Identification of a Negative Regulatory Role for Spi-C in the Murine B Cell Lineage

Stephen K. H. Li, Lauren A. Solomon, Patricia C. Fulkerson and Rodney P. DeKoter
J Immunol April 15, 2015, 194 (8) 3798-3807; DOI: https://doi.org/10.4049/jimmunol.1402432

Abstract

Spi-C is an E26 transformation-specific family transcription factor that is highly related to PU.1 and Spi-B. Spi-C is expressed in developing B cells, but its function in B cell development and function is not well characterized. To determine whether Spi-C functions as a negative regulator of Spi-B (encoded by Spib), mice were generated that were germline knockout for Spib and heterozygous for Spic (Spib−/−Spic+/−). Interestingly, loss of one Spic allele substantially rescued B cell frequencies and absolute numbers in Spib−/− mouse spleens. Spib−/−Spic+/− B cells had restored proliferation compared with Spib−/− B cells in response to anti-IgM or LPS stimulation. Investigation of a potential mechanism for the Spib−/−Spic+/− phenotype revealed that steady-state levels of Nfkb1, encoding p50, were elevated in Spib−/−Spic+/− B cells compared with Spib−/− B cells. Spi-B was shown to directly activate the Nfkb1 gene, whereas Spi-C was shown to repress this gene. These results indicate a novel role for Spi-C as a negative regulator of B cell development and function.

Introduction

B cell development occurs in the bone marrow of mammals, where committed progenitor B (pro-B) cells are generated from lymphoid progenitor cells. Pro-B cells develop into mature B cells through a series of defined stages marked by rearrangement of Ig H and L chain genes. Immature B cells leaving the bone marrow are termed transitional type 1 (T1) B cells, which migrate to the spleen and differentiate into transitional type 2 (T2) B cells (1). T2 B cells can mature into either follicular (FO) or marginal zone (MZ) B cells that go on to participate in Ab formation and immune regulation (1).

Transcription factors function as either activators or repressors of gene expression to govern B cell development. E26 transformation-specific (ETS) transcription factors PU.1 (encoded by Spi1) and Spi-B (encoded by Spib) function as transcriptional activators during B cell development. PU.1 is required to generate B cell progenitors, because Spi1−/− mice have a profound block in B cell development (2, 3). Spib−/− mice have reduced numbers of B cells that are defective in BCR signaling and are unable to sustain Ab responses to T-dependent Ags (4, 5). PU.1 and Spi-B activate transcription by interacting with the consensus core motif 5′-GGAA-3′ at specific sites within the genome (6). As a result of the similarities in DNA-binding specificities and overlapping expression patterns, many gene targets are functionally redundant between PU.1 and Spi-B, although there are also exclusive roles for each (4, 7, 8).

Transcriptional networks that control B cell differentiation include both transcriptional activators and repressors (9). Interestingly, few published examples of negative regulators of ETS transcription factor function exist. Spi-C is an ETS transcription factor that is a potential negative regulator. Spi-C was originally described as a PU.1-related protein containing an N-terminal acid-activation domain (10–12). Ectopic overexpression of Spi-C in either cultured pro-B cells using a retroviral vector (13) or in mice using a B cell–specific transgene (Eμ–Spi-C) (14) suggested that Spi-C functions as a negative regulator by opposing PU.1 activity. Transgenic Spi-C expression under the control of the B cell–specific IgH intronic enhancer resulted in reduced absolute numbers of T1, T2, MZ, and FO B cells in Eμ–Spi-C mice compared with wild-type (WT) mice (14). Recently a Spic-knockout mouse was generated, allowing for Spi-C loss-of-function analysis (15). Spi-C was revealed to be essential for generating red pulp macrophages in the spleen and is inducible by Heme (16). Spi-C is upregulated in Bach1/Bach2-knockout B cells and results in altered gene expression patterns in B cells (17). Therefore, Spi-C may have a regulatory role in B cells for development and function.

The goal of this study was to determine whether Spi-C plays a negative regulatory role in B cell development and function. A loss-of-function approach was used by crossing a Spic-null allele onto a Spib-knockout background (Spib−/−Spic+/− mice). The effect of Spic heterozygosity in Spib−/− mice might have no effect on the phenotype, further impair B cell development, or rescue the Spib−/− phenotype. It was observed that Spic heterozygosity substantially rescued both the development and function of B cells in Spib−/− mice. Taken together, our results identify a negative regulatory role for Spi-C in B cells.

