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.