/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Linfócitos T/citologiaRESUMO
BACKGROUND: Leprosy is an infectious disease in which the susceptibility to the pathogen Mycobacterium leprae and the clinical manifestations are attributed to host immune cell response. Receptor mediated events and signalling in the immune cells are mediated by protein phosphorylation. The main signalling pathways and protein kinases known to be involved in the regulation of immune cells are cAMP dependent kinases, calcium/calmodulin dependent kinases, protein kinase C and mitogen activated protein kinases. The cumulative consequence of alterations in signalling pathways can be evaluated by intrinsic cellular protein phosphorylation by gamma-P32 ATP. The present study was designed to assess the protein phosphorylation in the immune cells of leprosy patients as compared with normal individuals. METHODOLOGY: Lymphocyte protein phosphorylation was conducted in 15 leprosy patients and 9 normal individuals. Protein phosphorylation of lymphocytes was carried out in the presence/absence of protein kinase modulators. The phosphorylation patterns were documented and analysed consequent to SDS-PAGE, staining, destaining, drying and autoradiography. RESULTS: The major phosphorylated proteins in lymphocytes were of molecular weights 20-22, 24-29, 30-35, 43, 46-50 and 66-68 kDa. In general, the major phosphorylated proteins were similar in the controls and in the patients. The phosphorylatability of these proteins varied with different modulators. Variations in the phosphorylation pattern were observed in 25% of the leprosy patients where there was a decrease of the 66 kDa protein and a decrease of 20-22 kDa protein phosphorylation. CONCLUSION: The observed alterations in the protein phosphorylation pattern could be due to alteration in kinases and/or their substrates or due to the effect of M. leprae on immune cells.
Assuntos
Hanseníase/imunologia , Fosfoproteínas/metabolismo , Linfócitos T/metabolismo , Autorradiografia , Estudos de Casos e Controles , Humanos , Fosforilação , Proteínas Quinases/metabolismoRESUMO
CD1 has been clearly shown to function as a microbial recognition system for activation of T cell responses, but its importance for mammalian protective responses against infections is still uncertain. The function of the group 1 CD1 isoforms, including human CD1a, CDlb, and CDLc, seems closely linked to adaptive immunity. These CD1 molecules control the responses of T cells that are highly specific for particular lipid antigens, the best known of which are abundantly expressed by pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium leprae. Studies done mainly on human circulating T cells ex vivo support a significant role for group I CD1-restricted T cells in protective immunity to mycobacteria and potentially other pathogens, although supportive data from animal models is currently limited. In contrast, group 2 CD1 molecules, which include human CD1d and its orthologs, have been predominantly associated with the activation of CD1d-restricted NKT cells, which appear to be more appropriately viewed as a facet of the innate immune system. Whereas the recognition of certain self-lipid ligands by CD d-restricted NKT cells is well accepted, the importance of these T cells in mediating adaptive immune recognition of specific microbial lipid antigens remains controversial. Despite continuing uncertainty about the role of CD 1d-restricted NKT cells in natural infections, studies in mouse models demonstrate the potential of these T cells to exert various effects on a wide spectrum of infectious diseases, most likely by serving as a bridge between innate and adaptive immune responses.
Assuntos
Antígenos CD1/metabolismo , Doenças Transmissíveis/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Antígenos CD1/imunologia , Doenças Transmissíveis/etiologia , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , Camundongos , Linfócitos T/metabolismoRESUMO
Expression of the transcription factor Foxp3 (forkhead box P3) has been implicated as a key element for CD25(+) T regulatory cell function in mice. However, literature over similar involvement of FOXP3 expression in human T regulatory cells is limited. We found that, unlike murine cells, FOXP3 mRNA expression could be induced in human CD25(-) and CD8(+) peripheral blood mononuclear cells, which were both negative for FOXP3 mRNA expression after isolation. Expression of FOXP3 mRNA began as soon as 24-40 hours after stimulation, demonstrating a correlation between activation and FOXP3 mRNA expression in human cells. In order to determine whether FOXP3 expression is confined to CD4(+)CD25(+) T cells with a regulatory phenotype, we analyzed several well-defined T-cell clones and lines with various specificities. Surprisingly, expression of FOXP3 mRNA was detected in all clones and limited to the CD25(hi) populations. Nonetheless, the CD25(hi) fraction did not display regulatory properties because both the CD25(hi) and CD25(low) populations exhibited a similar proliferative- and interferon-gamma-secreting potential after antigenic stimulation. These results indicate that FOXP3 expression in humans, unlike mice, may not be specific for cells with a regulatory phenotype and may be only a consequence of activation status.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/fisiologia , Leucócitos Mononucleares/metabolismo , Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação a DNA/imunologia , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Células Jurkat , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Ativação Linfocitária/fisiologia , Camundongos , Mycobacterium leprae/metabolismo , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Receptores de Interleucina-2 , Linfócitos T/imunologiaRESUMO
Current knowledge on the structure of lipoarabinomannan (LAM) has resulted primarily from detailed studies on a few selected laboratory strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmatis. Our previous work was the first to report on the salient structural features of M. tuberculosis clinical isolates and demonstrated significant structural variations. A prime effort is to correlate a particular structural characteristic with observed differences in eliciting an immunobiological response, especially in the context of CD1-restricted presentation of LAM to T cells. T cell clones derived from the cutaneous lesions of leprosy patients have been shown to recognize specifically LAM from Mycobacterium leprae and not from M. tuberculosis Erdman or H37Rv. Herein we provide further fine structural data on LAM from M. leprae (LepLAM) and a tuberculosis clinical isolate, CSU20 (CSU20LAM), which was unexpectedly recognized by the supposedly LepLAM-specific CD1-restricted T cell clones. In comparison with the de facto laboratory LAM standard from M. tuberculosis H37Rv (RvLAM), LepLAM derived from in vivo grown M. leprae is apparently simpler in its arabinan architecture with a high degree of exposed, non-mannose-capped termini. On the other hand, CSU20, an ethambutol-resistant clinical isolate, makes a vastly heterogeneous population of LAM ranging from rather small and non-mannose-capped to full-length and fully capped variants. LepLAM and CSU20LAM contain a higher level of succinylation than RvLAM, which, in the context of truncated or less elaborated arabinan, may contribute to selective recognition by T cells. LAM from all species could be resolved into discrete forms by isoelectric focusing based apparently on their arabinan heterogeneity. In the light of our current and more recent findings, we reason that all immunobiological data should be cautiously interpreted and that the actual LAM variants that may be present in vivo during infection and pathogenesis need to be taken into consideration.
