Epigenetic Modulation of Class-Switch DNA Recombination to IgA by miR-146a Through Downregulation of Smad2, Smad3 and Smad4

IgA is the predominant antibody isotype at intestinal mucosae, where it plays a critical role in homeostasis and provides a first line of immune protection. Dysregulation of IgA production, however, can contribute to immunopathology, particularly in kidneys in which IgA deposition can cause nephropathy. Class-switch DNA recombination (CSR) to IgA is directed by TGF-β signaling, which activates Smad2 and Smad3. Activated Smad2/Smad3 dimers are recruited together with Smad4 to the IgH α locus Iα promoter to activate germline Iα-Cα transcription, the first step in the unfolding of CSR to IgA. Epigenetic factors, such as non-coding RNAs, particularly microRNAs, have been shown to regulate T cells, dendritic cells and other immune elements, as well as modulate the antibody response, including CSR, in a B cell-intrinsic fashion. Here we showed that the most abundant miRNA in resting B cells, miR-146a targets Smad2, Smad3 and Smad4 mRNA 3’UTRs and keeps CSR to IgA in check in resting B cells. Indeed, enforced miR-146a expression in B cells aborted induction of IgA CSR by decreasing Smad levels. By contrast, upon induction of CSR to IgA, as directed by TGF-β, B cells downregulated miR-146a, thereby reversing the silencing of Smad2, Smad3 and Smad4, which, once expressed, led to recruitment of Smad2, Smad3 and Smad4 to the Iα promoter for activation of germline Iα-Cα transcription. Deletion of miR-146a in miR-146a –/– mice significantly increased circulating levels of steady state total IgA, but not IgM, IgG or IgE, and heightened the specific IgA antibody response to OVA. In miR-146a –/– mice, the elevated systemic IgA levels were associated with increased IgA+ B cells in intestinal mucosae, increased amounts of fecal free and bacteria-bound IgA as well as kidney IgA deposition, a hallmark of IgA nephropathy. Increased germline Iα-Cα transcription and CSR to IgA in miR-146a –/– B cells in vitro proved that miR-146a-induced Smad2, Smad3 and Smad4 repression is B cell intrinsic. The B cell-intrinsic role of miR-146a in the modulation of CSR to IgA was formally confirmed in vivo by construction and OVA immunization of mixed bone marrow μMT/miR-146a –/– chimeric mice. Thus, by inhibiting Smad2, Smad3 and Smad4 expression, miR-146a plays an important and B cell intrinsic role in modulation of CSR to IgA and the IgA antibody response.


INTRODUCTION
IgA is the predominant antibody isotype at mucosal surfaces and the most abundant immunoglobulin (Ig) isotype in the body (1)(2)(3)(4)(5). It plays a critical role in mucosal homeostasis in the gastrointestinal, respiratory and urogenital tracts. IgA also provides a first line of immune protection against harmful commensals as well as airborne, ingested and sexually transmitted pathogens that come into contact with such mucosal surfaces (6). In the gut, IgA dimers are selectively transported across epithelia into the intestinal lumen, where they neutralize toxins and block the entry of pathogenic bacteria and viruses across the intestinal epithelium (4,7). Gut IgA effect both immune protection and immune exclusion in a noninflammatory manner, thereby playing a central role in hostmicrobial interaction. Intestinal IgA comprise low-affinity antibodies, mostly polyreactive, that prevent commensal bacteria from penetrating the mucosal barrier, as well as highaffinity IgA that neutralize toxins and microbial pathogens (8). Low affinity IgA would be generated in a T cell-independent fashion, generally in the lamina propria, while high affinity IgA would be generated in a T cell-dependent way, mainly in germinal centers of Peyer's patches.
Whether in T cell-dependent or independent fashion, IgA are produced through class-switch DNA recombination (CSR), a process that requires germline transcription of Ig heavy chain constant a gene (Iga), as activated by induction of the Ia promoter, and expression of activation-induced cytidine deaminase (AID, encoded by Aicda gene) (8)(9)(10)(11). Like CSR to other isotypes, CSR to IgA is a tightly controlled process. It is initiated by TLR ligand:TLR or CD154:CD40 engagement and TGF-b engagement of the TGF-b receptor. The latter activates (by phosphorylation) Smad2 and Smad3 transcription factors (12,13). Activated Smad2/Smad3 heterodimers form complexes with Smad4 for nuclear translocation and docking onto Smadbinding elements (SBEs) in the Ia promoter (8,(14)(15)(16). Here, in conjunction with co-factors, such as Runx3 and Pu.1, Smad2, Smad3 and Smad4 activate germline Ia-Ca transcription, which critically initiates CSR to IgA (17)(18)(19)(20). Accordingly, overexpression of Smad3 and Smad4 in B cells increased IgA expression (15) and B cell-specific Smad2 deletion reduced CSR to IgA (20). Other cytokines, such as IL-4 and IL-5, can enhance CSR to IgA, generally by boosting secretion of endogenous B cell TGF-b (21). Retinoic acid (RA), a vitamin A metabolite that is abundant in the gut, has been shown to induce gut homing receptors on activated T cells and CSR to IgA throughout gut-associated lymphoid tissue (GALT). Indeed, impaired GALT dendritic cells conversion of retinol to RA leads to decreased intestinal IgA, resulting in a noticeably decreased IgA in the circulating blood (22).
