Autophagy links antimicrobial activity with antigen presentation in Langerhans cells.

DC, through the uptake, processing, and presentation of antigen, are responsible for activation of T cell responses to defend the host against infection, yet it is not known if they can directly kill invading bacteria. Here, we studied in human leprosy, how Langerhans cells (LC), specialized DC, contribute to host defense against bacterial infection. IFN-γ treatment of LC isolated from human epidermis and infected with Mycobacterium leprae (M. leprae) activated an antimicrobial activity, which was dependent on the upregulation of the antimicrobial peptide cathelicidin and induction of autophagy. IFN-γ induction of autophagy promoted fusion of phagosomes containing M. leprae with lysosomes and the delivery of cathelicidin to the intracellular compartment containing the pathogen. Autophagy enhanced the ability of M. leprae-infected LC to present antigen to CD1a-restricted T cells. The frequency of IFN-γ labeling and LC containing both cathelicidin and autophagic vesicles was greater in the self-healing lesions vs. progressive lesions, thus correlating with the effectiveness of host defense against the pathogen. These data indicate that autophagy links the ability of DC to kill and degrade an invading pathogen, ensuring cell survival from the infection while facilitating presentation of microbial antigens to resident T cells.

Involvement of TNF-Producing CD8+ Effector Memory T Cells with Immunopathogenesis of Erythema Nodosum Leprosum in Leprosy Patients.

Type 2 reaction (T2R) or erythema nodosum leprosum (ENL), a sudden episode of acute inflammation predominantly affecting lepromatous leprosy patients (LL), characterized by a reduced cellular immune response. This possibly indicates a close relationship between the onset of T2R and the altered frequency, and functional activity of T lymphocytes, particularly of memory subsets. This study performed ex vivo and in vitro characterizations of T cell blood subpopulations from LL patients with or without T2R. In addition, the evaluation of activity of these subpopulations was performed by analyzing the frequency of these cells producing IFN-γ, TNF, and IL-10 by flow cytometry. Furthermore, the expression of transcription factors, for the differentiation of T cells, were analyzed by quantitative real-time polymerase chain reaction. Our results showed an increased frequency of CD8+/TNF+ effector memory T cells (TEM) among T2Rs. Moreover, there was evidence of a reduced frequency of CD4 and CD8+ IFN-γ-producing cells in T2R, and a reduced expression of STAT4 and TBX21. Finally, a significant and positive correlation between bacteriological index (BI) of T2R patients and CD4+/TNF+ and CD4+/IFN-γ+ T cells was observed. Thus, negative correlation between BI and the frequency of CD4+/IL-10+ T cells was noted. These results suggest that CD8+/TNF+ TEM are primarily responsible for the transient alteration in the immune response to Mycobacterium leprae in ENL patients. Thus, our study improves our understanding of pathogenic mechanisms and might suggest new therapeutic approaches for leprosy.

Mycobacterium indicus pranii protein MIP_05962 induces Th1 cell mediated immune response in mice.

Utility of Mycobacterium indicus pranii (MIP) as a multistage vaccine against mycobacterial infections demands identification of its protective antigens. We explored antigenicity and immunogenicity of a candidate protein MIP_05962 that depicts homology to HSP18 of M. leprae and antigen1 of Mycobacterium tuberculosis. This protein elicited substantial antibody response in immunized mice along with modulation of cellular immune response towards protective Th1 type. Both CD4+ and CD8+ subsets from immunized mice produced hallmark protective cytokines, IFN-γ, TNF-α and IL-2. This protein also enhanced the CD4+ effector memory that could act as first line of defence during infections. These results point to MIP_05962 as a protective antigen that contributes, in conjunction with others, to the protective immunity of this live vaccine candidate.

Debaryomyces hansenii CBS 8339 ß-glucan enhances immune responses and down-stream gene signaling pathways in goat peripheral blood leukocytes.

Debaryomyces hansenii-derived ß-glucan has shown immunostimulant effect on aquaculture species and recently on goat peripheral blood leukocytes. Moreover, the marine yeast D. hansenii CBS 8339 has demonstrated to enhance fish immune response. Nonetheless, the associated immune signaling pathways induced by ß-glucan from this marine yeast have not been characterized yet. This study described the effects of ß-glucan from D. hansenii CBS 8339 against challenge with Escherichia coli and activation of possible mechanisms on goat peripheral blood leukocytes. The proton nuclear magnetic resonance spectra showed that D. hansenii had ß-(1,3)(1,6)-glucan. The phagocytic ability enhanced after E. coli challenge, and nitric oxide production increased before and after challenge in leukocytes stimulated with D. hansenii ß-glucan. In addition, an early gene expression stimulation was found related to ß-glucan recognition by TLR2 and Dectin-1 receptors, intracellular regulation by Syk, TRAF6, MyD88 and transcription factor NFκB, and effector functions of pro-inflammatory cytokine, such as IL-1ß and TNF-α. Interestingly, simulation with D. hansenii-derived ß-glucan increased leukocyte viability after E. coli challenge. In conclusion, ß-glucan from D. hansenii CBS 8339 reduced cytotoxic effects of E. coli and modulated signaling pathways and innate immune response in goat peripheral blood leukocytes.

Characterization of a cross-reactive, immunodominant and HLA-promiscuous epitope of Mycobacterium tuberculosis-specific major antigenic protein PPE68.

