Hong Zan

MicroRNAs in lupus

Abstract Systemic lupus erythematosus is a prototypic autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies, which play 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, somatic hypermutation 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 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.

Epigenetics of Peripheral B-Cell Differentiation and the Antibody Response

Epigenetic modifications, such as histone post-translational modifications, DNA methylation, and alteration of gene expression by non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are heritable changes that are independent from the genomic DNA sequence. These regulate gene activities and, therefore, cellular functions. Epigenetic modifications act in concert with transcription factors and play critical roles in B cell development and differentiation, thereby modulating antibody responses to foreign- and self-antigens. Upon antigen encounter by mature B cells in the periphery, alterations of these lymphocytes epigenetic landscape are induced by the same stimuli that drive the antibody response. Such alterations instruct B cells to undergo immunoglobulin (Ig) class switch DNA recombination (CSR) and somatic hypermutation (SHM), as well as differentiation to memory B cells or long-lived plasma cells for the immune memory. Inducible histone modifications, together with DNA methylation and miRNAs modulate the transcriptome, particularly the expression of activation-induced cytidine deaminase, which is essential for CSR and SHM, and factors central to plasma cell differentiation, such as B lymphocyte-induced maturation protein-1. These inducible B cell-intrinsic epigenetic marks guide the maturation of antibody responses. Combinatorial histone modifications also function as histone codes to target CSR and, possibly, SHM machinery to the Ig loci by recruiting specific adaptors that can stabilize CSR/SHM factors. In addition, lncRNAs, such as recently reported lncRNA-CSR and an lncRNA generated through transcription of the S region that form G-quadruplex structures, are also important for CSR targeting. Epigenetic dysregulation in B cells, including the aberrant expression of non-coding RNAs and alterations of histone modifications and DNA methylation, can result in aberrant antibody responses to foreign antigens, such as those on microbial pathogens, and generation of pathogenic autoantibodies, IgE in allergic reactions, as well as B cell neoplasia. Epigenetic marks would be attractive targets for new therapeutics for autoimmune and allergic diseases, and B cell malignancies.

DNA Replication to Aid Somatic Hypermutation

Immunoglobulin (Ig) heavy and light chain loci undergo V, (D) and J gene recombination in bone marrow, giving rise to the diverse pre-immune repertoire of V(D)J segments of B cell receptors (BCR). After encountering antigen, naive B cells divide and differentiate in germinal centers of secondary lymphoid organs. Here, Ig V(D)J gene segments are the targets of point-mutations at a rate of 10−3 change per base per cell division. This rate is a million-fold higher than that of mutations occurring spontaneously in the genome at large; hence, the term somatic hypermutation (SHM). Mutated Ig V(D)J regions provide the structural substrate for positive selection by antigen of high affinity mutants, which are characteristic of a mature antibody response. The Ig locus of germinal center B cells also undergoes class switch DNA recombination (CSR), which replaces the constant μ (Cμ) region of the heavy chain with a downstream Cγ, Cα, Ce region, thereby endowing antibodies with new biological effector functions. n nActivation-induced cytidine deaminase (AID) initiates SHM and CSR (Muramatsu et al., 2000; Revy et al., 2000) by directly deaminating cytidine in DNA (dC), thereby yielding premutagenic uracil DNA lesions (dU) (Petersen-Mahrt et al., 2002; Rada et al., 2004; Neuberger et al., 2005). SHM preferential targets the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) mutational hotspot, which contains the preferred AID deamination motif WRC (Petersen-Mahrt and Neuberger, 2003; Pham et al., 2003; Yu et al., 2004). dU is a non-bulky DNA lesion that, if not repaired, can pair with dA without blocking the DNA replication fork, thereby giving rise to dG → dA and dC → dT transitions. Consistent with this prediction, mice with a double deficiency of Ung, a uracil-DNA glycosylase (UDG), and MutantS homolog 2 (Msh2), a mismatch sensor and initiator of the mismatch repair (MMR) cascade, display only dG → dA and dC → dT transitions, likely due to ablation of both the Ung-mediated base excision repair (BER) and the Msh2-mediated MMR pathways (Rada et al., 2004). Accordingly, in contrast to other DNA lesions, such as abasic sites or pyrimidine dimers, dU in the DNA template does not block transcriptional elongation by human RNA polymerase II, leading to incorporation of either G or A in nascent transcripts (Kuraoka et al., 2003). In B cells, this would lead to the introduction of same mutations in antibodies as those that would be generated from mutated DNA templates carrying dG → dA and dC → dT transitions. Thus, dU repair in Ig V(D)J DNA is not under the feedback pressure from faulty transcription; instead, it is dealt with by Ung and Msh2 in an actively mutagenic fashion during DNA synthesis, as effected by error-prone lesion-bypass or translesion DNA synthesis (TLS) polymerases. Here, we will discuss DNA repair factors and their assembly into a putative “mutasome”, which is centered on the proliferating cell nuclear antigen (PCNA), and their role in the SHM process.

Genome-Wide Analysis Reveals Selective Modulation of microRNAs and mRNAs by Histone Deacetylase Inhibitor in B Cells Induced to Undergo Class-Switch DNA Recombination and Plasma Cell Differentiation.

