Elevated IL-6R on CD4+ T cells promotes IL-6 driven Th17 cell responses in patients with T1R leprosy reactions

Th17 cells play vital role during pathogenesis of leprosy reactions. Previously, we have reported that IL-23 is involved in Th17 cells differentiation. Subsequently, our group also showed that IL-6 induces Th17 cell differentiation along with TGF-β in leprosy reactions. Here, we next asked the question that whether IL-6 or IL-23 induced Th17 cells are different in nature? In this study, Type 1 Reactions (T1R) showed significantly (p < 0.001) higher percentage of IL-17A producing CD4+IL6R+ T cells as compared to non-reaction (NR) patients. Furthermore, recombinant IL-6, IL-23 and TGF-β promoted IL-17A secretion by CD4+IL6R+ T cells. Subsequently, IL-6R and IL-23R blocking experiments showed significantly (p < 0.002) down regulated IL-17A in T1R reaction as compared to NR leprosy patients. The present study for the first time establishes that pathogenic Th17 cells produce IL-17 in an IL-6 dependent manner in leprosy T1R reactions. Thus, present approaches that specifically target Th17 cells and/or the cytokines that promote their development, such as IL-6, TGF-β and IL-23A may provide more focused treatment strategies for the management of Mycobacterium leprae and its reactions.

system and are also activated by various cytokines, such as IL-1β, IL-6, IL-23 and TGF-β. The differentiation of naive T cells into Th17 cells is regulated by several mechanisms. Mainly, it has been reported that TGF-β and IL-6 coordinately induce Th17 differentiation 11,12 via induction of transcription factor ROR-γt, which is a downstream target of signal transducer and activator of transcription 3 (STAT3) [13][14][15] . Saini et al. 8 , reported that IL-23 is also identified as an essential cytokine for Th17 cells differentiation in leprosy diseases. Furthermore, our group has also reported that rIL-23 modulates the plasticity of Tregs in leprosy patients which are converted into Th17 like cells 16 . Moreover, aryl hydrocarbon receptor (AHR) contributes to the Th17 cells differentiation through a cytokine-independent mechanism 17 . Among these differentiating cytokines, TGF-β is essential for polarizing naive T cells towards Th17 and Treg cells. Interestingly, our previous report showed synergistic effect of TGF-β with the pro-inflammatory cytokine IL-6 in inducing Th17 differentiation 18 . Moreover, IL-23 is also involved in Th17 differentiation in non-reaction (NR) leprosy patients. Surprisingly, the association of IL-23R and not IL-6R with IL-17 + cells in NR leprosy patients is not well studied. IL-6R and IL-23R are key players in the development and maintenance of Th17 cells 19 . A recent study demonstrated increased percentage of CD4 + T cells expressing IL-6R in chronic hepatitis B patients and higher levels of IL-17 upon stimulation with the HBV core antigen (HBcAg) in vitro 20 . IL-6 cytokine has the ability to polarize Th1/Th2 cells balance towards Th2 cell subset. Several studies have shown that murine naïve CD4 + T cells differentiate into Th17 cells via simultaneous treatment with IL-6 and low doses of TGF-β 21 .
Our second study on leprosy reactions demonstrated that elevated expression of IL-6 and lower TGF-β in leprosy reaction initiated synergistic effect in differentiation of Th17 cells 18 . In brief emergencies in reversal reactions is increased by Th17 cells which may be triggered by subsequently decreased Treg cell activity through low TGF-β producing FOXP3 + Treg cells. The synergetic effect of down regulated TGF-β and upregulated IL-6 in both reactions may play an important role in the balance of Treg and Th17 cell differentiation and thereby lead to the immunopathology associated with leprosy reactions 18 . Th17 and Treg cells are key players in immunopathology of leprosy reaction and leprosy as reported by us and others 8,10,18,22,23 . Their role in leprosy reactions and the differentiation and signaling pathways of these cells are yet to be fully determined. Therefore, by elucidating the molecular mechanisms that link Th17 differentiation and immuno-pathogenesis in leprosy reactions, a novel therapeutic strategy could be provided. Various kinds of cytokines and receptor can positively or negatively regulate the development of leprosy reactions. In contrast, interferon (IFN)-γ, a Th1 cytokine, is considered a positive regulator of leprosy, as disruption or low production of IFN-γ enhances the severity in leprosy 4 . These findings indicate that cytokine network determines the progression or regression of leprosy 24 . In the present study, using IL-6 receptor (IL-6R)-blocking antibody, we examined the effects of IL-6 blockade on leprosy reactions, in light of Th17 cell differentiation. Consequently, we report that IL-6 is essential for the initiation of leprosy reactions, rather than its progression, through Th17 differentiation. The present study thus for the first time establishes that specifically targeting Th17 cells along with its cytokine IL17 may provide more focused treatment strategies for the management of M. leprae and its reaction.

