Study On Ratio Imbalance Of Peripheral Blood Th17/Treg Cells In Patients With Lupus Nephritis

[Background]Systemic lupus erythematosus (SLE) is a very common and complex autoimmune disease which influences many organs with variety symptoms. The cause and pathogenesis of SLE is still unclear. It is revealed that the main pathogenesis of SLE is the failure of tolerance to autoantigens and the dysfunction of immune cells that regulate autoimmunity. Lupus nephritis is one of the most serious complications of SLE. According to the examination of renal tissue by electron microscopy and immunofluorescence, almost all of the patients with SLE are suffering lupus nephritis with different severity. Although renal biopsy is the most reliable way to diagnose and evaluate SLE, it is still restricted in clinic for its invasion. Therefore, it is necessary and important to explore the marker for early diagnosis and evaluation of lupus nephritis. Regulatory T cells are a subset of CD4+T cells which can suppress immune function and play an important role in maintaining the balance between defending pathogens and autoimmune tolerance. Thl7cells are a new kind of CD4+T cells, which have been found to play an important role in proinflammatory processes recently. Th17cells can induce a lot of proinflammatory cytokines and chemotatic factors to take part in inflammatory response by secreting IL-17. A recent breakthrough has revealed IL-17-secreting cells (Th-17) are the main pathogenic effector subset involved in the induction of inflammation and autoimmunity. Many studies revealed that the ratio imbalance of peripheral blood Thl7/Treg cells participate in the occurrence and progression of a lot of autoimmune diseases. To our best knowledge, few data are available on Thl7/Treg in patients with lupus nephritis. In our study, we choose the ratio of Thl7/Treg cells in peripheral blood as our main study point to explore the pathogenesis and clinical significance of the ratio imbalance of peripheral blood Thl7/Treg cells in lupus nephritis patients and to acquire new clue in evaluation and therapeutic reaction.[Objective]1. Explore the role of ratio imbalance of peripheral blood Th17/Treg cells and related cytokines in pathogenesis of lupus nephritic patients. Analysis the association between the ratio of Th17to Treg cells in peripheral blood, severity of lupus nephritis and AI. 2. Study the change of the ratio of Th17to Treg cells in peripheral blood and the related cytokines within the therapeutic procession to explore the significance of Th17/Treg in therapy.3. Explore the significance of the change of Th17to Treg cells in peripheral blood in prediction the therapeutic reaction of patients with lupus nephritis.[Methods]A total of60patients with lupus nephritis (All of them are in line with the1982revised American College of Rheumatology SLE classification criteria and are diagnosed with lupus nephritis through renal biopsy.) and20cases of healthy volunteers enrolled into this study as lupus nephritis group and control group. According to the systemic lupus erythematosus disease activity index (SLEDAI), the60patients with lupus nephritis were divided into active lupus nephritis group (SLEDAI>9) and inactive lupus nephritis group (SLEDAI≤9). The demographic data and disease activity data were recorded in detail and about2ml peripheral blood each participant was drawn. Then, we detected the frequency of Th17and Treg in lupus nephritis patients’and healthy controls’peripheral blood CD4+T lymphocytes by flow cytometry to calculate the ratio of Th17to Treg. Detect the expression of such cytokines as IL-6, IL-10, IL-17, IL-23, TGF-β1and so on in lupus nephritis patients’ and healthy controls’serum by ELISA. Analysis the differences of the ratio of Thl7to Treg and the related cytokines among active lupus nephritis group, inactive lupus nephritis group and control group and further analysis the correlation to disease activity. Study the change of the ratio of Th17to Treg and the related cytokines combined with SLEDAI through corticosteroids treatment within a two-month follow up.[Results]1. A total of60patients with LN enrolled in this study. Of them,90%were femal,10%were male, with an age of (35.60±5.79) years, LN duration of1.00(0.50-3.00) years, SLEDAI of9.50±4.61, anti-dsDNA level of128.00(60.50-259.91)IU/ml,24h urine protein excretion of (2.21±1.81)g/24h, serum creatinine of (95.34±34.30) μmol/l. Of them3.33%were type Ⅰ LN,5.00%were type Ⅱ,31.67%were type Ⅲ,51.67%were type Ⅳ,8.33%were type Ⅴ.65%were treated with corticosteroids combined with immunodepressant and35%were treated with corticosteroids alone.2. Compared with control group, the ratios of Thl7to CD4+T lymphocytes and Th17to Treg in lupus nephritis group were both significantly high (4.73±2.70%vs.1.90±0.77%;0.95±0.67vs.0.29±0.13, P<0.05). Compared with inactive lupus nephritis group, the