Mir-17–92 regulates bone marrow homing of plasma cells and production of immunoglobulin G2c

Xu, Shengli; Ou, Xijun; Huo, Jianxin; Lim, Kristen; Huang, Yuhan; Chee, Sheena; Lam, Kong-Peng

doi:10.1038/ncomms7764

Article
Published: 17 April 2015

Mir-17–92 regulates bone marrow homing of plasma cells and production of immunoglobulin G2c

Shengli Xu^1,2^na1,
Xijun Ou¹^na1,
Jianxin Huo¹,
Kristen Lim¹,
Yuhan Huang¹,
Sheena Chee¹ &
…
Kong-Peng Lam^1,2,3

Nature Communications volume 6, Article number: 6764 (2015) Cite this article

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Abstract

The polycistronic mir-17–92 cluster, also known as oncomir-1, was previously shown to be essential for early B lymphopoiesis. However, its role in late-stage B-cell differentiation and function remains unexplored. Here we ablate mir-17–92 in mature B cells and demonstrate that mir-17–92 is dispensable for conventional B-cell development in the periphery. Interestingly, mir-17–92-deficiency in B cells leads to enhanced homing of plasma cells to the bone marrow during T-cell-dependent immune response and selectively impairs IgG2c production. Mechanistically, mir-17–92 directly represses the expression of Sphingosine 1-phosphate receptor 1 and transcription factor IKAROS, which are, respectively, important for plasma cell homing and IgG2c production. We further show that deletion of mir-17–92 could reduce IgG2c anti-DNA autoantibody production and hence mitigate immune complex glomerulonephritis in Shp1-deficient mice prone to autoimmunity. Our results identify important roles for mir-17–92 in the regulation of peripheral B-cell function.

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Introduction

B cells and antibodies are central elements of adaptive humoral immunity and critical for host protection. B lymphopoiesis begins with committed haematopoietic stem cells differentiating into pro-B, pre-B and immature B cells in the bone marrow (BM) and cumulates with mature B cells seeding the peripheral lymphoid organs. On activation by foreign antigens, naive B cells undergo terminal differentiation into memory B and antibody-secreting plasma cells (PCs) that provide life-long protection^1,2. B-cell differentiation is governed by a network of transcription factors^3,4. Recently, microRNAs (miRNAs) are shown to exert additional control over B-cell differentiation and function^5,6.

MiRNAs are endogenously expressed short non-coding RNAs that fine-tune gene expression at the post-transcriptional level by either promoting the degradation or impeding the translation of target mRNAs^7,8,9. A ‘seed region’ of six to eight nucleotides at the 5′ end of a miRNA determines its target specificity¹⁰. The importance of miRNAs in B-cell biology is highlighted by the drastic defects in mice with global deletion of miRNAs caused by ablating Dicer, a RNAse III endonuclease critical for miRNA processing, in precursor¹¹, peripheral mature¹² and activated¹³ B cells. When Dicer was deleted at the earliest stage of B-cell maturation using mb-1 Cre, B lymphopoiesis was severely blocked at the pro-B to pre-B-cell transition¹¹. This defect was mainly attributed to the absence of mir-17–92 whose seed sequences were enriched in the 3′-untranslated regions (3′UTRs) of mRNAs upregulated in Dicer-deficient pro-B cells¹¹.

MiR-17–92, or oncomir-1, is a polycistronic miRNA cluster that yields six mature miRNAs, including mir-17, mir-18a, mir-19a, mir-20a, mir-19b and miR-92a. It was first identified as a potent miRNA oncogene on human chromosome 13q31, a region that was amplified in a number of B-cell malignancies^14,15. Overexpression of mir-17–92 in haematopoietic cells significantly accelerated c-Myc induced B-lymphomagenesis in mice¹⁶ and B-cell-specific mir-17–92 transgenic mice developed lymphoma with high penetrance¹⁷. On the other hand, germline deletion of mir-17–92 compromised early B lymphopoiesis suggesting that mir-17–92 plays essential roles in early B-cell development¹⁸. Pro-apoptotic BIM was identified as a direct target of mir-17–92 and its upregulation in mutant pro-B cells accounted for the impairment in B-lymphopoiesis¹⁸.

In this study, we deleted mir-17–92 in mature B cells using CD19-Cre to examine its role in late-stage B-cell differentiation and function. In contrast to its essential role in early B lymphopoiesis in the BM, mir-17–92-deficency does not perturb conventional B-cell populations in the periphery. Interestingly, during T-cell-dependent (TD) antibody response, there is increased PCs in the BM due to elevated expression of Sphingosine 1-phosphate receptor 1 (S1PR1), which mediates BM homing of PCs¹⁹. We show that five out of six members of mir-17–92 cluster directly target the 3′UTR of S1pr1 and removal of one S1pr1 allele could rebalance S1pr1 expression and normalize the enhanced BM homing phenotype of mir-17–92-deficient PCs. We also reveal that immunoglobulin G2c (IgG2c) production is impaired in the absence of mir-17–92 and this defect is due to increased expression of the transcription factor IKAROS, which is important for IgG2c production²⁰ and directly repressed by mir-92a (ref. 21). Furthermore, we demonstrate that mir-17–92 deletion in B cells could reduce IgG2c autoantibody production and attenuate immune complex glomerulonephritis in autoimmune Shp1-deficient mice. Taken together, our findings identify novel roles for mir-17–92 in regulating peripheral B-cell function by fine-tuning different target genes.

Results

B-cell development in mice lacking mir-17–92 in B cells

To address the role of mir-17–92 in peripheral B cells, we deleted loxP-flanked (floxed) mir-17–92 alleles in mice using CD19-Cre that is inefficient with floxed gene deletion in early B cells but mediates efficient gene deletion in mature B lymphocytes²². This allows us to bypass the pro-B to pre-B-cell block in mir-17–92 germline-deleted mice¹⁸ to investigate its role in the later stages of B-cell maturation and function.

