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.

Effect of apoptotic cell recognition on macrophage polarization and mycobacterial persistence.

Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-ß) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-ß. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection.

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.

Autophagy Impairment Is Associated With Increased Inflammasome Activation and Reversal Reaction Development in Multibacillary Leprosy.

Leprosy reactions are responsible for incapacities in leprosy and represent the major cause of permanent neuropathy. The identification of biomarkers able to identify patients more prone to develop reaction could contribute to adequate clinical management and the prevention of disability. Reversal reaction may occur in unstable borderline patients and also in lepromatous patients. To identify biomarker signature profiles related with the reversal reaction onset, multibacillary patients were recruited and classified accordingly the occurrence or not of reversal reaction during or after multidrugtherapy. Analysis of skin lesion cells at diagnosis of multibacillary leprosy demonstrated that in the group that developed reaction (T1R) in the future there was a downregulation of autophagy associated with the overexpression of TLR2 and MLST8. The autophagy impairment in T1R group was associated with increased expression of NLRP3, caspase-1 (p10) and IL-1ß production. In addition, analysis of IL-1ß production in serum from multibacillary patients demonstrated that patients who developed reversal reaction have significantly increased concentrations of IL-1ß at diagnosis, suggesting that the pattern of innate immune responses could predict the reactional episode outcome. In vitro analysis demonstrated that the blockade of autophagy with 3-methyladenine (3-MA) in Mycobacterium leprae-stimulated human primary monocytes increased the assembly of NLRP3 specks assembly, and it was associated with an increase of IL-1ß and IL-6 production. Together, our data suggest an important role for autophagy in multibacillary leprosy patients to avoid exacerbated inflammasome activation and the onset of reversal reaction.

Indoleamine 2,3-dioxygenase and iron are required for Mycobacterium leprae survival.

Our previous study has demonstrated that IL-10 may modulate both indoleamine 2,3-dioxygenase (IDO) and CD163 expression in lepromatous leprosy (LL) cells, favoring Mycobacterium leprae persistence through induction of regulatory pathways and iron storage. Here, we observed that in LL lesion cells there is an increase in the expression of proteins involved in iron metabolism such as hemoglobin (Hb), haptoglobin, heme oxygenase 1 and transferrin receptor 1 (TfR1) when compared to tuberculoid leprosy (BT) cells. We also found increased iron deposits and diminished expression of the iron exporter ferroportin 1 in LL lesion cells. Hemin, but not FeSO4 stimulation, was able to enhance M. leprae viability by a mechanism that involves IDO. Analysis of cell phenotype in lesions demonstrated a predominance of M2 markers in LL when compared with BT lesion cells. A positive correlation between CD163 and PPARG with the bacillary index (BI) was observed. In contrast, TNF, STAT1 and CSF2 presented a negative correlation with the BI. In summary, this study demonstrates that iron may regulate IDO expression by a mechanism that involves IL-10, which may contribute for the predominance of M2-like phenotype in LL lesions that favors the phagocytosis and maintenance of M. leprae in host cells.

Inflammatory Cytokines Are Involved in Focal Demyelination in Leprosy Neuritis.

Mycobacterium leprae (ML) infection causes nerve damage that often leads to permanent loss of cutaneous sensitivity and limb deformities, but understanding of the pathogenesis of leprous neuropathy that would lead to more effective treatments is incomplete. We studied reactional leprosy patients with (n = 9) and without (n = 8) acute neuritis. Nerve conduction studies over the course of the reactional episode showed the findings of demyelination in all patients with neuritis. Evaluation of patient sera revealed no correlation of the presence of antibodies against gangliosides and the clinical demyelination. In nerve biopsies of 3 patients with neuritis, we identified tumor necrosis factor (TNF), TNF receptors, and TNF-converting enzyme in Schwann cells (SCs) using immunofluorescence. To elucidate immunopathogenetic mechanisms, we performed experiments using a human SC line. ML induced transmembrane TNF and TNF receptor 1 expression in the SCs; TNF also induced interleukin (IL)- 6 and IL-8 production by the SCs; and ML induced IL-23 secretion, indicating involvement of this previously unrecognized factor in leprosy nerve damage. These data suggest that ML may contribute to TNF-mediated inflammation and focal demyelination by rendering SCs more sensitive to TNF within the nerves of patients with leprous neuropathy.

