Autophagy Is an Innate Mechanism Associated with Leprosy Polarization.

Leprosy is a chronic infectious disease that may present different clinical forms according to the immune response of the host. Levels of IFN-γ are significantly raised in paucibacillary tuberculoid (T-lep) when compared with multibacillary lepromatous (L-lep) patients. IFN-γ primes macrophages for inflammatory activation and induces the autophagy antimicrobial mechanism. The involvement of autophagy in the immune response against Mycobacterium leprae remains unexplored. Here, we demonstrated by different autophagic assays that LC3-positive autophagosomes were predominantly observed in T-lep when compared with L-lep lesions and skin-derived macrophages. Accumulation of the autophagic receptors SQSTM1/p62 and NBR1, expression of lysosomal antimicrobial peptides and colocalization analysis of autolysosomes revealed an impairment of the autophagic flux in L-lep cells, which was restored by IFN-γ or rapamycin treatment. Autophagy PCR array gene-expression analysis revealed a significantly upregulation of autophagy genes (BECN1, GPSM3, ATG14, APOL1, and TPR) in T-lep cells. Furthermore, an upregulation of autophagy genes (TPR, GFI1B and GNAI3) as well as LC3 levels was observed in cells of L-lep patients that developed type 1 reaction (T1R) episodes, an acute inflammatory condition associated with increased IFN-γ levels. Finally, we observed increased BCL2 expression in L-lep cells that could be responsible for the blockage of BECN1-mediated autophagy. In addition, in vitro studies demonstrated that dead, but not live M. leprae can induce autophagy in primary and lineage human monocytes, and that live mycobacteria can reduce the autophagy activation triggered by dead mycobacteria, suggesting that M. leprae may hamper the autophagic machinery as an immune escape mechanism. Together, these results indicate that autophagy is an important innate mechanism associated with the M. leprae control in skin macrophages.

Autophagy-Associated IL-15 Production Is Involved in the Pathogenesis of Leprosy Type 1 Reaction.

Leprosy reactional episodes are acute inflammatory events that may occur during the clinical course of the disease. Type 1 reaction (T1R) is associated with an increase in neural damage, and the understanding of the molecular pathways related to T1R onset is pivotal for the development of strategies that may effectively control the reaction. Interferon-gamma (IFN-γ) is a key cytokine associated with T1R onset and is also associated with autophagy induction. Here, we evaluated the modulation of the autophagy pathway in Mycobacterium leprae-stimulated cells in the presence or absence of IFN-γ. We observed that IFN-γ treatment promoted autophagy activation and increased the expression of genes related to the formation of phagosomes, autophagy regulation and function, or lysosomal pathways in M. leprae-stimulated cells. IFN-γ increased interleukin (IL)-15 secretion in M. leprae-stimulated THP-1 cells in a process associated with autophagy activation. We also observed higher IL15 gene expression in multibacillary (MB) patients who later developed T1R during clinical follow-up when compared to MB patients who did not develop the episode. By overlapping gene expression patterns, we observed 13 common elements shared between T1R skin lesion cells and THP-1 cells stimulated with both M. leprae and IFN-γ. Among these genes, the autophagy regulator Translocated Promoter Region, Nuclear Basket Protein (TPR) was significantly increased in T1R cells when compared with non-reactional MB cells. Overall, our results indicate that IFN-γ may induce a TPR-mediated autophagy transcriptional program in M. leprae-stimulated cells similar to that observed in skin cells during T1R by a pathway that involves IL-15 production, suggesting the involvement of this cytokine in the pathogenesis of T1R.

Encapsulation of ovarian allograft precludes immune rejection and promotes restoration of endocrine function in immune-competent ovariectomized mice.

