Distinct Roles of Th17 and Th1 Cells in Inflammatory Responses Associated with the Presentation of Paucibacillary Leprosy and Leprosy Reactions.

It is well established that helper T cell responses influence resistance or susceptibility to Mycobacterium leprae infection, but the role of more recently described helper T cell subsets in determining severity is less clear. To investigate the involvement of Th17 cells in the pathogenesis of leprosy, we determined the immune profile with variant presentations of leprosy. Firstly, IL-17A, IFN-γ and IL-10 were evaluated in conjunction with CD4+ T cell staining by confocal microscopy of lesion biopsies from tuberculoid (TT) and lepromatous leprosy (LL) patients. Secondly, inflammatory cytokines were measured by multiplex assay of serum samples from Multibacillary (MB, n = 28) and Paucibacillary (PB, n = 23) patients and household contacts (HHC, n = 23). Patients with leprosy were also evaluated for leprosy reaction occurrence: LR+ (n = 8) and LR- (n = 20). Finally, peripheral blood mononuclear cells were analysed by flow cytometry used to determine the phenotype of cytokine-producing cells. Lesions from TT patients were found to have more CD4+ IL-17A+ cells than those from LL patients. Higher concentrations of IL-17A and IL-1ß were observed in serum from PB than MB patients. The highest serum IFN-γ concentrations were, however, detected in sera from MB patients that developed leprosy reactions (MB LR+ ). Together, these results indicate that Th1 cells were associated with both the PB presentation and also with leprosy reactions. In contrast, Th17 cells were associated with an effective inflammatory response that is present in the PB forms but were not predictive of leprosy reactions in MB patients.

CD4+ TCRγδ+ FoxP3+ cells: An unidentified population of immunosuppressive cells towards disease progression leprosy patients.

This study, for the first time, reveals the role of M. leprae-specific CD4+ TCRγδ+ FoxP3+ cells in the progression and pathogenesis of leprosy. Co-culture with CD4+ CD25- cells suggested the immunosuppressive nature of CD4+ TCRγδ+ cells in dose-dependent manner. Isolation of CD4+ TCRγδ+ cells from leprosy patients and then culture in presence of M. leprae cell wall antigens (MLCwA) along with TGF ß, IPP and IL-2 suggested that these cells are M. leprae specific. TGF-ß-mediated SMAD3 signalling was turned out to be major factor towards the expression of FoxP3 in these cells. SMAD3 silencing during induction of these cells barely showed the induction of FoxP3. High density of SMAD3 binding at TGFßRII in CD4+ TCRγδ+ FoxP3+ furthermore suggested the TGF-ß-directed SMAD3 signalling in these cells. Taken together the above data, we can conclude that CD4+ TCRγδ+ FoxP3+ cells possess the potential to track the severity of the disease in leprosy patients.

Development of Type 2, But Not Type 1, Leprosy Reactions is Associated with a Severe Reduction of Circulating and In situ Regulatory T-Cells.

Leprosy is frequently complicated by the appearance of reactions that are difficult to treat and are the main cause of sequelae. We speculated that disturbances in regulatory T-cells (Tregs) could play a role in leprosy reactions. We determined the frequency of circulating Tregs in patients with type 1 reaction (T1R) and type 2 reaction (T2R). The in situ frequency of Tregs and interleukin (IL)-17, IL-6, and transforming growth factor beta (TGF)-ß-expressing cells was also determined. T2R patients showed markedly lower number of circulating and in situ Tregs than T1R patients and controls. This decrease was paralleled by increased in situ IL-17 expression but decreased TGF-ß expression. Biopsies from T1R and T2R patients before the reaction episodes showed similar number of forkhead box protein P3+ (FoxP3+) and IL-17+ cells. However, in biopsies taken during the reaction, T2R patients showed a decrease in Tregs and increase in IL-17+ cells, whereas T1R patients showed the opposite: Tregs increased but IL-17+ cells decreased. We also found decreased expansion of Tregs upon in vitro stimulation with Mycobacterium leprae and a trend for lower expression of FoxP3 and the immunosuppressive molecule cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in T2R Tregs. Our results provide some evidence to the hypothesis that, in T2R, downmodulation of Tregs may favor the development of T-helper-17 responses that characterize this reaction.

Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal.