Epigenetic factors and modifications, such as non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), DNA methylation and histone posttranslational modifications effect heritable changes in gene expression that are independent from genomic DNA sequence. As we have shown, epigenetic factors and modifications act in concert with transcription factors to regulate B cell CSR, somatic hypermutation (SHM), plasma cell differentiation and generation of memory B cells, thereby shaping the antibody response to foreign-and self-antigens (11,(23)(24)(25)(26)(27). In particular, select miRNAs silence B cell Aicda and Prdm1 genes, thereby downregulating AID enzyme and Blimp1 transcription factor, which critically mediate CSR/SHM and plasma cell differentiation (11,23,25,(28)(29)(30). Prior to modulating these peripheral B cell differentiation processes, miRNAs regulate B development by modulating the expression of genes at the pro-B and pre-B cell stages (31,32).
miR-146a is one of a small number of miRNAs, whose cell expression is strongly induced by challenge with bacterial endotoxins, and whose prolonged expression has been linked to immune tolerance, implying that it acts as a fine-tuning mechanism to prevent an overstimulation of the immune system and, possibly, inflammatory response (33)(34)(35). Indeed, miR-146a has been suggested to regulate immune cells, such as T cell, dendritic cells and macrophages, as well as myeloid cells by exerting immunosuppressive and anti-inflammatory functions (36)(37)(38)(39)(40)(41)(42). These would include suppression of gut immunity, autoimmunity and myeloproliferation (43)(44)(45). miR-146a is one of the most abundant miRNAs in B cells, particularly resting B cells (38,44), and its role in B cell function just starts getting attention. miR-146a has been shown to control germinal center responses by targeting multiple components of the CD40 signaling pathway in B cells (39). miR-146a expression has been suggested to reduce IgA production in the gut, and miR-146a deficiency has led to development of immune complex glomerulonephritis in mice (45,46). miR-146a G/C polymorphism rs2910164 has been shown to associate with pediatric hyper IgA and IgA nephropathy, a syndrome that is characterized by high serum IgA levels, and IgA deposition in glomeruli, as well as with diabetic nephropathy (47,48). Such a miR-146a G/C polymorphism seemingly reduced the expression level of pre-miR-146a, likely by affecting the binding efficiency of the Drosha/DGCR8 complex to pri-miR-146a, thereby eventually reducing the amount of mature miR-146a (49). Further, miR-146a was found to act as a negative regulator of TGF-b signaling in skeletal muscle cells (50). Finally, it was shown to target Smad4, as induced by the TGF-b signaling pathway in promyelocytic leukemia cells (51).
The above findings and considerations led us to hypothesize that miR-146a plays an important role in regulating B cell CSR to IgA by modulating TGF-b-induced Smad2, Smad3 and Smad4. We tested our hypothesis by analyzing miR-146a -/mice for basal level of circulating IgA as well as frequency of IgA + B cells in spleen, bone marrow, mesenteric lymph nodes (MLNs), lamina propria and Peyer's patches, fecal bacteria-bound and free IgA as well as IgA deposition in kidney glomeruli. We further analyzed the magnitude of a specific IgA antibody response in miR-146a -/mice as well as constructed mMT/ miR-146a -/mixed bone marrow chimera mice to prove that any miR-146a related dysregulation in IgA production was due to intrinsic B cell miR-146a. We also tested the miR-146a -/-B cell intrinsic ability to undergo CSR to IgA in vitro in response to T-dependent and T-independent CSR-inducing stimuli. In these in vitro CSR-induced B cells, we measured the expression of Smad2, Smad3, Smad4 transcripts, the recruitment of Smad2, Smad3 and Smad4 transcription factors to the Ia promoter and consequent germline Ia-Ca transcription. Finally, we enforced expressed miR-146 in B cells to analyze the impact of this miRNA on Smad2, Samd3 and Smad4 expression levels. We concluded that miR-146a plays an important role in keeping in check Smad2, Smad3 and Smad4 transcription in resting B cells, and inhibiting CSR to IgA. Upon exposure to IgA CSR-inducing stimuli, such as CD154 or LPS plus TGF-b, B cells physiologically downregulate miR-146a, thereby allowing for increased Smad2, Smad3 and Smad4 expression, germline Ia-Ca transcription and unfolding of IgA CSR.