Fluorescence-activated sorting (FACS) analyses revealed that the frequency and numbers of pro-B (B220⁺CD43⁺IgM⁻) and pre-B (B220⁺CD43⁻IgM⁻) cells were comparable in the BM of mir-17–92^+/+Cd19^Cre/+ and mir-17–92^fl/flCd19^Cre/+ (denoted, respectively, as mir-17–92 control and mir-17–92 bKO thereafter; Fig. 1a, Supplementary Fig. 1a). Quantitative reverse trancription–PCR (qRT–PCR) analyses indicated that the expression of individual mir-17–92 member was not significantly reduced in BM B220⁺IgM⁻ pro-B/pre-B cells of mir-17–92 bKO mice (Supplementary Fig. 1b), suggesting that the almost intact expression of mir-17–92 allows for normal progression of early B lymphopoiesis in these mice. However, the expression of individual mir-17–92 member was greatly reduced (>80%) in immature (IgM⁺B220⁺) and recirculating mature (IgM⁺B220^high(hi)) B cells in the BM of mir-17–92 bKO mice (Supplementary Fig. 1c,d) but these two B-cell populations were largely unperturbed (Fig. 1a).

**Figure 1: Analyses of conventional B cells in *mir-17*–92 bKO mice.**

The frequency of B220⁺IgM⁺ conventional B cells in the spleen and lymph nodes were also indistinguishable between mir-17–92 bKO and control mice (Fig. 1b). We further observed comparable percentage of follicular B (B220⁺CD93⁻CD23⁺CD21⁻), marginal zone B (B220⁺CD93⁻CD23⁻CD21⁺) and fraction I, II and III transitional B cells in the spleens of mir-17–92 bKO and control mice (Fig. 1c). B-cell follicles were maintained normally in mir-17–92 bKO mice (Fig. 1d). The mir-17–92^fl/fl alleles were indeed almost completely deleted (Supplementary Fig. 1e) and the expression of individual miRNAs significantly reduced (>80%) in various mutant peripheral B-cell populations (Supplementary Fig. 1f–h). However, mir-17–92 bKO mice had slightly reduced (∼20%) total B-cell numbers in their spleens and lymph nodes compared with control mice (Fig. 1e). As the enhanced expression of pro-apoptotic protein BIM, a known target of mir-17–92, was thought to account for the drastic reduction of mir-17–92-deficient pre-B cells¹⁸, we examined if the mild reduction of total peripheral B cells in mir-17–92 bKO mice could be ascribed to dysregulated BIM expression. Interestingly, Bim mRNA level was comparable between mir-17–92 bKO and control B cells (Fig. 1f), but BIM protein was enhanced in mir-17–92 bKO B cells (Fig. 1g,h), suggesting that mir-17–92 could modulate BIM expression at the protein level and the subtle decrease in total numbers of mutant peripheral B cells could be due to mildly increased BIM expression. Together, these results suggest that mir-17–92 cluster is not essential for conventional B-cell development in the periphery as it is for early B lymphopoiesis.

By contrast, both the frequency and total B-cell numbers were found to be reduced in the peritoneal cavities of mir-17–92 bKO mice compared with control mice (Fig. 2a,b). The reduction was ascribed to decreased B1-a (CD19⁺CD43⁺CD5⁺) and B1-b (CD19⁺CD43⁺CD5⁻) cells as their numbers were reduced by approximately seven- and twofold, respectively, whereas conventional B (B2) (CD19⁺CD43⁻CD5⁻) cells were not significantly affected. These data indicate that mir-17–92 is required for B1 cell development or maintenance.

**Figure 2: Reduced B1 cells and defective TI antibody response in *mir-17*–92 bKO mice.**

Antibody responses in mir-17–92 bKO mice

We determined if mir-17–92 is required for B-cell function by examining antibody responses in mutant mice. When immunized with T-cell-independent (TI) antigen 4-hydroxy-3-nitrophenylacetyl (NP) coupled to Ficoll, which elicits IgM and IgG3 antibody production, mir-17–92 bKO mice exhibited significantly decreased NP-specific IgM and IgG3 antibody production compared with control mice at day 7 and 14 post immunization (Fig. 2c). This defect is consistent with the drastic reduction of B1 cells in these mice, which are the major responders to TI antigens²³. Thus, mir-17–92 is required for TI antibody response.

We next examined TD antibody response in mir-17–92 bKO mice by challenging them with NP coupled to chicken globulin (NP-CGG). We observed that mir-17–92 bKO and control mice produced similar amounts of NP-specific IgM and IgG1 antibodies, the two major antibody isotypes elicited by NP-CGG immunization, at various time points post immunization (Fig. 3a). The CD19⁺Fas⁺CD38⁻ germinal centre (GC) B cells were comparably present in mir-17–92 bKO and control mice at day 10 post immunization, and class-switched antigen-specific NIP⁺IgG1⁺ (GC) B cells also appeared to be unperturbed in the mutant mice (Fig. 3b,c). We further stimulated B cells in vitro with anti-CD40 antibody and interleukin (IL)-4 and found IgG1 class switching to be indistinguishable between mutant and control B cells (Supplementary Fig. 2a). These data indicate that mir-17–92 is largely not required for GC response and the production of TD class-switched antigen-specific IgG1 antibodies.

**Figure 3: TD antibody response in *mir-17*–92 bKO mice.**

Increased homing of PCs to BM in mir-17–92 bKO mice

We also examined in mutant mice the generation of memory B cells and long-lived PCs, which are differentiated through the GC response^24,25. The frequency of NP-specific CD38⁺IgG1^high memory B cells was found to be similar in the spleen of mir-17–92 bKO and control mice at day 56 post immunization, suggesting that mir-17–92 is dispensable for memory B-cell generation and maintenance (Fig. 3d,e). However, when long-lived PCs in the BM were examined by enzyme-linked immunospot (ELISPOT) assays, we consistently observed an increase (20–30%) in the frequency of NP-specific IgG1 PCs in mir-17–92 bKO compared with control mice (Fig. 3f).

