Expression Of Regulatory B Cells And Related Cytokines And The Complication Of Osteonecrosis In Patients With Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus(SLE)is a chronic,systemic autoimmune inflammatory disease characterized by the production of numerous autoantibodies,particularly antinuclear antibodies(ANA),increased immune complex deposition,and multiple organ damage.Although both genetic and environmental factors may contribute to SLE development,the precise etiology of SLE is not fully understood.As a central player in SLE development,the immune system with both T and B cells contributes to the pathogenesis of human SLE.Historically,B cells are considered to be positive regulators of humoral immune responses because of their ability to terminally differentiate into plasma cells and produce antigen-specific antibodies.Specific B cell subsets,however,negatively regulate immune responses and have been termed regulatory B cells(Bregs).Bregs are associated with the CD19+CD24highCD38high phenotype and are characterized by the production of the immunoregulatory cytokines interleukin(IL)-10 and transforming growth factor(TGF)-?.Through the secretion of these immunosuppressive cytokines,Bregs suppress other immune cells and have been studied extensively for their potential role in the treatment of various autoimmune diseases.Recent studies indicate that Bregs are functionally impaired in SLE patients,suggesting their potential involvement in the pathogenesis of lupus.However,no studies have systematically characterized the status of Bregs,the levels of serum IL-10 or TGF-?,or IL-10 signaling.Moreover,their associations with the SLEDAI and other laboratory parameters in SLE patients have not been examined.Hence,we performed this study to address these questions in order to significantly improve our understanding of the pathogenesis of SLE as well as to justify targeting Bregs as a therapeutic approach for SLE and potentially other autoimmune diseases.ObjectiveThe aim of this study was to examine the status and clinical significance of CD 19+CD24thighCD38high regulatory B cells(Bregs),serum interleukin(IL)-10,serum transforming growth factor(TGF)-P,and IL-10 receptor(IL-10R)expression in peripheral blood from patients with systemic lupus erythematosus(SLE).Method1.A total of 56 SLE patients and 35 healthy individuals were recruited into this study.According to the presence of lupus nephritis(LN),the SLE patients were divided into two groups:those with lupus nephritis(LN group;N = 24)and those without(SLE group;N = 32).2.The SLE disease activity index(SLEDAI)was calculated,and other laboratory parameters were measured.Peripheral blood was collected from all participants.The frequency of CD19+CD24highCD38high Bregs and IL-10R+ expression on circulating lymphocytes was examined by flow cytometry.The serum levels of IL-10 and TGF-?were measured using the enzyme-linked immunosorbent assay(ELISA).The associations between these measurements and SLED AI or other laboratory parameters were analyzed by correlation analysis.a portion for laboratory tests as follows:routine blood tests for ESR,red blood cell(RBC)count,and lymphocyte count;indirect immunofluorescence assay for ANA titer;automated immunofluorescence assay for anti-dsDNA antibody titer;liver function tests for CRP,complement components C3 and C4,IgG,IgM,IgA,and aspartate aminotransferase(AST)levels.3.Statistical analysis was performed using SPSS 18.0 software.Data distribution was checked for normality using the Kolmogorov-Smirnov test.Normally distributed data were expressed as the mean ± standard deviation,and the differences were examined using two-tailed t-tests.Correlation coefficients and their significance were calculated by two-tailed Pearson correlation.For nonparametric data,the results are presented as medians[25th-75th percentile].The Mann-Whitney U-test was used to compare the data between different groups of patients and controls.Correlation coefficients and their significance were calculated by two-tailed Spearman's Rho correlation.P values of<0.05 were considered statistically significant.Results1.There was a higher percentage of CD19+CD24highCD38high Bregs among circulating lymphocytes from SLE patients than from healthy individuals(39.83 ±21.39%vs.8.74 ± 3.97%;P<0.001).Further analysis on the clinical significance of Bregs showed that the frequency of CD19+CD24highCD38high Bregs was not significantly correlated with the SLEDAI score(r =-0.111,P = 0.416)or the presence of LN in SLE patients(P>0.05,data not shown),but it was negatively