Materials and Methods

Generation and breeding of mice

Mice were housed at Western University’s Health Sciences animal facility and monitored under an approved animal use subcommittee protocol in accord with Western University Council on Animal Care. C57BL/6 (WT) mice were purchased from Charles River Laboratories (Pointe-Claire, QC, Canada). Spib+/−Spic+/− mice were backcrossed to C57BL/6 mice for five generations prior to the generation of Spib−/−Spic+/− mice. Spib−/−Spic+/− mice were generated by crossing male and female Spib−/−Spic+/− mice. Eμ–Spi-C Spib−/−Spic+/− mice were generated by crossing Eμ–Spi-C+ male mice with Spib−/−Spic+/− females. Genotyping was performed by PCR, as previously described (7, 15). All experiments were performed on mice aged 6–12 wk.

B cell enrichment and proliferation analysis

RBCs were removed from spleen cell suspensions by hypotonic lysis with ammonium chloride solution. B cells were enriched by negative selection using biotin-conjugated anti-CD43 (S7) Ab, streptavidin MicroBeads, LD depletion columns, and a QuadroMACS separation unit (Miltenyi Biotec, Bergisch Gladbach, Germany). B cells (2 × 105/well) were plated in 96-well flat-bottom plates and stimulated with LPS (10 μg/ml; List Biological Laboratories), anti-IgM Ab [50 μg/ml, affinity pure F(ab′)2 fragment], or goat anti-mouse IgM Ab (μ chain specific; Jackson ImmunoResearch Laboratories) in complete IMDM. Proliferation was assessed after 72 h of incubation at 37°C with a TACS MTT Cell Proliferation assay (Trevigen, Gaithersburg, MD), according to the manufacturer’s instructions. For CFSE analysis, 10 × 106 B cells were stained with 5 μM CFSE (BioLegend, San Diego, CA) for 5 min at room temperature prior to plating. Frequency of stained cells was assessed after 72 h of incubation at 37°C by flow cytometry.

Flow cytometry

Abs and reagents purchased from eBioscience (San Diego, CA), BD Bioscience (Franklin Lakes, NJ), or BioLegend included 7-aminoactinomycin D, CFSE, Brilliant Violet 421–conjugated anti-CD45R/B220 (Ra3-6B2), allophycocyanin-conjugated IgM (II/41), allophycocyanin-conjugated annexin V, biotin-conjugated anti-CD43 (S7), PE-conjugated CD93 (AA4.1), PE-conjugated anti–BP-1 (BP-1), PE-conjugated anti-CD19 (1D3), PE-conjugated phospho-Syk (moch1ct), FITC-conjugated CD24 (30-F1), FITC-conjugated anti-CD21/CD35 (eBio8D9), FITC-conjugated anti-CD23 (B3B4), and PE-Cy5–conjugated streptavidin. Cells were blocked with purified anti-CD16/CD32 (Mouse BD Fc Block). Intracellular staining was performed using a two-step protocol and an intracellular fixation and permeabilization buffer set obtained from eBioscience. Ab-stained cell analysis was performed using FACSCalibur and LSR II systems, and sorting was performed using a FACSAria III system (BD Biosciences). Sorted cells were determined to be >98% pure. Data analysis was performed using FlowJo software (TreeStar, Ashland, OR).

Reverse transcription–quantitative PCR

RNA was isolated with TRIzol reagent (Invitrogen, Burlington, ON, Canada). cDNA was synthesized using an iScript cDNA Synthesis Kit (Bio-Rad, Mississauga, ON, Canada), and quantitative PCR (qPCR) was performed with a Rotor-Gene 6000 instrument (Corbett Life Sciences, Valencia, CA). Relative mRNA transcript levels were normalized to β2-microglobulin and compared between samples using the comparative threshold cycle method. Calculations were performed using REST 2009 software (18). Primer sequences are listed in Supplemental Table I.

Immunoblot analysis

Lysates were prepared using Laemmli buffer and applied to 8–10% SDS polyacrylamide gels for electrophoresis. Proteins were transferred to nitrocellulose membranes using a Trans-Blot Semi-Dry system (Bio-Rad), and membranes were blocked for 1 h in 5% milk in TBST. Membranes were probed with anti–Spi-C (Aviva Systems Biology, San Diego, CA), anti-Flag (M2; Sigma-Aldrich, St. Louis, MO), anti-Syk (C-20; Santa Cruz Biotechnology, Dallas, TX), or anti–β-actin (I-19; Santa Cruz Biotechnology) Ab and diluted to the manufacturers’ recommended concentrations in 1% milk/TBST overnight at 4°C. A secondary HRP-conjugated anti-goat Ab was incubated in 1% milk/TBST. Membranes were washed and visualized with SuperSignal West Pico reagent (Thermo-Fisher Scientific).

ELISAs

Serum was collected and quantified for IgG2b and IgM. ELISA kits were purchased from eBioscience and performed according to the manufacturer’s instructions. ELISA plates were analyzed using an Epoch microplate spectrophotometer with Gen5 software (BioTek, Winooski, VT).