The increased long-lived PCs in the BM of mir-17–92 bKO mice prompted us to examine PCs in detail at early time points after immunization. Strikingly, at day 10 post immunization, the frequency of PCs was ∼2.5-fold higher in the BM of mutant mice compared with controls (Fig. 4a,b). However, the frequency of splenic PCs from the same mice was not higher but rather reduced (Fig. 4a,b). The increased presence of PCs in the BM and decreased frequency of PCs in the spleen of mir-17–92 bKO mice was evident as early as day 7 post immunization (Fig. 4c). These results suggest that the increased PCs in the BM of immunized mir-17–92 bKO mice is not due to exaggerated generation of PCs in the spleen but could be due to the enhanced homing of splenic PCs to the BM during immune response. We further assessed if PC differentiation was perturbed in the absence of mir-17–92 by culturing B cells with lipopolysaccharide (LPS) in vitro. After 3 days of LPS stimulation, the percentage of B220^lowCD138⁺ plasmablasts from mir-17–92 bKO and control B-cell cultures was comparable (Supplementary Fig. 2b), implying that mir-17–92 deficiency does not affect PC differentiation per se. Taken together, our data suggest that during an acute antibody response, PC differentiation is normal but homing to the BM is enhanced in the absence of mir-17–92.

**Figure 4: Enhanced BM homing of PCs in *mir-17*–92 bKO mice.**

Mir-17–92 directly targets S1pr1 expression in B cells

The homing of PCs from secondary lymphoid organs to BM is mainly mediated by sphingosine 1-phosphate (S1P) and chemokine CXCL12 through their engagements of S1PR1 (ref. 19) and chemokine-receptor CXCR4 (ref. 26), respectively. To understand how mir-17–92 modulates BM homing of PCs, we searched for possible targets of mir-17–92 using two miRNA target prediction software—TargetScan and MicroCosm, focusing on S1P–S1PR1 and CXCL12–CXCR4 signalling axes, and revealed that S1pr1 is a predicted target gene of mir-17–92 with five out of six members of the cluster (namely mir-17, mir-19a, mir-19b, mir-20a and mir-92a) targeting it.

We next determined if S1pr1 was indeed targeted by mir-17–92. The 3′UTR of S1pr1 contains four predicted binding sites with mir-19a and mir-19b targeting two sites and mir-17, mir-20a and mir-92a each targeting one site (Fig. 5a). A luciferase reporter construct with full-length S1pr1 3′UTR DNA fragment was cloned downstream of firefly luciferase coding sequence and transfected into HEK293 cells together with either the whole mir-17–92 cluster or individual relevant miRNAs. We observed significantly reduced luciferase activity with the overexpression of either the whole cluster or individual miRNAs (Fig. 5b). We also generated reporter constructs containing individual wild-type (A to D) or mutant (Am to Dm)-binding sites where the DNA sequences complementary to the miRNA seed regions were mutated (Supplementary Fig. 3). When transfected into HEK293 cells with individual miRNAs, the constructs containing wild-type 3′UTR yielded lower luciferase activities compared with the various mutant counterparts (Fig. 5c). Thus, our results suggest that S1pr1 is a bona fide target of mir-17–92, with five out of six members directly targeting its 3′UTR at four different sites.

**Figure 5: *Mir-17*–92 targets *S1pr1* in PCs.**

Modulation of S1PR1 expression in mir-17–92-deficient B cells

We proceeded to examine the effect of mir-17–92 deficiency on S1PR1 expression in B cells. First, splenic B220⁺IgM⁺ B cells were sorted and S1pr1 mRNA was examined by qRT–PCR. We found increased (∼30%) S1pr1 mRNA level in mir-17–92 bKO B cells compared with controls (Fig. 5d), which was correlated with a slight increase in cell surface S1PR1 expression as determined by FACS analyses (Fig. 5e). We also examined S1pr1 mRNA in sorted CD138⁺B220^lowplasmablasts differentiated in vitro using LPS and B220⁻CD138⁺NIP⁺ plasmablasts from the spleens of immunized mice. We found that mir-17–92 deficiency led to increase in S1pr1 mRNA (Fig. 5f,h), which was corroborated by increased S1PR1 surface levels in the mutant cells (Fig. 5g,i). Hence, our data suggest that mir-17–92 modulates S1PR1 expression in PCs.

Next we investigated if modulating S1PR1 activity or expression in mir-17–92-deficient PCs could influence their homing to BM. It is difficult to test S1P1 function in vitro due to receptor desensitization caused by non-physiological ligand exposure and the inability to model S1P concentration differential that exists across endothelial cells in vivo²⁷. Thus, we examined BM homing of PCs in immunized mir-17–92 bKO mice in response to different dosages of FTY720 (Fig. 6a), a potent S1P1 inhibitor^28,29 that impedes the BM homing of PCs from secondary lymphoid tissues¹⁹. We reasoned that higher S1PR1 expression level in mir-17–92-deficient PCs would render them less sensitive to FTY720 inhibition. Consistent with previous report¹⁹, when mice were given high dose of FTY720 (3.0 mg kg⁻¹), there was hardly any PCs detected in the BM of both mir-17–92 control and bKO mice at day 10 post immunization (Fig. 6b). Interestingly, when FTY720 dosage was reduced to 1.0 mg kg⁻¹, NP-specific PCs were found in the BM of mir-17–92 bKO but not control mice. When the dosage was further lowered to 0.3 mg kg⁻¹, we could detect NP-specific PCs in the BM of both mir-17–92 control and bKO mice but with the frequency of PCs significantly higher (approximately threefold) in the latter (Fig. 6b). These data are consistent with our hypothesis that a higher level of S1PR1 expression in mir-17–92-deficient PCs renders them less sensitive to FTY720 inhibition.

