RESUMO
BACKGROUND: Since macrophages are one of the major cell types involved in the Mycobacterium leprae immune response, roles of the M1 and M2 macrophage subpopulations have been well defined. However, the role of M4 macrophages in leprosy or other infectious diseases caused by mycobacteria has not yet been clearly characterized. This study aimed to investigate the presence and potential role of M4 macrophages in the immunopathology of leprosy. METHODS: We analyzed the presence of M4 macrophage markers (CD68, MRP8, MMP7, IL-6, and TNF-α) in 33 leprosy skin lesion samples from 18 patients with tuberculoid leprosy and 15 with lepromatous leprosy by immunohistochemistry. RESULTS: The M4 phenotype was more strongly expressed in patients with the lepromatous form of the disease, indicating that this subpopulation is less effective in the elimination of the bacillus and consequently is associated with the evolution to one of the multibacillary clinical forms of infection. CONCLUSION: M4 macrophages are one of the cell types involved in the microbial response to M. leprae and probably are less effective in controlling bacillus replication, contributing to the evolution to the lepromatous form of the disease.
Assuntos
Hanseníase/metabolismo , Macrófagos/metabolismo , Mycobacterium leprae/imunologia , Dermatopatias/metabolismo , Pele/metabolismo , Adulto , Biomarcadores/metabolismo , Brasil , Feminino , Humanos , Imuno-Histoquímica , Hanseníase/imunologia , Hanseníase/patologia , Hanseníase Virchowiana/imunologia , Hanseníase Virchowiana/metabolismo , Hanseníase Virchowiana/patologia , Hanseníase Tuberculoide/imunologia , Hanseníase Tuberculoide/metabolismo , Hanseníase Tuberculoide/patologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Pele/imunologia , Pele/patologia , Dermatopatias/imunologia , Dermatopatias/microbiologia , Dermatopatias/patologiaRESUMO
Mast cells (MCs) have important immunoregulatory roles in skin inflammation. Annexin A1 (ANXA1) is an endogenous anti-inflammatory protein that can be expressed by mast cells, neutrophils, eosinophils, monocytes, epithelial and T cells. This study investigated MCs heterogeneity and ANXA1 expression in human dermatoses with special emphasis in leprosy. Sixty one skin biopsies from 2 groups were investigated: 40 newly diagnosed untreated leprosy patients (18 reaction-free, 11 type 1 reaction/T1R, 11 type 2 reaction/T2R); 21 patients with other dermatoses. Tryptase/try+ and chymase/chy + phenotypic markers and toluidine blue stained intact/degranulated MC counts/mm2 were evaluated. Try+/chy+ MCs and ANXA1 were identified by streptavidin-biotin-peroxidase immunostaining and density was reported. In leprosy, degranulated MCs outnumbered intact ones regardless of the leprosy form (from tuberculoid/TT to lepromatous/LL), leprosy reactions (reactional/reaction-free) and type of reaction (T1R/T2R). Compared to other dermatoses, leprosy skin lesions showed lower numbers of degranulated and intact MCs. Try+ MCs outnumbered chy+ in leprosy lesions (reaction-free/reactional, particularly in T2R), but not in other dermatoses. Compared to other dermatoses, ANXA1 expression, which is also expressed in mast cells, was higher in the epidermis of leprosy skin lesions, independently of reactional episode. In leprosy, higher MC degranulation and differential expression of try+/chy+ subsets independent of leprosy type and reaction suggest that the Mycobacterium leprae infection itself dictates the inflammatory MCs activation in skin lesions. Higher expression of ANXA1 in leprosy suggests its potential anti-inflammatory role to maintain homeostasis preventing tissue and nerve damage.
Assuntos
Anexina A1/biossíntese , Anexina A1/imunologia , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/metabolismo , Hanseníase/imunologia , Hanseníase/metabolismo , Mastócitos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Brasil , Quimases/metabolismo , Epiderme/imunologia , Epiderme/patologia , Feminino , Humanos , Hanseníase/patologia , Hanseníase Virchowiana/metabolismo , Hanseníase Tuberculoide/metabolismo , Masculino , Mastócitos/patologia , Pessoa de Meia-Idade , Mycobacterium leprae/imunologia , Mycobacterium leprae/patogenicidade , Pele/patologia , Dermatopatias/metabolismo , Dermatopatias/patologia , Triptases/metabolismo , Adulto JovemRESUMO
Mycobacterium leprae causes leprosy, a dermatoneurological disease which affects the skin and peripheral nerves. One of several cellular structures affected during M. leprae infection is the endoplasmic reticulum (ER). Infection by microorganisms can result in ER stress and lead to the accumulation of unfolded or poorly folded proteins. To restore homeostasis in the cell, the cell induces a series of signaling cascades known as the unfolded protein response called UPR (unfolded protein response). The present work is aimed at investigating the in situ expression of these markers in cutaneous lesions of clinical forms of leprosy and establish possible correlation expression patterns and types of lesion. A total of 43 samples from leprosy patients were analyzed by immunohistochemistry with monoclonal antibodies against GRP78/BiP, PERK, IRE1α, and ATF6. A statistically significant difference between the indeterminate, tuberculoid, and lepromatous clinical forms was detected, with high expression of GRP78/BiP, PERK, IRE1α, and ATF6 in tuberculoid forms (TT) when compared to lepromatous leprosy (LL) and indeterminate (I) leprosy. These results represent the first evidence of ER stress in samples of skin lesions from leprosy patients. We believe that they will provide better understanding of the complex pathogenesis of the disease and facilitate further characterization of the cascade of molecular events elicited during infection.
