Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require AID, and plasma cell differentiation, which requires Blimp-1, are critical for the generation of class-switched and hypermutated (mature) antibody and autoantibody responses. We showed here that the histone deacetylase (HDAC) inhibitors (HDI) valproic acid (VPA) and butyrate upregulated miR-155, miR-181b and miR-361, which silenced AICDA/Aicda (AID) mRNA, and miR-23b, miR-30a and miR-125b, which silenced PRDM1/Prdm1 (Blimp-1) mRNA, in human and mouse B cells. This led to downregulation of AID, Blimp-1 and Xbp-1 expression, thereby dampening CSR, SHM and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of HDI-mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important inducers/modulators of AICDA/Aicda), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2 or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell intrinsic epigenetic mechanisms, VPA blunted class-switched and hypermutated T-dependent and T-independent antibody responses in C57BL/6 mice. In addition, it decreased class-switched and hypermutated autoantibodies, ameliorated disease and extended survival in lupus MRL/Faslpr/lpr mice. Our findings outline epigenetic mechanisms that modulate expression of an enzyme (AID) and transcription factors (Blimp-1 and Xbp-1) that critical to the B cell differentiation processes that underpin antibody and autoantibody responses. They also provide therapeutics proof-of-principle in autoantibody-mediated autoimmunity.

Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S–S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52−/− B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S–S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S–S recombination, as emphasized by the significantly greater CSR reduction in Rad52−/− versus Rad52+/+ B cells on Ku86 knockdown.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies, which plays an important role in disease development and progression. Lupus preferentially affects women during their reproductive years. The pathogenesis of lupus is contributed by both genetic factors and epigenetic modifications that arise from exposure to the environment. Epigenetic marks, including DNA methylation, histone post-translational modifications and microRNAs (miRNAs), interact with genetic programs to regulate immune responses. Epigenetic modifications influence gene expression and modulate B cell functions, such as class switch DNA recombination (CSR), somatic hypermutation (SHM) and plasma cell differentiation, thereby informing the antibody response. Epigenetic dysregulation can result in aberrant antibody responses to exogenous antigens or self-antigens, such as chromatin, histones and dsDNA in lupus. miRNAs play key roles in the post-transcriptional regulation of most gene-regulatory pathways and regulate both the innate and the adaptive immune responses. In mice, dysregulation of miRNAs leads to aberrant immune responses and development of systemic autoimmunity. Altered miRNA expression has been reported in human autoimmune diseases, including lupus. The dysregulation of miRNAs in lupus could be the result of multiple environmental factors, such as sex hormones and viral or bacterial infection. Modulation of miRNA is a potential therapeutic strategy for lupus.

Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require AID, and plasma cell differentiation, which requires Blimp-1, are critical for the generation of class-switched and hypermutated (mature) antibody and autoantibody responses. We show that histone deacetylase inhibitors (HDIs) valproic acid (VPA) and butyrate upregulated miR-155, miR-181b and miR-361 to silence AICDA/Aicda, and miR-23b, miR-30a and miR-125b to silence PRDM1/Prdm1, in human and mouse B cells. This led to downregulation of AID, Blimp-1 and Xbp-1, thereby inhibiting CSR, SHM and plasma cell differentiation. The selectivity of HDI-mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important AICDA/Aicda inducer/modulators), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2 or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell intrinsic epigenetic mechanisms, VPA blunted class-switched and hypermutated T-dependent and T-independent antibody responses in C57BL/6 mice. It also decreased class-switched and hypermutated autoantibodies, ameliorated disease and extended survival in lupus MRL/Faslpr/lpr mice. Our findings outline important epigenetic mechanisms of modulation of antibody responses and providing proof-of-principle therapeutics in autoantibody-mediated autoimmunity.

