Association Study Of T Follicular Heplper Cells With Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus (SLE) is a complicated autoimmune disorder which is characterized in generation of various autoantibodies and multi-organ damage. Generally it is believed that the incidence of SLE is related to abnormal immunoreactions like over-activation of T and B lymphocytes as well as increase of immunoglobulin, particularly represented by proliferation, differentiation and dysfunction B lymphocytes.In the interaction between T and B lymphocytes, people always believe that IFN-γand IL-4, which are produced by Th1 and Th2, play a significant role in assisting secretion of antibody by B lymphocytes. However, there are increasing evidences proving that T follicular helper (Tfh) is the major Th cells assisting B lymphocytes. Tfh is thus receiving more and more attention in terms of its function in autoimmune diseases, becoming the new hot issues for research,It has been founded in the research that an ectopic germinal centre (GC) is formed in the secondary lymphoid organs of BXSB mice, while Tfh abnormity participates in the mechanism of ectopic GC activation, secretion of Anti-ds-DNA and organ damage. To reduce Tfh will conduce to decrease of antibody generation and lupus symptom, suggesting Tfh's involvement in SLE.Normally Tfh cells are located in follicular B cells in lymphoid tissue, and its regulation on B cell proliferation and plasma cell differentiation is also completed within lymphoid follicles. Yet in a condition of autoimmune disorder, Tfh cells may transfer to non-lymphoid tissue, resulting in formation of resembled lymphoid tissues with ectopic GC. Some researchers have found similar Tfh cells which can regulate plasma cell differentiation in autoimmune reactions. Therefore, the function of Tfh cells is not limited within lymphoid follicle, it may also function beyond lymphoid follicle. To study the changes of Tfh cells in pathogenesis of systemic lupus erythematosus and its regulatory mechanism is helpful for us to understand the pathogenesis of SLE from new perspectives and offering basis for seeking for new therapeutic target.Presently there are relative few researches on the distribution changes of Tfh cells in SLE and its regulatory mechanism. Recently two independent researches have found that peripheral blood of SLE showed abnormity of Tfh cell, yet they drew opposite conclusions. The researches by Simpson et al. revealed that part of the peripheral blood of SLE showed increasing Tfh cells, whereas Wong et al. found that the expression of Tfh cells in the peripheral blood of SLE reduced. SLE is a kind of heterogeneous disease and the patients present different clinical symptoms. Moreover, patients with different damaged organ also display different autoimmune abnormalities, so there will be differences if the selected patients are heterogeneous. Besides, there might be a more important reason—different cell phenotypes selected in the research. Simpson's research selected CD4+CXCR5+ICOS+, while Wong used CD4+CXCR5+. Cells with different phenotypes also show differences in biologic functions. This might be another reason resulting in different research results. Even if giving the same phenotype, cell functions may vary with their orientations. For example, tonsils Tfh cell can facilitate the activation and proliferation of B cell, while the CD4+CXCR5+ cells in peripheral blood do not have such function. Therefore, it still needs to be studied further if there are Tfh cell changes in the peripheral blood of SLE, and which celle phonotype can represent Tfh cell.In fact, the differentiation of Tfh cells is influenced by multiple factors such as IL-21, Bcl-6, B cells and other Th cells, and there are diversified way of differentiation, while Bcl-6 plays the decisive role in regulating the differentiation of Tfh cells since all kinds of means regulate Tfh differentiation through influencing Bcl-6 expression. Any T cells without Bcl-6 can not be dedifferentiated into Tfh cells. Consequently, the regulatory mechanism influencing Bcl-6 expression might participate in the differentiation of Tfh cells. In recent years, it has also been found that in the regulation mechanism of protein expression, miRNA plays a very important role as it can influence the differentiation of Th cells like Th1, Th2, Th17 and Treg through regulating key protein expression. Thus it is inferred that miRNAs might also be involved in differentiation