Th17-Mediated Immunodulation In Patients With MDR-TB And During Immunotherapy With RhIL-2

Background:Multidrug-resistant tuberculousis(MDR-TB), defined as resistance to at least isoniazid and rifampin, has emerged as a lethal global threat today. In notably countries in central Asia and eastern Europe 9-32% of new cases have MDR-TB and more than 50% of previously treated cases have MDR-TB. A total of 107 countries reported outcomes for treatment success in MDR-TB patients notified was only 48%, with the Global Plan’s target for 2015 of achieving at least 75% according to WHO report 2012. The treatment outcome of MDR-TB often showing higher rates of treatment failure and deaths than drug-sensitive diseases due to the use of classic second-line drugs in treatment of MDR-TB is made a worldwide challenge as to control of TB, which was evidently attributed to the insufficient recovery of disordered immunoregulationTuberculousis is widely recoganized to result from an interaction between a potent immune response and a chronically persistent pathogen. The effective cell-mediated immune responses(CMI) is known as critical protective immune mechanism for controlling mycobacterium. The impaired CMI lead to immunopathological tissue damage. The adaptive immune response against TB requires activation of CD4+T cells, which was pivotal for an effective specific immune response to Mtb infection. Naive CD4+T cells may differentiate into one of several lineages of T helper (Th) cells upon their cytokine profile and transcription, including Thl, Th2 and regulatory T cells (Treg), Th17 population was identified recentely with IL-17 as their signature cytokins to induce pro-inflammatory responses and to interact with other effector T cells. Emerging data reported that Th17 cells played an important role in host defense against specific antigen with expanding of interleukin (IL)-17 expression detected during early stage of Mycobacterium tuberculosis infection,suggesting that Th17 responses are triggered by specific pathogens which are not efficiently cleared by Thl and Th2-type immunity. However little is known about the role of Th17 play in MDR-TB patients as well as their dynamics during MDR-TB treatment, moreover we have not yet fully defined either their and or capacity to mediate protection or pathogenesis.IL-2 cytokines play a pivotal role in CMI, with inducing proliferation and differentiation of the activated effector T cells, promoting the secretion of γ-interferon(IFN-γ), and enhancing the ability of the anti-Mycobacterium tuberculosis. A former study reported that optimized therapy with low-dose adjunctive rhIL-2 for MDR-TB patients enhanced the reaction to treatment with releasing the bacterial load with improving the incidence and speed of sputum conversion, as well as increased expression of IFN-y mRNA and IL-2mRNA detected at the site of PPD into the body after 28 days of adjunctive rhIL-2 administation, suggesting that adjunctive rhIL-2 immunotherapy activated effective CMI. Until now, rare progress at home and abroad was reported on large-scale clinical studies involving immunotherapy with rhIL-2 for MDR-TB, yet the mechanisms underlying is unclear, therefore, to clarify the immunomodulation mechanisms triggered by MDR-TB infection helping to find a therapy focusing on transition from immunopathologic pathway toward the protective immune pathway,which would improve treatment success for MDR-TB, our present study including three part respectively focusing on the clinical effects of immunotherapy with adjunctive rhIL-2, the role of Th17 in immunomodulation and the alteration of Th17 response during treatment with adjunctive rhIL-2,all of which were based on MDR-TB patients.Methods and resultsPart 1 The clinical effects of immunotherapy with adjunctive rhIL-2 for patients with MDR-TBMethods:The randomized case-control prospective study was employed to recall the MDR-TB cases and observe their efficacy of adjuvant therapy and their prognoses. Based on the multiple center collaboration with 101 valid cases enrolling,51 cases in control group were administered the standard chemotherapy within a course of 24 months while 50 valid cases in treated group were administered recombinant human interleukin-2(rhIL-2) in combination with the standard chemotherapy within a total course of 18 months. RhIL-2 was administered during the first eight month of the total course. Bacteriological and