Materials and methodology
Patient recruitment and ethics statement. A total of 56 new patients (Untreated; without MDT and steroid) were investigated. Samples were obtained from patients who were clinically and histologically proven for leprosy type according to Ridley-Jopling classification 25

Isolation of peripheral blood mononuclear cells (PBMCs) from blood samples. Six ml heparin-
ized venous blood samples were layered on Ficoll-Hypaque (Sigma Aldrich, USA) after diluting with 1:1 in RPMI 1640. PBMCs were isolated by density gradient centrifugation at 800×g for 20 min. Cells in the interphase were collected into a new tube and washed thrice in sterile 1 × HBSS by centrifugation at 1,600 rpm for 10 min. After the last wash cells were re-suspended in RPMI 1640 along with 10% FBS (Sigma, USA), cell viability and enumeration were estimated by 0.2% trypan blue exclusion using hemocytometer.
Cell culture. 2 × 10 6 cells/ml were stimulated with or without Mycobacterium leprae sonicated antigen (MLSA; 10 μg/ml) and recombinant-IL-6 (10 ng/ml; Peprotech NJ, USA), r-TGF-β (2.0 ng/ml, Peprotech NJ, USA) and r-IL-23 (6.0 ng/ml, R&D, USA) were added with different combinations. Cultures were also stimulated with phytohemagglutinin (PHA; 5 μg/ml, Sigma, USA). Cells were stimulated in tissue culture media containing 10% FBS (Sigma, USA) and RPMI 1640 (Gibco, USA) and incubated in 5% CO 2 incubator at 37 °C for 48 h. In  After gating, CD4 + T cells were further gated as CD25 + FOXP3 + and CD25 neg FOXP3 neg cells, deriving each gate as a daughter of the gate set up in the previous step as FSC vs. SSC. Two 15 ml centrifuge tubes were prepared by adding 2 ml complete medium as collection tubes for sorting and recorded ~ 20,000 events from each staining control sample. Gates, if necessary were adjusted, based upon the staining controls. Once the gates were standardized and satisfactory, samples were loaded, and sorting performed. Sorted cells were resuspended in complete medium. To assess the purity of the sorted cells, a small aliquot was run on flowcytometry, under the same template as was used for the sorting. Purity of more than 96.2% was routinely observed. The sorted CD4 + T cells were later cryo-preserved for qPCR analysis.
RNA isolation and reverse transcriptase PCR reaction. RNeasy Mini Kit (Qiagen, USA) used for RNA isolation from sorted CD4 + CD25 neg FOXP3 neg cells according to manufacturer's instructions. Nanodrop spectrophotometer (Nanodrop Technologies, USA) was used for quantification of RNA. RNA purity at 260/280 from 1.8 to 2.0 was considered to be best. RNA (28S and 18S) was also checked for quality, RIN value of ≥ 7 was considered to be optimum by using Bioanalyzer (Agilent Technologies, Singapore). For reverse transcriptase PCR reaction, 500 ng total RNA was transcribed into cDNA using RT First strand kit (SA Biosciences, USA). RT-PCR was performed according to the manufacturer's instructions and cDNA stored at − 20 °C till further use. www.nature.com/scientificreports/ (anti-mouse antibody) was added, and plates were further incubated at room temperature for 1 h. After 5 times washing, 100 µl/well of Avidin-HRP diluted in 1× assay diluent was added and incubated at room temperature for 30 min. Color development step was done with peroxidase color substrate TMB (Tetra Methyl Banzedine). After developing, the plate reaction was stopped by stopping solution containing 1 N H 2 SO 4 . The optical density (OD) was taken at 450 nm.

Results
Isolated CD4 + T helper cells were investigated in 19 of each Type 1 reactions (T1R) patients and non-reactions (NR) leprosy patients by Real Time PCR (qPCR) for RNA estimation. 48 h, MLSA stimulated PBMCs cultures, were studied in patients with Type 1 reactions (T1R, n = 10) and non-reactions (BT, n = 10) leprosy patients and further evaluated by flow cytometry for Th17 cell characterization. Furthermore, ELISA was performed for measuring cytokines in culture supernatants. The enrolled patients details of the T1R reaction and NR clinical groups of leprosy are listed in Table 1.

