DISCUSSION AND CONCLUSION
In the present study, we show that Brd4 regulates plasma cell differentiation and immunoglobulin production by, at least in part, modulating BLIMP1 expression at the transcriptional level but does not affect the proliferation or apoptosis of activated primary CD19+ B cells from healthy controls or mice with B cell-specific deletion of Brd4. Brd4 inhibition also reduces the in vitro differentiation of plasma cells and the production of immunoglobulin in patients with SLE. The Brd4 inhibitor PFI-1 reduces hypergammaglobulinemia and the percentage of plasma cells and attenuates nephritis in MRL/lprmice. The inhibitory effect of Brd4 deficiency on nephritis and the percentages of plasmablasts and plasma cells is also shown in B cell-specific deletion of Brd4 mice with pristane-induced lupus. Collectively, our findings suggest that Brd4 plays a key role in regulating plasma cell differentiation and that Brd4 inhibitors have therapeutic potential for B cell-associated autoimmune disorders such as SLE.
Antibody production is dependent on the generation and maintenance of antibody-secreting cells, including plasmablasts and plasma cells, from their activated B cells. Short-lived plasmablasts are rapidly secreted effector cells of the early antibody response, whereas long-lived plasma cells are critical mediators of the lasting humoural immune response. However, the regulation of plasma cell differentiation is clearly complex and remains to be defined. Recent studies indicate that BET proteins are important in regulating Th17 cell differentiation (Cheung, Zhang et al., 2017; Mele et al., 2013). Brd4 has also been related to the IGH locus in B-cell lymphomas(Donato, Croci et al., 2017). These findings prompted us to speculate whether Brd4 is involved in B cell differentiation. In this work, we found that Brd4 suppression with a specific inhibitor or shRNA reduced the percentages of plasmablasts and plasma cells and the production of immunoglobulin in activated CD19+ B cells from healthy humans. By using mice with B cell-specific deletion of the Brd4 gene, we further observed that Brd4 deficiency reduced plasma cell differentiation and immunoglobulin secretion. These findings suggest a critical role of Brd4 in modulating plasma cell differentiation. Consistent with our results, a previous report showed that Brd4 plays an important role in regulating immunoglobulin gene expression from human CLNH11.4 B cells(Shim, Lee et al., 2017). Since the induction of apoptosis and the inhibition of proliferation in activated B cells may contribute to the reduction in plasma cell differentiation and immunoglobulin production, we determined the effect of Brd4 inhibition on the proliferation and apoptosis of B cells. However, we observed that Brd4 inhibition did not influence the proliferation and apoptosis of activated CD19+ B cells, suggesting that the effect of Brd4 inhibition on plasma cell differentiation is not associated with the proliferation and apoptosis of B cells. However, inconsistent with our findings, a recent report indicated that JQ1 affects the survival and proliferation of murine B cells(Kong, Rimes et al., 2020).
The differentiation of activated B cells into plasma cells is associated with alterations in gene expression that result in the loss of B cell identity and the gain of protein secretion functions. The master transcription factor BLIMP-1 plays a critical role in controlling B lymphocytes to a plasma cell fate(Angelin-Duclos et al., 2000; Nutt et al., 2011). In the B cell lineage, BLIMP1 is almost exclusively expressed in ASCs, with higher expression in long-lived plasma cells and lower expression in plasmablasts(Kallies, Hasbold et al., 2004). In the present study, we observed that costimulation with anti-IgM, rCD40L, CpG ODN2006, rIL-10 and rIL-15 for 7 days could upregulate BLIMP1 expression in CD19+ B cells from human heathy controls, and this increase was reduced by Brd4 inhibition. A similar result was obtained in CD19+ B cells from Brd4-CKO mice compared with WT mice. These data suggest that Brd4 modulates plasma cell differentiation through, at least in part, regulating BLIMP1 expression. Furthermore, we investigated how Brd4 regulates BLIMP1 expression and found that Brd4 directly binds and activates the endogenous BLIMP1 promoter and thereby promotes its expression. These findings are consistent with previous studies showing that Brd4 binds directly and regulates other transcription factors, such as acetyl-310 RelA of nuclear factor kappa B (Sun, Wang et al., 2015) and c-Myc (Ba, Long et al., 2018). It is well known that Brd4 can interact with positive transcription elongation factor b (P-TEFb) and recruit P-TEFb to the promoter (Z. Yang, Yik et al., 2005; Zhang, Prakash et al., 2012). Elevated Brd4 expression induces an increase in the P-TEFb-dependent phosphorylation of RNA polymerase II CTD and the transcriptional activation of genes(Devaiah, Lewis et al., 2012). Therefore, based on our findings, we infer that Brd4 recruits P-TEFb to the BLIMP1 promoter, then results in the P-TEFb-dependent phosphorylation of RNA polymerase II, and finally promotes BLIMP1 expression. Collectively, our findings suggest that Brd4 controls plasma cell differentiation by, at least in part, directly regulating BLIMP1 transcription.
It has been established that B cells play a central role in both the onset and maintenance of autoimmunity in SLE. Recent studies indicate the significant clinical benefit of B cell depletion on the disease (Leandro, Edwards et al., 2002; Looney, Anolik et al., 2004; Reddy et al., 2015). An increased frequency of plasmablasts and plasma cells in peripheral blood has been demonstrated in active lupus patients (Arce, Jackson et al., 2001; Odendahl, Jacobi et al., 2000; C. Wei, Anolik et al., 2007). In the present study, we observed that Brd4 inhibition decreased the frequency of plasma cells and the production of immunoglobulin in costimulation-stimulated CD19+ B cells from patients with SLE, indicating the role of Brd4 inhibition in regulating plasma cell differentiation in patients with SLE. Furthermore, to determine thein vivo effect of Brd4 inhibition on SLE, PFI-1 was used to administer the lupus animal model MRL/lpr mice. We found that PFI-1 treatment decreases hypergammaglobulinemia and the levels of autoantibodies and attenuates nephritis in MRL/lpr mice, which is consistent with a previous report that JQ1 treatment improves nephritis in MRL/lpr mice (S. Wei, Sun et al., 2015). More interestingly, the percentages of plasma cells and the expression of BLIMP1 were significantly reduced in B cells isolated from lymph nodes and spleens of PFI-1-treated MRL/lpr mice compared with the control group. We also demonstrate that nephritis is improved in B cell-specific deletion of Brd4 mice in a pristane-induced lupus model. Autoreactive plasma cells contribute to the maintenance of autoimmunity in SLE by the persistent secretion of autoantibodies (Hiepe, Dörner et al., 2011); therefore, our findings suggest that the suppression of Brd4 in plasma cell differentiation may contribute to its therapeutic efficacy in MRL/lpr mice. However, we do not rule out the possibility that Brd4 inhibition may have other molecular and cellular effects in vivo , since previous observations have shown the role of Brd4 inhibitors in modulating the differentiation and activation of Th17 and dendritic cells (Mele et al., 2013; Toniolo et al., 2015).
In conclusion, we identified Brd4 as an essential factor in regulating plasmablasts –mediated plasma cell differentiation and suggest that Brd4 is a target for the treatment of B cell-associated autoimmune disorders.