BACKGROUND: Acute inflammatory reactions are a frequently occurring, tissue destructing phenomenon in infectious- as well as autoimmune diseases, providing clinical challenges for early diagnosis. In leprosy, an infectious disease initiated by Mycobacterium leprae (M. leprae), these reactions represent the major cause of permanent neuropathy. However, laboratory tests for early diagnosis of reactional episodes which would significantly contribute to prevention of tissue damage are not yet available. Although classical diagnostics involve a variety of tests, current research utilizes limited approaches for biomarker identification. In this study, we therefore studied leprosy as a model to identify biomarkers specific for inflammatory reactional episodes. METHODS: To identify host biomarker profiles associated with early onset of type 1 leprosy reactions, prospective cohorts including leprosy patients with and without reactions were recruited in Bangladesh, Brazil, Ethiopia and Nepal. The presence of multiple cyto-/chemokines induced by M. leprae antigen stimulation of peripheral blood mononuclear cells as well as the levels of antibodies directed against M. leprae-specific antigens in sera, were measured longitudinally in patients. RESULTS: At all sites, longitudinal analyses showed that IFN-γ-, IP-10-, IL-17- and VEGF-production by M. leprae (antigen)-stimulated PBMC peaked at diagnosis of type 1 reactions, compared to when reactions were absent. In contrast, IL-10 production decreased during type 1 reaction while increasing after treatment. Thus, ratios of these pro-inflammatory cytokines versus IL-10 provide useful tools for early diagnosing type 1 reactions and evaluating treatment. Of further importance for rapid diagnosis, circulating IP-10 in sera were significantly increased during type 1 reactions. On the other hand, humoral immunity, characterized by M. leprae-specific antibody detection, did not identify onset of type 1 reactions, but allowed treatment monitoring instead. CONCLUSIONS: This study identifies immune-profiles as promising host biomarkers for detecting intra-individual changes during acute inflammation in leprosy, also providing an approach for other chronic (infectious) diseases to help early diagnose these episodes and contribute to timely treatment and prevention of tissue damage.

Serum Th17 cytokines in leprosy: correlation with circulating CD4(+) CD25 (high)FoxP3 (+) T-regs cells, as well as down regulatory cytokines.

Leprosy is not only a bacteriological disease but also an immunological disease, in which T helper17 and CD4(+) CD25(high)FoxP3(+) regulatory T cells (T-regs), among others, may play a role. We aimed to evaluate serum levels of interleukin (IL)-17, IL-22 (Th17 cytokines), IL-10 and transforming growth factor (TGF)-ß (down regulatory cytokines) in 43 untreated leprosy patients and 40 controls by enzyme-linked immunosorbent assay, and to assess circulating CD4(+) CD25(high)FoxP3(+)T-regs in patients using flow cytometry. Patients were grouped into tuberculoid, pure neural, borderline, lepromatous, type 1 reactional leprosy, and erythema nodosum leprosum. IL-10 and TGF-ß were significantly higher in patients as compared to controls (p < 0.001), while IL-17, but not IL-22, was significantly lower (p < 0.001), with no significant difference comparing patients' subgroups. Significantly higher CD4(+) CD25(high)FoxP3(+)T-regs levels was detected in tuberculoid, type 1 reaction and pure neural leprosy, while the lowest levels in erythema nodosum leprosum (p < 0.001). TregsFoxP3 expression% was significantly lower in pure neural leprosy than other patients' subgroups (p < 0.05). T-regs/T-effs was lowest in erythema nodosum leprosum (p < 0.05). TGF-ß correlated negatively with TregsFoxP3 expression% and T-effs% (p = 0.009 and 0.018 respectively). Leprosy is associated with defective IL-17 and overproduction of IL-10 and TGF-ß. Tuberculoid, type 1 reaction and pure neural leprosy express significantly higher circulating T-regs, consistent with effector immune mechanisms activation, but with lower TregsFoxP3 expression (in pure neural leprosy). Erythema nodosum leprosum is characterized by deficient T-regs and increased TregsFoxP3 expression%. The present study pinpointed a potential role of Th17, CD4(+) CD25(high)FoxP3(+)T-regs, and probably CD4(+) CD25(+)IL-10(+) T regulatory cells 1 (Tr1), and Th3 in leprosy.

Estimation of serum level of interleukin-17 and interleukin-4 in leprosy, towards more understanding of leprosy immunopathogenesis.