ELISAs and ELISPOTs
Titers of IgG1 and IgA in cell culture supernatants of in vitrostimulated B cells or IgM, IgG1, IgA or IgE in vivo titers of circulating/fecal total and/or OVA-specific-IgM, IgG1, IgA and IgE were measured using specific ELISAs, as we described (28,(54)(55)(56). For antibody secreting cell (ASC) analysis, MultiScreen ® ELISPOT plates (MAIPS4510; Millipore) were activated with ethanol (35%), washed four times with PBS and coated with unlabeled rabbit polyclonal antibodies against mouse IgM, IgG1 or IgA in PBS overnight at 4°C. The plates were then washed six times with PBS, blocked with BSA (0.5%) in RPMI/HEPES plus L-glutamine for 1 h at room temperature. The plates were then used to culture single cell suspensions from MLNs, Peyer's patches, bone marrow and spleen cells at 37°C for 16 h in 10% FBS RPMI medium containing 50 mM 2-ME and 1× antibioticantimycotic mixture (15240-062; Invitrogen) (FBS-RPMI) at 150,000 cells/ml. The culture fluids were then removed, the plates were washed six times, incubated with biotin-anti-IgM, IgG1, or IgA Ab for 2 h on a shaker at room temperature, washed, incubated with horseradish peroxidase (HRP)streptavidin (Santa Cruz Biotech) for 1 h on a shaker at room temperature, washed again and developed using the Vectastain AEC peroxidase substrate kit (SK-4200, Vector Laboratories) following manufacturer's protocol. ASCs were imaged and quantified using a CTL-ImmunoSpot Analyzer and software.

Quantitative RT-PCR (qRT-PCR) of mRNAs and miRNAs
For quantification of germline I H -C H , post-recombination Im-Ca and circular Ia-Cm transcripts, as well as Smad2, Smad3, Smad4, Aicda, Prdm1, Traf6 and Irak1 transcripts, RNA was extracted from 0.2-5.0 × 10 6 cells using either Trizol ® Reagent (Invitrogen) or RNeasy Plus Mini Kit (Qiagen). Residual DNA was removed from using gDNA eliminator columns (Qiagen). cDNA was synthesized from total RNA with the SuperScript ™ III First-Strand Synthesis System (Invitrogen) using oligo-dT primer. Transcript expression was measured by qRT-PCR with the appropriate primers (Supplementary Table 1

Fluorescence Microscopy
To analyze IgM and IgA-producing cells in the lamina propria, the intestine was folded into a "Swiss-roll", fixed with PFA (4%), and embedded in paraffin. Ten mm sections were cut and heated at 80°C to adhere to the slide, washed four times in xylene for 2 min, dehydrated two times with 100% ethanol for 1 min, two times with 95% ethanol for 1 min and washed two times in water for 1 min. Antigens were unmasked using 2 mM EDTA in 100°C for 40 mins followed by a cooling step at 25°C on the bench top, 3 times washing with 1x TBS and blocking using 10% BSA for 15 min. Slides were again washed three times with 1x TBS and stained with PE-anti-IgM, primary-rabbit anti mouse-IgA mAb (PA-1-30826, Thermo fisher) followed by Alexa Fluor 488 ® -anti rabbit-IgG (H+L) F(ab') 2 mAb (4412; Cell Signaling) for 2 h in a moist dark chamber. To analyze IgA-producing cells in MLNs, 10 mm MLN sections were prepared by cryostat and loaded onto positively charged slides, fixed in cold acetone and stained with rabbit anti mouse-IgA mAb (PA-1-30826, Thermo fisher) followed by Alexa Fluor 488 ® -anti rabbit-IgG (H+L) F(ab') 2 mAb (4412, Cell Signaling) for 1 h in a moist dark chamber. After washing three times with Triton X-100 (0.1%) in TBS, slides were air-dried and coverslips were mounted with ProLong ® Gold Antifade Reagent using DAPI (Invitrogen). Fluorescence images were captured using a 10 x objective lens with a Zeiss Axio Imager Z1 fluorescence microscope.

Kidney Pathology
To analyze kidney IgA deposition, kidneys from miR-146a -/and miR-146a +/+ mice were fixed in 4% formaldehyde and paraffin-embedded for hematoxylin and eosin staining. For immunofluorescence analysis, 5-mm cryostat sections were loaded onto positively charged slides, fixed in cold acetone, and stained with FITC-labeled rat anti-mouse IgA mAb (clone C10-3, BD Biosciences). Cover slips were mounted using ProLong Gold Antifade Reagent for microscopy analysis.

Microbiota Analysis
Microbial DNA were extracted from mouse feces using Quick-DNA ™ Fecal/Soil Microbe Microprep Kit (Zymo Research) according to the manufacturer's instructions. To determine the composition of the bacterial phyla present in mouse feces, isolated bacterial DNA was tagged and sequenced using miSeq platform. The V3-V4 hypervariable region of the bacteria 16S rRNA gene was amplified by PCR using tagged bact-341F primer 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCT ACGGGNGGCWGCAG-3', bact-805R primer 5'-GTCTCGT GGGCTCGGAGATGTGTATAAGAGACAGGACTACHVG GGTATCTAATCC-3' and Phusion DNA polymerase. Multiplexing indices and Illumina sequencing adapters were then added to the amplicons by limited-cycle amplification using the Nextera XT Index Kit (Illumina). The libraries were normalized, pooled and sequenced using the MiSeq system (Illumina). Sequencing and quality assessment were performed by the UT Health Genome Sequencing Facility. Bacterial taxonomy was assigned using the Ribosomal Database Project (RDP) classifier (57).