radiological improvement were compared between the two groups at the end of each month during the course of treatment Results:By end of the treatment, the treatment group show lower cases of treatment failure, missed and deaths than the control group. The cure rate of the treatment group was significantly 28% higher than the treatment group with 53% versus 25%(p= 0.02). Course of treatment was shortend with adjuvant rhIL-2 in treatment for at least 6 months. Conclusion:Standard anti-tuberculousis chemotherapy in combination with rhIL-2, for improving the treatment success and course,was potentially effective therapy for MDR-TB.Part 2 The role of Th17 in immunomodulation of patients with MDR-TBMethods:Flow cytometry and the Cytometric Bead Array (CBA) were used respectively from the cell and protein levels to measure the frequency of Th17,Treg and Th1, Th2 cells as well as the concentration of their associated cytokines in circulation of 25 MDR-TB cases from control group as compared to their healthy controls and DS-TB controls. Dynamic change were observed and compared before and during the chemotherapy of these 25 cases analysed with their clinical data contemporary. Results:MDR-TB patients show higher levels of Th17 proportion (3.28 ± 0.55)% (p<0.01) and Treg cell proportion (p<0.01) as well as lower levels of Thl proportion(p<0.01),while higher levels of IL-17, IL-4,IL-6 pg/ml (p<0.05), and lower level of IFN-γ (p<0.05) also found in circulation before treatment in comparison with their DS-TB patients and healthy controls. During the treatment course MDR-TB patients show decreasing of Th17 proportion (p<0.01) and increasing of Th1 proportion (p <0.01) up to the level in healthy subjects (p>0.05) as well as decreasing of IL-17,IL-6, IL-4 and increasing IFN-yconcentrations in circulation(p<0.05) according to the data after 6 months and after 12months when compared to their baseline,moreover a positive correlation between the expressions of IL-17 and IL-6 were found statistically in MDR-TB patients during treatment (p <0.05, r= 0.25 [0.20-0.31]). Much lower levels of Th17 and Thl expression was found in patients with improved lung radiologic picture than the patients with no change or deterioration radiologically after 12 month treatment (p<0.01). Conclusion: Th17-mediated response play a critical role in the immune defence response triggered by MDR-TB for producing pro-inflammatory effect probably to compensate for impaired Thl response. Imbalance of Th17/Thl responses was restored as improvement in focus of infection.Part 3 The alteration of Th17 response during treatment with adjunctive rhIL-2 for patients with MDR-TBMethods:Flow cytometry and real-time quantitative PCR (RT-qPCR) were used respectively from the cell and gene levels to measure dynamic expression of Thl7, Treg and Thl cells as well as relative level of RORyt mRNA, Foxp3mRNA and oth、 er associated cytokines mRNA in 23 cases from treatment group before,during and after the adjunctive rhIL-2 administeration as compared with contemporary parallel data of the 25 control cases. Results:Cases administerd with adjunctive rhIL-2 treatment presented expanding in Thl expression and striking decrease in Th17 and Treg expressions during and after rhIL-2 course as compared with the parallel data of the cases from control group (p<0.05), while the relative level of IL-17A mRNA, ROR-γt mRNA and Foxp3mRNA much lower, IFN-γ mRNA significantly elevated (p <0.05). The value of Th17/Treg was found decreasing during treatment however with no contemporary difference between the patients from the two groups.Conclusion:Th17-mediated responses probably for producing excessive inflammation was weakened with the Thl-mediated response with protective effect strengthened in patients with MDR-TB after immunotherapy with adjunctive rhIL-2. Th17 was involved in the modulation of balance between protective mechanisms and immunopathologic mechanism induced by CMI in pattens of balancing Th17/Th1 responses and Th17/Treg responses induced.Summary:Our study suggested that rhIL-2 improved the treatment success for MDR-TB and that Th17 population would be potential markers in immune monitoring, as well as providing clues for the further study on immunodulation mechanisms and theoretical basis for the the immunotherapy for MDR-TB patients. Further study is required for elucidating the mechanisms of involvement of Th17 in immunoregulation network.