Patients with localized tuberculoid and generalized lepromatous leprosy show respectively Th1 and Th2 cytokine profile. Additionally, other patients in both types of leprosy also show a non discriminating Th0 cytokine profile with both interferon-γ and IL-4. The present study investigated the role of Th17 cells which appear to be a distinct subtype of Th subtypes in 19 tuberculoid and 18 lepromatous leprosy patients. Five healthy subjects with long term exposure to infection and 4 skin biopsies from healthy subjects undergoing cosmetic surgery were used as controls. An array of Th17 related primers for cytokines, chemokines and transcription factors was used in real time reverse transcribed PCR to evaluate gene expression, ELISA for cytokine secretion in the supernatants of antigen stimulated PBMC cultures and flow cytometry for establishing the phenotype of the IL-17, IL-21 producing cells. IL-17 isoforms showed significantly higher expression and release in supernatants of antigen stimulated PBMC cultures and dermal lesions of healthy contacts and tuberculoid leprosy as compared to lepromatous leprosy (p<0.003). This was further confirmed by Th17 associated transcription factor RORC, cytokines IL-21, IL-22, and IL-23, chemokines MMP13, CCL20, CCL22. Of interest was the association of IL-23R and not IL-6R with IL-17⁺ cells. The Th17 cells were CD4⁺ CCR6⁺ confirming their effector cell lineage. Polarized Th1 cytokines were seen in 3/7 tuberculoid and Th2 cytokines in 5/10 lepromatous leprosy patients. Of importance was the higher association of Th17 pathway factors with the non-polarized Th0 types as compared to the polarized Th1 and Th2 (p<0.01). Our study draws attention to a third type of effector Th cell that may play a role in leprosy. Leprosy caused by Mycobacterium leprosy continues to be a public health challenge in developing countries. It manifests as a leprosy spectrum with varied clinical forms of localized (tuberculoid) and generalized (lepromatous) disease. Consensus on the immunological basis for leprosy spectrum is lacking. T helper subsets of Th1 and Th2 based on mutually exclusive cytokines IFN-γ and IL-4 respectively were considered as a basis for the inverse relationship between T cell functions and antibody responses seen in this disease. However, there are some patients who show Th0 subset which is non discriminatory and produce both cytokines. In the present study, we provide evidence for the presence of the recently described third subset of Th17 in both antigen stimulated PBMC and skin lesions of tuberculoid leprosy. They appear to be associated with Th0 profile and may represent a third type of effector cell in leprosy.

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. 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-β 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. 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-β 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. Reversal reactions (RR; Type 1) and Erythema Nodosum Leprosum (ENL; Type 2) are two types of leprosy reactions which appear episodically in a proportion of leprosy patients and lead to high morbidity and peripheral nerve damage that require immediate medical attention, ENL seen in anergic lepromatous patients, show immune complexes as well as transient emergence of T cell functions. Lesions of RR with borderline tuberculoid background show pathological features associated with delayed-type hypersensitivity. The present study shows increased Th17 cell show activity in reaction patients as compared to the matching stable leprosy type and which may contribute to the inflammation/immunopathology observed in the lesions. This increase is accompanied by reduction in Treg cells and their inhibitory activity. The balance between Th17 and Treg cells may be influenced by the combined effects of reduction in TGF-β and increase in IL-6, IL-21 cytokines. Of interest was the expression of more genes associated with T cells, cytokines, chemokines, signaling and transcription factors by RR as compared to ENL patient.

Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25⁺FOXP3⁺ regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples. Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3⁺, TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4⁺CD25⁺FOXP3⁺ T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25⁺ cells of the CD4⁺ but not that of CD8⁺ T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients. Our results indicate that FOXP3⁺ iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy. Lepromatous leprosy is a generalized infectious disease caused by Mycobacterium leprae with the patients showing T cell mediated unresponsiveness to the pathogen and chronicity of lesions. The causation of unresponsiveness and anergy in this form of leprosy is not fully understood. The recent discovery of CD25⁺FOXP3⁺ cells with regulatory functions (Tregs) in mice and man have made it possible to study their role in the dampening of T cell responses in lepromatous leprosy. We investigated both skin and PBMC from leprosy patients for lineage specific molecular, and phenotypic markers of Tregs as well as cytokines in situ and in in vitro M.leprae stimulated PBMC cultures (iTreg). Our studies find an increase in lineage specific CD4⁺ iTregs in lepromatous leprosy as compared to the limited form of borderline tuberculoid leprosy. Such cells secrete TGF-β, an inhibitory cytokine and may play a role in negatively regulating the T cell immune responses in lepromatous disease.

Regulatory B cells (Bregs) are known to exhibit their regulatory functions through interleukin-10 (IL-10) cytokine which suppress inflammation. There are only a few studies explaining the phenotype and functioning of these cells in contribution to host immunity in leprosy. Here, we evaluated the role of IL-10 producing Bregs in the pathogenesis of leprosy and assessed their immunoregulatory effects on Tregs and effector T cells. We found an increased frequency of Bregs and increased expression of their immune modulatory molecules (IL-10, FoxP3, and PDL-1) in leprosy patients. The potential immunoregulatory mechanism of Bregs was also investigated using MACS sorted Teff (CD4⁺CD25⁻) and Treg (CD4⁺CD25⁺) cells were cocultured with Bregs to elucidate the effects of Bregs on effector T and regulatory T cells. Cell coculture results showed that purified Bregs cells from leprosy patients convert CD4⁺CD25⁻ cells into CD4⁺CD25⁺ cells. Cell coculture experiments also demonstrated that leprosy derived IL-10 producing Bregs enhance FoxP3 and PD-1 expression in Tregs and enhanced Tregs activity. Blocking of IL-10 receptor confirmed that IL-10 producing Breg has immunomodulatory effect on Tregs and effector T cells as effector T cells are not converted into Tregs and enhanced expression of FoxP3 and PD-1 was not observed on Tregs. Collectively, these findings demonstrate that IL-10 producing Breg cells play an important mechanism in controlling the immunopathogenesis of leprosy and have an immunomodulatory effect on Tregs and effector T cells. Our findings may pave way for novel targets of IL-10 producing Bregs for immunotherapy in leprosy patients.

Osteoporosis is a systemic skeletal disease that leads to lower bone mineral density and intensifies the risk of unexpected fractures. Recently, our group reported that numerical defect in the frequencies of Bregs along with their compromised tendency to produce IL-10 cytokine further aggravates inflammatory bone loss in post-menopausal osteoporosis (PMO). Dysbiosis induced mucosal injury and leaky gut are the predominant contributors involved in the progression of inflammatory diseases including PMO. Furthermore, several evidence suggest that gut microbial composition plays a crucial role in the development and differentiation of Bregs. Nevertheless, the potential role of dysbiotic gut microbiota (GM) and Bregs under estrogen deficient PMO conditions has never been deciphered. Here, we evaluated the role of GM in the onset and progression of PMO along with its role in modulating the anti-osteoporotic potential of Bregs. We found that enhancement in the endotoxin producing bacteria and concomitant reduction in the short chain fatty acids producing bacteria, both under pre-clinical and clinical osteoporotic condition augment inflammatory bone loss. This suggests that dysbiosis of GM potentially exacerbates bone deterioration under estrogen deficient PMO conditions. Remarkably, supplementation of probiotic Bacillus coagulans significantly improved the bone mineral density, bone strength, and bone microarchitecture by modulating the anti-osteoclastogenic, immunosuppressive and immunomodulatory potential of Bregs. The present study delves deeper into the role of immune homeostasis ("Breg-Treg-Th17" cell axis) and GM profile in the pathophysiology of PMO. Altogether, findings of the present study open novel therapeutic avenues, suggesting restoration of GM composition as one of the viable therapeutic options in mitigating inflammatory bone loss under PMO conditions via modulating the "Gut-Immune-Bone" axis.