Hierarchical clustering identified inflammation associated genes correlation with IL-6R.
Gene expression of isolated CD4 + T cells of leprosy patients (NR vs T1R) were analyzed for 21 signature genes of Th17 lineage and associated molecules with a rationale to understand the differences between the immunopathology/ inflammation associated with localized T1R reactions via qPCR assay (Fig. 1). Heat maps of gene expression showed significantly high intensity in T1R as compared to NR patients.  (Fig. 2). Table 3 lists correlation between IL-6R and IL-23R gene expression with other Th17 related inflammatory markers. Moreover, IL-6R www.nature.com/scientificreports/ gene expression was also found to have significant (p < 0.01-p < 0.005) positive correlation with IL-17 isomers in T1R but not in NR patients. On the other hand, IL-23R showed significant (p < 0.0001) positive correlation with IL-17 isomers in NR but not in T1R patients. Surprisingly, IL-17C showed positive correlation in both T1R and NR patients (Table 3).This may be due to variation in kinetics of secretion and thus further study is warranted.
Type 1 reactions showed high CD4 + IL6R + T cells. Figure 3A flowcytometry dot plot is showing FMO control for IL6R + and IL23R + with respective flurochrome. Figure 3B,E shows analyzed 48 h MLSA stimulated PBMC by flow cytometry in T1R and NR patients. Of interest, we have also compared in-vitro MLSA stimulated with unstimulated cultures to know how IL-6R and IL-23R expression respond under these conditions. Interestingly, we didn't find any significant difference (data not shown). Dot plot shows CD4 + IL-6R + (Fig. 3B)  Table 2).Groups of genes were associated with hierarchical clustering using the complete-linkage clustering (pcrdataanalysis.sabiosciences.com). Each horizontal row represents the same gene product and each vertical row the same patient. The fluorescence range from high (red) to low (green) is indicated by the colored bar and fluorescence intensity/gene expression. In general, T1R patients show higher expression of several genes representing, cytokines, cytokine receptors, and chemokines as compared to NR patients (see also    (19)  www.nature.com/scientificreports/ and IL23R + cells (Fig. 3E). CD4 + IL-6R + cells (Fig. 3C) were significantly high (p < 0.0003) in T1R (mean% ± SD; 7.9 ± 1.0) as compared to NR (5.2 ± 1.0) patients. However, CD4 + IL23R + (Fig. 3F) cells were detected without any difference between T1R and NR leprosy patients. Moreover, we further analyzed co-expression of IL-6R + and IL-23R + on CD4 + cells but no significant co-expression was detected (data not showed). Of interest, gene expression of IL-6R and IL-23R in T1R and NR patients in 48 h MLSA stimulated PBMC (Fig. 3D,G) showed a significant increase (p < 0.01, p < 0.002) in T1R as compared with NR leprosy patients. IL-6R and IL-23R showed high delta threshold cycles (ΔCt) (mean ± SD; 2.9 ± 0.4) and (3.5 ± 0.8) in T1R group as compared to (3.7 ± 0.6) and (5.0 ± 0.8) NR patients respectively.

