Study On Th22 Cells In Systemic Lupus Erythematosus And Their Relationship With Pathogenesis Of Lupus Nephritis

Background: T helper 22(Th22)cells producing IL-22 cytokines,play a critical role in host defense against external infectious pathogens and are important in the pathogenesis of various autoimmune and inflammatory diseases.However,their relations with the development of systemic lupus erythematosus(SLE)and target organ injuries of SLE,such as lupus nephritis(LN)still need to be unraveled.Aim: Using immunology,molecular biology,cytology,as well as other techniques in vivo and in vitro experiments,our study aims at the following aspects: 1.the expression and distribution of Th22 cells in SLE patients;2.the relationships between Th22 cells and disease indicators and the organs impairment in SLE patients;3.the expression and distribution of Th22 cells and their target cells in the biospy of SLE patients with lupus nephritis and skin diseases;4.the Th22 cells secreting function and the potential influence of Th22 cells on the growth and inflammatory response of human renal mesangial cells(HRMC),to explore the potential mechanic role of Th22 cells in the pathogenesis of SLE and LN.Methods: 1.Flow cytometry analysis was performed to determine the expression and distribution of Th22 cells and other Th cell subsets in peripheral blood mononuclear cells(PBMC)of SLE patients and healthy controls and real-time quantitative PCR detecting system(q PCR)was applied to validate the phenotypes.We further analyzed the function of IL-22 producing Th cells with flow cytometric analysis of stain for intracellular IL-22.The levels of IL-22,IL-17,IFN-γ,and TNF-α were determined by enzyme linked immunosorbent assay(ELISA)and cytometric bead array(CBA).2.The study explored the relation between Th22 cells and IL-22 and the disease progression and target organ injuries in SLE by applying stratified analysis according to target organ involvement,such as kidey and skin,and clinical parameters,such as SLE Disease Activity Index(SLEDAI),Revised Cutaneous Lupus Erythematosus Disease Area andSeverity Index(RCLASI),levels of eythrocyte sedimentation rate(ESR),complements,urinary protein.SLE patients with sole kidney and skin involvement underwent immunotherapy were followed up for 12 weeks to observe the effect of immunotherapy on the levels of Th22 cells and IL-22.3.The expression and distribution of Th22 cells and IL-22R1 were determined by immunofluorescence assay in the biopsy tissues of LN patients and SLE patients with skin diseases.4.In vitro experiments,a transwell co-culture system was established to simulate the microenvironment of Th22 cells and their target cells,to determine the secreting function of Th22 cells during HRMC cell existence and the impact on HRMC.ELISA was utilized to determined the levels of IL-22,IL-6 and TNF-α in the supernate of the culture and CCK-8 assay was applied to evaluate the proliferation of HRMC cells.Results: 1.The frequencies of circulating Th22 cells,Th17 cells and Th17.1 cells in SLE patients were elevated significantly than those in healthy controls(HC).Especially,the frequencies of circulating IL-22+Th22 cells,and the percentages of IL-22+Th22 cells in total Th22 cells were elevated in SLE.The results of q PCR analysis evidenced increased levels of IL-22 m RNA,the specific transcription factor Ah R of Th22 cells,the specific transcription factor Rorc of Th17 cells in SLE patients.The frequencies of Th22 cells correlated with the levels of IL-22 m RNA,the specific transcription factor Ah R of Th22 cells and there are positive correlations of Th17 cells with Rorc,and Th1 cells with T-bet.Plasma levels of IL-22,IL-17,IFN-γ and TNF-α were elevated in SLE patients,and IL-22 positively correlated with Th22 cells and IL-22+Th22 cells.Positive correlations were also found in IL-17 levels with Th17 cells.2.The correlational analysis with clinical parameters and stratified analysis showed that the elevated frequencies of circulating Th22 cells and other Th subsets from SLE patients were correlated with SLEDAI and other indicators.Higher levels of circulating Th22 cells and IL-22 were observed in patients with sole skin involvement(SS)compared with none kidney or skin involvement(NKS).In SS,Th22 cells correlated with SI,DAI and Ig G positively,but C3 negatively,and IL-22 correlated Th22 cells and SI positively.In patients with sole kidney impairment,elevated Th22 cells and IL-22 were observed compared with NKS,and elevated Th22 cells correlated with ESR.After 12 weeks immunotherapy,the levels of Th22 cells and IL-22 decreased complying with the alleviation of the disease.3.Immunofluorescence assay found that IL-22R1 were expressed on the membrane ofmesangial cells in kidney and keratinocyte in skin,Th22 cells preferentially gathered in the lesion area adjacent to target cells in kidney and skin biopsy tissues.The levels of Th22 cells in the tissues of SLE patients were higher than those in healthy controls.4.Transwell co-culture experiments revealed that when contacting with HRMC,the secreting function of Th22 cells increased.Th22 cells induced the proliferation of HRMC by secreting IL-22,in a non-contact-dependent way,however,Th22 cells induced the HRMC producing IL-6 and TNF-α by secreting IL-22 and contacting.Conclusions: 1.The levels and functions of Th22 cells and levels of IL-22 are elevated in SLE.2.After immunotherapy,the levels of Th22 cells and IL-22 decreased in SLE.3.Th22 cells may infiltrate into the kidney and skin tissues and produce IL-22 cytokines to influence the target cells,which contributes to the organ injuries of SLE.4.Th22/IL-22 plays an important role in the pathogenesis of LN by inducing the proliferation and production of inflammatory cytokines of HRMC.