Macrophage immunophenotypes in Jorge Lobo's disease and lepromatous leprosy- A comparative study.

Jorge Lobo's disease (JLD) and lepromatous leprosy (LL) share several clinical, histological and immunological features, especially a deficiency in the cellular immune response. Macrophages participate in innate and adaptive inflammatory immune responses, as well as in tissue regeneration and repair. Macrophage function deficiency results in maintenance of diseases. M1 macrophages produce pro-inflammatory mediators and M2 produce anti-inflammatory cytokines. To better understand JLD and LL pathogenesis, we studied the immunophenotype profile of macrophage subtypes in 52 JLD skin lesions, in comparison with 16 LL samples, using a panmacrophage (CD68) antibody and selective immunohistochemical markers for M1 (iNOS) and M2 (CD163, CD204) responses, HAM56 (resident/fixed macrophage) and MAC 387 (recently infiltrating macrophage) antibodies. We found no differences between the groups regarding the density of the CD163, CD204, MAC387+ immunostained cells, including iNOS, considered a M1 marker. But HAM56+ cell density was higher in LL samples. By comparing the M2 and M1 immunomarkers in each disease separately, some other differences were found. Our results reinforce a higher M2 response in JLD and LL patients, depicting predominant production of anti-inflammatory cytokines, but also some distinction in degree of macrophage activation. Significant amounts of iNOS + macrophages take part in the immune milieu of both LL and JLD samples, displaying impaired microbicidal activity, like alternatively activated M2 cells.

T-Regulatory Cells in Erythema Nodosum Leprosum: An Immunohistochemical and Image Morphometric Study.

ABSTRACT: Erythema nodosum leprosum (ENL) occurs as an immune-inflammatory complication of multibacillary leprosy (MBL), precipitated by an interaction between the host, bacilli, and the environment. This complication often causes significant morbidity due to systemic involvement and needs to be treated aggressively. T-regulatory cells (T-regs) are the immunomodulatory subset of T cells that are hypothesized to play a role in ENL. We have performed immunohistochemistry for FoxP3 (T-reg), CD3 (pan-T), CD4 (helper T), and CD8 (cytotoxic T) on 50 biopsy-proven cases of ENL along with 84 biopsy-proven cases of paucibacillary leprosy (PBL) (n = 49) and MBL (n = 35). Image morphometry was applied to objectively assess the relative preponderance of these subsets of T cells. The area fraction of T-regs showed a trend of reduction from PBL to MBL to ENL (P = 0.068), whereas the FoxP3:CD3 (T-reg: pan-T) ratio showed a significant reduction across these groups (P = 0.023). However, there was no significant difference of T-regs or FoxP3:CD3 ratio between MBL and ENL. The T-regs showed a significant positive correlation (P = 0.007) with the cytotoxic T cells in the skin biopsy. The presence of dermal eosinophils in ENL showed a trend association with the FoxP3:CD3 ratio (P = 0.05). Various histopathological parameters including epidermal spongiosis, dermal stromal edema, dermal ill-formed granuloma, and the presence of bacilli within the endothelium and vascular smooth muscle correlated with various T-cell subsets. Our study, one of the largest on this topic, objectively assessed the role of T-regs in the spectrum of leprosy. Nevertheless, the precipitation of ENL from MBL is probably not associated with the T-reg subset alone.

Association of IL-10 Gene Polymorphism With IL-10 Secretion by CD4 and T Regulatory Cells in Human Leprosy.