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.

Avaliação dos marcadores fenotípicos e funcionais da reação reversa na hanseníase / Evaluation of phenotypic and functional markers in leprosy`s type 1 reaction

Na hanseníase, a reação reversa (RR) é considerada um fenômeno de reativação imune, no qual uma resposta inflamatória abrupta se inicia, comprometendo a pele e os nervos periféricos.A RR ocorre ao longo do espectro da hanseníase, sendo descrita, inclusive, nas formas anérgicas lepromatosas (LL) subpolares. É sugerido que nas formas reacionais, células imunes da pele como macrófagos e células dendríticas, se tornam ativadas espontaneamente e iniciam uma resposta imune contra componentes do Mycobacterium leprae (ML), o que levaria a quebrada imunossupressão pré-existente. O objetivo desse estudo é caracterizar as populaçõescelulares que constituem as lesões de pele L-lep (BL e LL) antes e no início da RR, assim como,determinar a programação genética envolvida na quebra da anergia tecidual nesse grupo durante esse episódio. O início da RR altera drasticamente a organização e morfologia da lesãoL-lep, levando ao aparecimento de novas estruturas e tipos celulares, como células epitelioides,granulomas e uma grande diversidade fenotípica de macrófagos e células dendríticas. Omarcador de células dendríticas plasmocitoides, CD123, foi mais expresso em lesões de pele RRdo que no tecido não reacional, com a população CD123+ exibindo tanto marcadores fenotípicosde macrófagos quanto de células dendríticas. Por sua vez, o ML pôde induzir a expressão dessa molécula in vitro.

A análise de expressão gênica mostrou um aumento dos níveis de RNA mensageiro da interleucina-3 (IL-3), fator estimulador de colônias de macrófagos (M-CSF), fatorestimulador de colônias de macrófagos e granulócitos (GM-CSF), fator de necrose tumoral-alfa(TNFalfa), interferon-gama (IFN-gama), indoleamina-2,3-dioxigenase (IDO), interleucina-15 (IL-15), receptorde vitamina D (VDR), quimiocina CXCL10 e receptor Toll-like 2 (TLR2) nas lesões RR quandoem comparação com o grupo L-lep. Em síntese, nosso estudo demonstrou que o microambienteda lesão RR favorece a diferenciação e plasticidade celular, além de exibir uma variedade depopulações mielomonocíticas, destacando o papel das células CD123+ e de um eixo de ativaçãodependente de IL-3 durante a RR. (AU)

In leprosy, type 1 reaction (T1R) is considered a Th1 immune reactivation in which a suddeninflammatory response takes place that compromises the skin and peripheral nerves. T1R occursacross the leprosy clinical spectrum and has even been described in the anergic subpolarlepromatous (LL) forms. It is hypothesized that in the T1R forms, skin immune cells such asmacrophages and dendritic cells (DCs) become activated, which in turn could initiate a localinnate immune response against the existing Mycobacterium leprae (ML) components,overwhelming the predominant immunosuppressive state. The aim of the present study is tocharacterize the skin cellular populations that constitute the BL and LL (L-lep) skin lesion beforeand during the T1R episode, as well as to determine the gene programming involved in thedisruption of the tissue immunosuppression brought about by this phenomenon. The outset ofT1R drastically alters the morphological landscape of the LL skin lesion, leading to theappearance of new structures and cell types such as epithelioid granulomas and a wide variety ofmacrophagic and DC phenotypes. The plasmacytoid DC marker, CD123, was more intenselyexpressed in T1R skin lesions than in non-reactional tissue, with CD123+ cells exhibiting bothmacrophagic and DC phenotypic markers. In turn, ML, but not TNF-alpha, was able to increaseCD123 expression while gene expression analyses demonstrated IL-3, M-CSF, GM-CSF, TNFalpha,IFN-gama, IDO, IL-15, VDR, CXCL10, and TLR2 up regulation in the T1R lesions in comparison to thenon-reactional group. In summary, our study showed that the T1R lesion environment favors celldifferentiation and plasticity in addition to displaying a high diversity of myelomonocyticpopulations and highlighting the role of CD123+ cells and the IL-3 axis during the progression ofT1R. (AU)