**Figure 6: Effects of FTY720 inhibition and *S1pr1* haploinsufficiency on *mir-17*–92 bKO PCs.**

To further determine if increased S1PR1 expression indeed contributed to enhanced PCs homing to the BM in mir-17–92 bKO mice, we generated mir-17–92^fl/flS1pr1^fl/+Cd19^Cre/+ mice that have one S1pr1 allele specifically deleted in B cells. We reasoned that S1pr1 haploinsufficiency could reduce the elevated expression caused by mir-17–92 deficiency and therefore normalize the PC homing phenotype in these mice. First, we examined S1pr1 expression in mir-17–92^fl/flS1pr1^+/+Cd19^Cre/+ and mir-17–92^fl/flS1pr1^fl/+Cd19^Cre/+ B cells and found that S1pr1 was reduced ∼45% when one S1pr1 allele was ablated (Fig. 6c). We next immunized the mice with NP-CGG and examined NP-specific PCs in the BM at day 10 post immunization by ELISPOT assay. As expected, the number of NP-specific IgG1 PCs in the BM of mir-17–92^fl/flS1pr1^+/+Cd19^Cre/+ mice was increased compared with mir-17–92^+/+S1pr1^+/+Cd19^Cre/+ mice (Fig. 6d,e). Interestingly, when one S1pr1 allele was ablated, the number of NP-specific IgG1 PCs in the BM of mir-17–92^fl/flS1pr1^fl/+Cd19^Cre/+ mice was reduced compared with mir-17–92^fl/flS1pr1^+/+Cd19^Cre/+ mice and became similar to that found in mir-17–92^+/+S1pr1^+/+Cd19^Cre/+ mice. These results indicate that increased S1PR1 expression indeed contributes to the increased BM homing of PCs in mir-17–92 bKO mice. Taken together, our data suggest that mir-17–92 restrains BM homing of PCs by directly suppressing S1PR1 expression.

Impaired IgG2c antibody production in mir-17–92 bKO mice

During our initial characterization of mir-17∼92 bKO mice, we found normal levels of IgM, IgG1, IgG3 and IgG2b, but significantly reduced IgG2c antibodies, in the sera of unchallenged mutant mice compared with controls (Fig. 7a). It is known that inbred mouse strains such as C57BL/6 and SJL with the Igh-1b allele possess only the gene encoding the IgG2c isotype, whereas BALB/c and 129/Sv strains with the Igh-1a allele express only IgG2a (ref. 30). The allele-specific PCR analysis confirmed the presence of IgG2c but not IgG2a gene alleles in mice used in our study (Supplementary Fig. 4), which have been backcrossed to C57BL/6 background for more than 10 generations, and therefore ruled out the possibility that the reduced serum IgG2c in mutant mice was due to different genetic backgrounds.

**Figure 7: *MiR-17*–92 is required for IgG2c production.**

We next examined if the production of antigen-specific IgG2c antibodies would be affected during acute antibody response. Interestingly, mir-17–92 bKO mice had markedly reduced NP-specific IgG2c antibodies at various time points post immunization compared with control mice (Fig. 7b). This is in contrast to the normal production of NP-specific IgG1 antibodies in mir-17–92 bKO mice (Fig. 3a). To exclude the possibility that defective IgG2c production in mir-17–92 bKO mice could be due to the modest reduction of peripheral B-cell numbers in these mice, we generated and analysed mir-17–92^fl/flAicda^Cre/+ mice in which the floxed mir-17–92 allele was deleted only in antigen-activated but not naïve B cells (Supplementary Fig. 5a). Unchallenged mir-17–92^fl/flAicda^Cre/+ mice had normal B-cell populations in their spleens and lymph nodes compared with mir-17–92^+/+Aicda^Cre/+ mice (Supplementary Fig. 5b,c). NP-specific IgG1 antibodies were also not drastically affected in mir-17–92^fl/flAicda^Cre/+ mice, especially at later time points post immunization (Supplementary Fig. 5d). In contrast, the production of NP-specific IgG2c antibodies was significantly reduced in mir-17–92^fl/flAicda^Cre/+ mice (Fig. 7c), suggesting that mir-17–92 is indeed important for IgG2c antibody production.

To further determine if mir-17–92-deficient B cells were intrinsically defective in IgG2c production, we purified B cells from mir-17–92 bKO and control mice and stimulated them in vitro with LPS+IFN-γ, which induces preferential Ig class switching to IgG2c/2a, and assessed the presence of IgG2c⁺ cells by FACS analysis. After 3 days, mir-17–92 bKO B-cell culture generated significantly reduced IgG2c⁺ cells (∼30% of control; Fig. 7d,e). However, the generation of IgG3⁺ and IgG1⁺ B cells after LPS and LPS+IL-4 stimulations, respectively, was comparable between mir-17–92 bKO and control cultures. The switching to IgG2b in response to LPS+TGFβ+IL-5 stimulation appeared to be reduced but it was not statistically significant (Fig. 7d,e). These data suggest that mir-17–92 is intrinsically required for IgG2c production in B cells.

Mir-17–92 regulates GLT and histone acetylation at Cγ2c locus

We explored how mir-17–92 could regulate IgG2c production. As AID is absolutely required for Ig class switch recombination (CSR), we first assessed if the gene expression of Aicda was affected in mir-17–92-deficent B cells using qRT–PCR. We found comparable Aicda expression between stimulated mir-17–92 bKO and control B cells (Supplementary Fig. 6), thus excluding the possibility that the defective IgG2c production was due to dysregulated AID expression. Next we examined germline transcription (GLT) in IgH switch (S) regions, which is important for AID to access ssDNA targets³¹. When germline transcripts for γ3, γ1, γ2c and γ2b in various B-cell cultures were determined by qRT–PCR, we detected marked reduction in γ2c GLT in mir-17–92 bKO cells stimulated with LPS+IFNγ compared with controls, whereas γ3, γ1 and γ2b GLT levels were largely comparable between mutant and control cells (Fig. 7f).