Class switch DNA recombination (CSR) diversifies the biological effector functions of antibodies and is critical for the maturation of the immune response. It is initiated by AID-mediated generation of DSBs in the switch (S) regions that will undergo recombination, mainly through Ku70/Ku86-mediated nonhomologous end-joining. It has been shown, however, that Ku-deficient B cells can undergo CSR, suggesting that other DNA repair mechanisms are at work. Herein, we show that deficiency of Rad52, an important component of the homologous recombination DNA repair pathway, resulted in significantly increased CSR and is associated with increased recruitment of Ku70/Ku86 to the S region DSB ends. In Rad52-/- B cells, the increased role of Ku70/Ku86 in S region DNA recombination was further emphasized by decreased occurrence of microhomologies in S-S junctions and inter-chromosomal c-Myc/IgH translocations. Conversely, enforced expression of Rad52 in B cells impaired CSR and decreased Ku70/Ku86 recruitment to S region DNA, as revealed by ChIP assays. Accordingly, EMSAs using Sμ probes with blunt-ends or short overhangs, revealed that in CSR, Ku70/Ku86 and Rad52 bind to different types of DSB ends. Our findings show that Rad52 modulates CSR by binding to S region DSB ends. This may facilitate a Ku-independent repair pathway, which would resolve AID-mediated S region DSBs, thereby explaining the residual CSR that has been shown to occur in a conditional Ku70/Ku86 B cell knockout.

Class switch DNA recombination (CSR) plays a central role in the maturation of the antibody response. CSR entails the generation of double-strand DNA breaks (DSBs) mediated by activation-induced cytidine deaminase (AID). Resolution of these DSBs can occur through Ku70/Ku86 dependent classical non-homologous end-joining (C-NHEJ), or through an undefined alternative-end joining (A-EJ) that introduces microhomologies in the S-S junctions. Here we investigate the role of Rad52, an important DSB repair factor in CSR. We found that Rad52 deficiency elicits an increase in NP-specific IgG1 and IgG3 responses to T-dependent and T-independent antigens. The increased antibody response is not due to alterations in lymphoid differentiation, B cell proliferation or cell division but rather a direct effect on the CSR machinery. This phenomenon is B cell intrinsic, as CSR to IgG1, IgG2a, IgG3 and IgA virtually double in Rad52-/- B cells stimulated with LPS and respective cytokines. In CSR, both Rad52 and Ku70/Ku86 are specifically recruited to S region DNA in an AID-dependent manner. In the absence of Rad52, the recruitment of Ku70/Ku86 to the S regions was significantly increased. Finally, the frequency and length of microhomologies in the S-S junctions was significantly reduced in Rad52-/- B cells. Altogether, our findings suggest that Rad52 competes with Ku70/Ku86 in binding S region to repair DSBs in CSR possibly utilizing an alternative end-joining pathway.

As we suggested, epigenetic factors such as miRNAs, can interact with genetic programs to regulate B cell functions and inform antibody response. We have shown that histone deacetylase inhibitors (HDIs) valproic acid and butyrate, inhibited class-switch DNA recombination (CSR), somatic hypermutation (SHM) and plasma cell differentiation by modulating B cell intrinsic mechanisms. They repressed AID and Blimp-1 expression, which are critical for CSR/SHM and plasma cell differentiation, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm-1 mRNAs, as demonstrated by qRT-PCRs. To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed miRNA-Seq and mRNA-Seq analysis in B cells stimulated with LPS plus IL-4 followed by VPA treatment. Consistent with our recent findings (J. Immunol. 193:5933-5950, 2014), these B cells showed reduced Aicda and Prdm1 expression, and increased expression of miR-155, miR-181b and miR-361, which target Aicda, and miR-23b, miR-30a and miR-125b, which target Prdm1,. The HDI-mediated modulation of mRNA was very selective. Among over 22,000 mRNAs analyzed, less than 20 mRNAs, including Aicda and Prdm1, significantly expressed in activated B cells were reduced by over two fold. Our findings indicate that, in B cells, HDI selectively modulate mRNAs, possibly through miRNA upregulation and as a result of HDACs existing in unique contexts of HDAC/co-factor complexes,.