Enforced Expression of miR-146a
The miR-146a expression retroviral construct pMSCV-PIG-miR-146a (58) Figures 1A-C). In addition, they showed a higher frequency of IgA-but not IgM-or IgG-secreting cells in spleen and MLNs ( Figure 1D) as well as IgA deposition in kidney glomeruli and glomerular sclerosis ( Figure 1E). Thus, deletion of miR-146a increases systemic and local IgA.

miR-146a Knockout Increases Total and OVA-Specific IgA in Blood and Feces
To define the role of miR-146a in the modulation of an antigenspecific response, miR-146a +/+ and miR-146a -/mice were administered OVA via intragastric route once a week for three consecutive weeks. Serum and feces were collected from each mouse one week after the third OVA administration for analysis of systemic and fecal total and OVA-specific antibody titers, at which time mice were sacrificed for all other studies. miR-146a -/mice displayed significantly higher total and OVA-specific IgA (p < 0.05) in serum and feces than their miR-146a +/+ counterparts (Figures 2A-C). By contrast, miR-146a -/mice showed no significant changes in total or OVA-specific IgM, IgG1 or IgE titers in serum or feces. Thus, miR-146a deletion increases total and antigen-specific IgA in the circulation and the gut.
To determine whether the increased IgA production in miR-146a -/mice stemmed solely from B cell-intrinsic miR-146a deficiency, we constructed µMT/miR-146a +/+ and µMT/miR-146a -/chimera mice using myeloablated C57BL/6 mice grafted with (80%) bone marrow cells from µMT (B6.129S2-Ighm tm1Cgn /J) mice specifically deficient in B cells and (20%) purified bone marrow B cells from µMT/miR-146a -/mice. Chimeric µMT/miR-146a +/+ and µMT/miR-146a -/mice were administered OVA via intragastric route once a week for three consecutive weeks. Serum and feces were collected one week after the third administration for systemic and gut titles of total and   OVA-specific antibodies analysis, at which time mice were sacrificed (6 wks after bone marrow transplantation) for all other analyses. µMT/miR-146a -/mice show significantly elevated systemic and gut IgA (fecal) total and OVA-specific IgA but not IgM, IgG1 or IgE as compared to their µMT/miR-146a +/+ counterparts ( Figures 3A-C). Accordingly, µMT/miR-146a -/mice showed significantly greater amounts of bacteriabound IgA in feces, greater proportions of IgA + B cells in peripheral blood and Peyer's patches as well as higher numbers of OVA-specific ASCs in bone marrow, spleen and MLNs with no changes in the proportion of CD19 + IgG1 + B cells or CD138 + plasma cells (Figures 3D-F). Thus, intrinsic B cell miR-146a deficiency per se underpins increased IgA CSR and increased IgA levels in vivo.
Altered Gut Microbiota in Intrinsic B Cell miR-146a Deficient (µMT/miR-146a -/-) Chimeric Mice As IgA is critical for microbiome homeostasis at the intestinal mucosa and as miR-146a deletion resulted in major increases of IgA-producing B cells as well as total and specific IgA, we hypothesized that miR-146a deficiency would impact the composition of gut bacteria, thereby further emphasizing the role of miR-146a in IgA response. We analyzed the fecal microbiota in µMT/miR-146a -/mice by 16S rRNA gene amplicon sequencing. In all three µMT/miR-146a -/mice analyzed, the increased IgA production associated with an altered gut bacterial composition as compared to the three µMT/miR-146a +/+ mouse counterparts. We found that Verrucomicrobiaceae, Bacteroidaceae, Porphyromonadaceae and Lachnospiraceae, which are among the most abundant bacterial taxa in feces, were the major families represented in the gut (fecal) microbiome of µMT/miR-146a +/+ mice ( Figures 4A-C).