Chromatin remodelling is required for many DNA-directed processes, such as transcription, recombination and replication. Acetylation (Ac) on amino-terminal tails of histone H3 and H4 at S regions has been shown to be important for Ig GLT and CSR^32,33. We therefore performed chromatin immunoprecipitation (ChIP)-qPCR to assess the acetylation of histone H3 (H3Ac) and H4 (H4Ac) at the S region and Cγ2c I exon of LPS+IFNγ-treated B cells using specific antibodies. When measured by ChIP–qPCR, mutant cells had lower H3Ac levels were lower at the S region and Cγ2c I exon compared with control cells at 48 h after LPS+IFNγ stimulation (Fig. 8a), correlating with decreased IgG2c GLT in mutant B cells (Fig. 7f). Consistent with the normal IgG1 GLT (Fig. 7f), H3Ac levels at Sγ1/Iγ1 loci were comparable between mutant and control B cells stimulated with LPS+IL-4 (Fig. 8b). We also examined H4Ac at Cγ2c S region, which is known to reflect AID accessibility and activity at S regions and important for CSR³². We showed that H4Ac level at Sγ2c was lower in LPS+IFNγ-stimulated mir-17–92 bKO cells compared with similarly treated control cells (Fig. 8c), indicating reduced AID accessibility at Sγ2c region of mutant cells. However, H4Ac level at the Sγ1 region was comparable in LPS+IL-4-treated mir-17–92 bKO and control cells (Fig. 8d). Taken together, these results suggest that mir-17–92 deficiency affects chromatin remodelling at Cγ2c/2a locus and consequently, GLT, AID accessibility and CSR.

**Figure 8: Reduced histone acetylation at Cγ2c locus and increased IKAROS expression in B cells lacking *miR-17*–92.**

Mir-92a directly targets Ikzf1 and regulates IgG2c production

As miRNAs negatively regulate gene expression, we hypothesized that mir-17–92 could target negative regulator(s) of IgG2c CSR that likely also regulates chromatin remodelling. Hence, the absence of mir-17–92 would lead to the derepression of negative regulator(s) and result in reduced IgG2c production. It was previously shown that IKAROS deficiency led to increased and ectopic switching to IgG2c/2a (ref. 20). IKAROS was also showed to maintain repressive chromatin at Cγ2c/2a and suppress GLT, AID accessibility and CSR of IgG2a/2c (ref. 20). More recently, Ikzf1, the gene encoding IKAROS, was demonstrated to be directly targeted by mir-92a in T cells²¹. Given that mir-17–92-deficient B cells exhibited reduced GLT and hypoacetylation of histone at Sγ2c region, we postulated that mir-17–92 likely regulated IgG2a/2c switching by modulating IKAROS expression in activated B cells. We found that Ikzf1 was indeed targeted by mir-17–92 or mir-92a but not by other members of the cluster using luciferase reporter assays (Supplementary Fig. 7). We demonstrated that mir-17–92-deficient B cells had higher (∼60%) Ikzf1 mRNA levels than control cells when treated with LPS+IFNγ (Fig. 8d). Consistent with increased Ikzf1 mRNA, the protein level of IKAROS was also enhanced in stimulated mutant cells as determined by western blotting (Fig. 8e,f). These results together indicate that mir-17–92 directly targets Ikzf1 and modulates IKAROS expression in B cells.

We next asked if elevated IKAROS expression could account for the defective IgG2c production in mir-17–92-deficient B cells. We used short interfering RNA (siRNA) to knock-down IKAROS in the mir-17–92-deficient cells with nucleofection, which can efficiently transfect activated B cells in vitro (Supplementary Fig. 8). Western blotting analyses showed that IKAROS protein level was noticeably reduced in mutant B cells transfected with Ikzf1-targeting siRNA compared with control siRNA (Fig. 9a). More importantly, Ikzf1-silenced mutant B cells had increased IgG2c class switching compared with non-silenced counterparts (Fig. 9b,c). We further demonstrated that introduction of mir-92a mimics but not others could enhance IgG2c production in mir-17–92-deficient and control B cells (Fig. 9d). Taken together, our data suggest that mir-92a from mir-17–92 cluster directly represses IKAROS expression and regulates IgG2c production.

**Figure 9: *MiR-92* modulates IgG2c antibody production via targeting *Ikzf1*.**

Mir-17–92 deficiency attenuates murine autoimmunity

IgG2c/2a is the most prevalent autoantibody isotype found in murine lupus^34,35. These autoantibodies bind self DNA and ribonucleoproteins to form immune complexes that cause tissue damage. The systemic autoimmunity in mir-17–92-transgenic mice^36,37 and selective IgG2c defect in mir-17–9^fl/flCd19^Cre/+ mice led us to explore if mir-17–92 ablation could attenuate B-cell-mediated autoimmunity. Hence, we introduced mir-17–92 deficiency into Ptpn6^fl/flCd19^Cre/+ mice that had Shp1 tyrosine phosphatase deleted in B cells and developed autoimmunity resembling that of human lupus³⁸.

Compared with age-matched Ptpn6^+/+mir-17–92^+/+Cd19^Cre/+ control mice, 10-week-old Ptpn6^fl/flmir-17–92^+/+Cd19^Cre/+ mice manifested increased sera titres of IgM and IgG2c antibodies reactive to single-stranded (ss) and double-stranded (ds) DNA. Interestingly, sera titres of anti-ssDNA and anti-dsDNA IgM antibody were diminished by 50–60% in Ptpn6^fl/flmir-17–92^fl/flCd19^Cre/+ mice (Fig. 10a,b), which could be due to the reduction of IgM antibody-producing B1 cells resulting from mir-17–92-deficiency (Fig. 2). Strikingly, anti-DNA antibodies of the IgG2c isotype, which were abundantly present in Ptpn6^fl/flmir-17–92^+/+Cd19^Cre/+ mice, were significantly reduced in Ptpn6^fl/flmir-17–92^fl/flCd19^Cre/+ mice. Immunofluorescence staining of renal sections (Fig. 10c) revealed the presence of prominently coarse granular and segmental IgG depositions in the mesangium of the glomeruli of Ptpn6^fl/fl mir-17–92^+/+Cd19^Cre/+ mice, which was indicative of immune complex glomerulonephritis. Interestingly, the amount of IgG immune complex deposition was significantly reduced in the kidneys of Ptpn6^fl/flmir-17–92^fl/flCd19^Cre/+ mice (Fig. 10c,d). These results suggest that the ablation of mir-17–92 could potentially suppress IgG2c autoantibody production and therefore alleviate symptoms of systemic autoimmunity in mice.