BACKGROUND: Combating Mycobacterium leprae is known to be via T-helper1 response. However, other T-helper effector cells; T-helper17 and T-helper2; play a role, particularly in the context of disease type. AIMS: We aimed to evaluate serum levels of interleukin (IL)-17 (T-helper17 cytokine) and IL-4 (T-helper2 cytokine) in untreated patients with different types of leprosy, compared to controls. METHODS: Using enzyme-linked immunosorbent assay, serum IL-17 and IL-4 levels were estimated in 43 leprotic patients and 43 controls. Patients were divided into six groups; tuberculoid, borderline cases, lepromatous, erythema nodosum leprosum (ENL), type 1 reactional leprosy, and pure neural leprosy. Patients were also categorized according to bacillary load and the presence or absence of reactions. RESULTS: Serum IL-17 was significantly lower in cases (4-61.5 pg/mL; median 19), compared to controls (26-55 pg/mL; median 36) (P < 0.001), and was significantly lower in each type of leprosy compared to controls, with the lowest level in lepromatous leprosy (4-61.5 pg/mL; median 12.5). Significantly elevated serum IL-4 was found in patients (1.31-122.4 pg/mL; median 2.31) compared to controls (1.45-5.72 pg/mL; median 2.02) (P = 0.008), with the highest level among lepromatous leprosy patients (2-87.2 pg/mL; median 28.9), and the lowest in type 1 reactional leprosy (1.4-2.5 pg/mL; median 1.87) (P = 0.006). CONCLUSION: Defective secretion of IL-17 is related to disease acquisition as well as progression toward lepromatous pole in leprosy patients. The overproduction of IL-4 in patients with lepromatous leprosy may infer their liability to develop ENL. Nevertheless, the small number of the studied population is a limitation.

Cortisol and proinflammatory cytokine profiles in type 1 (reversal) reactions of leprosy.

PURPOSE: Cortisol levels in the circulation and at the sites of peripheral inflammation regulate type 1 (Reversal) reactions in leprosy akin to delayed type hypersensitivity reactions (DTH). In this study we determine the extent to which the differential mRNA expression of genes encoding cortisone-cortisol shuttle enzymes (11 ß hydroxysteriod dehydrogenase I & II (11 ß HSD I & II)), circulatory levels of proinflammatory cytokines (IL-6, IL-7, IP-10, IL-17F, IL-23, TNF-α, IL-1ß, PDGF BB and CRP) and cortisol are associated with development of type 1 reactions in leprosy. METHODS: Urine, blood and incisional skin biopsy samples from site of lesions were collected from 49 newly diagnosed untreated leprosy cases in T1R and 51 cases not in reaction (NR). mRNA expression levels of genes encoding 11 ß HSD I & II in skin biopsy samples were determined by realtime PCR. Cortisol levels from the lesional skin biopsies, serum and urine samples and serum proinflammatory cytokine levels were measured using ELISA. RESULTS: The mean expression ratios of 11 ß HSD I & II are significantly lower in leprosy cases with T1R when compared to the NR leprosy cases. Cortisol levels in lesional skin biopsies and in urine are significantly lower (p=0.001) in leprosy cases with T1R. Serum cytokine levels of IP-10, IL-17F, IL-IL-6 and TNF-α are significantly higher (p<0.05) in leprosy cases with T1R when compared the NR leprosy cases. CONCLUSION: Our study indicated an association of urinary and lesional skin cortisol levels with the manifestation of T1R in leprosy. IP-10, IL-17F, IL-6 and TNF-α can be potential prognostic serological markers and gene expression markers for early detection of type 1 reactions in leprosy.

Increased serum circulatory levels of interleukin 17F in type 1 reactions of leprosy.

PURPOSE: Leprosy is a chronic infectious disease caused by Mycobacterium leprae affecting mainly skin and peripheral nerves. Acute inflammatory episodes in the borderline immunological spectrum of the disease cause severe nerve and tissue damage leading to deformities. Finding of any serological marker for leprosy reactions will help in prediction of reactions and in early treatment intervention. The objective of this study was to measure the serum circulatory levels of Interleukin 17F (IL 17F) and to correlate the levels with type 1 and type 2 reactional states and with clinico-histopathological spectrum of leprosy. We studied IL 17F to delineate its role and its clinical implications in leprosy reactions. METHODS: Patients were classified based on the Ridley DS and Jopling WH Classification and blood samples (5 ml each) were collected from 80 active untreated leprosy cases in Type 1 reaction (T1R), 21 cases in Type 2 (Erythema Nodosum Leprosum ENL) reaction (T2R), 80 cases without reaction (NR), and 94 non-leprosy cases (NL). Serum was separated and measured for IL 17F levels using ELISA (Commercial Kits, R&D Systems Inc., USA). RESULTS: IL 17F levels were significantly higher in the T1R group when compared to the NR group (p < 0.001). The borderline lepromatous group showed the highest levels of IL 17F among the other groups in the disease spectrum. Bacteriological index (BI) showed negative correlation with the IL 17F levels. CONCLUSION: The results specify that serum circulatory levels of IL 17F are elevated during T1Rs in the borderline spectrum of the disease and thus may play a role in the regulation of inflammatory responses associated with reactions in leprosy.