Avaliação dos marcadores fenotípicos e funcionais da reação reversa na hanseníase / Evaluation of phenotypic and functional markers in leprosy`s type 1 reaction

Na hanseníase, a reação reversa (RR) é considerada um fenômeno de reativação imune, no qual uma resposta inflamatória abrupta se inicia, comprometendo a pele e os nervos periféricos.A RR ocorre ao longo do espectro da hanseníase, sendo descrita, inclusive, nas formas anérgicas lepromatosas (LL) subpolares. É sugerido que nas formas reacionais, células imunes da pele como macrófagos e células dendríticas, se tornam ativadas espontaneamente e iniciam uma resposta imune contra componentes do Mycobacterium leprae (ML), o que levaria a quebrada imunossupressão pré-existente. O objetivo desse estudo é caracterizar as populaçõescelulares que constituem as lesões de pele L-lep (BL e LL) antes e no início da RR, assim como,determinar a programação genética envolvida na quebra da anergia tecidual nesse grupo durante esse episódio. O início da RR altera drasticamente a organização e morfologia da lesãoL-lep, levando ao aparecimento de novas estruturas e tipos celulares, como células epitelioides,granulomas e uma grande diversidade fenotípica de macrófagos e células dendríticas. Omarcador de células dendríticas plasmocitoides, CD123, foi mais expresso em lesões de pele RRdo que no tecido não reacional, com a população CD123+ exibindo tanto marcadores fenotípicosde macrófagos quanto de células dendríticas. Por sua vez, o ML pôde induzir a expressão dessa molécula in vitro...

A análise de expressão gênica mostrou um aumento dos níveis de RNA mensageiro da interleucina-3 (IL-3), fator estimulador de colônias de macrófagos (M-CSF), fatorestimulador de colônias de macrófagos e granulócitos (GM-CSF), fator de necrose tumoral-alfa(TNFalfa), interferon-gama (IFN-gama), indoleamina-2,3-dioxigenase (IDO), interleucina-15 (IL-15), receptorde vitamina D (VDR), quimiocina CXCL10 e receptor Toll-like 2 (TLR2) nas lesões RR quandoem comparação com o grupo L-lep. Em síntese, nosso estudo demonstrou que o microambienteda lesão RR favorece a diferenciação e plasticidade celular, além de exibir uma variedade depopulações mielomonocíticas, destacando o papel das células CD123+ e de um eixo de ativaçãodependente de IL-3 durante a RR...

In leprosy, type 1 reaction (T1R) is considered a Th1 immune reactivation in which a suddeninflammatory response takes place that compromises the skin and peripheral nerves. T1R occursacross the leprosy clinical spectrum and has even been described in the anergic subpolarlepromatous (LL) forms. It is hypothesized that in the T1R forms, skin immune cells such asmacrophages and dendritic cells (DCs) become activated, which in turn could initiate a localinnate immune response against the existing Mycobacterium leprae (ML) components,overwhelming the predominant immunosuppressive state. The aim of the present study is tocharacterize the skin cellular populations that constitute the BL and LL (L-lep) skin lesion beforeand during the T1R episode, as well as to determine the gene programming involved in thedisruption of the tissue immunosuppression brought about by this phenomenon. The outset ofT1R drastically alters the morphological landscape of the LL skin lesion, leading to theappearance of new structures and cell types such as epithelioid granulomas and a wide variety ofmacrophagic and DC phenotypes. The plasmacytoid DC marker, CD123, was more intenselyexpressed in T1R skin lesions than in non-reactional tissue, with CD123+ cells exhibiting bothmacrophagic and DC phenotypic markers. In turn, ML, but not TNF-alpha, was able to increaseCD123 expression while gene expression analyses demonstrated IL-3, M-CSF, GM-CSF, TNFalpha,IFN-gama, IDO, IL-15, VDR, CXCL10, and TLR2 up regulation in the T1R lesions in comparison to thenon-reactional group. In summary, our study showed that the T1R lesion environment favors celldifferentiation and plasticity in addition to displaying a high diversity of myelomonocyticpopulations and highlighting the role of CD123+ cells and the IL-3 axis during the progression ofT1R...