Verrucomicrobiaceae and particularly its Akkermansia genus bacteria have been shown to be physiologically bound by IgA and to positively correlate with IgA level in the gut (59)(60)(61). Accordingly, Verrucomicrobiaceae and Akkermansia bacteria were significantly increased in µMT/miR-146a -/mice, in which IgA were elevated. Bacteria of the Bacteroidaceae family and particularly its Bacteroides genus bacteria, which are typically abundant in the colon but not generally bound by IgA (59,60), were decreased in µMT/miR-146a -/mice ( Figures 4A-C). Bacteria of the Clostridia class, including those of the Lachnospiraceae family and the Blautia genus, members of the phyla Firmicutes, which are also not bound by IgA in vivo (59,60) were not altered in µMT/miR-146a -/mice ( Figures 4A-C). Rikenellaceae and Prevotellaceae family bacteria, which may or may not be bound by IgA, were decreased, while Sutterellaceae and Enterobacteriaceae, which also may or may not be bound by IgA, were increased in µMT/ miR-146a -/mice. Overall, fecal bacteria families and genera were consistently different in µMT/miR-146a -/and µMT/miR-146a +/+ mice, as revealed by principal component analysis (PCA) ( Figure 4D). Thus, intrinsic B cell miR-146a deficiency, as in µMT/miR-146a -/chimeric mice, leads to increased IgA levels and altered composition of the gut microbiota.  Figure 5A). Increased CSR to IgA in miR-146a -/-B cells was specific, as these B cells showed no alteration in CSR to IgG1 or differentiation to plasmablasts/ plasma cells (as determined by proportion of CD19 + IgG + B cells and CD138 + cells). The specificity of increased IgA CSR in miR-146a -/-B cells stimulated by LPS or CD154 plus IL-4, IL-5, TGFb, anti-d mAb and RA was emphasized by the increased germline Ia-Ca transcripts, post-recombination Im-Ca transcripts, postrecombination circle Ia-Cm transcripts and increased IgA secretion as compared to miR-146a +/+ B cells, as well as by the unchanged IgG1 secretion, and germline Ig1-Cg1 and Iϵ-Cϵ transcripts in the very miR-146a -/-B cells ( Figures 5B, C). Finally, the specific impact of the miR-146a on Iga locus transcription and recombination was further underlined by the normal level of Aicda transcripts in miR-146a -/-B cells undergoing CSR to IgA. Thus, intrinsic B cell miR-146a deficiency specifically enhances CSR to IgA while not affecting Aicda expression or plasma cell differentiation.
The Stimuli That Induce CSR to IgA Repress B Cell miR-146a, Thereby Upregulating Smad2, Smad3 and Smad4 Expression Initiation of CSR to IgA requires TGF-b signaling which induces expression of Smad2, Smad3 and Smad4. Activated Smad2 and Smad3 dimerize and together with Smad4 bind to SBEs in the Ia promoter, thereby initiating germline Ia-Ca transcription, the first step in CSR to IgA (62). Prompted by luciferase reporter assays showing miR-146a targeting of SMAD4 mRNA 3'UTR (63-65), we used miRNA-mRNA complementarity prediction tools (TargetScan.org, miRNA.org and miRbase.org) to identify miR-146a-specific target sites in the 3'UTRs of human and mouse Smad2/SMAD2, Smad3/SMAD3 and Smad4/SMAD4 ( Figure 6A), suggesting a potentially silencing of Smad2, Smad3 and Smad4 by miR-146a in both human and mouse B cells. This implies that the miR-146a expression level, which is high in resting B cells, must be dampened by those stimuli that induce CSR to IgA to free Smad2/SMAD2, Smad3/SMAD3 and Smad4/SMAD4 genes from miR-146a silencing for CSR to IgA to unfold. Toward addressing this postulated miR-146a modulatory function on CSR to IgA, C57BL/6 mouse B cells were stimulated with LPS or CD154 plus IL-4, TGF-b with or without RA, as well as human B cells with CD154 or CpG plus TGF-b to induce CSR to IgA. miR-146a expression was  Figure 6B). The downregulation of miR-146a in B cells undergoing CSR to IgA in vitro mimicked the downregulation of miR-146a in B cells undergoing IgA CSR in vivo, as in MLNs and Peyer's patches, as opposed to the high level of miR-146a in spleen B cells among which the B cells switching to IgA are a minor proportion of the overall B cell population (in non-intentionally immunized mice) ( Figure 6B).
In mouse B cells induced to undergo CSR to IgA by LPS plus IL-4, TGF-b and RA, miR-146a downregulation was concomitant with increased Smad2, Smad3 and Smad4 expression ( Figure 6C). Thus, the stimuli that induce B cells to undergo CSR to IgA downregulate miR-146a, thereby freeing Smad2/SMAD2, Smad3/SMAD3 and Smad4/SMAD4 from miR-146a inhibition and promoting expression of these transcription factors.