**Figure 10: Ablation of *mir-17*–92 in B cells attenuates autoimmunity in Shp1-deficient mice.**

Discussion

We examined the role of mir-17–92 in late-stage B-cell differentiation and function by ablating it using CD19-cre. We show that mir-17–92 is not absolutely required for conventional B-cell maturation in the periphery in contrast to its essential role in early B lymphopoiesis. However, we unveil two novel roles for mir-17–92 in peripheral B cells, namely the modulation of PC homing to the BM and selective regulation of IgG2c production. We further identify S1PR1 and IKAROS as direct physiological targets of mir-17–92 responsible for these two outcomes.

Previous studies have postulated that mir-17–92 could largely account for Dicer’s function in early B-lymphoiesis by targeting BIM^11,18. Here we demonstrate that mir-17–92 is largely dispensable for the maturation of conventional B cells in the periphery, suggesting that it plays a more important role in early B-lymphoiesis than in peripheral B-cell maturation. However, we demonstrate that mir-17–92 is required for B1 cell development and TI immune response. We also show that BIM protein but not mRNA level is increased in mir-17–92-deficient B cells and could account for the modestly decreased total peripheral B cells in mir-17–92 bKO mice. Previous studies of mir-17–92-deficient or -overexpressing B cells^11,39 have demonstrated either enhanced or decreased BIM protein expression in mutant B cells, but Bim mRNA level was not examined in those studies. Our results suggest that mir-17–92 regulation of BIM mainly occurs at the protein level.

In contrast to the absolute requirement for Dicer in GC response¹³, mir-17–92 bKO mice have normal NP-specific IgM and IgG1 antibody production and unperturbed GC during TD antibody response, suggesting that miRNAs other than mir-17–92 are important for these processes. In support of this view, a previous miRNA deep-sequencing study has shown that mir-17–92 family members are not amongst the most highly expressed miRNAs in GC B cells⁴⁰.

Interestingly, we show that mir-17–92 is important for modulating PC homing to BM where survival niches are provided for their long-term persistence^41,42,43. We show that five out of six members of mir-17–92 cluster target the 3′UTR of S1pr1 at four different sites and the absence of mir-17–92 leads to higher S1pr1 expression in PCs. It is possible that S1pr1 could be synergistically and simultaneously targeted by these five miRNAs in PCs. The enhanced BM homing of PCs in mir-17–92 bKO mice can be normalized by removing one copy of S1pr1 gene. Thus, our data suggest that mir-17–92-mediated fine-tuning of S1PR1 expression is another means to control BM homing of PCs, and this insight could be useful for the development of miRNA-based drugs to interfere with PC homing to BM survival niches in PC-related diseases such as multiple myeloma and autoimmunity.

The mir-17–92-mediated modulation of S1PR1 expression and enhanced homing of PCs in mutant mice raise the possibility that the slightly decreased peripheral B cells in mutant mice might be due to increased egress of B cells from secondary lymphoid organs into circulation. However, our analyses showed that the distribution of naïve B cells in blood and secondary lymphoid organs was comparable between mir-17–92 bKO and control mice (Supplementary Fig. 9), suggesting that the mildly increased S1PR1 level in mutant naïve B cells does not significantly shift the equilibrium distribution of B cells in favour of blood. The noticeable homing phenotype of mutant PCs but not naïve B cells could be due to the different expression levels of mir-17–92, which is upregulated in PCs (Supplementary Fig. 10), or the differential expression of other S1PR1-targeting miRNAs in these cells.

We also show that mir-17–92 deficiency in B cells impairs IgG2c antibody production, which is accompanied by abnormal GLT and chromatin remodelling at Cγ2a/2c locus. We further demonstrate that mir-17–92 can inhibit the expression of IKAROS, the transcription factor known to maintain repressive chromatin at Cγ2C/2a, and therefore suppress transcription, AID accessibility and CSR of IgG2a/2c²⁰. It was recently demonstrated that IKAROS expression in B cells could be modulated by the transcription factor IRF5 (ref. 44). Thus, our current study uncovers a post-transcriptional layer of IKAROS regulation by miRNAs. Interestingly, in IKAROS hypomorphic mice that have 10-fold less IKAROS protein than wild-type mice²⁰, the production of IgG2b was also enhanced. However, in our study, we did not observe a significant change in IgG2b production in mir-17–92-deficient B cells. This could be due to the mild (approximately twofold) increase in IKAROS expression in these cells. In agreement with this viewpoint, IgG2b was also not affected in IRF5-deficent B cells, which manifested modest changes in IKAROS expression^44,45. It is possible that IgG2c/2a CSR is more sensitive to subtle changes in IKAROS levels than that of other Ig isotypes.

Another interesting finding from our study is that mir-17–92 deletion in B cells can significantly reduce the amount of pathogenic IgG2c anti-DNA antibodies in autoimmune Shp1-deficient mice. In addition, IgG immune complex glomerulonephritis in Shp1-deficient mice could be attenuated by mir-17–92 deficiency. Murine IgG2c/IgG2a and its analogue isotype IgG1 in humans are the most prevalent and potent Ig isotypes found in autoimmune diseases^46,47,48,49. Therefore, our results suggest that mir-17–92 could be a possible target for therapeutic intervention by providing an avenue to control pathogenic IgG2c autoantibody production in lupus.

Methods

Mice

Mir-17–92^fl/fl, S1pr1^fl/fl, Ptpn6^fl/fl and Cd19^Cre/+ mice were obtained from The Jackson Laboratory. Aicda^Cre/+ mouse was generated and described previously¹³ and maintained in C57BL/6 background. Mir-17–92^fl/fl mice were backcrossed >10 generations into C57BL/B background before intercrossing with Cd19^Cre/+ and Aicda^Cre/+ mice to generate mir-17–92^fl/flCd19^Cre/+ and mir-17–92^fl/flAicda^Cre/+ mice. Genotyping of IgG2a and IgG2c alleles was performed by PCR using allele-specific primers as described³⁰ previously. Mice of 10 to 16 weeks old of both genders were used according to the protocols approved by the Institutional Animal Care and Use Committees (IACUCs) based on guidelines from the National Advisory Committee on Laboratory Animal Research (NACLAR) of Singapore.