Plasmids and cloning

Spi-C cDNA was cloned into the hemagglutinin tag-containing pcDNA3 vector, as previously described (13). The Nfkb1 promoter was amplified from C57BL/6 genomic DNA by PCR using LA Taq (TaKaRa; Clontech Laboratories, Mountain View, CA), purified, and cloned into the pSC-A vector using a PCR cloning kit (Agilent Technologies, La Jolla, CA). A 468-bp Nfkb1 promoter was PCR amplified using a forward primer containing a HindIII site and subcloned. The promoter was HindIII digested from pSC-A and ligated into the HindIII site of the pGL3-basic (Promega, Madison, WI) luciferase reporter vector. ETS binding sites were mutated using the QuikChange Lightning Site-Directed Mutagenesis Kit, according to the manufacturer’s instructions (Agilent Technologies). Constructs were verified by DNA sequencing. Plasmid DNA was prepared by growth in DH5α bacteria and purified with a Maxi Plasmid Kit (Geneaid, New Taipei City, Taiwan). All restriction enzymes were purchased from New England Biolabs (Ipswich, MA). Primer sequences used for cloning are listed in Supplemental Table I.

Transient transfection

WEHI-279 MIGR1, MIG–3XFLAG–Spi-B, and MIG–3XFLAG–Spi-C cells were generated, as previously described (7, 19). Cells in mid to early log-phase growth were washed three times with serum-free DMEM (4.5 g/l; Wisent, St-Bruno, QC, Canada) and incubated for 10 min at room temperature with 10 μg each luciferase reporter plasmid and 1 μg pRL-TK (Promega). Cell–DNA mixtures were electroporated at 220 V and 950 mF using 4-mm gap cuvettes with a Gene Pulser II with Capacitance Extender (Bio-Rad), incubated at room temperature for 10 min, transferred to six-well culture plates in complete DMEM, and incubated at 37°C for 24 h. A Dual-Luciferase Reporter Assay System (Promega) was performed on cell lysates. Light production was measured using a Lumat LB 9507 tube luminometer (Berthold Technologies, Oak Ridge, TN).

Chromatin immunoprecipitation experiments

Chromatin was prepared from enriched B cells, as previously described (7, 19). Cells were cross-linked and lysed, followed by sonication. Sonicated chromatin was incubated overnight at 4°C with anti–Spi-C (generated by the Fulkerson laboratory) and anti-FLAG (M2; Sigma-Aldrich) conjugated to protein G Dynabeads (Invitrogen). Bead complexes were enriched using a MagneSphere Technology Magnetic Separation Stand (Promega) and washed. Immunocomplexes were eluted, and cross-links were reversed overnight at 65°C. DNA was purified using a QIAquick PCR Purification Kit (QIAGEN, Limburg, The Netherlands). Enrichment was measured using qPCR of immunoprecipitated DNA with the primers indicated in Supplemental Table I.

Statistical analysis

Statistical significance was determined using one-way or two-way ANOVA analysis, with a Bonferroni posttest, unless otherwise indicated. The p values ≤ 0.05 were considered significant. Statistical analyses were performed using Prism 5.0 (GraphPad).

Results

Restored splenic B cell numbers in Spib−/−Spic+/− mice compared with Spib−/−Spic+/+ mice

To determine the roles of Spi-C in B cell development, mice homozygous for germline-null alleles of Spib (4) and heterozygous Spic (15) were mated. Spib−/−Spic+/− mice were generated in Mendelian ratios (Table I) and were healthy and fertile. However, few Spib−/−Spic−/− mice were generated (Table I). Therefore, these studies focused on analysis of Spib−/−Spic+/+ and Spib−/−Spic+/− mice.

View this table:
Table I. Expected and actual frequencies of mouse genotypes from offspring generated by mating male and female Spib−/−Spic+/− mice

To determine that the haploid state of Spi-C is insufficient in Spib−/−Spic+/− mice, immunoblot analysis was performed on WT, Spib−/−, and Spib−/−Spic+/− spleen lysates to detect protein levels of Spi-C. A reduction in Spi-C was measured in Spib−/−Spic+/− spleen lysates compared with those from WT or Spib−/− mice (Fig. 1A). As previously noted, Spib−/− mice had reduced total numbers of splenocytes compared with WT mice (Fig. 1B). However, total numbers of splenocytes were substantially rescued in Spib−/−Spic+/− mice compared with Spib−/− mice (Fig. 1B). To assess the population of mature B cells, frequencies of B220+CD93− cells were determined using flow cytometry (Fig. 1C). Spib−/− spleens contained decreased frequencies of mature B cells and decreased absolute numbers of mature B cells. In contrast, Spib−/−Spic+/− spleens had substantially rescued frequencies and absolute numbers of mature B cells compared with Spib−/− spleens (Fig. 1D). Overall, these results showed that knocking out one allele of Spic on a Spib−/− background rescued absolute number of mature B cells in Spib−/− spleens. These results suggest that Spi-C levels are involved in regulating B cell differentiation.