Premature ovarian insufficiency (POI) is a significant complication of cytotoxic treatments due to extreme ovarian sensitivity to chemotherapy and radiation. POI is particularly devastating for young girls reaching puberty, because it irreversibly affects their physical and cognitive development. Changes occurring during puberty determine their height, bone health, insulin responsiveness, lipid metabolism, cardiovascular health and cognition. The only available treatment for POI during puberty is hormone replacement therapy (HRT), which delivers non-physiological levels of estrogen, lacks other ovarian hormones and pulsatility, and is not responsive to feedback regulation. Here we report that ovarian allografts encapsulated in a hydrogel-based capsule and implanted in ovariectomized mice restore ovarian endocrine function in immune competent mice. Ovarian tissue from BALB/c mice was encapsulated in poly(ethylene-glycol) (PEG) hydrogels, with a proteolytically degradable core and a non-degradable shell. The dual capsules were implanted subcutaneously in immune competent ovariectomized C57BL/6 mice for a period of 60 days. As expected, non-encapsulated ovarian allografts implanted in a control group sensitized the recipients as confirmed with donor-specific IgG in the serum, which increased 26-fold in the 3 weeks following transplantation (p = 0.02) and infiltration of the graft with CD8 T cells consistent with allo-immunity. In contrast, encapsulation in the Dual PEG capsules prevented sensitization to the allograft in all the recipients with no evidence of lymphocytic infiltration. In summary, the approach of hydrogel-based immunoisolation presents a minimally invasive and robust cell-therapy to restore hormonal balance in ovarian insufficiency. This report is the first to demonstrate the application of a tunable PEG-based hydrogel as an immunoisolator of allogeneic ovarian tissue to restore endocrine function in ovariectomized mice and prevent cell-mediated immune rejection in immune competent mice.

Genetic polymorphisms of the IL6 and NOD2 genes are risk factors for inflammatory reactions in leprosy.

The pathways that trigger exacerbated immune reactions in leprosy could be determined by genetic variations. Here, in a prospective approach, both genetic and non-genetic variables influencing the amount of time before the development of reactional episodes were studied using Kaplan-Meier survival curves, and the genetic effect was estimated by the Cox proportional-hazards regression model. In a sample including 447 leprosy patients, we confirmed that gender (male), and high bacillary clinical forms are risk factors for leprosy reactions. From the 15 single nucleotide polymorphisms (SNPs) at the 8 candidate genes genotyped (TNF/LTA, IFNG, IL10, TLR1, NOD2, SOD2, and IL6) we observed statistically different survival curves for rs751271 at the NOD2 and rs2069845 at the IL6 genes (log-rank p-values = 0.002 and 0.023, respectively), suggesting an influence on the amount of time before developing leprosy reactions. Cox models showed associations between the SNPs rs751271 at NOD2 and rs2069845 at IL6 with leprosy reactions (HRGT = 0.45, p = 0.002; HRAG = 1.88, p = 0.0008, respectively). Finally, IL-6 and IFN-γ levels were confirmed as high, while IL-10 titers were low in the sera of reactional patients. Rs751271-GT genotype-bearing individuals correlated (p = 0.05) with lower levels of IL-6 in sera samples, corroborating the genetic results. Although the experimental size may be considered a limitation of the study, the findings confirm the association of classical variables such as sex and clinical forms with leprosy, demonstrating the consistency of the results. From the results, we conclude that SNPs at the NOD2 and IL6 genes are associated with leprosy reactions as an outcome. NOD2 also has a clear functional pro-inflammatory link that is coherent with the exacerbated responses observed in these patients.

Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum.

Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates-a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably contributing to the immunopathogenesis of the disease.

Type 1 reaction in leprosy: a model for a better understanding of tissue immunity under an immunopathological condition.

Type 1 reaction (T1R) or reversal reaction is the leading cause of physical disabilities and deformities in leprosy. Leprosy patients, even after being considered cured and released from treatment, may suffer from reactional episodes for long periods of time. Early diagnosis is a great challenge for effectively treating and managing T1R. There is an urgent need to identify the most significant biomarkers to prevent recurrent T1R and to differentiate late T1R from relapse. T1R continues to be treated with corticosteroids and complications due to iatrogenic treatment remain frequent. This review aims to provide a framework from which to approach the great challenges that still persist in T1R management and debate key issues in order to reduce the distance between basic research and the clinic.