Immunization and FTY720 administration

Mice were immunized intraperitoneally with 100 μg NP-CGG₃₈ (Biosearch Technologies) precipitated in Imject alum (Pierce, Biotechnology). For the in vivo inhibition of S1PR1, immunized mice were given various doses (3.0, 1.0 and 0.3 mg kg⁻¹) of FTY720 (Cayman Chemical) twice by intraperitoneal injection.

Flow cytometry

Cells were harvested from BM, spleen, inguinal lymph nodes, peritoneal cavities and peripheral blood of mice and treated with Fc-block and stained with biotin- or fluorescent-labelled monoclonal antibodies (mAbs) as described¹³. The following mAbs were from BD Pharmingen or as specified: fluorescein isothiocyanate (FITC) anti-Fas (Cat. No: 554257), FITC anti-CD19 (Cat. No: 553785), FITC anti-IgG1 (Cat. No: 553443), FITC anti-CD21 (Cat. No: 553818), FITC-IgM (Cat. No: 553408); phycoerythrin (PE) anti-Fas (Cat. No: 554258), PE anti-CD138 (Cat. No: 553714), PE anti-B220 (Cat. No: 553090), PE anti-CD5 (Cat. No: 553023), PE anti-CD93 (ebioscience, Cat. No:12–5892); biotinylated (bio-) anti-CD38 (Cat. No: 553762), bio-anti-IgM (Cat. No: 553406), bio-anti-IgD (Cat. No: 553506), bio-anti-CD138 (Cat. No: 553717), bio-anti-CD43 (Cat. No: 553269), anti-CD62L (Cat. No: 553149), bio-anti-Thy1.2 (Cat. No: 553002), bio-anti-CD3 (Cat. No: 663060), bio-anti-Gr-1 (Cat. No: 553125), bio-anti-F4/80 (Biolegend; Cat. No:108404), bio-anti-CD5 (Cat. No:553019), bio-anti-Ter119 (Cat. No: 553612), bio-anti-DX5 (Cat. No: 553856), bio-anti-NK1.1 (Cat. No: 553163), bio-anti-CD93 (Cat. No: 550434), bio-anti-IgG3 (Cat. No: 553401), bio-anti-IgG1 (Cat. No: 553441), bio-anti-IgG2c (Cat. No:553504) and bio-anti-IgG2b (Cat. No: 553393); allophycocyanin (APC) anti-CD19 (Cat. No: 550992), APC anti-CD38 (ebioscience; Cat. No:17–0381), APC-Cy7 anti-B220 (Cat. No: 552094), APC Cy7anti-CD19 (Cat. No: 557655). Biotin conjugates were visualized with Streptavidin-conjugated peridinin chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5; Cat. No:551419), Streptavidin-conjugated APC (Cat. No: 554067) or APC-Cy7 (Cat. No: 554063). Fluorescein-labelled Peanut agglutinin (PNA) and 4′,6-Diamidino-2-phenyindole (DAPI) were purchased from Sigma-Aldrich. PE-labelled NIP-BSA (Biosearch Technologies) were prepared using R-PE labelling kit-SH (Dojindo). All the antibodies were used in 1:100 dilution except specified elsewhere. Dead cells were excluded electronically using DAPI. Exclusion staining (Dump) for antigen-specific GC B cells or PCs included anti-IgM, anti-IgD, anti-CD138, anti-CD3, anti-Th1.2, anti-Gr-1, anti-F4/80, anti-Ter119, anti-Dx5, anti-NK1.1, anti-AA4.1 and anti-CD5 mAbs; or anti-IgD, anti-IgM, anri-CD4, anti-CD8, anti-F4/80 and anti-Gr-1 mAbs. Cell surface staining of S1PR1 was performed using rat anti-S1PR1 antibody (R&D System)⁵⁰. In brief, cells were stained with rat anti-S1PR1 (5 μg/ml) followed by donkey anti-rat IgG biotin and streptavidin-APC-Cy7. Data were collected on LSR II flow cytometer (BD Pharmingen) and analysed with Flowjo (Treestar).

Cell sorting and qRT–PCR

For cell isolation, pro-B/pre-B (IgM⁻B220⁺), immature B (IgM⁺B220⁺) and mature B (IgM⁺B220^hi) cells from BM and transitional B (B220⁺AA4.1⁺), follicular B (B220⁺AA4.1⁻CD23⁺CD21⁻), marginal zone B (B220⁺AA4.1⁻CD23⁻CD21⁺) and total B (IgM⁺B220⁺) cells from spleens of unchallenged mice and GC B (CD19⁺CD38⁻Fas⁺), non-GC B (CD19⁺CD38⁺Fas⁻) cells and PCs (Dump⁻B220^lo/−CD138⁺NIP⁺) from spleens of immunized mice were sorted directly into Trizol LS with FACSAria (BD Pharmingen). Total RNA was prepared from sorted cells and subjected to cDNA synthesis with reverse transcriptase using random hexamers as primers (Research Instruments). Platinum SYBR Green Supermix (Life Technologies) was used in quantitative real-time PCR (qRT–PCR) for Bim, S1pr1 and Ikzf1 using primers as follow: Bim (forward: 5′-CGACAGTCTCAGGAGGAACC-3′, reverse: 5′-CAATGCCTTCTCCATACCAGA-3′), S1pr1 (forward: 5′-GAGTCCTGCGGACGCAGCTT-3′, reverse: 5′-AGCCAGGTCAGCGAGCAATCC-3′) and Ikzf1 (forward: 5′-GGATCATTCTTGGCCCCCAAA-3′, reverse: 5′-ACCCTCATCGACATCCATTGT-3′). TaqMan MicroRNA system (Life Technologies) was used for miRNA quantification. GLT levels for γ3, γ1, γ2c and γ2b were determined as described²⁰ previously.

