Distribution of CD4+RORg-T Th17 and CD25+ FOXP3+ Treg in leprosy patients with reversal reaction

Objective To compare the distribution of CD4+ Th17 and CD25+ FOXP3 T regulatory cell (Treg) between type I reversal reaction (RR) and type II reversal reaction i.e. erythema nodosum leprosum (ENL) patient groups. Methods A total of 50 samples, consisted of 27 samples of reversal reaction (RR) and 23 samples of ENL, were collected. Observation of CD4+ RORg-T Th17 and CD25+ FOXP3 Treg were conducted with immunohistochemistry staining technique using anti FOX-P3 and anti RORg-T. Expression of CD4+ ROR-g Th17 and CD25+ FOXP3 Treg in percentage were analyzed using Ttest. Results There is a significant difference in mean CD4+ ROR-g Th17 and IL17 cell distribution for RR patient group (14.96% and 10.72%) compared with ENL (9.12% and 4.28%). No significant difference were found between mean CD25+ FOXP3 Treg and TGF-β cell distribution in RR patient group (6.12% and 5.44%) compared with ENL group (6.16% and 5.96%). Conclusion There is a significant increment od CD+RORg-T Th17 and IL17 in RR patients group compared with ENL patients group. however, the distribution of CD25+ FOXP3+ Treg and TGF beta in RR has no significant difference compared with ENL.


INTRODUCTION
Leprosy is a frightening disease for community which cause ulceration, mutilation, and deformit ies to the patients.The disease has variety of manifestations depend on patient's immune response towards the infection.Two key purposes from leprosy management are recovery and disability prevention fro m the disease.WHO reco mmended a Multi Drug Therapy (MDT) consists of rifamp icin and dapsone for PB leprosy, and rifampicin, dapsone, and clofazimine to treat MB leprosy.Therapy is given for 6 months duration for PB leprosy and 12 months duration for MB leprosy.MDT has been proven effective to eradicate M. leprae in most of the patients. 1Even though it has been proven effective, Index by : the most difficult issue in leprosy management is to manage the leprosy reaction.
Leprosy reaction is caused by patient's immunological dynamic response to M. leprae which could happen before, during, and after MDT administration. 2This reaction is an acute episode of the disease with constitutional symptoms, activation, sometimes accompanied with new skin efflorescence.Two types of leprosy reaction are type 1, known as reversal reaction (RR), caused by acute changes in cellu lar immun ity, and type 2 reaction known as eritema nodosum leprosum (ENL), caused by humoral immun ity process. 3Reversal reaction is associated with cell immun ity (CMI) activit ies, consists of T-helper cell (Th 1) and (Th2).It is characterized with granulo ma expansion, edema, CD4+ recru it ment, increasing IL-2, IL-10, and IL-12 receptors, and expression of human leukocyte antigens-D in cellular infiltrate. 4nother CD4+ Th subset that shows divergence in leprosy is the Th17 populationassociated with inflammat ion. 5 The Th17 subset is highest in healthy contacts exposed to the diseases compared with patients, indicating its importance in innate immun ity. 6,7Th17 cells appeared in the leprosy patients who showed non-polarised Th0 subset. 6herefore it would appear that Th17 plays a ro le in the immune responses to M. leprae infection and may be an alternate pathway for bacillary clearance both in the early and later stages of infection.The associated chemokines may help in its migrat ion to lesional sites.
Another distinct lineage of T cells that has excit ing implications for dampening inflammatory responses is the T regulatory cell (Treg), which has a CD4+CD25+ nuclear FOXP3+ phenotype and shares a similar d ifferentiation pathway to Th17 cells, although they have opposite effects.Several types of Tregs have been described; some are natural Tregs derived fro m the thymus and act via contact with target cells. 8Others are inducible and mediate inhibit ion through cytokines such as transforming growth factor-β (TGF-β) and IL-10 (induced Treg [iTreg]). 9Transcription factor FOXP3 is thought to be the primary requirement for the suppressive function, though low and transient expression has been reported in activated human T cells with and without suppressor function.Though Tregs in mice express CD25 constitutively, in humans only those with CD25 show suppressive function. 10eprosy reactions in contrast showed a decrease in Treg cells which paralleled the increase in Th 17 population. 11Moreover, there was downregulation of intracellular TGF-β. 11,12Most reports showed a reduction in Treg cells in patients with ENL reactions compared with non-reaction L-lep counterparts.Akan tetapi pada reaksi kusta tipe I (RR) bagaimana distribusi Th17 dan Treg masih belum jelas. 11,12However, in type I leprosy reaction (RR), the distribution of Th17 and Treg has not yet been identified.Thus, this study aimed to compare the distribution between Th17 and FOXP3 Treg in RR and ENL patient groups.Biopsy samples were collected at the lesion area or lower arm extensor side.Then, the area was disinfected with alcohol 70% and local anesthetic Lidocaine 0.25 ml was administered subcutaneously near the biopsy area.Biopsy were conducted using punch method with diameter of 3 mm.Tissue sample was inserted to a 1.5 ml tube filled with formalin 10%.The after-biopsy skin was cleaned using NaCl 0.9%, then topical antibiotic fusidic acid was applied and the skin was covered with sterile gauze.The sample underwent a fixation and processed in paraffin.Microscopic slide was made using rotary microto me with 4µm slicing.Stain ing with Hemato xylin-Eosin was applied to confirm the structure and leprosy type.Observation of CD4+ RORg -T Th17 and C25+ FOXP3+ Treg were using immunohistochemistry single staining technique with anti-FOX-P3 anti RORg-T retrieved fro m Santa Cruz Biotech (USA ) and immunohistochemistry kit of D-Bio Sys Immunostaining Kit (Netherland).The staining result was then photomicrographed using Nio kon E-100 M icroscope with ICLEA7 Sony Camera 400x magnification, then expression analysis was done using immunoratio software (Freeware).Percentage of expression CD4+ RORg-T Th17 and C25+ FOXP3+ Treg were inserted to table and analyzed using T-test in IBM -SPSS 21 for W indows.