miR-146a Ablation Leads to Further
Smad2, Smad3 and Smad4 Upregulation in B Cells Induced to Undergo CSR to IgA Having shown that IgA CSR-inducing stimuli downregulated miR-146a resulting in increased Smad2, Smad3 and Smad4 expression, germline Ia-Ca transcription, followed by postrecombination Im-Ca, circle Ia-Cm transcripts, and CSR to IgA, we wanted to prove that ablation of miR-146a leads to further Smad2, Smad3 and Smad4 expression (critical for Ia-Ca transcription and initiation of CSR to IgA). To this end, we stimulated miR-146a -/-B cells and for comparison miR-146a +/+ B cells with IgA CSR-inducing stimuli LPS or CD154 plus IL-4, IL-5, TGF-b, anti-d mAb and RA. Whether stimulated by LPS or CD154, miR-146a -/-B cells upregulated Smad2, Smad3 and Smad4 transcripts as well as Smad2, Smad3 and Smad4 proteins at higher levels than their equally stimulated miR-146a +/+ B cell counterparts ( Figures 7A, B). They also underwent greater CSR to IgA, as indicated by the significantly higher expression of germline Ia-Ca and post-recombination  Im-Ca transcripts, as well as Traf6 and Irak1, both genes whose proteins are involved in TGF-b activation signaling pathways (66). miR-146a specificity for Iga locus CSR regulation was further emphasized by the comparable levels of induced Aicda and Prdm1 expression in miR-146a -/and miR-146a +/+ B cells ( Figures 7A, B). Thus, ablation of miR-146a significantly increases Smad2, Smad3 and Smad4 expression.  (67)] in the Ia promoter, the DNA region immediately upstream of the Ia transcription initiation site. Of such five SBEs, four were CAGAC and one was a GGCCG, overall, highly conserved in mouse and human Ia promoter region ( Figure 8A). As we hypothesized, ablation of miR-146a, which resulted in significantly increased Smad2, Smad3 and Smad4 levels, led to significantly increased recruitment of Smad2, Smad3 and Smad4 transcription factors to the Ia promoter in B cells activated by LPS or CD154 plus IL-4, IL-5, TGF-b, anti-d mAb and RA, and as analyzed by specific ChIP ( Figure 8B). Thus, as freed from miR-146a silencing, increased Smad2, Smad3 and Smad4 expression led to significantly increased recruitment of Smad2, Smad3 and Smad4 to the Ia promoter SBEs ( Figure 8C).

Enforced Expression of B Cell miR-146a Reduces CSR to IgA
Having shown that mouse and human B cells physiologically downregulate miR-146a in response to IgA CSR-inducing stimuli to free Smad2, Smad3 and Smad4 genes from silencing by this miRNA and, therefore, allowing for greater expression of Smad2, Smad3 and Smad4 and recruitment of these transcription factors to the Ia promoter to effect germline Ia-Ca transcription, we reasoned that CSR to IgA would be inhibited by high levels of miR-146a. To this end, we enforced expression of miR-146a in LPS-primed B cells by transducing them with retroviral vector pMSCV-PIG-miR-146a (58) expressing GFP and miR-146a or a control pMSCV-PIG vector expressing GFP only. The transduced B cells were subsequently activated by LPS plus IL-4, IL-5, TGF-b, anti-d mAb and RA, and cultured before being analyzed for expression of IgA, AID, Smad2, Smad3 and Smad4 proteins. The pMSCV-PIG-miR-146a retroviral vector-transduced B cells showed a significant reduction in Smad2, Smad3 and Smad4 expression and CSR to IgA as compared to the pMSCV-PIG retroviral vectortransduced B cells in the face of unchanged AID levels ( Figure 9). Thus, miR-146a inhibits Smad2, Smad3 and Smad4 expression, thereby dampening CSR to IgA.

DISCUSSION
IgA is the predominant antibody isotype found in mucous secretions. By maintaining a homeostatic environment at the mucosal surfaces, IgA provides a protective barrier against microbial pathogens and immune evasion. Dysregulation of CSR to IgA, however, can lead to IgA overproduction, such as in hyper IgA syndrome in which IgA kidney deposition leads to immunopathology. While many factors and trans-regulatory elements have been shown to control CSR, there is more to be learned about direct regulation of CSR to IgA, particularly with respect to the role of epigenetics factors, such as non-coding RNAs. Regulation of CSR to IgA must involve Smad2, Smad3 and Smad4. Indeed, these transcription factors provide the critical link between TGF-b signaling and the downstream activation of germline Ia-Ca transcription, the initiating event of CSR to IgA. Here we showed that miR-146a plays an important role in the epigenetic modulation of CSR to IgA by inhibiting Smad2, Smad3 and Smad4 expression. By using miR-146a -/mice, we showed the lack of miR-146a leads to an enhancement of IgA responses at intestinal and systemic levels. By using mixed bone marrow mMT/miR-146a -/chimeric mice, we further showed that the modulation of CSR to IgA by miR-146a is B cell-intrinsic. As miR-146a can be downregulated by Smad proteins (50), TGF-b-mediated activation of Smad proteins in B cells would lead to miR-146 reduction, which in turn would result in further expression of Smad proteins themselves. This positive Smads-miR-146a feedback would amplify TGF-b-directed CSR to IgA. miR-146a would regulate the proliferation of certain immune cells, as suggested by the development of tumors in secondary lymphoid organs and myeloproliferation in aged miR-146a knock-out mice (44). Aberrant miR-146a expression has been identified in autoimmune diseases and disorders, including systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome and inflammatory bowel disease (68). miR-146a would play a physiological role in cells of the innate and adaptive response (34,38). It is highly expressed in Tfh and germinal center B cells and thought to restrain the responsiveness of these two immune cell populations (69). miR-146a has been shown to