correlated with the blood complement C3(r =-0.432,P = 0.002),complement C4(r =-0.497,P<0.001),and serum indirect bilirubin(r =-0.335,P = 0.035)levels.2.The percentage of IL-10R+ cells among circulating lymphocytes was dramatically lower in SLE patients than in healthy individuals(23.76 ± 0.62%vs.51.01 ± 16.03%;P<0.001).Furthermore,the expression level of IL-10F,based on the mean fluorescence intensity(MFI)of these cells,was also significantly lower in SLE patients compared to healthy individuals(20.18 ± 3.88 vs.23.23±3.66;P<0.001).Further correlation analysis showed that the MFI of IL10-R1+ lymphocytes did not significantly correlate with the presence of LN in SLE patients(P>0.05,data not shown),the SLEDAI(r = 0.093,P = 0.494),serum alanine transaminase(ALT)(r?0.254,P = 0.075),or serum alkaline phosphatase(ALP)(r = 0.252,P = 0.077),but it negatively correlated with the ESR(r =-0.389,P = 0.016).3.The serum IL-10 concentration was significantly higher in the SLE patients than in the healthy controls(3.65[2.22-9.17]pg/mL in the SLE patients vs.1.04[0.43-1.46]pg/mL in the healthy controls;P<0.001).Correlation analysis showed that the serum IL-10 level did not correlate with the presence of LN in SLE patients(P>0.05),or the SLEDAI,but it positively correlated with the ESR,ANA titer,IgG,IgM,and AST and negatively correlated with the complement C3 level as well as red blood cell and lymphocyte counts.4.The serum TGF-?1 level was not significantly different between the SLE patients and the healthy controls(11262.02 ± 7784.17 pg/mL vs.13143.27 ± 5559.21 pg/mL,P-0.239).However,the serum TGF-?l level in the SLE patients with LN was significantly lower than in the healthy controls(9382.07 ± 7558,16 pg/mL vs.13143.27 ± 5559.21 p.g/mL,P=0.038),although there was also not a dramatic difference in the serum TGF-?1 level between the SLE patients with or without LN(9382.07 ± 7558.16 pg/mL vs.12671.98 ± 7767.39 pg/mL,P = 0.118).Correlation analysis showed that the serum TGF-?1 level was not significantly correlated with the SLEDAI score,but it was positively correlated with the red blood cell count and negatively correlated with globulin,24-h urine protein,IgM,and AST.5.The percentage of CD19+CD24highCD38high Bregs was positively correlated with the percentage of IL-10R+ lymphocytes,the MFI of IL-10R+ lymphocytes,and the serum IL-10 level.In addition,the percentage of IL-10R+ lymphocytes was positively correlated with its expression level(MFI),while the serum TGF-?1 level was negatively correlated with the serum IL-10 level.Conclusion1.Compared with normal control group,CD19+CD24higherCD38high Bregs and IL-10 in the peripheral blood of patients with SLE increased significantly,and IL-10 receptor decreased significantly.Deficient IL-10R expression may result in the compensatory enhanced expression of IL-10,expansion of Bregs,and/or compromise the functions of Bregs and IL-10,contributing to SLE development.2.The serum TGF-?1 level was not significantly different between the SLE patients and the healthy controls.However,the serum TGF-?1 level in the SLE patients with LN was significantly lower than in the healthy controls.The dysfunction of TGF-?1 in regulating the immune system leads to autoimmune disease.3.These results suggest that the expression of these immunological factors have correlations with clinical parameters and disease activity.They may play essential roles in SLE pathogenesis and be potential therapeutic targets or biomarkers in SLE.BackgroundSystemic lupus erythematosus(SLE)is a heterogeneous disease with many complications.The bone damage of SLE includes osteopenia,osteoporosis(OP),osteonecrosis(ON)and fractures.OP is a systemic disease characterized by bone loss and disruption of the microstructure.ON is the death of cellular elements of the bone,which leads to collapse of the bony structure,culminating in joint pain and loss of function.Another feature of SLE is immune disorders,including the production of antibodies and cytokines.Studies have suggested that interleukin 10(IL-10)and transforming growth factor ?1(TGF-?1)are inhibitory cytokines which can mediate immune tolerance,inhibit excessive inflammation.They may be involved in the regulation of bone metabolism and bone damage.But the research about IL-10 and TGF-?1 in SLE with bone damage is not much.Hence,we performed this study to detect and compare the differences in IL-10 and TGF-?1 expression in peripheral blood of SLE patients with bone damage.We performed this study also in order to significantly improve our understanding of the inhibitory cytokines