Immunofluorescence histology

Splenic and renal sections of 8 μm thickness were prepared from tissues frozen in Tissue-Tek (Sakura) as described previously¹³. After blocking, sections were stained at room temperature for 30 min with a cocktail of antibodies and mounted with CYTOSEAL 60 (Electron Microscopy Sciences) and analysed on a FV1000 confocal microscope (Olympus).

ELISA and ELISPOT assay

Sera were collected from unchallenged or immunized mice on various days. Total or NP-specific antibodies of various isotypes in sera were measured via enzyme-linked immunosorbent assay (ELISA). The relative titres of NP-specific serum antibodies are shown as the relative of dilutions that give absorbance reading within the linear range of the assay⁵¹. The frequency of NP-specific IgG1 antibody-secreting cells was determined by ELISPOT assay¹³.

Plasmid construction and luciferase reporter assay

Mir-17, mir-18a, mir-19a, mir-19b, mir-20a, mir-92a and mir-17–92 cluster were cloned into MSCV retroviral vector. The 3′UTR fragments of mouse S1pr1 and Ikzf1 with predicted binding sites for individual miRNAs and their mutants were amplified from genomic DNA and inserted into pmirGLO plasmid (Promega) and confirmed by sequencing. For luciferase reporter assay, HEK 293 cells were plated into 48-well plates at 1 × 10⁵ cells per well 16 h before transfection. pmirGLO (0.2 μg) reporter plasmid containing the S1pr1 and Ikzf1 3′UTR fragments or their mutants and 0.2 μg different miRNA-expressing plasmids were transfected into each well using Lipofectamine 2000 transfection reagent (Invitrogen) in triplicate. Luciferase assays were performed 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega) on a SpectraMax Luminescence Microplate Reader (Molecular Devices). Luciferase activity was expressed as ratio between firefly and renilla luciferase activities.

Cell culture and nucleofection of B cells

Splenic B cells were purified with anti-CD43 mAb-coupled magnetic beads using magnetic-activated cell sorting negative selection protocol (Miltenyi Biotech, Bergisch Gladbach, Germany). Cells were stimulated for 3 days with 10 μg ml⁻¹ LPS (Sigma) in DMEM supplemented with the necessary additives. IL-4 (10 ng ml⁻¹), IL-5 (5 ng ml⁻¹), IFNγ (10 ng ml⁻¹) and TGFβ (2 ng ml⁻¹) from R&D Systems were used to induce preferential Ig class switching to various isotypes. For nucleofection, purified B cells were stimulated with 10 μg ml⁻¹ LPS and 10 ng ml⁻¹ IFNγ overnight and transfected with siRNA or miRNA mimics from Life Technologies using P4 Primary Cell 4D-Nucleofector X Kit on an Amaxa 4D-nucleofector (Lonza). Transfected cells were recultured and analysed 48 h later.

Western blotting

Cells were lysed and protein concentrations were determined as described previously⁵². Equal amount of proteins was loaded and subjected to SDS–polyacrylamide gel electrophoresis, transferred and blotted with specific antibodies. BIM-specific antibody (C34C5) was from Cell Signalling. The IKAROS-specific antibody (M20) was from Santa Cruz. Western blot images shown in Figs 1, 8 and 9 have been cropped for presentation. Full-sized images are presented in Supplementary Fig. 11.

ChIP–qPCR

ChIP was performed using EZ-ChIP kit (Millipore) according to the manufacturer’s protocol. In brief, 1 × 10⁷ stimulated B cells were crosslinked in 1% formaldehyde. Chromatin was sonicated to 500–1,000 bp using Misonix Sonicator 3000 Ultrasonic Cell Disruptor (QSonica). Sonicated chromatin was precleared with Protein G Agarose and subsequently subjected to immunoprecipitation with anti-H3Ac, anti-H4Ac or control antibodies (Millipore). One per cent of the precleared chromatin was saved as input. Protein/DNA complexes were eluted and reverse crosslinked to free DNA, which was further purified using spin columns (Millipore). The input DNA was diluted 10 times. Two μl of DNA samples was analysed by qPCR using Iγ1, Sγ1, Iγ2c, Sγ2c and Gapdh primers^20,53. The acetylation index was calculated described previously²⁰.

Statistics

Unpaired two-tailed Student’s t-test was used for statistical analyses.

Additional information

How to cite this article: Xu, S. et al. Mir-17–92 regulates bone marrow homing of plasma cells and production of immunoglobulin G2c. Nat. Commun. 6:6764 doi: 10.1038/ncomms7764 (2015).

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Acknowledgements

We thank Kenny Boey for the maintenance of mice and Ke Guo for helpful suggestions with histology. This work was funded by the Biomedical Research Council of the Singapore Agency for Science Technology and Research.

Author information

Shengli Xu and Xijun Ou: These authors contributed equally to this work

Authors and Affiliations

Bioprocessing Technology Institute, Agency for Science, Technology and Research (A-STAR), Singapore, 138668
Shengli Xu, Xijun Ou, Jianxin Huo, Kristen Lim, Yuhan Huang, Sheena Chee & Kong-Peng Lam
Department of Physiology, National University of Singapore, Singapore, 117597, Singapore
Shengli Xu & Kong-Peng Lam
Department of Microbiology, National University of Singapore, Singapore, 117545
Kong-Peng Lam

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Shengli Xu
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Xijun Ou
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Jianxin Huo
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Kristen Lim
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Contributions

S.X. and K.-P. L conceptualized the research and wrote the manuscript; S.X., X.O. and K.-P. L. designed the experiments and analysed the data. S.X., X.O., J.H., K.L., Y.H. and S.C. performed the experiments. All authors discussed the results and commented on the manuscript.

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Correspondence to Shengli Xu or Kong-Peng Lam.

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Xu, S., Ou, X., Huo, J. et al. Mir-17–92 regulates bone marrow homing of plasma cells and production of immunoglobulin G2c. Nat Commun 6, 6764 (2015). https://doi.org/10.1038/ncomms7764

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Received: 11 June 2014
Accepted: 25 February 2015
Published: 17 April 2015
DOI: https://doi.org/10.1038/ncomms7764

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