Assuntos
Biomarcadores/metabolismo , Estresse do Retículo Endoplasmático , Hanseníase Virchowiana/diagnóstico , Hanseníase Tuberculoide/diagnóstico , Fator 6 Ativador da Transcrição/metabolismo , Diagnóstico Diferencial , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Hanseníase/classificação , Hanseníase/metabolismo , Hanseníase Virchowiana/metabolismo , Hanseníase Tuberculoide/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Regulação para Cima , eIF-2 Quinase/metabolismoRESUMO
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.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/fisiologia , Ferro/fisiologia , Mycobacterium leprae/fisiologia , Adulto , Feminino , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Ferro/metabolismo , Hanseníase Virchowiana/metabolismo , Hanseníase Virchowiana/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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 FeSO 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.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Immunoblotting , Hanseníase Virchowiana/metabolismo , Hanseníase Virchowiana/microbiologia , Técnicas Imunoenzimáticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Indolamina-Pirrol 2,3,-Dioxigenase/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Ferro/fisiologia , Ferro/metabolismo , Mycobacterium leprae/fisiologia , Mycobacterium leprae/metabolismoRESUMO
Leprosy is an infectious-contagious disease whose clinical evolution depends on the immune response pattern of the host. Adhesion molecules and leukocyte migration from blood to tissue are of the utmost importance for the recognition and elimination of infectious pathogens. Selectins are transmembrane glycoproteins that share a similar structural organization and can be divided into three types according to their site of expression. The biopsies were cut into 5µm thick sections and submitted to immunohistochemistry using antibodies against E-selectin and P-selectin. The number of E-selectin-positive cells was significantly higher in the tuberculoid form than in the lepromatous form. The immunostaining pattern of P-selectin differed from that of E-selectin. Analysis showed a larger number of endothelial cells expressing CD62P in the lepromatous form compared to the tuberculoid form. The presence of these adhesins in the endothelium contributing to or impairing the recruitment of immune cells to inflamed tissue and consequently influences the pattern of immune response and the clinical presentation of the disease.
Assuntos
Selectina E/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Hanseníase Virchowiana/metabolismo , Hanseníase Tuberculoide/metabolismo , Selectina-P/metabolismo , Pele/metabolismo , Moléculas de Adesão Celular , Endotélio Vascular/citologia , Humanos , Imuno-HistoquímicaRESUMO
Histoid leprosy is a rare multibacillary form that presents with disseminated papule-nodular cutaneous lesions. To study the inflammatory infiltrate of the histoid form and to compare it with other lepromatous forms, we performed histological and immunohistochemical analysis on skin biopsies. Fifteen patients were included for histopathological analysis (10 histoid and five lepromatous) via the haematoxylin-eosin and Ziehl-Neelsen-Faraco stains. Thus, immunohistochemical techniques using immunoperoxidase assay were performed for: anti-BCG, anti-M. leprae, anti-CD8, anti-CD3, anti-CD20, anti-S100, anti-CD1a, anti-CD68 and antivimentin. Spindle cells were present in all histoid patients. A pseudocapsule was observed in half of both studied forms. A comparison using the Ziehl-Neelsen-Faraco stain to evaluate anti-BCG and anti-M.leprae showed no major differences. The CD3+ cells were more pronounced in the histoid form than the lepromatous form. There was greater immunoreactivity toward CD8+ cells in the histoid form, as well as the CD20+ cell count. A similar count of S100+ cells in the epidermis of both leprosy forms was observed. There was a slight increase of dendritic cells in the histoid patients in the superficial and deep dermis. For CD1a marker, we observed expression in the epidermis and superficial dermis in both forms. A diffuse and intense infiltrate of CD68+ cells was also observed in the histoid and lepromatous forms. The high positivity for vimentin did not allow for a positive cell count. We concluded that the activation of both the cellular and humoral response is more pronounced in the histoid form because the T and B cells showed greater infiltration than those in the lepromatous form. The activation of dendritic and Langerhans cells is similar in both forms. The spindle cells likely belong to the macrophage population, thus maintaining phagocytic ability. The quantities of pseudocapsules and bacilli are similar and cannot serve as criteria for diagnosis.