Leprosy is a chronic bacterial disease caused by Mycobacterium leprae. Cytokines are known to play vital role as a peacekeeper during inflammatory and other immunocompromised conditions such as leprosy. This study has tried to bridge the gap of information on cytokine gene polymorphisms and its potential role in the pathogenesis of leprosy. Interleukin-10 (IL-10) is an immunosuppressive cytokine, found to be elevated in leprosy that accounted for the suppression of host's immune system by regulating the functions of other immune cells. T helper cells and T regulatory (Tregs) cells are the major source of IL-10 in lepromatous leprosy patients. In this study, we have documented the association of IL-10 cytokine gene polymorphism with the disease progression. A total of 132 lepromatous leprosy patients and 120 healthy controls were analyzed for IL-10 cytokine gene polymorphisms using PCR-SSP assay and flow cytometry was used to analyze IL-10 secretion by CD4 and Tregs in various genotype of leprosy patients. The frequencies of IL-10 (-819) TT and IL-10 (-1082) GG genotypes were significantly higher in leprosy patients as compared to healthy controls. This observation advocates that these genotypes were associated with the susceptibility and development of the disease. In addition, flow cytometry analysis demonstrated an increased number of IL-10 producing CD4 and Treg cells in IL-10 (819) TT genotype compared to CT and CC genotypes. These observations were further supported by immunohistochemical studies. Therefore, we can conclude that IL-10 cytokine gene polymorphisms by affecting its production can determine the predilection and progression of leprosy in the study population.

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.

γδ T cells are associated with inflammation and immunopathogenesis of leprosy reactions.

BACKGROUND: Leprosy reactions appear episodically in leprosy patients, which lead to high inflammation, morbidity and peripheral nerve damage. The role of Th17 cell has been well studied in leprosy reactions but the role of γδ or unconventional T cells which is an other major source of IL-17 in many diseases, not studied in leprosy reactional episodes. OBJECTIVE: The aim of the present study to elucidate the role of γδ T cells in leprosy reactions. METHODOLOGY: A total of 40 untreated non-reaction and reactions patients were recruited. PBMCs were isolated and stimulated with M. leprae sonicated antigen (MLSA) for 48 h and immuno-phenotyping was done using flow cytometry. Moreover, γδ T cells were isolated by Magnetic beads technology and mRNA expression of IL-17, IFN-γ, TGF-ß and FOXP3 were analyzed by real-time PCR (qPCR) and cytokine was estimated in the culture supernatant by ELISA. RESULTS: γδ T cells were significantly increased in both Reversal reaction (RR) and Erythema nodosum leprosum (ENL) reaction patients. These cells produced significant amount of IL-17 and IFN-γ. Furthermore, CD3+TCRγδ+ T cells expressed transient FOXP3 with a low amount of TGF-ß in both reactions as compared to stable patients. Moreover, low TGF-ß producing TCR-γδ cells were associated with low phosphorylation of STAT5A. CONCLUSION: This study will add to our understanding of the immunological features that mediate and regulate the pathogenesis of leprosy and may helpful to reduce the immuno-pathogenesis of leprosy reaction by targeting these cells.

Regulatory T cells: Friends or foe in human Mycobacterium leprae infection?

Regulatory T cells (Tregs) are known to control immune responses by suppressing the antigen-presenting and effector T cells. Some mechanisms adopted by Tregs in combating Mycobacterium infections have been proposed. Nevertheless, in M. leprae infection, also known as leprosy or Hansen's disease, the role of Tregs has not been completely elucidated. Using multicolor flow cytometry, we evaluated the expression of different cell surface and intracellular molecules present in Tregs from peripheral blood samples of leprosy patients. Before initiating treatment, thirteen new cases of leprosy were grouped according to the Ridley-Jopling classification in to the paucibacilary (PB) or multibacilary (MB) group. Fifteen non-infected individuals (NI) were included as control subjects. Tregs were higher in the MB group than in the NI group. Tregs also co-expressed high amounts of PD1 and PDL-1, indicating that these cells could induce apoptosis of effector cells and simultaneously prevent their own apoptosis. Our data showed that compared to the NI group, Tregs from the PB group expressed higher levels of CD95L, which may be associated with other apoptotic pathways that may decrease Tregs in these patients. Correlation analysis reinforced that PD1 and CD95L are efficient apoptosis' pathway that decreased levels of Tregs in the NI and PB groups. We also observed significant differences in cytokine expression of Tregs from the PB and MB groups. Compared to the NI group, Tregs from the MB group showed higher IL-17 expression; however, compared to the PB group, the expression of IL-10 in Tregs from the MB group was lower, suggesting inefficient control of inflammation. Therefore, we concluded that different pathways were involved in Treg-induced suppression of leprosy. Moreover, Treg-mediated regulation of inflammation via IL-10 and IL-17 expression in leprosy patients was inefficient. Thus, we propose that during M. leprae infection, Tregs may impair the immune responses elicited against this bacillus, favor bacterial replication, and aid in persistence of a disseminated multibacillary disease.