Discussion
The role of inflammatory Th17 cells in pathogenesis have already been explored in many bacterial [27][28][29] and viral infections 30 along with autoimmunity 27 and cancer 31,32 . Differentiation of inflammatory Th17 cells depends upon a specific cytokine milieu and specific signaling via receptors. Our earlier study Saini et al. 8 showed that when T helper cells polarization is not occurring against M. leprae antigen, Th17 cells play a protective role in stable leprosy (NR) patients. Moreover IL-23 and its receptor are involved in differentiation of Th17 cells in NR patients 8 . M. leprae specific T-cell mediated immunity is the main cause of T1R and it has already been assessed by in-vitro studies against M. leprae antigens 33 . T1R leprosy reactions occur for clearing bacteria and such increased cellular immune responses may be beneficial because they promote bacterial killing mechanisms. However, the accompanying inflammation in and around the infected nerve tissue can result in severe and irreversible damage and is a matter of debate. Our group earlier reported Saini et al. 18 that Th17 response was significantly high in T1R patients as compared to NR. Moreover, the present study showed that its not IL-23 and TGF-β but IL-6 and its receptor IL-6R which are high in T1R. Taken together, all these data provided evidence that Th17 cells are involved in cell mediated inflammation. In the present study, we have analyzed 21 genes related to Th17 signatures and involved in inflammation. IL-6R was showed to be significantly high in T1R and correlated with IL-17 isomers. On the other hand, IL-23R was correlated with IL-17 isomers in NR patients but not in T1R patients. These finding also indicates that both IL-6 and IL-6R are important for T1R leprosy reactions. Moreover, two patients have shown high intensity of gene expression as they had erythematous plaques with hyperesthesia and tenderness clinically. The correlation also supports that inflammation occurs due to IL-6 and its receptor in T1R patients. www.nature.com/scientificreports/ The present study attempts to investigate the pathway underlying the induced Th17 response in T1R patients, we first observed cytokine milieu which was involved in IL-17 production and found that IL-6 and IL-23 play a major role in immunopathology of T1R patients. Additionally, decreasing or absence of Th1 associated IL-12/ IFN-γ promotes IL-23/IL-17 for protective immune response observed in primary infection by M. tuberculosis and M. leprae 34,35 . IL-6 is a pleiotropic cytokine which can control immune regulation in infectious diseases. In the context of autoimmune disease, IL-6 in combination with TGFβ1 promotes the differentiation of Th17 cells. On the other hand IL-6 also suppresses the differentiation of Treg cells. A report by Heink et al. 36 showed many sources of IL-6 production via different mechanisms involved in generation of pathogenic Th17 cells and the inhibition of Treg cells in-vivo. Another significant report considered Th17 lineage with protective nature in infectious diseases. Some important reports found that IL-17 + cells are a part of the innate T-cell immune response like neutrophils activation during the primary response to intracellular pathogen 9,37,38 . However, findings diverge on the association of traditional CD4 + IL-17 + T cells and showed adaptive immune response to these infections 39 . Many studies showed that IL-17 helps to create bridge between innate and adaptive immune responses with the help IFN-γ. TGF-β plays distinct role during Th17 immune responses in M. leprae infection, by assisting the differentiation and production of Th17 responses in leprosy. However, TGF-β is associated with FOXP3 + Treg differentiation and stability and also our previous study have already reported that TGF-β is low in T1R as compared to NR leprosy patients 18 . The absolute function of TGF-β in regulating and balancing Th17 and Treg response in T1R patients remains to be elucidated. Subsequently, TGF-β was maintained in homeostatic differentiation of Th17 cells with IL-6. This can be another homeostatic kinetics in T1R and NR patients and it Figure 6. Combination of IL-6 + TGF-β showed significantly down regulated IL-17A protein as compare to all combinations in T1R and NR patients. Representative scatter plot is showing mean% ± SD of IL-17 (pg/ml) in 48 h MLSA and diffrent combinations of recombinants IL-6, IL-23 and TGF-β stimulated culture supernatant in T1R (10) and NR (10). Abbreviations: Mann Whitney T-test was performed for the statistical difference, p < 0.05 value was considered as significant NR; Non-reactions, T1R; Type 1 reactions), MLSA; Mycobacterium leprae sonicated antigen. experiments showed that PBMCs produced significant amount of IL-17A. Thus, further investigations are necessary to understand the mechanism underlying the increased IL-6R expression on CD4 + T cells, as well as the induced Th17 responses in T1R patients. Subsequently, consistent with our previous findings in patients with T1R leprosy, we found IL-6R and IL-6 expression in stimulated PBMC cells, correlated with the Th17 response in T1R. However, we did not find any difference unlike M. leprae antigens which down-regulated IL-6R expression on CD4 + T cells from patients with NR patients. Among the IL-6R and IL-23R, increased CD4 + IL-6R + T cells showed T cell activation via T cell receptor (TCR) engagement or upon binding of IL-6 to IL-6R 40,41 . Because proliferation of M. leprae antigen-specific Th17 cells depend on TCR engagement with high IL-6R expression and is correlated with specific IL-17A response in our study, this mechanism explains the increased IL-6R expression in T1R as compared to NR leprosy individuals in this study. Therefore, increased IL-6R expression might be due to engagement of IL-6, the production of which is able to reduce Mycobacterium infection 42,43 . More significantly, our in-vitro data showed that blocking of IL-6R signaling on CD4 + T cells significantly inhibited  www.nature.com/scientificreports/ non-specific (PHA-stimulated) and MLSA specific production of IL-17A, but not IFN-γ as IL-6 is not involved in Th1 cell differentiation. As expected, TGF-β production was significantly enhanced after blocking with IL-6R and IL-23R along with significantly down regulated IL-17. These findings further validate our previous findings thereby establishing synergistic and opposite regulation between Treg and Th17 cells 18 . We found no significant co-expression between the IL-6R and IL-23R in MLSA stimulated cells (data not shown).Also, these cells are different in nature and further investigation is needed.
In summary, our present study for the first time reports that CD4 + IL6R + T cells are involved in IL-17A production in T1R leprosy patients. Thus, it can be a key mechanism for Th17 responses in patients with T1R. Firstly, CD4 + IL-6R + and not CD4 + IL-23R + T cells increase the production of IL-17A in T1R. Secondly, M. leprae (MLSA) antigen activates IL-6R to produce IL-17A by CD4 + T cells from T1R leprosy patients. Thirdly, the production of IL-17A depends on IL-6R signaling. Thus, understanding the immuno-pathogenic nature of Th17 cell responses in T1R patients is of prime importance, and would thus lead to opening of novel avenues in the treatment and management of inflammatory response which are lethal for T1R patients.