regulate germinal center response by targeting multiple genes associated with CD40 signaling pathway in B cells (39). In aged CD21-cre miR-146a fl/fl mice, or CD21-cre miR-146a fl/fl mice immunized (i.p.) with SRBCs or NP-  KLH, miR-146a ablation in B cells led to increased germinal center formation and enhanced IgG production. miR-146a has been suggested to restrict the expansion of intestinal T cells and to reduce gut luminal IgA production (45). Indeed, as we showed here, miR-146a expression is lower in gut-associated lymphoid tissues, such as Peyer's patches and mesenteric lymph nodes, which contain a large portion of IgA-producing cells, than in the spleen which is poorer in IgA-producing cells. miR-146a is generally an LPS-responsive gene, as induced by the NF-kB signaling pathway, particularly in innate immune cells, such as granulocytes, monocytes, macrophages and NK cells. Excessive induction miR-146a dampens Irak1 and Traf6 (34,68,70). Irak1 and Traf6 play a significant role in TLR signaling and, possibly, NF-kB activation (71,72), leading to expression of AID or other genes central to peripheral B cell differentiation. Consistent with a role of miR-146a in targeting Irak1 and Traf6 mRNAs, our data showed that expression of Irak1 and Traf6 was increased in miR-146a -/-B cells. This, however, did not lead to alteration in Aicda or Prdm1 expression or germline Ig1-Cg1 and Iϵ-Cϵ transcripts, further emphasizing that miR-146a directly regulates CSR to IgA but not IgG1 or IgE. The direct modulation of CSR to IgA by miR-146a would provide a novel mechanism in miR-146amediated regulation of the antibody response in addition to miRNA146a role in regulation of germinal center formation (39). Upon immunization with OVA via intragastric gavage, miR-146a -/or mMT/miR-146a -/mice displayed significant increased IgA but not IgG1. In these miR-146a deficient mice, the lack of alteration in IgG1 production was associated with  unchanged germline Ig1-Cg1 transcripts and might be due to the type of immunization, which is known to skew the antibody response toward IgA and not to elicit full spleen germinal centers. TGF-b directs CSR to IgA by activating Smad2 and Smad3 transcription factors, which dimerize to Smad3/Smad3. Activated Smad3/Smad3 dimers recruit Smad4 and in conjunction with co-factors, such as Runx3 and Pu.1, would engage the Ia promoter of the IgH Ca gene through the five conserved SBEs we identified in this study, thereby inducing germline Ia-Ca transcription, the first and critical step leading to CSR to IgA (15,(17)(18)(19)(20)(21). Here we showed that miR-146a silences Smad2, Smad3 and Smad4 expression in B cells, thereby inhibiting CSR to IgA. We also showed that B cell miR-146a is profoundly downregulated by the stimulus that specifically directs CSR to IgA, i.e., TGF-b, thereby increasing Smad2/ Smad3 and Smad4. The specificity of miR-146a downregulation by stimuli that induce Smad proteins and CSR to IgA was emphasized by high levels of miR-146a in human and mouse resting B cells as well as B cells activated by stimuli that are known not to direct CSR to IgA. It was further confirmed by the low levels of miR-146a in B cells undergoing CSR to IgA in vivo, as in MLNs and Peyer's patches, as opposed to the high level of miR-146a in spleen B cells, among which B cells switching to IgA are a minor proportion of the overall B cell population. Besides miR-146a, Smad2, Smad3 and Smad4 may also be targeted by other miRNAs, such as miR-23a, miR-23b, miR-18, miR-27, miR-214 and miR-21 (73). However, virtually all these miRNAs are expressed at low level and are unchanged in B cells undergoing CSR to IgA (our unpublished high throughput miRNA-Seq data), and, therefore, may not play a significant role in the regulation of such CSR.
The mechanism underlying TGF-b-mediated downregulation of miR-146a is not clear. We suggest here that such downregulation is effected by Smad proteins. These have been shown to modulate miRNA expression through both transcriptional and post-transcriptional mechanisms (74)(75)(76), and overexpression of Smad3 and Samd4 has been reported to suppress miR-146 (50). We would argue that in B cells undergoing CSR to IgA, Smad2/Smad3 and Smad4 suppression of miR-146 levels frees Smad2, Smad3 and Smad4 from this miRNA-mediated gene silencing, thereby further increasing Smad2, Smad3 and Smad4 expression. This would create an actively self-potentiating Smads-miR-146a loop starting with (B) Expression of Smad2, Smad3 and Smad4 proteins as analyzed by immunoblotting. Densitometry quantification of immunoblotting signals was normalized to b-Actin levels and depicted as ratios of readings in miR-146a -/and miR-146a +/+ B cells to the average value in miR-146a +/+ B cells. Data are ratios to stimulated miR-146a +/+ B cells (set as 1; means ± SEM of three independent experiments). ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant, unpaired t-test. TGF-b-induction of activated Smads, Smads-mediated miR-146 repression, which results in increased Smad expression, leading to further miR-146a repression, increased Smad expression, and further potentiation of germline Ia-Ca transcription and CSR to IgA ( Figure 10). This actively self-potentiating Smads-miR-146a loop would not rule out the possibility that repression of miR-146a in B cells induced to undergo CSR to IgA may also occur independently of Smad proteins, perhaps through "noncanonical" TGF-b signaling factors such as ERK, p38, RhoA and phosphoinositide 3 kinase (PI3K), which can also be activated by TGF-b (66,77,78). Smad-mediated and Smadindependent pathways of repression of miR-146 would not be mutually exclusive, while both dependent on and initiated by TGF-b signaling.