Immunohistochemical assessment of cell populations in leprosy-spectrum lesions and reactional forms.

In situ immunophenotyping of leprosy lesions can improve our understanding of the biology of inflammatory cells during the immune response to Mycobacterium leprae antigens. In the present study, biopsies from 10 healthy controls and 70 leprosy patients were selected, 10 for each of the following conditions: clinical tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL), lepromatous (LL), reversal reaction (R1), and erythema nodosum leprosum (R2). Qualitative and quantitative immunohistochemical analyses were performed to detect CD3, CD4, CD8, FoxP3, CD20, CD138, CD1a, CD57, CD15, CD117, CD68, and CD163. In addition, histochemistry was employed to identify eosinophils. The amount of CD3+ and CD4+ T cells was higher in TT than in LL patients. CD8+ T cells were predominant in T lymphocyte infiltrations in the basal layer of the epidermis. The number of FoxP3+ cells was similar among different forms of the disease, but was higher in BL and LL than in R2 individuals. CD20+ lymphocytes were most abundant in TT samples, while CD138+ plasma cells displayed no detectable differences. Epithelioid macrophages from the center of TT and R1 granulomas exhibited the M1 phenotype (CD68+CD163-), whereas those in LL granulomas showed the M2 phenotype (CD68+CD163+). There was a gradual decrease in the amount of CD1a+ cells from the TT towards the LL form of the disease. A significant increase in the number of neutrophils was observed only in R2 samples. All the cells investigated, except eosinophils, participated in the immunopathogenesis of leprosy.

Immunohistochemical assessment of cell populations in leprosy-spectrum lesions and reactional forms

In situ immunophenotyping of leprosy lesions can improve our understanding of the biology of inflammatory cells during the immune response to Mycobacterium leprae antigens. In the present study, biopsies from 10 healthy controls and 70 leprosy patients were selected, 10 for each of the following conditions clinical tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL), lepromatous (LL), reversal reaction (R1), and erythema nodosum leprosum (R2). Qualitative and quantitative immunohistochemical analyses were performed to detect CD3, CD4, CD8, FoxP3, CD20, CD138, CD1a, CD57, CD15, CD117, CD68, and CD163. In addition, histochemistry was employed to identify eosinophils. The amount of CD3+ and CD4+ T cells was higher in TT than in LL patients. CD8+ T cells were predominant in T lymphocyte infiltrations in the basal layer of the epidermis. The number of FoxP3+ cells was similar among different forms of the disease, but was higher in BL and LL than in R2 individuals. CD20+ lymphocytes were most abundant in TT samples, while CD138+ plasma cells displayed no detectable differences. Epithelioid macrophages from the center of TT and R1 granulomas exhibited the M1 phenotype (CD68+CD163-), whereas those in LL granulomas showed the M2 phenotype (CD68+CD163+). There was a gradual decrease in the amount of CD1a+ cells from the TT towards the LL form of the disease. A significant increase in the number of neutrophils was observed only in R2 samples. All the cells investigated, except eosinophils, participated in the immunopathogenesis of leprosy.

Leprosy Reactions Show Increased Th17 Cell Activity and Reduced FOXP3+ Tregs with Concomitant Decrease in TGF-ß and Increase in IL-6.