RES ULT
In this study, there were 27 samp les of RR patients and 23 patients of ENL patiens.Each cases were classified based Hemato xy lin-Eosin (HE) staining microscope examination, as the figure below.Immunohistochemistry study result showed a significant difference in mean CD4+RORg-T Th 17 and IL17 cell distribution in RR patient group (14.96% and 10.72%) co mpared with ENL patient group (9.12% and 4.28%).Ho wever, no significant difference in mean CD25+ FOXP3+ Treg and TGF-β cell d istribution in RR patient group (6.12% and 5.44%) compared with ENL patient group (6.16% and 5.96%).

DISCUSS ION
In this study, exp ression of FOXP3+ was evaluated from the skin with T lymphocyte infiltrate.We found that Treg cells were produced in reactive state (either type I or II).Treg cell has an important role in controlling the overactive immune response towards microbe's antigen, especially pathogens which caused persistent infection in this study.Prior study has explained a significant decrement of FOXP3+ Treg in ENL co mpared with stabilized leprosy patients, although it was not found in RR patients. 12In this study, we found FOXP3+ Treg in all skin specimens of RR and ENL with a low nu mber.Fro m all of the case, positive FOXP3 cell was strongly related with epithelio id or macrophage, showing a functional interaction between Treg and histiocyte.We observed no statistically significant differences between FoxP3+ Treg cells between type I reaction (RR) and type II reaction (ENL), even the number is almost the same.
Other study showed that CD+ FOXP3+ T-Reg cell produced TGF-β and increased in stabilized lepro matous patients and also could explain issues with this type of leprosy. 11our study showed that TGFb 1 occurred in type I leprosy reaction (RR) and type II (ENL), where the presentation tends to be lower in RR thatn ENL, but this is not statistically significant.
This study emphasized an inhibition of FOXP3 cell expression and also TGF-β in RR condition compared with ENL, wh ich related to an unresponsiveness of T cell observed in RR patients.One interesting finding in this study is an increasing RORgT Th 17 positive cells found in RR leprosy compared with ENL patients, and it was statistically significant.Also, RR patients showed a higher IL-17A expression than ENL patients.
The present study was undertaken with a view to understanding the inflammation and or immunopathology seen in patients under going episodes of leprosy reactions which are a cause of severe morbidity and nerve damage.Previous studies had shown that Th17 cells formed a third subset in leprosy and were seen in stable leprosy patients in the absence of Th1 and Th2 polarization. 11Our study showed a disturbed equilibriu m between Th17 cells and Treg cells in leprosy reaction, especially increased Th17 cells and decreased Treg cells in number.Th is imbalance was mediated by cytokines, showed by decreasing TGF-β number in the same time.Increasing Th17 activ ities with IL-17A expression would explain the inflammatory and immunopathology process caused by leprosy reaction.
Overall, our results provide some evidence to the hypothesis that, in T1R, down modulation of Tregs cells would favor the development of Th-17 responses that characterize this type of reaction.We thus believe that better understanding of the role played by Tregs cells in reaction episodes can possibly provide a new target for the treatment of this still-challenging complication of leprosy.
A total of 50 samples were used in this study, consisted of 27 samp les of RR patients and 23 samples of ENL patients.The samples were collected fro m outpatient clin ic in Donorojo Hospital and Ministry of Health in Jepara fro m June 2014-August 2014.Patients were 20-60 years old and had agreed to sign the standard informed consent from Ethical Co mmittee of Airlangga University.