of Tfh cells through regulating Bcl-6 expression. Yet there is no relevant research which can prove this point.Whereas ectopic Tfh cell may occur to SLE patients and involve in SLE pathogenesis, along with the regulative function of Bcl-6 and miRNA to Tfh cell differentiation, this research intends is design to detect the changes of different phenotypes of Tfh cells in the peripheral blood and lymph nodes based on a comparison between SLE patient and healthy people, and explore the function of Tfh cells in SLE pathogenesis. Meanwhile, proceeding from Bcl-6, bioinformatics is used for screening miRNAs which regulates Bcl-6 expression and examining the expression levels of Bcl-6 and miRNAs as well as the influences of their changes on Tfh cell differentiation. In addition, the regulatory mechanism Tfh cells differentiation is discussed preliminarily.Objective1. To find out the changes of Tfh in SLE patients'lymphoid node and peripheral blood as well as its role in SLE pathogenesis.2. To discuss the influences of Bcl-6 and related miRNAs on Tfh cell differentiation in SLE patients'peripheral blood.Methods1. Eight new-onset SLE patients and eight normal voluntary controls were selected. Immunohistochemical double staining method was used to examine CD4~+Bcl6~+ cells in the lymphoid tissue of the SLE patients and controls. Then the relation between the changes of the CD4~+Bcl6~+ cells in the lymphoid tissue and the incidence of SLE was analyzed. 2. 47 patients with active SLE, 48 with stable SLE and 59 normal voluntary controls were selected. By employing flow cytometry, the percentage of Tfh cells (CD4~+CXCR5~+ICOS~+, CD4~+CXCR5~+, CD4~+ICOS~+) in the peripheral blood was tested, while the relation of its changes with the incidence of SLE and organ damage was analyzed as well.3. After selecting part of the above samples, flow cytometry was used to detect the CD19~+B cells, and ELISA method for detection of serum IL-21 and CXCL13. Moreover, all kinds of clinical data of the SLE patients were collected. According to the correlation between Tfh cells and CD19~+B cells, IL-21, CXCL13 and autoantibody, the relation between CD4~+CXCR5~+ICOS~+, CD4~+CXCR5~+, CD4~+ICOS~+ and Tfh cells was analyzed.4. Taking Bcl-6 as the target gene, bioinformatics method was adopted in order to screen miRNA which controls the expression of Bcl-6. By selecting 8 new-onset SLE patients and 7 normal controls, Q-PCR was used to test the expression levels of Bcl-6 mRNA and screened miRNAs in the peripheral blood. Then a comparison on the difference between the expression levels of Bcl-6 mRNA and miRNAs of SLE patients and normal controls. After increasing the sample to 19 SLE patients and 13 normal controls, the correlation between the expression of Bcl-6 mRNA and miRNAs and its influences on the differentiation of CD4~+CXCR5~+ICOS~+, CD4~+CXCR5~+, CD4~+ICOS~+ in peripheral blood were analyzed as well.Results1. The changes of Tfh cells in lymph nodes: the CD4~+Bcl6~+ cells in SLE patients were higher than that in normal controls, but showing no significant difference (P=0.058).2. The changes of Tfh cells in peripheral blood: a comparison of the percentage changes of the CD4~+CXCR5~+, CD4~+ICOS~+ and CD4~+CXCR5~+ICOS~+ in peripheral blood is made between active SLE Group, stable SLE group and normal control group, and the result showed that the percentage changes of CD4~+ICOS~+ and CD4~+CXCR5~+ICOS~+ revealed significant difference among these three groups (χ~2=49.635, P﹤0.001;χ~2=32.150, P﹤0.001), while no significance difference was perceived in terms of percentage changes of CD4~+CXCR5~+ cell group (χ~2=4.421, P=0.110). By using Mann-Whitney U rank sum test, comparisons was made between three groups, and the results showed that there was a significant increase in percentage of CD4~+ICOS~+ cell subset in activate SLE group by contrast with stable SLE group and normal control group (P﹤0.001) and in stable SLE group compared with normal control group (P﹤0.001), yet no significant difference was displayed in percentage of CD4~+CXCR5~+ICOS~+ between stable SLE group and normal control group(P=0.090).3. Correlation between Tfh cell changes in peripheral blood and organ damage of SLE patients: according to different clinical features, a comparison on the percentage changes of CD4~+CXCR5~+ICOS~+, CD4~+CXCR5~+ and CD4~+ICOS~+ in PBMC was performed. The result shown that SLE patients with tetter has significantly low percentage of CD4~+CXCR5~+ compared with SLE