ratio of Treg to CD4+T lymphocytes in active lupus nephritis group was significantly low (4.90±2.05%vs.6.71±2.37%,P<0.01), but the ratios of Th17to CD4+T lymphocytes and Thl7to Treg were significantly high (5.94±3.00%w.3.48±1.57%;1.33±0.71vs.0.57±0.33, P<0.01).3. The ration of Treg to CD4+T lymphocytes in the peripheral blood of lupus nephritis patients was negatively correlated with SLEDAI and AI (r=-0.328, P<0.05; r=-0.496, P<0.01). The ration of Th17to CD4+T lymphocytes was positively correlated with SLEDAI and AI (r=0.576, P<0.001; r=0.651, P<0.01). The ration of Th17to Treg was positively correlated with SLEDAI and AI (r=0.650, P<0.001; r=0.650, P<0.01).4. There is no correlation between the anti-dsDNA level, C3level and the SLEDAI (P>0.05) and there is also no correlation between the anti-dsDNA level, C3level and AI(P>0.05).5. Compared with control group, the levels of IL-10and TGF-β1in LN patients were both significantly low (9.60±2.17pg/ml vs.11.93±0.47pg/ml3972.22±910.33pg/ml vs.4695.99±545.01pg/ml, P<0.01), the levels of serum IL-17and IL-23in active LN patients were both significantly high (9.56±2.73pg/ml vs.6.33±0.39pg/ml；174.36±57.13pg/ml vs.111.58±44.77pg/ml, P<0.05). Compared with inactive LN patients, the level of IL-10in active LN patients was significantly low (8.78±2.39pg/ml vs.10.74±0.78pg/ml, P<0.01), the levels of serum IL-17and IL-23were both significantly high (9.56±2.73pg/ml vs.6.21±0.63pg/ml;174.36±57.13pg/ml vs.135.93±49.11pg/ml, P<0.05).The level of IL-6is positively correlated with the anti-dsDNA level(r=0.341, P<0.05) and there is no correlation between the level of IL-6and SLEDAI and AI. The level of IL-10is negatively correlated with SLEDAI and AI(r=-0.567, P<0.001, r=-0.422, P<0.01). The level of IL-17is positively correlated with SLEDAI and AI(r=0.559, P<0.001, r=0.479, P<0.01). The level of IL-23is positively correlated with SLEDAI and AI(r=0.339, P<0.05, r=0.350, P<0.05). The TGF-β1level has no correlation with SLEDAI, AI and the anti-dsDNA level.6. As the diagnostic marker of active LN(SLEDAI>9), the AUC of Thl7/Treg, IL-17, IL-23was0.92(0.83-1.00),0.91(0.81-1.00) and0.67(0.51-0.83),respectively; as the diagnostic marker of AI>8, the AUC of Thl7/Treg and IL-17was0.87(0.76-0.99) and0.84(0.69-0.99); Thl7/Treg was the most accurate diagnostic marker. IL-10, the anti-dsDNA level and C3level have no value in diagnosis of both active LN and AI>8; IL-23also has no value in diagnosis of AI>8.7. The frequency of Treg in the peripheral blood was positively correlated with the serum IL-10level (r=0.445,P<0.01) and the serum TGF-β1level (r=0.324,P<0.05). The frequency of Th17in the peripheral blood was positively correlated with the serum IL17level (r=0.701,P<0.001) and the serum IL-23level (r=0.2825,P<0.05).8. After a two-month treatment of corticosteroids combined with immunodepressant or alone, the SLEDAI and the ratio of Th17to Treg in the peripheral blood of31active lupus nephritis patients were significantly lower (13.60±2.68vs.8.60±3.32;1.44±0.85vs.0.82±0.66,P<0.001); the serum IL-10level and TGF-β1level were significantly higher (12.99±1.77pg/ml vs.16.53±1.26pg/ml;3408.37±711.94pg/ml vs.4482.24±912.68pg/ml, P<0.05); the serum IL-17level and IL-23level were significantly lower (6.68±0.99pg/ml vs.5.75±0.78pg/ml;199.89±56.98pg/ml vs.135.74±51.74pg/ml, P<0.001).9. Through the two-month follow-up, we found that compared with the starting time, the ratios of Thl7/Treg at the end of the first month and the second month were both significantly lower (1.30±0.37vs.0.54±0.14;1.30±0.37vs.0.63±0.10, P<0.05), meanwhile there was no significant change of the SLEDAI at the end of the first month (13.00±1.67vs.12.67±1.75, P>0.05), but SLEDAI at the end of the second month were significantly lower (13.00±1.67vs.8.00±2.10, P<0.05).(Conclusions]1. Compared with normal group, the ratio of Th17to Treg in the peripheral blood was significant high in both active LN patients and inactive LN patients which was also positively correlated with disease activity and AI.2. Compared with the serum anti-dsDNA level C3level and the level of related serum cytokines, the ratio of Th17to Treg in the peripheral blood was a better marker for evaluating the severity of SLE.3. In contrast with the serum anti-dsDNA level C3level IL-10, IL-17and IL-23, the ratio of Thl7to Treg is the most accurate marker in diagnosis of active LN and AI>8.4. The change of the ratio of Th17to Treg is earlier than the change of SLEDAI.5. The ratio of Th17to Treg might be a good marker to predict the efficiency of corticosteroids combined with immunodepressant treatment.6. corticosteroids combined with immunodepressant or alone abatement clinical symptoms of LN patients maybe associate with the improving of the ratio of Thl7to Treg, the decrease of proinflammatory cytokines (IL-23, IL-17) and the increase of anti-inflammatory cytokines (IL-10, TGF-β1).