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.

Immunology of leprosy and diagnostic challenges.

Leprosy, caused by noncultivable Mycobacterium leprae (ML), has varied manifestations, which are associated with the host immune responses. The dermal involvement is accompanied by peripheral nerve damage, which leads to sensory motor loss and deformities. Both innate and acquired immune responses are involved. The main cell to be compromised is the CD4 + T helper cell, which shows antigen specific unresponsiveness to only ML and not to other common antigens in the bacilliferous generalized lepromatous form of the disease. In contrast, the paucibacillary localized tuberculoid form shows appropriate T cell functions and poor antibody response. The dichotomy between T cell functions and antibodies are discussed against the current information on cytokines, Th subsets, and regulatory T cells. During lepromatous reactions, there is a temporary, heightened T cell immunity, even in lepromatous subjects. The dermal lesions confirm many features observed with peripheral blood mononuclear cells and give additional information on local immune responses. Nerve damage involves both immune and nonimmune mechanisms. Leprosy is a model disease for understanding host immune responses to intracellular bacilli. There are challenges in diagnosing early leprosy. In spite of intensive efforts by many groups, consensus on a universal test suitable for endemic areas is awaited.

Immunology fo leprosy and diagnostic challenges

Leprosy, caused by noncultivable Mycobacterium leprae (ML), has varied manifestations, which are associated with the host immune responses. The dermal involvement is accompanied by peripheral nerve damage, which leads to sensory motor loss and deformities. Both innate and acquired immune responses are involved. The main cell to be compromised is the CD4 + T helper cell, which shows antigen specific unresponsiveness to only ML and not to other common antigens in the bacilliferous generalized lepromatous form of the disease. In contrast, the paucibacillary localized tuberculoid form shows appropriate T cell functions and poor antibody response. The dichotomy between T cell functions and antibodies are discussed against the current information on cytokines, Th subsets, and regulatory T cells. During lepromatous reactions, there is a temporary, heightened T cell immunity, even in lepromatous subjects. The dermal lesions confirm many features observed with peripheral blood mononuclear cells and give additional information on local immune responses. Nerve damage involves both immune and nonimmune mechanisms. Leprosy is a model disease for understanding host immune responses to intracellular bacilli. There are challenges in diagnosing early leprosy. In spite of intensive efforts by many groups, consensus on a universal test suitable for endemic areas is awaited.

Increase in TGF-ß secreting CD4⁺CD25⁺ FOXP3⁺ T regulatory cells in anergic lepromatous leprosy patients.

BACKGROUND: 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. METHODOLOGY/PRINCIPLE FINDINGS: 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-ß 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-ß which was further confirmed by increase in TGF-ß in culture supernatants (p<0.003). Furthermore, TGF-ß in iTreg cells was associated with phosphorylation of STAT5A. TGF-ß 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. CONCLUSIONS/SIGNIFICANCE: Our results indicate that FOXP3(+) iTregs with TGF-ß may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.

CD4+ Th17 cells discriminate clinical types and constitute a third subset of non Th1, Non Th2 T cells in human leprosy.

BACKGROUND: 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. METHODOLOGY/PRINCIPLE FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Lsr2 of Mycobacterium leprae and its synthetic peptides elicit restitution of T cell responses in erythema nodosum leprosum and reversal reactions in patients with lepromatous leprosy.

The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742-752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs.

Genetic evidence of TAP1 gene variant as a susceptibility factor in Indian leprosy patients.

The heterodimeric transporter associated with antigen processing (TAP) gene loci is known to play a vital role in immune surveillance. We investigated a possible association of gene polymorphisms both in TAP1 and TAP2 in a cohort of clinically classified leprosy patients (n=222) and in ethnically matched controls (n=223). The TAP1 and TAP2 genes were genotyped for four single nucleotide polymorphisms TAP1 (rs1057141 Iso333Val and rs1135216 Asp637Gly) and TAP2 (rs2228396 Ala565Thr and rs241447 Ala665Thr) by direct sequencing and ARMS-PCR. The minor allele of TAP1 637G contributes to an increased risk to leprosy compared to controls (OR: 1.68, 95% CI 1.2-2.36, P=0.0057). An increased risk for the variant minor allele of the TAP1 637G to multibacillary (BL+LL) or paucibacillary (BT+TT) infections was also observed [multibacillary vs. controls (OR: 1.56, 95% CI 1.07-2.28, P=0.054); paucibacillary vs. controls (OR: 1.92, 95% CI 1.21-3.01, P=0.013)]. In the dominant model, the genotypes of the TAP1 rs1135216AG+GG additionally contributed to an increased risk. Overall our findings demonstrate that the TAP1 gene variant (rs1135216 Asp637Gly) influences the susceptibility to clinically classified leprosy patients in Indian population.