PPE68 (Rv3873), a major antignic protein encoded by Mycobacteriun tuberculosis-specific genomic region of difference (RD)1, is a strong stimulator of peripheral blood mononuclear cells (PBMCs) obtained from tuberculosis patients and Mycobacterium bovis bacillus Calmette Guerin (BCG)-vaccianted healthy subjects in T helper (Th)1 cell assays, i.e. antigen-induced proliferation and interferon-gamma (IFN-γ) secretion. To confirm the antigen-specific recognition of PPE68 by T cells in IFN-γ assays, antigen-induced human T-cell lines were established from PBMCs of M. Bovis BCG-vaccinated and HLA-heterogeneous healthy subjects and tested with peptide pools of RD1 proteins. The results showed that PPE68 was recognized by antigen-specific T-cell lines from HLA-heteregeneous subjects. To further identify the immunodominant and HLA-promiscuous Th1-1 cell epitopes present in PPE68, 24 synthetic peptides covering the sequence of PPE68 were indivdually analyzed for HLA-DR binding prediction analysis and tested with PBMCs from M. bovis BCG-vaccinated and HLA-heterogeuous healthy subjects in IFN-γ assays. The results identified the peptide P9, i.e. aa 121-VLTATNFFGINTIPIALTEMDYFIR-145, as an immunodominant and HLA-DR promiscuous peptide of PPE68. Furthermore, by using deletion peptides, the immunodominant and HLA-DR promiscuous core sequence was mapped to aa 127-FFGINTIPIA-136. Interestingly, the core sequence is present in several PPE proteins of M. tuberculosis, and conserved in all sequenced strains/species of M. tuberculosis and M. tuberculosis complex, and several other pathogenic mycobacterial species, including M. leprae and M. avium-intracellulalae complex. These results suggest that the peptide aa 121-145 may be exploited as a peptide-based vaccine candidate against tuberculosis and other mycobacterial diseases.

Avaliação da participação de TGF-beta1 na regulação da proliferação de células de Schwann infectadas pelo Mycobacterium leprae / TGF-beta1 evaluation to participate in the regulation of proliferation of Schwann cells infected by Mycobacterium leprae

A hanseníase é uma doença infecciosa causada pelo Mycobacterium leprae que invade prioritariamente as células de Schwann presentes no sistema nervoso periférico. Essas células quando infectadas sofrem alterações funcionais, interferindo, em última instância, no processo de regeneração dos nervos periféricos. Na tentativa de elucidar mecanismos específicos pelos quais a invasão pelo Mycobacterium leprae leva a alterações celulares, alguns mediadores químicos vem sendo estudados com mais ênfase. Entre eles, TGF-B1 que, além de seu envolvimento na modulação da resposta imune e de seu papel crítico no processo de reparo, exerce influência sobre os processos de mielinização, diferenciação e proliferação celular. Nesse estudo, nos propusemos a avaliar o perfil de produção de TGF-B1 em cultura primária de células de Schwann provenientes de nervos ciáticos de camundongos BALB-c foram divididas em grupos controle e experimentais, nos quais se procedeu a infecção com Mycobacterium leprae vivo, nas multiplicidades de infecção 50:1 e 100:1. Todas as células foram marcadas com fluoróforo CFSE para avaliação de proliferação por imunofluorescência em microscopia confocal. Nos sobrenadantes abtidos, foi quantificada a produção de TGF-B1 por ELISA, que aos análise de dados, não apresentou diferenças estatisticamente significantes. Havendo, no entanto, tendência a menor produção do referido fator nos grupos experimentais em relação ao controle, principalmente em 48 e 96 horas. A proliferação celular, identificada pelo marcador CFSE, foi estatisticamente significante nos períodos de 168 a 240 horas, para a MOI 50:1, bem como em 96 e 240 horas, para MOI 100:1. Observou-se ainda, correlação negativa entre produção de TGF-B1 e expressão de CFSE. Sob as condições ensaiadas, concluímos que a elevação na proliferação de células de Schwann resulta da infecção por Mycobacterium leprae propriamente dita, sem necessariamente haver correlação direta com a produção de TGF-B1 nas mesmas.

Leprosy is an infectious disease caused by Mycobacterium leprae that primarily invades Schwann cells present in the peripheral nervous system. These cells when infected suffer functional changes, interfering, ultimately, the process of regeneration of peripheral nerves. In an attempt to elucidate specific mechanisms by which the invasion by Mycobacterium leprae leads to cellular changes, some chemical mediators have been studied with more emphasis. Among them, TGF-B1 that in addition to its involvement in the modulation of the immune response and its critical role in the repair process, influences the myelination process, cell differentiation and proliferation. In this study, we set out to evaluate the TGF-B1 production profile in primary culture of Schwann cells from sciatic nerves of BALB-c mice were divided into control and experimental groups, in which we proceeded infection with Mycobacterium leprae live, the multiplicities of infection 50: 1 and 100: 1. All cells were labeled with CFSE fluorophore for proliferation assessment by immunofluorescence confocal microscopy. In abtidos supernatants was quantified the production of TGF-B1 by ELISA that the data analysis, no statistically significant differences. Having, however, tend to lower production of that factor in the experimental groups compared to the control, especially at 48 and 96 hours. The cell proliferation by CFSE-labeled tracer, was statistically significant in periods 168-240 hours for at MOI 50: 1 and at 96 and 240 hours to MOI 100: 1. There was also a negative correlation between TGF-B1 and expression of CFSE. Under the conditions tested, we concluded that the increase in Schwann cell proliferation results from the infection by Mycobacterium leprae itself, without necessarily have direct correlation with TGF-B1 in the same.