in the pathogenesis of SLE with bone damage,as well as to justify targeting them as a therapeutic approach for SLE with bone damage and potentially other autoimmune diseases.ObjectiveThe aim of this study was to investigate the expression of IL-10 and TGF-?1 in peripheral blood of patients with SLE complicated with bone lesions and to analyze their clinical significance.Method1.A total of 56 SLE patients were recruited into this study.Serum IL-10 and TGF-?1 were detected by ELISA.2.According to different bone mass,the SLE patients were divided into 3 groups:bone normal group(14 cases),bone loss group(26 cases),osteoporosis group(16 cases).According to whether the mergers of osteonecrosis,the SLE patients were divided into two groups:none osteonecrosis group(49 cases)and osteonecrosis group(7 cases).3.Statistical analysis was performed using SPSS 18.0 software.Data distribution was checked for normality using the Kolmogorov-Smirnov test.Normally distributed data were expressed as the mean ± standard deviation,and the differences were examined using two-tailed t-tests.For nonparametric data,the results are presented as medians[25th-75th percentile].The Mann-Whitney U-test was used to compare the data between different groups of patients and controls.P values of<0.05 were considered statistically significant.Results1.For TGF-?1 expression,bone mass normal group>osteopenia group>osteoporosis group,but only the difference between normal bone mass group and.osteoporosis group(14778.93 ± 5188.10 vs 9289.53± 6752.73 pg/mL,P = 0.020)was statistically significant.For IL-10 expression,bone mass normal group<osteopenia group<osteoporosis group,but only the difference between the normal bone group and osteoporosis group(0.12[0.01-2.74]vs 2.57[0.52-8.52]pg/mL,P =0.012)has statistical significance.2.For TGF-?1 expression,none osteonecrosis group(11960.43 ± 768.55 pg/mL)>bone necrosis group(6373.13 ± 5315.75 pg/mL),but the difference was not statistically significant(P = 0.075).For IL-10 expression,none osteonecrosis group(0.87[0.21-4.56]pg/mL)<bone necrosis group(8.03[1.61-9.56]pg/mL),the difference was statistically significant(P = 0.017).Conclusion1.The level of serum TGF-?1 in peripheral blood of SLE patients with osteoporosis was significantly lower than that of SLE patients with normal bone mass.Although the difference was not statistically significant,the expression of TGF-?1 in SLE patients with osteonecrosis has the trend of reduce.Suggesting that the reduction of TGF-?1 expression may be one of the pathogenesis of SLE complicated with abnormal bone metabolism.2.The level of serum IL-10 in peripheral blood of SLE patients with osteoporosis was significantly higher than that of SLE patients with normal bone mass.Serum IL-10 in peripheral blood of SLE patients with bone necrosis was higher than that of SLE patients without bone necrosis.This increase of IL-10 expression may be compensatory synthesis under chronic inflammation.BackgroundSystemic lupus erythematosus(SLE)is a heterogeneous disease,and differences in the clinical features may be risk factors for osteonecrosis(ON)in addition to treatment with glucocorticoids.ObjectiveTo assess the major risk factors for ON in SLE,and provide evidence for decision-making on prevention.MethodThe Cochrane library,PubMed,Ovid,and Science Direct were searched for published case-control studies on the risk factors of ON in SLE.A meta-analysis of 23 case-control studies(1,071 cases and 23,065 controls)that met the inclusion criteria was conducted using Revman 5.3 software.After analysis of homogeneity,the pooled odds ratios(OR)and 95%confidence intervals(CI)of each risk factor were calculated.ResultsThe pooled OR and 95%CI of each risk factor of ON in the patients with SLE were as follows:arthritis 1.69[1.32,2.17],central nervous system(CNS)involvement 1.34[1.06,1.71],diabetes mellitus 1.59[1.03,2.46],hypertension 1.69[1.42,2.02],oral ulcer 1.48[1.06,2.08],renal involvement 1.53[1.27,1.83],vasculitis 2.45[1.54,3.89],smoking history 1.64[1.01,2.65],leucopenia 1.54[1.11,2.13],thrombocytopenia 1.63[1.14,2.32],cytotoxic drugs 1.79[1.25,2.57],cyclophosphamide 3.13[1.58,6.21]and anti-Sm antibodies 0.48[0.27,0.85].Conclusion:In addition to glucocorticosteroids,other factors,including arthritis,CNS involvement,diabetes mellitus,hypertension,oral ulcer,renal involvement,vasculitis,smoking history,leucopenia,thrombocytopenia,cytotoxic drugs and cyclophosphamide are major risk factors of ON in patients with SLE.Antimalarial drugs are not protective factor against ON in patients with SLE.