C57BL/6 (CD45.2⁺, stock No. 000664), C57/CD45.1⁺ (B6.SJL-Ptprc^aPepc^b/BoyJ, stock No. 002014), miR-146a^–/– (B6.Cg-Mir146^tm1.1Bal /J, CD45.2⁺, stock No. 016239) (52) and μMT (B6.129S2-Ighm^tm1Cgn /J, CD45.2⁺, stock No. 002288) (53) mice were purchased from the Jackson Laboratory. miR-146a^–/– and miR-146a^+/+ mice (8-12 wk of age) were analyzed for fecal free and bacteria-bound IgA using our published procedure (23). They were also analyzed for spleen, bone marrow, MLNs, lamina propria and Peyer’s patches IgA⁺ B cells using an anti-mouse IgA mAb (clone C10-3, BD Bioscience) and mAb to mouse CD19 (clone 1D3, BD Bioscience). All mice were housed and maintained in the University of Texas Health Science Center San Antonio pathogen–free vivarium and provided with autoclaved food and deionized water. The Institutional Animal Care and Use Committees (IACUC) of the University of Texas Health San Antonio approved all animal research protocols.

To construct mixed bone marrow chimeric B cell-specific miR-146a^–/– (μMT/miR-146a^–/– ) mice and controls μMT/miR-146a^+/+ mice, sex-matched recipient C57/CD45.1⁺ mice (8-12 wks of age) from the same breeding batch were treated with neomycin sulfate (2 mg/ml in drinking water) for one week before being γ-irradiated for complete myeloablation (1000 Rad or 10 Gy, 12.5 min from a ¹³⁷cesium source). After 24 h, the mice were randomized into two groups and injected through the tail vein with 2.5 x 10⁶ mixed bone marrow cells as comprised of 80% (2 x 10⁶) cells from a μMT mouse and 20% (0.5 x 10⁶) cells from either a miR-146a^–/– mouse or its miR-146a^+/+ littermate. Prior to mixing, bone marrow cells isolated form tibia and fibula of donor miR-146a^–/– or miR-146a^+/+ mice were depleted of T cells by incubation with biotinylated anti-CD3 mAb (Magnisort^® Streptavidin Negative Selection Beads (eBioscience). Chimeric mice were monitored by flow cytometry for CD45.1 (clone A20, 110471, BioLegend), CD45.2 (clone 104, 109830, BioLegend), CD19 (clone 1D3, 20-0193, Tonbo), CD4 (clone RM4-5, 45-0042-82, eBioscience), CD8 (clone H35-17.2, 12-0083-81, eBioscience), CD11b (clone M1/70, 101212, BioLegend), and/or CD11c (clone N418, 117323, BioLegend) expression on circulating mononuclear cells for at least 6 wks to confirm the immune system reconstitution.

For antigen-specific IgA response experiments, miR-146a^–/– and miR-146a^+/+ mice as well as μMT/miR-146a^–/– and μMT/miR-146a^+/+ mice were given ovalbumin (OVA, 20 mg) with cholera toxin (OVA CT) via intragastric gavage (23) at d 0, 7 and 14. Sera and feces were collected at d 21 (for total and OVA-specific IgM, IgG1, IgA and IgE analysis), at which time mice were sacrificed for all other studies.

Titers of IgG1 and IgA in cell culture supernatants of in vitro-stimulated 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, 54–56). 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× antibiotic-antimycotic 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.

Naive IgM⁺IgD⁺ B cells were isolated from 8- to 12-wk-old C57BL/6 mice, as described (9). B cells were resuspended in FBS-RPMI at 37°C in 48-well plates and stimulated with LPS (5 μg/ml) from Escherichia coli (055:B5; Sigma-Aldrich) for CSR to IgG3; LPS (3 μg/ml) or CD154 [1 U/ml; obtained from membrane fragments of baculovirus-infected Sf21 insect cells (45)] plus IL-4 (5 ng/ml; R&D Systems) for CSR to IgG1/IgE and plasma cell differentiation; or LPS (3 μg/ml) or CD154 (1 U/ml) plus TGF-β (2 ng/ml; R&D Systems), IL-4 (5 ng/ml), IL-5 (3 ng/ml; R&D Systems), RA (10 nM, Sigma) and anti-Igδ mAb for CSR to IgA. After 96 h, the cells were stained with FITC-labeled rat mAb to mouse IgG1 (clone A85-1), mouse IgG2a/c (clone R19-15), mouse IgG3 (clone R40-82), mouse IgA (clone C10-3), or PE-labeled rat mAb to mouse CD19 (clone 1D3), all from BD Biosciences, 7-Aminoactinomycin D (7-AAD; A1310, Invitrogen) and PE-Cy7-anti-CD138 mAb (142513, Biolegend). The same mAbs were used to stain mouse mononuclear cells ex vivo. Cell analyses were performed using a LSR-II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (TreeStar). All experiments were performed in triplicates.