Mecanismos inatos associados à imunopatogênese da forma multibacilar e ao estabelecimento da reação reversa em pacientes com hanseníase

Os macrófagos lepromatosos têm um fenótipo regulatório semelhante a M2 que contribui para a imunossupressão observada na hanseníase multibacilar. Nosso estudo anterior demonstrou que a IL-10 pode modular tanto a expressão de indoleamina 2,3-dioxigenase (IDO) quanto a de CD163 em células de pacientes lepromatosos, favorecendo a persistência de Mycobacterium leprae através da indução de vias regulatórias e armazenamento de ferro. Aqui investigamos o fenótipo celular relacionado à patogênese da hanseníase lepromatosa (LL). O aumento da expressão gênica dos genes MSRA, PPARG e CD163, relacionados ao fenótipo M2, foi observado em células de lesão LL, acompanhada da expressão aumentada de hemoglobina (Hb), haptoglobina, heme oxigenase 1 e receptor de transferrina 1 quando comparada à hanseníase tuberculóide (BT). Também encontramos aumento dos depósitos de ferro e diminuição da expressão da ferroportina 1 exportadora de ferro, ambas características-chave dos macrófagos M1. As concentrações de hemoglobina estavam diminuídos no soro de pacientes LL. O estímulo com M. leprae induziu um aumento na expressão gênica de TFRC e IL10 em monócitos humanos. O estímulo com hemina foi capaz de aumentar a viabilidade de M. leprae por um mecanismo que envolve IDO. Em resumo este estudo demonstra que o ferro não somente regula a expressão de IDO, mas também a sua função como um importante elemento para a sobrevivência de M. leprae no hospedeiro. Adicionalmente, o estudo das lesões de pele dos pacientes lepromatosos que desenvolveram ou não episódios reacionais, demonstrou diferenças significativas de expressão de genes das vias de autofagia e inflamassoma ainda no momento do diagnóstico da hanseníase. Pacientes multibacilares que não desenvolveram reação reversa (RR) (grupo SR) apresentaram uma superexpressão dos genes da via da autofagia e um acúmulo de pro-Caspase 1 (p45), enquanto os pacientes que desenvolveram RR (grupo RR) apresentaram uma superexpressão dos genes da via dos inflamassomas e de Caspase 1 ativa (p10). A análise das vias estudadas pode revelar candidatos à biomarcadores de RR capazes de identificar o risco do desenvolvimento de reação meses antes dela ocorrer, permitindo uma maior vigilância desses doentes e talvez, futuramente, um tratamento profilático para evitar a ocorrência de RR em pacientes com hanseníase.

O envolvimento do ferro na modulação da resposta imune em pacientes multibacilares da hanseníase.