Lsr2 peptides of Mycobacterium leprae show hierarchical responses in lymphoproliferative assays, with selective recognition by patients with anergic lepromatous leprosy.

Lsr2 protein of Mycobacterium leprae was shown earlier to elicit B and T cell responses in leprosy patients (20, 28). Lymphoproliferation to M. leprae and Lsr2 antigens was observed in >70% of tuberculoid (T) patients and in 16 and 34% of lepromatous (L) patients, respectively. We focused on the M. leprae nonresponders in the lepromatous group using 22 synthetic Lsr2 peptides (end-to-end peptides A to F and overlapping peptides p1 to p16) in in vitro T cell responses. A total of 125 leprosy and 13 tuberculosis patients and 19 healthy controls from the area of endemicity (here, healthy controls, or HC) were investigated. The highest responses were observed (67 to 100%) in HC for all peptides except p1 to p3, and the lowest was observed in tuberculosis patients. Significant differences in lymphoproliferation were observed in T, L, and HC groups (analysis of variance [ANOVA], P = 0.000 to 0.015) for all end-to-end peptides except B and for p5 and p7 to p10. Hierarchical recognition between lepromatous and tuberculoid leprosy was noted for p8 (P < 0.05) and between the HC and L groups for p7 to p10, p15, and p16 (P < 0.005 to P < 0.02). Significant lymphoproliferation was observed to peptides A to F and p1 to p9, p11, p12, p15, p16 (P = 0.000 to 0.001) with 40% responding to peptides C and p16 in L patients. Lepromatous patients also showed significantly higher levels of a gamma interferon (IFN-γ) response to peptide C than to other peptides (P < 0.05). Major histocompatibility complex (MHC) class II bias for peptide recognition was not observed. These studies indicate that Lsr2 has multiple T cell epitopes that induce in vitro T cell responses in the highly infective lepromatous leprosy patients.

Transmission of dapsone-resistant leprosy detected by molecular epidemiological approaches.

Drug resistance surveillance identified six untreated leprosy patients in the Philippines with Mycobacterium leprae folP1 mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked by M. leprae variable-number tandem repeat (VNTR) strain types. In India, folP1 mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target gene rpoB. These findings indicate that dapsone resistance is being transmitted.

Increased CXC ligand 10 levels and gene expression in type 1 leprosy reactions.

Type 1 reaction (T1R) is a systemic inflammatory syndrome causing substantial morbidity in leprosy. T1R results from spontaneously enhanced cellular immunity in borderline types of leprosy, but there are no established laboratory markers for the reaction. Preliminary studies have identified elevated circulating CXC ligand 10 (CXCL10) during T1R. Correlation of CXCL10 with clinical T1R was studied in repeated serum specimens obtained before, during, and after T1R. CXCL10 gene expression was assessed in biopsy specimens taken before and during T1R, and sections were stained for the cytokine using monoclonal antibodies. Sequential serum specimens revealed elevation of circulating CXCL10 associated with episodes of T1R (P = 0.0001) but no evidence of an earlier, predictive change in the level of the chemokine. Reverse transcriptase (RT)-PCR revealed elevated expression of CXCL10 transcripts during T1R, but not in patients who did not have T1R. No significant correlation between CXCL10 and gamma interferon (IFN-γ) mRNA levels was observed. Immunohistochemical staining of the skin biopsy specimens suggested an overall increase in CXCL10 but did not identify a particular strongly staining population of leukocytes. Increased CXCL10 in lesions and serum is characteristic of T1R. CXCL10 measurement offers new possibilities for laboratory diagnosis and monitoring of T1R. Studies of the regulation of CXCL10 may provide insight into the mechanisms of T1R and identify potential new drug targets for treatment.