For quantification of germline I_H-C_H , post-recombination Iμ-Cα and circular Iα-Cμ transcripts, as well as Smad2, Smad3, Smad4, Aicda, Prdm1, Traf6 and Irak1 transcripts, RNA was extracted from 0.2-5.0 × 10⁶ 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 ) using a Bio-Rad MyiQ™ Real-Time PCR Detection System (Bio-Rad Laboratories) to measure SYBR Green (IQ™ SYBR^® Green Supermix, Bio-Rad Laboratories) incorporation with the following protocol: 95°C for 15. sec, 40 cycles of 94°C for 10 sec, 60°C for 30 sec, 72°C for 30 sec. Data acquisition was performed during 72°C extension step. Melting curve analysis was performed from 72-95°C.

For quantification of mature miRNA transcripts, RNA was extracted from 0.2-5.0 × 10⁶ cells using miRNeasy^® Mini Kit (Qiagen) and reverse-transcribed with miScript II RT Kit (Qiagen) using miScript HiSpec buffer. A Bio-Rad MyiQ™ Real-Time PCR Detection System was used to measure SYBR Green (miScript SYBR Green PCR Kit; Qiagen) incorporation according to manufacturer’s instructions. Mature miRNA forward primers ( Supplementary Table 1 ) were used at 250 nM in conjunction with the Qiagen miScript Universal Primer and normalized to expression of small nuclear/nucleolar RNAs Rnu6/RNU61/2, Snord61/SNORD61, Snord68/SNORD68, and Snord70/SNORD70. The ΔΔCt method was used for miRNA qRT-PCR data analysis.

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 μm 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 μm 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.

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-μm 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.

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’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’, bact-805R primer 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-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).

After stimulation by CSR-inducing stimuli and culture, B cells were lysed in Laemmli buffer. Cell extracts containing equal amounts of protein (20 μg) were fractionated through SDS-PAGE (10%). The fractionated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories) overnight (30 V) at 4°C. After blocking and overnight incubation at 4°C with anti-Smad2 (A7699, Abclonal), anti-Smad3 (A19115, Abclonal) and anti-Smad4 (A5657, Abclonal) rabbit Abs, or anti-β-Actin mAb (AC-15, Sigma-Aldrich), the membranes were incubated with HRP-conjugated secondary Abs. After washing with PBS-Tween 20 (0.05%), bound HRP-conjugated Abs were revealed using Western Lightning^® Plus-Enhanced Chemiluminescence reagents (PerkinElmer Life and Analytical Sciences).

ChIP assays were performed as described (23, 24, 30). Briefly, B cells (5 × 10⁶) were treated with 1% formaldehyde for 10 min at 25°C to cross-link chromatin. After washing with cold PBS containing protease inhibitors (Roche Applied Science), chromatin was separated using nuclear lysis buffer (10 mM Tris-HCl, 1.0 mM EDTA, 0.5 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.5% Sarkosyl, pH 8.0) and resuspended in IP-1 buffer (20 mM Tris-HCl, 200 mM NaCl, 2.0 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, protease inhibitors). Chromatin was sonicated to yield ~0.2 – 1.0 kb DNA fragments, precleared with agarose beads bearing protein G (Santa Cruz Biotechnology), and then incubated with anti-Smad2 (A7699, Abclonal), anti-Smad3 (A19115, Abclonal) or anti-Smad4 (A5657, Abclonal) rabbit Abs at 4°C. After overnight incubation, immune complexes were isolated using agarose beads bearing protein G, eluted with elution buffer (50 mM Tris-HCl, 0.5% SDS, 200 mM NaCl, 100 μg/ml Proteinase K, pH 8.0), and then incubated at 65°C overnight to reverse formaldehyde cross-links. DNA was purified using QIAquick PCR Purification Kit (Qiagen). The purified DNA was used as a template for qPCR analysis of the Iα promoter using specific primers ( Supplemental Table 1 ).

The miR-146a expression retroviral construct pMSCV-PIG-miR-146a (58) was a gift from Joshua Mendell (Addgene plasmid # 64234) and the pMSCV-PIG (Puro IRES GFP) empty vector was a gift from David Bartel (Addgene plasmid # 21654). To generate the retrovirus, pMSCV-PIG-miR-146a or empty pMSCV-PIG vector were used together with the pCL-Eco retrovirus-packaging vector (Imgenex) to transfect HEK293T cells by the calcium phosphate-mediated method (ProFection Mammalian Transfection System; Promega). Viral supernatants were harvested and used to transduce spleen B cells from C57BL/6 mice, as we reported (24, 30, 54), after a 12 h activation by LPS. Transduced B cells were then stimulated by LPS plus TGF-β, IL-4, IL-5, RA and anti-Igδ mAb for 96 h before analyzing GFP⁺ B cells for surface IgA expression as well as intracellular AID, Smad2, Smad3 and Smad4 expression.