BACKGROUND: 50% of leprosy patients suffer from episodes of Type 1/ reversal reactions (RR) and Type 2/ Erythema Nodosum Leprosum (ENL) reactions which lead to morbidity and nerve damage. CD4+ subsets of Th17 cells and CD25+FOXP3+ regulatory T cells (Tregs) have been shown to play a major role in disease associated immunopathology and in stable leprosy as reported by us and others. The aim of our study was to analyze their role in leprosy reactions. METHODOLOGY AND PRINCIPLE FINDINGS: Quantitative reverse transcribed PCR (qPCR), flowcytometry and ELISA were used to respectively investigate gene expression, cell phenotypes and supernatant levels of cytokines in antigen stimulated PBMC cultures in patients with stable disease and those undergoing leprosy reactions. Both types of reactions are associated with significant increase of Th17 cells and associated cytokines IL-17A, IL-17F, IL-21, IL-23 and chemokines CCL20, CCL22 as compared to matching stable forms of leprosy. Concurrently patients in reactions show reduction in FOXP3+ Treg cells as well as reduction in TGF-ß and increase in IL-6. Moreover, expression of many T cell markers, cytokines, chemokines and signaling factors were observed to be increased in RR as compared to ENL reaction patients. CONCLUSIONS: Patients with leprosy reactions show an imbalance in Th17 and Treg populations. The reduction in Treg suppressor activity is associated withhigherTh17cell activity. The combined effect of reduced TGF-ß and enhanced IL-6, IL-21 cytokines influence the balance between Th17 or Treg cells in leprosy reactions as reported in the murine models and autoimmune diseases. The increase in Th17 cell associated cytokines may contribute to lesional inflammation.

Células CD3+, CD4+, CD8+, CD3-CD16+CD56+ e CD19+ em sangue periférico de pacientes com hanseníase e indivíduos saudáveis

A hanseníase é uma doença granulomatosa, infecto-contagiosa causada pelo Mycobacterium leprae. Trata-se de uma infecção crônica com amplo espectro de respostas imunes celulares em humanos. Possui alto poder infectante e baixo poder patogênico. Este estudo tem como objetivo quantificar e comparar leucócitos e sub populações de linfócitos T totais (CD3+), T auxiliares (CD3+CD4+), T citotóxicos (CD3+CD8+), B (CD19+) e NK (CD3-CD16+CD56+) em sangue periférico de indivíduos com hanseníase e controles saudáveis.Os pacientes foram provenientesdo Centro de Dermatologia D. Libânia, Fortaleza-CE, Brasil. A determinaçãodo número de linfócitos em cada subpopulação foi realizada porcitometria de fluxo.Aanálise estatísticafoi realizadapelo programa Grap hPad Prism 5.0para Windows comsignificância estabelecida para valores de p<0,05.É um estudo do tipo caso controle de caráter observacional, realizado a partir da análise do sangue periférico de indivíduos com diagnóstico de hanseníase e de indivíduos saudáveis.A população de pacientes com hanseníase, sem tratamento foi composta de 15 pessoas. A população de controles saudáveis foi composta por 29 pessoas...

Leprosy is an infectious and granulomatous disease caused by Mycobacterium leprae. The aim of this study was to quantify and compare levels of leucocytes and lymphocyte subpopulations (CD3+, CD3+CD4+, CD3+CD8+, CD19+, CD3-CD16+CD56+) in peripheral blood ofpatients with leprosy and healthy controls. Patients were followed at Centro de Dermatologia D. Libânia, Fortaleza-CE, Brasil. Flow cytometry was used to determine numbers of lymphocytes. Statistical analisys was done with GraphPad Prism 5.0 software for windows. P values under 0.05 were considered siginificant.This was an observational case-control study. Fifteen leprosy patients without treatment were evaluated and 29 healthy individuals were included in control group...

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization.

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

T-cell regulation in lepromatous leprosy.

Regulatory T (Treg) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25+ cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients' skin lesions. Depletion of CD25+ cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25+ cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25+ cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25+ T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25+ cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25+ cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3+ CD8+CD25+ T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68+CD163+ as well as FoxP3+ cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25+ Treg cells play a role in M. leprae-Th1 unresponsiveness in LL.

Longitudinal immune responses and gene expression profiles in type 1 leprosy reactions.

