Induction And Acceleration Of Auto-antibodies In Healthy And SLE-prone Mice By Human Cytomegalovirus Pp65

The relationship between SLE and infection by viruses, bacteria, or parasites, has been well known in medical literature. Some viruses, such as human cytomegalovirus (HCMV). Epstein-Barr virus (EBV). and parvovirus B19. may play a pivotal role in SLE. The potential role of HCMV in the etiology of SLE has been extensively studied. Nevertheless, the etiologic effect of HCMV on SLE remains unclear. In particular, the lower matrix phosphoprotein65(pp65), a dominant tegument protein of the HCMV virion, plays a significant role in immune evasion, and thereby, in controlling the innate and adaptive immune responses via HCMV-specific cytotoxic T lymphocytes.To further clarify the possible role of HCMV in SLE, we chose the HCMV-pp65recombinant plasmid as the vaccine to study its effect in C57BL/6or SLE-prone BXSB mice. HCMV-pp65DNA vaccine was constructed and HCMV-pp65protein was achieved by prokaryotic expression in E. coli strain BL21(DE3) and purification using Ni-NTA magnetic beads. A series of SLE-related serological makers and microRNAs (miRNAs; miRNA (miR)-125a, miR-126, and miR-146) were then determined in mice immunized with pp65vaccine. Afterwards, the potential role of human cytomegalovirus lower matrix phosphoprotein65(HCMV pp65) in murine systemic lupus erythematosus (SLE) was investigated.In this study, the recombinant pp65was produced by expression in Escherichia coli expression system via the prokaryotic plasmid pET-28b-pp65. and purified by Ni2+affinity chromatography. BXSB mice and C57BL/6mice were inoculated with pp65eukaryotic vector (pcDNA3.0-pp65) intramuscularly5times at2-week intervals and were subsequently bled via the retro-orbital vein. Indirect enzyme-linked immunosorbent assays (ELISAs) were used to evaluate the concentration of anti-pp65IgG. anti-dsDNA. and antinuclear antibodies (ANA). Interleukin (IL)-1β and tumor necrosis factor (TNF)-α were also determined by competitive ELISA. ANA were also detected by Indirect immunofluorescence (HF). and intensity of immunofluorescence is scored as negative, weakly, medium. or strongly positive.3major SLE-related circulating microRNAs (miR-125a, miR-146a, miR-126) were examined by real-time reverse transcription polymerase chain reaction (RT-PCR). All data are expressed as mean±SEM. The differences between2groups in antibody production and other indicators were compared by Mann-Whitney U test using the Prism program. A p value of<0.05was considered to be statistically significant.Antibody responses elicited by immunization with the recombinant plasmid pcDNA3.0-pp65(BP, CP groups) and blank plasmid (BC, CC groups) were all assessed2weeks after each immunization. It is showed that significant antibody responses were induced by pcDNA3.0-pp65vaccine6weeks after the first incubation in both strains of mice, while the serum of blank plasmid groups presented negative reactivity. The levels of anti-dsDNA and ANA in BP group were significant higher than those in the BC group after the fourth incubation. Similarly, the serum in the CP group had higher levels of the2auto-antibodies than the CC group did after the same time of immunization. These findings suggested that humoral autoantibody responses were induced by pcDNA-3.0-pp65plasmid in both healthy and lupus-prone mice. BXSB mice stimulated by pp65had higher IL-1β and TNF-α production. However. IL-1β and TNF-α were negative in C57BL/6mice (CC group), and were not detected in pp65-treated C57BL/6mice (CP group). Furthermore. qRT-PCR analysis clearly indicated that the expression of miR-125a, miR-126. and miR-146a was significantly downregulated in PBMC from pp65-immunized BXSB and C57BL/6mice compared with control BXSB and C57BL/6mice, respectively.In conclusion, HCMV pp65contributes to the onset and aggravation of SLE in C57BL/6and BXSB mice, respectively. HCMV pp65also acts as a suppressor of SLE-related miR-125a. miR-146a, and miR-126in both C57BL/6and BXSB mice. Particularly, the autoimmune effect caused by pp65in BXSB mice was more potent than that in C57BL/6mice. Therefore, the vaccine of pcDNA3.0-pp65could effectively induce the pp65-specific humoral immune response, as well as trigger the SLE-associated autoimmune responses in both healthy and SLE-prone mice. Inhibition of active pp65effects may be helpful in the treatment of SLE patients, and the miRNAs can potentially be applied as novel targets for SLE treatment.