Assuntos
Hanseníase Virchowiana/metabolismo , Hanseníase Virchowiana/patologia , Hanseníase Multibacilar/metabolismo , Hanseníase Multibacilar/patologia , Adulto , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Pele/química , Pele/metabolismo , Pele/patologiaRESUMO
OBJECTIVE: We investigated the in vitro and skin lesions production of cytokines in non-treated borderline tuberculoid (BT) and borderline lepromatous (BL) patients. PATIENTS AND METHODS: Seven untreated, non-reactional BT patients and 12 untreated, non-reactional BL patients were studied. Levels of the cytokines IFN-gamma, IL-10, TGF-beta1 and TNF-alpha were measured in supernantant of peripheral blood mononuclear cells (PBMC) cultures, stimulated with specific M. leprae antigen (sonicated and whole). The cytokines iNOS, IL-10 and TGF-beta1 were detected by immunohistochemistry in skin biopsies. RESULTS: BT patients produced higher levels of IFN-gamma than BL patients; iNOS expression in skin lesions was also higher in BT patients. TGF-beta1 was detected in more cells in BL patients; IL-10 expression was similar in both groups. There was a negative correlation between iNOS and TGF-beta1 expression in skin biopsies, positive correlation between TGF-beta1 in skin lesions and bacillary index, as well as positive correlation between iNOS detected in skin biopsies and PBMC IFN-gamma production. CONCLUSIONS: The BT patients had a mainly a Th1-profile of cytokines in their skin lesions and BL patients had a Th2 profile.
Assuntos
Citocinas/metabolismo , Hanseníase Dimorfa/metabolismo , Hanseníase Virchowiana/metabolismo , Hanseníase Tuberculoide/metabolismo , Biomarcadores/metabolismo , Biópsia , Brasil/epidemiologia , Feminino , Humanos , Imuno-Histoquímica , Interferon gama/metabolismo , Interleucina-10/metabolismo , Hanseníase Dimorfa/epidemiologia , Hanseníase Virchowiana/epidemiologia , Hanseníase Tuberculoide/epidemiologia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo II/metabolismo , Estatísticas não Paramétricas , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Tegumentary leishmaniasis and leprosy display similar spectra of disease phenotypes, which are dependent on cell-mediated immunity to specific antigens. Diffuse cutaneous leishmaniasis and lepromatous leprosy represent the anergic end of the spectrum, whereas mucocutaneous leishmaniasis and tuberculoid leprosy are associated with marked antigen-specific cellular immune response. METHODS: We characterized and compared the cell-mediated response to Leishmania and Mycobacterium leprae antigens in a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, which are at opposite ends of the immunopathological spectra of these diseases. This was done by performance of skin tests and by assessment of the cell proliferation and cytokine production of peripheral blood mononuclear cells (PBMCs). RESULTS: Strong skin-test reactions and PBMC proliferation were observed in response to Leishmania antigens but not to M. leprae antigens. The stimulation of PBMCs with Leishmania and M. leprae antigens induced comparable levels of tumor necrosis factor- alpha , interleukin-5, and interleukin-10. However, the interferon- gamma response to Leishmania antigens was remarkably high, and that to M. leprae antigens was almost nil. CONCLUSIONS: We found that concomitant leprosy and tegumentary leishmaniasis can produce opposite polar forms associated, respectively, with absent or exaggerated cell-mediated immune responses to each pathogen. This suggests that independent mechanisms influence the clinical outcome of each infection. Moreover, interferon- gamma appears to play a major role in the clinical expression of these intracellular infections.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Protozoários/imunologia , Interferon gama/metabolismo , Leishmaniose Mucocutânea/imunologia , Hanseníase Virchowiana/imunologia , Animais , Antiprotozoários/uso terapêutico , Proliferação de Células , Humanos , Leishmania/imunologia , Leishmaniose Mucocutânea/tratamento farmacológico , Leishmaniose Mucocutânea/metabolismo , Hansenostáticos/uso terapêutico , Hanseníase Virchowiana/tratamento farmacológico , Hanseníase Virchowiana/metabolismo , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Testes CutâneosRESUMO
The effects of reactional episodes on the cutaneous nerve fibers of leprosy patients was assessed in six patients (three with reversal reactions and three with erythema nodosum leprosum). Cryosections of cutaneous biopsy of reactional lesions taken during the episode and of another sample during the remission period were immunostained with anti-NGFr and anti-PGP 9.5 (indirect immunofluorescence). We found no significant statistical difference in the number of NGFr- and PGP 9.5-positive fibers between the reactional and post-reactional groups. A significant difference was detected between the number of NGFr and PGP 9.5-stained fibers inside of the reactional group of biopsy cryosections but this difference was ascribed to the distinct aspects of the nerve fibers displayed whether stained with anti-NGFr or with anti-PGP 9.5; NGFr-positive branches looked larger and so interpreted as containing more fibers. In addition, a substantial number NGFr-positive fibers were PGP 9.5-negative. No differences in the number of stained fibers among the distinct cutaneous regions examined (epidermis + upper dermis, mid and deep dermis) was detected. In conclusion, the number of PGP- and NGFr-positive fibers were not significantly different in the reactional and post-reactional biopsies in the present study. NGFr-staining of the nerve fibers is different from their PGP-imunoreactivity and the evaluation of the nerve fiber status on an innervated target organ should be carried out choosing markers for both components of nerve fibers (Schwann cells and axons).