| PartⅠObjective: The present study was to explore the correlation between HCMV infection and BAFF signal in renal transplantation recipients.Methods: ①113 follow-up renal transplantation recipients were enrolled in the study. Their peripheral blood(anticoagulated by EDTA-Na2) and urine were collected. Urine HCMV-DNA was detected by real-time PCR. ②Expression levels of BAFF, BAFF-R, TLR9, NLRC5, TACI transcript in peripheral blood mononuclear cells were detected by real-time PCR. Total of 88 concerned genes involved in Toll like receptor signal pathway, NF-κB signal pathway and cytokine-cytokine receptor signal pathway were detected by real-time PCR Gene Chip array to identify differentially expressed genes between positive group(HCMV DNA copies >10, 000 copy/ml urine) and negative group(HCMV DNA copies <10, 000 copy/ml urine); healthy volunteers were set as control group. ③Soluble BAFF and total Ig G in serum were detected by enzyme-linked immunoabsorbentassay(ELISA); anti-HCMV pp65 Ig G and Ig M antibody in serum were detected by indirect immunoinfluscent assay(IFA); anti-HLA-I&II&MICA antibody in serum were detected by Luminex assay. ④Data were analysed by SPSS17.0 software. The means of two independent samples were compared by t-test; correlation analysis were analysed by Spearman correlation test and Pearson correlation test; Statistical significance was defined as P <0.05; all P-values were two-sided.Results: ①Urine HCMV DNA copies >10, 000 copy/ml in 12 recipients were detected; HCMV-DNA copies in 83 recipients was below 10, 000 copy/ml, and no HCMV DNA was amplified in 18 cases. Recipients with urine HCMV DNA copies >10, 000 copy/ml were as positive group; recipients with urine HCMV DNA copies <10, 000 copy/ml were as negative group. ②ELISA results showed that s BAFF and total Ig G level all significantly increased in positive group(P <0.05) compared with negative group(928±360 pg/ml vs 511±298 pg/ml, P <0.05; 26, 260±15, 960 μg/ml vs 9, 944±7, 365 μg/ml, P <0.05. In the positive group, HCMV DNA copies and total Ig G level positively correlated with s BAFF(r =0.988 and 0.625, respectively)(P <0.05). ③ Luminex assay results showed that the incidence of anti-HLA-I&II&MICA antibody obviously increased in positive recipients(46.2% vs 12.5%). ④Real-time PCR results showed BAFF m RNA increased in positive recipients; BAFF-R, TLR-9, NLRC5, TACI showed no difference. Real-time PCR chip assay showed that 46 genes were differentially expressed. Compared with the negative group, two genes(BAFF-R and ICAM1) were significantly up-regulated, and 22 genes(TLR-9, NLRC5, LIGHT, etc) were significantly down-regulated in the positive group. Compared with the healthy controls, two genes(IFN-γ and ICAM1) were significantly up-regulated, and eight genes(CCL3L1, IL12 B, ILIR1, etc) were significantly down-regulated in the positive group. Compared with the healthy controls, 11 genes(IFN-γ, IRAK4, NF-κB2, etc) were significantly up-regulated, and BAFF-R was significantly down-regulated in negative recipients.Conclusion: The activation of HCMV would induce or enhance the activation of BAFF code in renal transplantation recipients, which may independently or cooperatively participate in renal allograft injury.PartⅡObjective: Our aim was to investigate the potential molecular mechanism and the outcome of the crosstalk between HCMV-TLR9 and BAFF signaling pathways in vitro.Methods: ① After 1, 3, 5, 7 days of HCMV infection in vitro, cytopathologic effects(CPE) on HFL-1 cells were observed; and NLRC5, TLR9, TRAF6 and IRAK4 expression in HFL-1 cells at the schedule timepoint were detected by Western Blot. ②After directly infected by HCMV or co-cultured with HCMV infected HFL-1 cells for 1, 3, 5, 7 days, the apoptosis rates of tonsil B cells, and the expression rates of CD19+, BAFF+, BAFF-R+, TACI+ and BCMA+ on B cells were analyzed by FACS; Ig G levels in cell culture supernatant were detected by ELISA. B cells treated with 5 μg/ml Cp G ODN2006 and its negative Control, 2.5 μg/ml Cp G ODN4084-F, 50 μg/ml Poly(I:C) and 200 ng/ml rh BAFF were set as control groups. ③Anti-BAFF-R antibody was added to the B cells culture medium before or after HCMV or ODN2006 stimulation. After cultured for more five days, apoptosis rates of B cells and Ig G concentration were detedcted, respectively. B cells treated with HCMV, Cp G ODN2006, anti-BAFF-R antibody alone were set as control groups. ④Data were analysed by SPSS17.0 software. The means of two independent samples were compared by t-test; Differences among multi-groups were evaluated by the ANOVA test; Statistical significance was defined as P <0.05; all P-values were two-sided.Results: ①After infection by HCMV, TLR9 expression in HFL-1 cells significantly increased and later significantly decreased over time; TRAF6 and IRAK4 expression showed decreasing tendencies; no significant fluctuations were detected in NLRC5 expression. However, the expression of TLR9, TRAF6, IRAK4 and NLRC5 did not show significant fluctuations in the control group. ②The apoptosis rate of B cells co-cultured with normal HFL-1 cells exhibited an almost linear increase. Compared with the control group, two platform stages of apoptosis rates appeared in B cells co-cultured with HCMV-infected HFL-1 cells(from day0 to day1 and from day3 to day5). Compared with the medium-only group, B cell apoptosis in the directly HCMV-treated group did not significantly increase from day1 to day5. In the Cp G DN2006-stimulated group, B cell apoptosis was down-regulated from day0 to day1, and then slowly increased until day5. In contrast, no changes were observed in the Cp G ODN2006 control and rh BAFF-stimulated groups. In the Cp G ODN4084-F and Poly(I:C)-stimulated groups, B cell apoptosis persistently increased over time and higher than the control group at every time point. ③Higher and longer BAFF-R expression was maintained on B cells infected with HCMV; a similar effect was observed in Cp G ODN2006 and rh BAFF-treated cells while BAFF, TACI, BCMA expression on B cells showed no difference. ④Blockade of BAFF signal prior to HCMV addition increased the apoptosis rate of B cells but did not significantly influence Ig G production. However, blockade of the BAFF signal prior to TLR9 agonist addition not only significantly increased the B cell apoptosis rate but also significantly decreased Ig G production. Individual HCMV, Cp G ODN2006, anti-BAFF-R antibody treatment showed no significant effect on B cells apoptosis; individual HCMV, Cp G ODN2006 treatment up-regulated the Ig G secretion by B cells.Conclusion: The activation of the HCMV/TLR9 signal on B cells induced the activation of BAFF/BAFF-R and inhibited B cell apoptosis in a degree. |
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