Avaliação dos marcadores fenotípicos e funcionais da reação reversa na hanseníase

Na hanseníase, a reação reversa (RR) é considerada um fenômeno de reativação imune, no qual uma resposta inflamatória abrupta se inicia, comprometendo a pele e os nervos periféricos. A RR ocorre ao longo do espectro da hanseníase, sendo descrita, inclusive, nas formas anérgicas lepromatosas (LL) subpolares. É sugerido que nas formas reacionais, células imunes da pele como macrófagos e células dendríticas, se tornam ativadas espontaneamente e iniciam uma resposta imune contra componentes do Mycobacterium leprae (ML), o que levaria a quebra da imunossupressão pré-existente. O objetivo desse estudo é caracterizar as populações celulares que constituem as lesões de pele L-lep (BL e LL) antes e no início da RR, assim como, determinar a programação genética envolvida na quebra da anergia tecidual nesse grupo durante esse episódio. O início da RR altera drasticamente a organização e morfologia da lesão L-lep, levando ao aparecimento de novas estruturas e tipos celulares, como células epitelioides, granulomas e uma grande diversidade fenotípica de macrófagos e células dendríticas. O marcador de células dendríticas plasmocitoides, CD123, foi mais expresso em lesões de pele RR do que no tecido não reacional, com a população CD123+ exibindo tanto marcadores fenotípicos de macrófagos quanto de células dendríticas. Por sua vez, o ML pôde induzir a expressão dessa molécula in vitro A análise de expressão gênica mostrou um aumento dos níveis de RNA mensageiro da interleucina-3 (IL-3), fator estimulador de colônias de macrófagos (M-CSF), fator estimulador de colônias de macrófagos e granulócitos (GM-CSF), fator de necrose tumoral-\03B1 (TNF\03B1), interferon-\03B3 (IFN-\03B3), indoleamina-2,3-dioxigenase (IDO), interleucina-15 (IL-15), receptor de vitamina D (VDR), quimiocina CXCL10 e receptor Toll-like 2 (TLR2) nas lesões RR quando em comparação com o grupo L-lep. Em síntese, nosso estudo demonstrou que o microambiente da lesão RR favorece a diferenciação e plasticidade celular, além de exibir uma variedade de populações mielomonocíticas, destacando o papel das células CD123+ e de um eixo de ativação dependente de IL-3 durante a RR

Mycobacterium leprae e o efeito do TNF_ na ativação de células de Schwann humanas

A hanseníase é uma das doenças mais antigas a acometer o homem e tem como agente etiológico o Mycobacterium leprae (ML), um patógeno intracelular obrigatório que infecta principalmente, macrófagos e células de Schwann (CS). As CS estão envolvidas na imunomodulação da lesão neurológica causada pelo ML, podendo, assim, desencadear uma resposta inflamatória frente à presença da bactéria. Entre as citocinas que participam dessa resposta está o Fator de Necrose Tumoral (TNF- ), molécula envolvida na patogênese de diversas doenças que acometem os sistemas nervosos central (SNC) e periférico (SNP). Foi descrito recentemente que o TNF- pode atuar como um mediador autócrino na lesão neural, sendo capaz de ativar CS na ausência do patógeno. Nesse trabalho foi avaliada a contribuição do TNF- e do ML para a injúria nervosa da hanseníase, através da análise da participação do efeito autócrino dessa citocina na resposta a infecção pelo ML e no processo de ativação sustentada das CS, utilizando a linhagem humana ST88-14. Nossos resultados sugerem que no dano neural da hanseníase, a infecção pela micobactéria pode contribuir para ativação sustentada das CS, ativando vias de sinalização com participação de ERK 1/2, e induzindo a produção de TNF- , que por sua vez, promove seus diversos efeitos através da interação com outras células do espaço endoneural, não sendo, imediatamente secretada. Além disso, foi abservada a participação do mecanismo de retroalimentação positiva dessa citocina na ativação dessas células, que pode não só aumentar os níveis dessa molécula no meio, como também a secreção das citocinas pró-inflamatórias IL-1 , IL-6 e IL-8, que cooperam com a resposta imune e com o estado de ativação prolongado das CS.