In the intestinal lumen, secretory IgA exist in dimeric form. Dimeric IgA bind to and 'coat' commensal bacteria, thereby playing a critical role in shaping the composition and functions of the gut microbiome (79). IgA can limit the access of bacteria to intestinal epithelial cells, facilitate bacterial clearance, regulate bacterial colonization and neutralize disease-associated bacteria (80). Humans and mice lacking IgA display gut microbiota dysbiosis, including reduced overall microbial diversity, altered bacterial composition and increased bacterial translocation (60,(81)(82)(83)(84). The role of IgA in shaping the gut microbiota was originally thought to be limited to restriction of growth of specific organisms. Recent data, however, have suggested that IgA can also promote colonization and expansion of select bacterial species (85,86). We show here that B cell-intrinsic miR-146a ablation, as in µMT/miR-146a -/mice, led to increased production of systemic and intestinal IgA including bacteriabound IgA (in feces), as compared to µMT/miR-146a +/+ mice, thereby resulting in a skwesed selection of gut microbial populations in the absence of miR-146a. Verrucomicrobiae class bacteria, in particularly, those of the Akkermansia genus, are common members of the human intestinal microbiota and were significantly increased in µMT/miR-146a -/mice, in which IgA were elevated. Indeed, such bacteria and have been shown to be positively correlate with IgA level and to be highly coated with     IgA (59)(60)(61)83). However, Bacteroidaceae family bacteria, particularly those of the Bacteroides genus, which, in contrast with Akkermansia bacteria, are not generally bound by IgA and are typically abundant in the colon (59,60), were also decreased in µMT/miR-146a -/micesome Bacteroides species have been shown to effectively induce production of gut IgA (87), which in turn, could possibly create a negative feedback to the very IgAinducing bacteria. Similarly, the class Clostridia bacteria, including those of the Lachnospiraceae family and the Blautia genus, members of the phyla Firmicutes, which are also not bound by IgA antibodies in vivo (59, 60), were also not altered in µMT/miR-146a -/mice. Thus, through regulation of IgA levels, B cell-intrinsic miR-146a can play an important role in modulating the steady state of the gut microbiome. Dysregulated IgA levels and antibody responses are often associated with disease (88). IgA deficiency can lead to severe respiratory tract infections or increase the risk of adverse reactions to blood products (89). Abnormally high IgA levels may cause immune disorders, such as chronic rheumatic disease or chronic gastrointestinal inflammation (88). Here, we demonstrated that miR-146a plays an important role in keeping in check Smad2, Smad3 and Smad4 expression and, thereby, inhibiting the TGF-b signaling pathway-dependent induction of CSR to IgA, which results in a significant modulation of the IgA antibody response. Conversely, deletion of miR-146a, as in miR-146a −/− mice, increased systemic and gut IgA, thereby leading to IgA deposition in kidney glomeruli and immunopathology. This is highly evocative of the occurrence of miR-146a single nucleotide polymorphism rs2910164 in childhood hyper IgA syndrome and IgA nephropathy (47). Similarly, high serum IgA levels, as in "hyper IgA" mice, have been shown to result in IgA nephropathy, due to IgA kidney deposition leading to chronic inflammation (90). Interestingly, high IgA levels also occur in most cases of hyperimmunoglobulinemia D syndrome (HIDS), an autosomal recessive disorder characterized by abdominal, articular and skin manifestations in conjunction with recurrent febrile attacks (91). In HIDS patients, high IgA concentrations stem from abnormally high IgA1 levels, which correlate with systemically high IgD levels. While mutations in the mevalonate kinase gene underpin the vast majority of HIDS cases, the root of systemic hyper IgA in HIDS patients remains unknown. By providing a first mechanistic and important insight into epigentic regulation of CSR to IgA expression, our findings open new avenues of investigation of the role of miR-146amediated IgA dysregulation in a variety of hyper IgA conditions, such as those in HIDS, "idiopathic" hyper IgA, autoimmune and non-autoimmune inflammatory diseases, cancer and select metabolic disorders.

DATA AVAILABILITY STATEMENT
16S rRNA gene-sequencing data have been deposited in NCBI Sequence Read Archive (SRA) under BioProject PRJNA777603. All the other data needed to evaluate the conclusions in the paper are present in the paper. Additional data related to this paper may be requested from the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by The Institutional Animal Care and Use Committees (IACUC) of the University of Texas Health San Antonio.

AUTHOR CONTRIBUTIONS
PC conceived and designed the study, planned the experiments, analyzed data, supervised the work, implemented the execution of the overall experimental plan, created figures and wrote the manuscript. SL, GM, CD, DC, and AF, each performed one-two experiments. HZ designed the study, planned the experiments, analyzed data, supervised the work, created figures and wrote the manuscript. All authors contributed to the article and approved the submitted version.