VNTR typing of Mycobacterium leprae in South Indian leprosy patients.

OBJECTIVES: To study the suitability, stability and diversity of short tandem repeat (STR) genomic markers to elicit strain variation in the Mycobacterium leprae isolates within leprosy patients from Andhra Pradesh and Tamil Nadu states in South India. MATERIALS AND METHODS: Slit skin smear (SSS) samples were collected from lesions and various body sites of newly diagnosed leprosy patients. The SSSs from each patient were pooled, except in the case of five patients. Total DNA was extracted from SSS samples. M. leprae STRs were amplified from the DNA either by multiplex PCR (MP) or single PCR methods. The number of repeats for each STR locus (the STR allele) was obtained either by fragment length analysis (FLA) or by DNA sequencing of the PCR amplicons. RESULTS AND CONCLUSION: Multiplex PCR minimised the use of DNA and reagents, and together with FLA, was time and cost effective for STR strain typing. After examination of the isolates of South Indian origin at 13 STR loci, it was determined that the alleles for (AC)8b, (GGT)5, 6-3a (rpoT), 21-3, 27-5, and 23-3 were conserved in two study populations. In a family from Andhra Pradesh, the M. leprae STR patterns in two patients were identical in 16 of 18 loci which indicate a common source of infection. Fourteen of 15 STR loci showed no intra-patient variation in the five patients tested in Tamil Nadu. Altogether, these studies indicate the suitability of STR strain typing for assessing short-range transmission chains.

High-resolution sonography: a new technique to detect nerve damage in leprosy.

BACKGROUND: Leprosy is the most common treatable peripheral nerve disorder worldwide with periods of acute neuritis leading to functional impairment of limbs, ulcer formation and stigmatizing deformities. Since the hallmarks of leprosy are nerve enlargement and inflammation, we used high-resolution sonography (US) and color Doppler (CD) imaging to demonstrate nerve enlargement and inflammation. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] We performed bilateral US of the ulnar (UN), median (MN), lateral popliteal (LP) and posterior tibial (PT) nerves in 20 leprosy patients and compared this with the clinical findings in these patients and with the sonographic findings in 30 healthy Indian controls. The nerves were significantly thicker in the leprosy patients as compared to healthy controls (p<0.0001 for each nerve). The two patients without nerve enlargements did not have a type 1 or type 2 reaction or signs of neuritis. The kappa for clinical palpation and nerve enlargement by sonography was 0.30 for all examined nerves (0.32 for UN, 0.41 for PN and 0.13 for LP). Increased neural vascularity by CD imaging was present in 39 of 152 examined nerves (26%). Increased vascularity was observed in multiple nerves in 6 of 12 patients with type 1 reaction and in 3 of 4 patients with type 2 reaction. Significant correlation was observed between clinical parameters of grade of thickening, sensory loss and muscle weakness and US abnormalities of nerve echotexture, endoneural flow and cross-sectional area (p<0.001). CONCLUSIONS/SIGNIFICANCE: We conclude that clinical examination of enlarged nerves in leprosy patients is subjective and inaccurate, whereas sonography provides an objective measure of nerve damage by showing increased vascularity, distorted echotexture and enlargement. This damage is sonographically more extensive and includes more nerves than clinically expected.

Clinical Utility of LSR/A 15 Gene for Micobacterium leprae Detection in Leprosy Tissues Using the Polymerase Chain Reaction

Resumo: Skin biopsy and slit-skin smears from 46 leprosy patients and 4 nonleprosy patients were tested for the presence of Mycobacterium leprae by the polymerase chain reaction (PCR) using primers based on the sequence of the LSR/15 kD gene. The PCR was found to be specific and sensitive, with a detection level of 10 and 100 bacilli. PCR using skin biopsies gave a higher detection rate than did slit-skin smears, probably due to the higher density of bacilli in a 4-mm punch biopsy. Dot blot hybridization with radioactive probes was 10-fold more sensitive than the ethidium bromide staining. Eight patients who did not show acid-fast bacilli in tissues by the conventional methods were shown to have PCR-amplified M. leprae DNA. False-negative results were obtained in 3 cases even though formal evidence for tissue inhibitors was absent