Assuntos
Etambutol/farmacologia , Lipopolissacarídeos/química , Mycobacterium leprae/metabolismo , Mycobacterium tuberculosis/metabolismo , Western Blotting , Divisão Celular , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Lipopolissacarídeos/metabolismo , Espectroscopia de Ressonância Magnética , Manose/química , Monossacarídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Frações Subcelulares/metabolismo , Linfócitos T/metabolismo , alfa-Manosidase/metabolismoRESUMO
Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model, we investigated whether expression of CD1a and langerin, an LC-specific C-type lectin, imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin, placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease.
Assuntos
Apresentação de Antígeno , Antígenos CD1/fisiologia , Antígenos de Superfície/fisiologia , Células de Langerhans/fisiologia , Lectinas Tipo C/fisiologia , Lectinas de Ligação a Manose/fisiologia , Linfócitos T/metabolismo , Antígenos CD , Antígenos CD1/metabolismo , Antígenos de Superfície/metabolismo , Divisão Celular , Relação Dose-Resposta a Droga , Epiderme/imunologia , Sangue Fetal/metabolismo , Humanos , Imuno-Histoquímica , Células de Langerhans/metabolismo , Lectinas/química , Lectinas Tipo C/metabolismo , Hanseníase/imunologia , Lectinas de Ligação a Manose/metabolismo , Microscopia de Fluorescência , Mycobacterium leprae/metabolismo , Fenótipo , Receptores de Antígenos/químicaRESUMO
Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT-6). Both ESAT-6 and CFP-10 are encoded by the RD1 region that is deleted from all tested M. bovis bacille Calmette-Guérin (BCG) strains but present in M. leprae, M. tuberculosis, M. bovis, M. kansasii, M. africanum and M. marinum. In this study, the homologue of CFP-10 in M. leprae (ML0050) is identified and characterized. Interferon-gamma production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP-10 of M. leprae is a potent antigen that crossreacts with CFP-10 of M. tuberculosis at the T-cell level. This crossreactivity has implications for the use of CFP-10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.
Assuntos
Proteínas de Bactérias/imunologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Reações Cruzadas/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Homologia de Sequência , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
In several human pathologies (e.g. cancer, rheumatoid arthritis, AIDS and leprosy) oxidative stress induces T cell hyporesponsiveness. Hyporesponsive T cells often appear to display impaired expression of some (e.g. TCR-zeta, p56(lck) and LAT) but not all (e.g. TCR-alphabeta and CD3-epsilon) crucial TCR-proximal signaling molecules but the underlying mechanisms have as yet not been identified. Using an in vitro system for oxidative-stress-induced T cell hyporesponsiveness we here report two sequential effects of oxidative stress on TCR signaling molecules: protein alterations and proteasomal degradation. We have identified the C-terminal part of TCR-zeta and the membrane-proximal domain of p56(lck) as potential targets for modifications induced by reactive oxygen species. Oxidative-stress-exposed proteins were differentially susceptible to proteasomal degradation: whereas modified TCR-zeta was relatively resistant, reactive oxygen species (ROS)-altered LAT and p56(lck) were much more susceptible. Importantly, we found that T cell hyporesponsiveness best correlated with ROS-dependent protein alteration since inhibition of proteasomal degradation did not restore function. Finally, our data provide an explanation for the paradox of reduced TCR-zeta signals combined with unaltered TCR-alphabeta and CD3-epsilon expression levels: the TCR-zeta chain in hyporesponsive T cells is still expressed but no longer detectable by certain mAb recognizing ROS-sensitive epitopes.
Assuntos
Acetilcisteína/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Membrana , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Acetilcisteína/farmacologia , Proteínas de Transporte/metabolismo , Técnicas de Cocultura , Cisteína Endopeptidases/metabolismo , Humanos , Tolerância Imunológica , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Neutrófilos/imunologia , Estresse Oxidativo , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Transdução de Sinais , Linfócitos T/efeitos dos fármacosAssuntos
Hanseníase Virchowiana/imunologia , Mycobacterium leprae/patogenicidade , Linfócitos T/metabolismo , Fator de Transcrição AP-1/metabolismo , Adulto , Núcleo Celular , Feminino , Humanos , Interleucina-2/biossíntese , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Linfócitos T/patologiaRESUMO
We examined the effect of IL-12 and IL-18 on bactericidal activities of mouse peritoneal cell (PC) against Mycobacterium leprae (M. leprae). We demonstrated that IL-12 and IL-18 synergistically induced the NO-dependent bactericidal activity of PC by stimulating Natural Killer (NK) cells and T-cells through IFN-gamma production. IL-12 and IL-18 induced host cell death through NK-cells and T-cells. Therefore. IL-12 and IL-18 play an important role on direct killing of intracellular M. leprae and on indirect killing of them through inducing host cell death.