PURPOSE: Leprosy, a chronic disease initiated by Mycobacterium leprae, is often complicated by acute inflammatory reactions. Although such episodes occur in at least 50% of all leprosy patients and may cause irreversible nerve damage, no laboratory tests are available for early diagnosis or prediction of reactions. Since immune- and genetic host factors are critical in leprosy reactions, we hypothesize that identification of host-derived biomarkers correlated to leprosy reactions can provide the basis for new tests to facilitate timely diagnosis and treatment thereby helping to prevent tissue damage. METHODS: The longitudinal host response of a leprosy patient, who was affected by a type 1 reaction (T1R) after MDT-treatment, was studied in unprecedented detail, measuring cellular and humoral immunity and gene expression profiles to identify biomarkers specific for T1R. RESULTS: Cytokine analysis in response to M. leprae revealed increased production of IFN-γ, IP-10, CXCL9, IL-17A and VEGF at diagnosis of T1R compared to before T1R, whereas a simultaneous decrease in IL-10 and G-CSF was observed at T1R. Cytokines shifts coincided with a reduction in known regulatory CD39(+)CCL4(+) and CD25(high) T-cell subsets. Moreover, RNA expression profiles revealed that IFN-induced genes, (V)EGF, and genes associated with cytotoxic T-cell responses (GNLY, GZMA/B, PRF1) were upregulated during T1R, whereas expression of T-cell regulation-associated genes were decreased. CONCLUSIONS: These data show that increased inflammation, vasculoneogenesis and cytotoxicity, perturbed T-cell regulation as well as IFN-induced genes play an important role in T1R and provide potential T1R-specific host biomarkers.

Sarcoidosis Th17 cells are ESAT-6 antigen specific but demonstrate reduced IFN-γ expression.

RATIONALE: Sarcoidosis is a granulomatous disease of unknown etiology. Many patients with sarcoidosis demonstrate antigen-specific immunity to mycobacterial virulence factors. Th-17 cells are crucial to the immune response in granulomatous inflammation, and have recently been shown to be present in greater numbers in the peripheral blood and bronchoalveolar lavage (BAL) fluid (BALF) of sarcoidosis patients than healthy controls. It is unclear whether Th-17 cells in sarcoidosis are specific for mycobacterial antigens, or whether they have similar functionality to control Th-17 cells. METHODS: Flow cytometry was used to determine the numbers of Th-17 cells present in the peripheral blood and BALF of patients with sarcoidosis, the percentage of Th-17 cells that were specific to the mycobacterial virulence factor ESAT-6, and as well as to assess IFN-γ expression in Th-17 cells following polyclonal stimulation. RESULTS: Patients with sarcoidosis had greater numbers of Th-17 cells in the peripheral blood and BALF than controls and produced significantly more extracellular IL-17A (p = 0.03 and p = 0.02, respectively). ESAT-6 specific Th-17 cells were present in both peripheral blood and BALF of sarcoidosis patients (p < 0.001 and p = 0.03, respectively). After polyclonal stimulation, Th-17 cells from sarcoidosis patients produced less IFN-γ than healthy controls. CONCLUSIONS: Patients with sarcoidosis have mycobacterial antigen-specific Th-17 cells peripherally and in sites of active sarcoidosis involvement. Despite the Th1 immunophenotype of sarcoidosis immunology, the Th-17 cells have reduced IFN-γ expression, compared to healthy controls. This reduction in immunity may contribute to sarcoidosis pathogenesis.

Immunophenotype of skin lymphocytic infiltrate in patients co-infected with Mycobacterium leprae and human immunodeficiency virus: a scenario dependent on CD8+ and/or CD20+ cells.

BACKGROUND: Leprosy occurs rarely in human immunodeficiency virus (HIV)-positive patients. In contrast to tuberculosis, there has been no report to date of an increase in HIV prevalence among patients with leprosy or of differences in leprosy's clinical spectrum. While several studies describe the systemic immune response profile in patients co-infected with HIV and leprosy, the local immune skin response has been evaluated in only a small number of case reports and limited series of patients. OBJECTIVE: To investigate the interaction between Mycobacterium leprae and HIV infection in the skin. METHODS: We investigated the presence and frequency of cells positive for CD4, CD8, CD20, TIA-1, FOXP3 and CD123 in lymphocytic infiltrates from 16 skin biopsies taken from 15 patients with HIV-leprosy co-infection. RESULTS: CD4+ cells were absent in infiltrates from 6 (38%) skin biopsies and present in 10 (62%) cases at low levels (<1·16%) of the lymphocytic infiltrate. CD8+ was the predominant phenotype in the infiltrate (99·4%), followed by TIA-1, expressed by >75% of CD8+ cells. FOXP3+ cells were also present, representing 3·4% of the lymphocytic infiltrate. CD20+ cells were detected in 75% of the cases; however, in two cases (12%) these cells represented 25-50% of the infiltrate, while in the other 10 cases (62%) they were present only focally (<25% of the infiltrate). CD123+ cells were not observed in any of the studied specimens. CONCLUSIONS: Data presented here suggest that cell-mediated immune responses to M. leprae are preserved at the site of disease and that in the absence of CD4+ cells, CD8+FOXP3+ and CD20+ cells may be involved in granuloma formation.

