Unveiling the role of NK cells, NKT-like cells, and γδ cells in pathogenesis of type 1 reactions in leprosy.

Leprosy is a disease with spectral clinical manifestations along with two types of reactions, type 1 reaction (T1R) and type 2 reaction (T2R). T1R especially occurs because of the defensive upgradation of cell-mediated immunity (CMI) to M. leprae antigens. T1R is the main cause of disability in leprosy. The role of conventional adaptive T cells has been well studied to understand T1R. A comprehensive understanding of the role of unconventional T cells in the manifestation of inflammation during T1R is crucial and has not been studied. In our study, we found significantly higher plasma levels of TNFα, IL1ß, IL17, and IP10 in T1R when compared to non-reaction (NR). Gene expression for cytokines in blood circulation by qPCR showed significantly higher expression of IFNγ, IP10, TNFα, IL6, IL17A and chemokines CCL3, CCR1, CCR5, and CXCR3 in T1R as compared to NR. Frequencies of NKT-like cells (48.7 %) and NK cells (22.3 %) were found significantly higher in T1R in comparison to NR (36.9 %, 18.3 %, respectively) (p = 0.0001). Significantly lower levels of γδT cells (3.32 %) were observed in T1R in comparison to NR (5.16 %). The present study has provided evidence for the first time on the role of plausible unconventional T cells in the immunopathogenesis of T1R in leprosy.

Transcription factors STAT-4, STAT-6 and CREB regulate Th1/Th2 response in leprosy patients: effect of M. leprae antigens.

BACKGROUND: Leprosy is an ideal human disease to study T cell regulation as patients show correlation between cytokine skewed Th1-Th2 responses and clinical forms of the disease. The Role of transcription factors on the modulation of Th1 and Th2 responses by M. leprae antigens has not been adequately studied. In the present study, we studied the effect of M. leprae antigens on transcription factors STAT-4, STAT-6 and CREB and their correlation with Th1/Th2 cell mediated immune responses in leprosy. METHODS: Leprosy patients of both categories of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were selected from the OPD of NJ1L & OMD, (ICMR), Agra and healthy individuals (H) were chosen from the staff and students working in the institute. Peripheral blood mononuclear cells (PBMCs) of the study subjects were stimulated with M. leprae antigens (WCL, MLSA, and PGL-1). Sandwich ELISA was done in the culture supernatants of healthy and leprosy patients to detect IL-4, IL-10 and IFN-γ. Further, expression of IFN-γ and IL-4 and activation of STAT4, STAT6 and CREB transcription factors in CD4+ T cell with or without stimulation of M. leprae antigens was investigated by flow cytometry. RESULTS: Lepromatous leprosy patients showed significantly lower IFN-γ and higher IL-4 levels in culture supernatant and significantly low expression of IFN-γ and higher expression of IL-4 by CD4+ T cells than healthy individuals with or without antigenic stimulation. Antigenic stimulation significantly increased IL-10 in BL/LL patients but not in BT/TT patients or healthy individuals. PGL-1 stimulation led to significantly higher activation of STAT-6 in BT/TT and BL/LL patients in comparison to healthy individuals. All the three antigens led to activation of CREB in healthy and BT/TT patients but not in BL/LL patients. CONCLUSION: Our findings show that M. leprae antigens differentially modulate activation of T cell transcription factors STAT-4/STAT-6 and CREB. These transcription factors are well known to regulate Th1 and Th2 mediated immune response which in turn could play vital role in the clinical manifestations of leprosy. These observations may help to determine how these T cell transcription factors affect the development of immune dysfunction and whether these new pathways have a role in immunomodulation in intracellular diseases like leprosy and TB.

Molecular mimicry between HSP 65 of Mycobacterium leprae and cytokeratin 10 of the host keratin; role in pathogenesis of leprosy.

Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (p<0.05) higher in all the groups of leprosy patients except TT/BT in comparison to HC. Significant positive correlation was found between number of lesions and level of AkAbs in leprosy patients. Highest lympho-proliferation for keratin protein was observed in T1R, followed by BL/LL, TT/BT, ENL. Lympho-proliferation was significantly (p<0.05) higher in all groups of leprosy patients except ENL in comparison to HC. Interestingly, it was noted that hyperimmunization of inbred strains of female BALB/c mice and rabbit with M. leprae soluble antigen (MLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ≈74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ≈65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy.

Phenolic-glycolipid-1 and lipoarabinomannan preferentially modulate TCR- and CD28-triggered proximal biochemical events, leading to T-cell unresponsiveness in mycobacterial diseases.

BACKGROUND: Advanced stages of leprosy show T cell unresponsiveness and lipids of mycobacterial origin are speculated to modulate immune responses in these patients. Present study elucidates the role of phenolicglycolipid (PGL-1) and Mannose-capped lipoarabinomannan (Man-LAM) on TCR- and TCR/CD28- mediated signalling. RESULTS: We observed that lipid antigens significantly inhibit proximal early signalling events like Zap-70 phosphorylation and calcium mobilization. Interestingly, these antigens preferentially curtailed TCR-triggered early downstream signalling events like p38 phosphorylation whereas potentiated that of Erk1/2. Further, at later stages inhibition of NFAT binding, IL-2 message, CD25 expression and T-cell blastogenesis by PGL-1 and Man-LAM was noted. CONCLUSION: Altogether, we report that Man-LAM and PGL-1 preferentially interfere with TCR/CD28-triggered upstream cell signalling events, leading to reduced IL-2 secretion and T-cell blastogenesis which potentially could lead to immunosupression and thus, disease exacerbation, as noted in disease spectrum.

Mycobacterial antigen(s) induce anergy by altering TCR- and TCR/CD28-induced signalling events: insights into T-cell unresponsiveness in leprosy.

Present study investigates the role of Mycobacterium leprae (M. leprae) antigens on TCR- and TCR/CD28-induced signalling leading to T-cell activation and further correlates these early biochemical events with T-cell anergy, as prevailed in advanced stages of leprosy. We observed that both whole cell lystae (WCL) and soluble fraction of M. leprae sonicate (MLSA) not only inhibited TCR, thapsigargin and ionomycin induced calcium fluxes by diminishing the opening of calcium channels, but also TCR- or TCR/CD28-induced proximal signalling events like phosphorylation of Zap-70 and protein kinase-C (PKC) activity. Study of TCR- and TCR/CD28-induced downstream signals revealed that M. leprae antigens curtail phosphorylation of both Erk1/2 and p38MAPK, consequently altering terminal signalling events like reduced binding of NFAT on IL-2 promoter and transcription of IL-2 gene, diminished expression of activation markers (CD25 and CD69). Furthermore, M. leprae fractions significantly inhibited IL-2 secretion and T-cell blastogenesis in healthy individuals. Altogether, results suggest that M. leprae interferes with TCR/CD28-induced upstream as well as downstream signalling events resulting in reduced IL-2 production and thus inhibition in T-cell proliferation, which might be responsible for T-cell unresponsiveness leading to stage of immunosuppression and consequently, for the progression of disease.