A forma lepromatosa da Hanseníase caracteriza-se pela baixa ou ausente resposta imune específica aos antígenos do Mycobacterium leprae e intensa proliferação bacilar. Dentre as células hospedeiras, os macrófagos parecem ter um papel crucial no direcionamento do perfil de resposta ao M. leprae. Muitas das vias de imunossupressão da resposta imune celular parecem estar envolvidas com o metabolismo do ferro e seus carreadores, como a expressão aumentada de IDO em macrófagos lepromatosos e de heme-oxigenase 1 em macrófagos M2. Anteriormente, o nosso grupo demonstrou que macrófagos de pacientes lepromatosos apresentam uma maior expressão do receptor scavenger CD163, quando comparados aos tuberculóides. O CD163 reconhece o complexo Hemoglobina-Haptoglobina e nós hipotetizamos que o M. leprae adaptou-se para aumentara expressão de CD163, a fim de aumentar estoques de ferro em macrófagos, contribuindo para sua persistência. Assim, o objetivo do presente trabalho foi investigar o envolvimento de ferro na imunopatogênese da hanseníase. Nós observamos que biópsias de lesão de pele de pacientes lepromatosos apresentam uma maior expressão de proteínas relacionadas à captação e metabolismo de ferro, assim como uma maior deposição de ferro na forma de ferritina e hemossiderina nos macrófagos espumosos, onde se localizam os bacilos. A adição de ferro exógeno, na forma de tratamento com sulfato ferroso, foi capaz de reduzir a expressão e atividade de indoleamina 2,3-dioxigenase induzida pelo M. leprae em monócitos de doadores saudáveis. A adição de ferro livre também foi capaz de modular a produção de citocinas, aumentando a produção de IL-12p70 e IL-10 em culturas de monócitos, e de IL-6 em culturas de células mononucleares do sangue periférico. O aumento dos receptores CD163 e receptor de transferrina 1 em macrófagos lepromatosos foram associados com o aumento dos estoques de ferro em biópsias de pele de pacientes lepromatosos. Em adição, o tratamento com ferro livre foi capaz aumentar vias proinflamatórias em leucócitos estimulados com M. leprae. Isto indica que a homeostase do ferro no hospedeiro no momento da infecção é importante para a definição do desfecho da Hanseníase, e que o ferro pode ter um papel duplo dependendo do momento e da forma que este está apresentado, podendo criar um ambiente favorável para a micobactéria ou induzindo a produção de citocinas próinflamatórias que podem contribuir para ativação de vias antimicrobianas em macrófagos.

Mecanismos inatos associados à imunopatogênese da forma multibacilar e ao estabelecimento da reação reversa em pacientes com hanseníase / Inborn mechanisms associated with the immunopathogenesis of the multibacillary form and the establishment of the reverse reaction in patients with leprosy

Os macrófagos lepromatosos têm um fenótipo regulatório semelhante a M2 que contribui para a imunossupressão observada na hanseníase multibacilar. Nosso estudo anterior demonstrou que a IL-10 pode modular tanto a expressão de indoleamina 2,3-dioxigenase (IDO) quanto a de CD163 em células de pacientes lepromatosos, favorecendo a persistência de Mycobacterium leprae através da indução de vias regulatórias e armazenamento de ferro. Aqui investigamos o fenótipo celular relacionado à patogênese da hanseníase lepromatosa (LL). O aumento da expressão gênica dos genes MSRA, PPARG e CD163, relacionados ao fenótipo M2, foi observado em células de lesão LL, acompanhada da expressão aumentada de hemoglobina (Hb), haptoglobina, heme oxigenase 1 e receptor de transferrina 1 quando comparada à hanseníase tuberculóide (BT). Também encontramos aumento dos depósitos de ferro e diminuição da expressão da ferroportina 1 exportadora de ferro, ambas características-chave dos macrófagos M1. As concentrações de hemoglobina estavam diminuídos no soro de pacientes LL. O estímulo com M. leprae induziu um aumento na expressão gênica de TFRC e IL10 em monócitos humanos. O estímulo com hemina foi capaz de aumentar a viabilidade de M. leprae por um mecanismo que envolve IDO. Em resumo este estudo demonstra que o ferro não somente regula a expressão de IDO, mas também a sua função como um importante elemento para a sobrevivência de M. leprae no hospedeiro

Adicionalmente, o estudo das lesões de pele dos pacientes lepromatosos que desenvolveram ou não episódios reacionais, demonstrou diferenças significativas de expressão de genes das vias de autofagia e inflamassoma ainda no momento do diagnóstico da hanseníase. Pacientes multibacilares que não desenvolveram reação reversa (RR) (grupo SR) apresentaram uma superexpressão dos genes da via da autofagia e um acúmulo de pro-Caspase 1 (p45), enquanto os pacientes que desenvolveram RR (grupo RR) apresentaram uma superexpressão dos genes da via dos inflamassomas e de Caspase 1 ativa (p10). A análise das vias estudadas pode revelar candidatos à biomarcadores de RR capazes de identificar o risco do desenvolvimento de reação meses antes dela ocorrer, permitindo uma maior vigilância desses doentes e talvez, futuramente, um tratamento profilático para evitar a ocorrência de RR em pacientes com hanseníase