As we have suggested, epigenetic factors, such as microRNAs (miRNAs), can interact with genetic programs to regulate B cell functions, thereby informing antibody and autoantibody responses. We have shown that histone deacetylase (HDAC) inhibitors (HDI) inhibit the differentiation events critical to the maturation of the antibody response: class-switch DNA recombination (CSR), somatic hypermutation (SHM), and plasma cell differentiation, by modulating intrinsic B cell mechanisms. HDI repress the expression of AID and Blimp-1, which are critical for CSR/SHM and plasma cell differentiation, respectively, in mouse and human B cells by upregulating selected miRNAs that silenced AICDA/Aicda and PRDM1/Prdm1 mRNAs, as demonstrated by multiple qRT-PCRs (J Immunol 193:5933–5950, 2014). To further define the selectivity of HDI-mediated modulation of miRNA and gene expression, we performed genome-wide miRNA-Seq and mRNA-Seq analysis in B cells stimulated by LPS plus IL-4 and treated with HDI or nil. Consistent with what we have shown using qRT-PCR, these HDI-treated B cells displayed reduced expression of Aicda and Prdm1, 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. In B cells induced to undergo CSR and plasma cell differentiation, about 23% of over 22,000 mRNAs analyzed were expressed at a significantly high copy number (more than 20 copies/cell). Only 18 (0.36%) of these highly expressed mRNAs, including Aicda, Prdm1, and Xbp1, were downregulated by HDI by 50% or more. Further, only 16 (0.30%) of the highly expressed mRNAs were upregulated (more than twofold) by HDI. The selectivity of HDI-mediated modulation of gene expression was emphasized by unchanged expression of the genes that are involved in regulation, targeting, or DNA repair processes of CSR, as well as unchanged expression of the genes encoding epigenetic regulators and factors that are important for cell signaling or apoptosis. Our findings indicate that, in B cells induced to undergo CSR and plasma cell differentiation, HDI modulate selected miRNAs and mRNAs, possibly as a result of HDACs existing in unique contexts of HDAC/cofactor complexes, as occurring in B lymphocytes, particularly when in an activated state.

Small Molecule Inhibition of Rab7 Impairs B Cell Class Switching and Plasma Cell Survival To Dampen the Autoantibody Response in Murine Lupus

IgG autoantibodies mediate pathology in systemic lupus patients and lupus-prone mice. In this study, we showed that the class-switched IgG autoantibody response in MRL/Faslpr/lpr and C57/Sle1Sle2Sle2 mice was blocked by the CID 1067700 compound, which specifically targeted Ras-related in brain 7 (Rab7), an endosome-localized small GTPase that was upregulated in activated human and mouse lupus B cells, leading to prevention of disease development and extension of lifespan. These were associated with decreased IgG-expressing B cells and plasma cells, but unchanged numbers and functions of myeloid cells and T cells. The Rab7 inhibitor suppressed T cell–dependent and T cell–independent Ab responses, but it did not affect T cell–mediated clearance of Chlamydia infection, consistent with a B cell–specific role of Rab7. Indeed, B cells and plasma cells were inherently sensitive to Rab7 gene knockout or Rab7 activity inhibition in class switching and survival, respectively, whereas proliferation/survival of B cells and generation of plasma cells were not affected. Impairment of NF-κB activation upon Rab7 inhibition, together with the rescue of B cell class switching and plasma cell survival by enforced NF-κB activation, indicated that Rab7 mediates these processes by promoting NF-κB activation, likely through signal transduction on intracellular membrane structures. Thus, a single Rab7-inhibiting small molecule can target two stages of B cell differentiation to dampen the pathogenic autoantibody response in lupus.

Editorial: Epigenetics of B Cells and Antibody Responses

Epigenetics is the study of changes in gene activity that are heritable but not caused by changes in the DNA sequence. By modulating gene activities, epigenetic changes regulate cell functions. They include DNA methylation, histone post-translational modifications, and gene silencing by the action of non-coding RNAs, particularly microRNAs. It is now clear that epigenetic changes regulate B cell development. By acting in concert with networks of transcription factors, they modulate the activation of B cell lineage-specific gene programs and repress inappropriate gene transcription in particular B cell differentiation states (1). The hallmark of B cell development in the bone marrow is the assembly of the B cell receptor (BCR) for antigen through rearrangement of immunoglobulin heavy (IgH) and light (IgL) chain V(D)J genes, as mediated by RAG1/RAG2 recombinases. Ig V(D)J rearrangement critically times the progression from pro-B cell to pre-B cell and, eventually, transitional and mature B cell. Such progression is modulated by epigenetic marks, such as DNA methylation and histone posttransla-tional modifications, which increase chromatin accessibility and target RAG1/RAG2 to V, D, and J DNA (2). It is also dependent on the expression of epigenetic factors, such as microRNAs. In mice deficient in Ago2, which is essential for microRNA biogenesis and function, B cell development is arrested at the pro-B cell stage (Danger et al.). Accordingly, B cell-specific ablation of microRNAs by B cell-specific knockout of Dicer virtually blocks B cell differentiation at the pro-B to pre-B cell transition (Danger et al.). After mature B cells encounter antigen, changes of the epigenetic landscape are induced by the same stimuli that drive the antibody response. Such epigenetic changes underpin the maturation of the antibody response itself. They instruct those B cell differentiation processes, somatic hyper-mutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation that are central to the maturation of the antibody response as well as the differentiation of memory B cells. Inducible histone modifications, together with DNA methylation and microRNAs, modulate the transcriptome, particularly the expression of activation-induced cytidine deaminase (AID), central to SHM and CSR, and B lymphocyte-induced maturation protein-1 (Blimp-1), the master transcription factor in plasma cell differentiation (1, 3–5) (Shen et al. and Zan and Casali). Combinatorial histone modifications also function as histone codes in targeting the CSR and, possibly, the SHM machinery to the Ig locus by recruiting specific adaptors (histone code readers) that can in turn target and/or stabilize CSR/SHM factors (6). Epigenetic …