patients free from tetter (P=0.021). After multiple comparisons among the three groups of SLE patients with tetter, SLE patients without tetter and normal control group, significant differences in percentage of CD4~+CXCR5~+ sub-set were displayed among these groups (χ~2=3.421, P=0.036). Mann-Whitney U rank sum test showed that the SLE patients with tetter had significantly lower percentage of CD4~+CXCR5~+ by contrast with that of normal control group (P=0.003).4. Correlation between Tfh cells in peripheral blood and Cytokines, autoantibody, CD4~+B cell: the result of bivariate correlation analysis indicated that the percentages of CD4~+CXCR5~+ICOS~+ and CD4~+ICOS~+ were significantly positively correlated with IL-21(r=0.235, P=0.030; r=0.218, P=0.044), meanwhile, such significant positive correlation was also found between the percentages of CD4~+CXCR5~+ICOS~+ and CD4~+CXCR5~+ and anti-nucleosome (r=0.377, P=0.008; r=0.314, P=0.030), and between the percentage of CD4~+ICOS~+ and anti-β2GP1(rs=0.412, P=0.007), or between the percentage of CD4~+CXCR5~+and ANA, anti-SSA (rs=0.428, P=0.003; rs=0.366, P=0.011), whereas a significant negative correlation was revealed between the percentage rate of CD4~+ICOS~+ and anti-RNP(rs=-0.388, P=0.007).5. The changes of Bcl-6 mRNA and miRNAs expressions: the expression of Bcl-6 mRNA in SLE group and normal control group shown no significant difference (P=0.478), while the expressions of miR-302a, miR-339-5p, and miR-346 in SLE group were significantly higher(P=0.027, P=0.003, P=0.036).6. Inter-influences among different miRNAs: Bivariate correlation analysis on the expression levels of different miRNAs was conducted, and the result revealed that miR-346 was significantly positively correlated with miR-302a and miR-10a (r=0.532, P=0.002; r=0.622, P=0.013), while miR-127-3p also had a significant positive correlation with miR-9(r=0.860, P﹤0.001).7.Influence of miRNAs on Bcl-6 mRNA expression: Bivariate correlation analysis on different miRNAs and Bcl-6 mRNA expression level was performed, while the results shown that miR-30a-5p and Bcl-6 mRNA were significantly positively correlated (r=0.585, P=0.022). Taking Bcl-6 Mrna as dependent variable and miRNAs as independent variable, by using linear regression analysis, it was found that the expression levels of miR-30a-5p, miR-127-3p, miR-10a and miR-302a were correlated with Bcl-6 mRNA. The regression equation was Yi=1.487~+0.145 X1i-0.342 X2i~+0.720 X3i-0.202 X4i (Yi stands for Bcl-6 mRNA expression level, X1i, X2i, X3i, X4i represent the expression level of miR-30a-5p, miR-127-3p, miR-10a and miR-302a respectively.8. Correlation of Bcl-6 with immunoglobulin and autoantibody: Bivariate correlation analysis was performed for analyzing the correlation of Bcl-6 mRNA with IgG, IgA, IgM, anti-C1q, anti-β2-GP1 and anti-nucleosome antibodies. The result showed that Bcl-6 mRNA was significantly positively correlated with anti-nucleosome antibodies (r=0.522, P=0.022).9. Influence of Bcl-6 on Tfh cell differentiation: Bivariate correlation analysis was carried out on Bcl-6 mRNA and the percentages of different Tfh cell sub-set. The analytical result indicated that there was no correlation between Bcl-6 mRNA and the percentages of different Tfh cell sub-set. By virtue of the influence of IL-21 and CXCL13 on Tfh cell, taking IL-21and CXCL13 as the control-variant, Bivariate correlation analysis on the Bcl-6 mRNA and the percentages of different Tfh cells. The result was Bcl-6 mRNA and the percentages of CD4~+CXCR5~+ICOS~+ cell sub-set showed a significant partial correlation (r=0.451, P=0.046).10. Influence of miRNAs on Tfh cell differentiation: Bivariate correlation analysis was carried out on expression levels of different miRNAs and the percentage of Tfh cells. The analytical results indicated that miR-339-5p is significantly negatively correlated to the percentage of CD4~+CXCR5~+ICOS~+ cells (r=-0.409, P=0.042).ConclusionsThere might be proliferation of Tfh in the peripheral blood of SLE patients, which may be involved in the onset of SLE through promoting the secretion of IL-21 and autoantibody. Moreover, the differentiation of Tfh cells in the peripheral blood may be regulated by IL-21, Bcl-6 and miR-339-5p, while Bcl-6 expression is influenced by multiple miRNAs such as miR-127-3p. Besides, the enhanced expressions of miR-302a, miR-339-5p and miR-346 in the peripheral blood of SLE patients may be involved in the onset of SLE through a variety of approaches.