All statistical analyses were performed using Excel (Microsoft) or GraphPad Prism^® software. Differences in Ig titers, CSR and RNA transcript expression were analyzed by Student’s paired (in vitro) and unpaired (in vivo) t-test assuming two-tailed distributions.

We investigated the impact of miR-146a deficiency on IgA expression and IgA-producing cells in non-intentionally immunized 22-wk-old miR-146a ^–/– mice. These mice showed higher levels of serum IgA (n = 6, 146.0 ± 10.2 vs. 78.4 ± 16.6 μg eq/ml, p = 0.014) and fecal IgA (n = 6, 271.3 ± 134.2 vs. 115.1 ± 28.2 μg eq/ml, p = 0.032) as well as fecal bacteria-bound IgA (68.25 vs. 23.2%) as compared to their miR-146a ^+/+ mouse counterparts. miR-146a ^–/– mice also showed higher frequency of IgA⁺ B cells in spleen, bone marrow, mesenteric lymph nodes (MLNs), intestinal lamina propria and Peyer’s patches ( 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.

To define the role of miR-146a in the modulation of an antigen-specific 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 bacteria-bound 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.

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 (59–61). 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.

To further define the role of miR-146a in CSR, we stimulated miR-146a ^–/– and miR-146a^+/+ B cells with LPS or CD154 plus IL-4 (to induce CSR to IgG1), LPS alone (CSR to IgG3), LPS or CD154 plus IL-4, IL-5, TGF-β, anti-δ mAb and RA (CSR to IgA). In addition to CSR, all these stimuli also induce plasma cell differentiation, albeit at different degrees. After a 96 h culture, CSR to IgA in miR-146a ^–/– B cells was significantly increased as compared to miR-146^+/+ B cells (27.0% vs 19.3% upon stimulation by LPS plus IL-4, IL-5, TGF-β, anti-δ mAb and RA; 26.6% vs 18.4% upon stimulation with CD154 plus IL-4, IL-5, TGF-β, anti-δ mAb and RA) ( 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, TGF-β, anti-δ mAb and RA was emphasized by the increased germline Iα-Cα transcripts, post-recombination Iμ-Cα transcripts, post-recombination circle Iα-Cμ transcripts and increased IgA secretion as compared to miR-146a^+/+ B cells, as well as by the unchanged IgG1 secretion, and germline Iγ1-Cγ1 and Iϵ-Cϵ transcripts in the very miR-146a ^–/– B cells ( Figures 5B, C ). Finally, the specific impact of the miR-146a on Igα 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.

Initiation of CSR to IgA requires TGF-β signaling which induces expression of Smad2, Smad3 and Smad4. Activated Smad2 and Smad3 dimerize and together with Smad4 bind to SBEs in the Iα promoter, thereby initiating germline Iα-Cα 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-β with or without RA, as well as human B cells with CD154 or CpG plus TGF-β to induce CSR to IgA. miR-146a expression was significantly downregulated (p < 0.01) in such mouse and human B cells stimulated to undergo CSR to IgA as compared to (mouse) B cells stimulated by nil or (human) B cells stimulated by CD154 or CpG alone (in absence of TGF-β), which does not induce CSR to IgA ( 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-β 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.

Having shown that IgA CSR-inducing stimuli downregulated miR-146a resulting in increased Smad2, Smad3 and Smad4 expression, germline Iα-Cα transcription, followed by post-recombination Iμ-Cα, circle Iα-Cμ transcripts, and CSR to IgA, we wanted to prove that ablation of miR-146a leads to further Smad2, Smad3 and Smad4 expression (critical for Iα-Cα 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-β, anti-δ 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 Iα-Cα and post-recombination Iμ-Cα transcripts, as well as Traf6 and Irak1, both genes whose proteins are involved in TGF-β activation signaling pathways (66). miR-146a specificity for Igα 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.

Having shown that induction of CSR to IgA in miR-146a ablated B cells leads to even higher levels of Smad2, Smad3 and Smad4, we hypothesized that this would result in increased Smad2, Smad3 and Smad4 recruitment to the Iα promoter, a requirement for activation of germline Iα-Cα transcription and CSR to IgA. Using TargetScan.org, miRNA.org and miRbase.org, we identified five SBEs [CAGAC and GGC(GC)|(CG) (67)] in the Iα promoter, the DNA region immediately upstream of the Iα transcription initiation site. Of such five SBEs, four were CAGAC and one was a GGCCG, overall, highly conserved in mouse and human Iα 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 Iα promoter in B cells activated by LPS or CD154 plus IL-4, IL-5, TGF-β, anti-δ 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 Iα promoter SBEs ( Figure 8C ).

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 Iα promoter to effect germline Iα-Cα 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-β, anti-δ 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 vector-transduced 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.