Alteration of the relative levels of iNKT cell subsets is associated with chronic mycobacterial infections.

CD1d-restricted invariant natural killer T cells (iNKT cells) have been identified as an important type of effector and regulatory T cell, but their roles in the chronic infectious diseases caused by Mycobacterium tuberculosis and Mycobacterium leprae remain poorly defined. Here, we studied circulating human iNKT cells in blood samples from tuberculosis (TB) and leprosy patients. We found that the percentages of iNKT cells among total circulating T cells in TB and leprosy patients were not significantly different from those in normal controls. However, both TB and leprosy patients showed a selective reduction of the proinflammatory CD4(-)CD8beta(-) (DN) iNKT cells with a proportionate increase in the CD4(+) iNKT cells. Similar phenotypic alterations in circulating iNKT cells were observed in a mouse model of M. tuberculosis infection. Taken together, these findings indicate that the selective reduction of circulating DN iNKT cells is associated with chronic infections caused by M. tuberculosis and M. leprae.

Impaired maturation and function of dendritic cells by mycobacteria through IL-1beta.

Dendritic cells (DC) are pivotal for initiation and regulation of innate and adaptive immune responses evoked by vaccination and natural infection. After infection, mycobacterial pathogens first encounter monocytes, which produce pro-inflammatory cytokines, including IL-1beta, TNF-alpha and IL-6. The role of these cytokines in DC maturation remains incompletely understood. Here, we show that maturation of DC from monocytes was impaired by pretreatment of monocytes with low doses of IL-1beta. Under these conditions, Mycobacterium leprae-infected DC failed to stimulate antigen-specific T cell responses. Expression of CD86 and CD83 and production of IL-12 in response to lipopolysaccharide and peptidoglycan were diminished. In contrast, these DC functions were not impaired by pretreatment with TNF-alpha, IL-6 or IL-10. When monocytes were infected with M. bovis Bacillus Calmette-Guérin, and subsequently differentiated to DC, the activity of these DC was suppressed as well. Thus, IL-1beta acts at early stages of differentiation of DC and impairs biological functions of DC at later stages. Therefore, production of IL-1beta by mycobacteria-infected antigen-presenting cells counteracts effective stimulation of innate and adaptive immune responses.

TUNEL and limited immunophenotypic analyses of apoptosis in paucibacillary and multibacillary leprosy lesions.

Some mycobacterial infections, such as tuberculosis, are characterized by apoptosis of infected or by-stander mononuclear immune cells. For localized (paucibacillary, PB) and disseminated (multibacillary, MB) leprosy, characterized by polarized Th1-like vs. Th2-like immune responses, respectively, little is known about lesional apoptosis. We analyzed sections of paraffin-embedded, untreated leprosy lesions from 21 patients by an indirect immunofluorescent terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. Some TUNEL (+) PB sections were then reacted with phycoerythrin-conjugated (red) antibodies against T cells, monocytes, or antigen-presenting (Langerhans) cells. TUNEL (+) bodies were detected in 9 of 16 PB lesions (56%) and in 1 of 5 MB lesions (20%). Some TUNEL (+) bodies in PB disease were CD3+ (T cell), as well as CD4+ (T-helper) or CD8+ (T-cytotoxic). Apoptosis characterizes PB and MB leprosy lesions and may be more frequent in PB disease. In PB disease, some TUNEL (+) bodies may derive from T cells.