FIGURE 1.
FIGURE 1.

Spi-C heterozygosity rescues absolute numbers of FO B cells in the spleens of Spib−/− mice. (A) Spi-C protein expression measured in WT, Spib−/−, and Spib−/−Spic+/− spleen lysates. Immunoblot was performed using anti–Spi-C Ab and anti–β-actin as a loading control. Data shown are a representative of three individual experiments. (B) Cell counts based on total cells isolated from the spleens of WT, Spib−/−, and Spib−/−Spic+/− mice. (C) Mature B cells were quantified by flow cytometry based on B220 and CD93. The dashed box represents the mature B cell population. (D) Quantitation of the frequency and absolute number of B220+CD93− splenocytes identified in WT, Spib−/−, and Spib−/−Spic+/− mice. (E) Mature FO and MZ B cell populations were quantified based on CD21 and IgM surface expression. (F) Quantitation of the frequency and absolute number of MZ (CD21highIgMhigh) and FO (CD21lowIgMint) mature B cells (B220+CD93−) in the spleen. (G) Mature FO and MZ B cell populations were quantified based on CD23 and IgM surface expression. Cells were isolated from WT, Spib−/−, and Spib−/−Spic+/− spleens and analyzed by flow cytometry. (H) Quantitation of the frequency and absolute number of MZ (CD23lowIgMhigh) and FO (CD23highIgMint) mature B cells (B220+CD93−) in the spleen. Data in (B) and (D) are mean and SD of 14 individual mice; data in (F) and (H) are mean and SD of 9 individual mice. *p < 0.05, **p < 0.01, ***p < 0.001.

Rescued absolute number of FO B cells in Spib−/−Spic+/− spleens compared with Spib−/− spleens

Spib−/− mice were reported to have reduced frequencies of FO B cells (7). To determine whether mature B cell populations were restored in Spib−/−Spic+/− spleens, flow cytometry was performed on splenocytes from WT, Spib−/−, and Spib−/−Spic+/− mice. Mature B cells were gated on B220+CD93−, followed by analysis of CD21 and IgM surface expression to identify FO (CD21lowIgMint) and MZ (CD21highIgMhi) B cells (Fig. 1E). Absolute numbers of MZ B cells were unaffected by either reduced Spi-B or Spi-C expression. However, FO B cells were decreased in both frequency and absolute number in Spib−/− spleens compared with WT spleens (Fig. 1F). Spib−/−Spic+/− spleens contained restored absolute numbers of FO B cells compared with Spib−/− spleens (Fig. 1F). Next, MZ and FO B cell populations were quantified by CD23 and IgM surface expression (Fig. 1G). Absolute numbers of MZ B cells (IgMhighCD23−/low) were unchanged among WT, Spib−/−, and Spib−/−Spic+/− spleens. However, frequencies of FO B cells (IgMintCD23+) were reduced in Spib−/− spleens, and this reduction was rescued in Spib−/−Spic+/− spleens (Fig. 1H). Taken together, these results suggest that the rescue of the absolute numbers of mature B cells in Spib−/−Spic+/− spleens was primarily a result of increased FO B cells.

Increased transitional B cells in Spib−/−Spic+/− spleens compared with Spib−/− spleens

Because Spib−/−Spic+/− spleens had increased numbers of FO B cells compared with Spib−/− spleens, it was hypothesized that there were increased immature B cells developing in the bone marrow of these mice. To test this, flow cytometry analysis was performed using the “Hardy” staining scheme for bone marrow cells (20) (Fig. 2A). Although Spib−/− mice had increased fraction D and reduced fraction F populations compared with WT B cells, no significant differences in the frequencies of fractions A through F were observed between Spib−/− and Spib−/−Spic+/− mice (Fig. 2B, 2C). These results suggested that restored FO B cell numbers were not due to an increase in the frequency of B cell progenitors derived from the bone marrow.

FIGURE 2.
FIGURE 2.

No differences in B cell composition between Spib−/− and Spib−/−Spic+/− mice in the bone marrow. (A) Bone marrow analysis was performed on WT, Spib−/−, and Spib−/−Spic+/− mice. Cells were analyzed by flow cytometry and gated using the Hardy scheme for quantifying B cell populations during development. Data shown are a representative of eight individual mice. (B) Quantitation of individual fractions A–F located in the bone marrow calculated as a percentage of total B220+ cells in the bone marrow. (C) Quantitation of individual fractions A–F located in the bone marrow calculated as a percentage of total cells in the bone marrow. Fractions were gated using the Hardy scheme. Data in (B) are mean and SD of eight individual mice. **p < 0.01, ***p < 0.001.

To assess whether rates of apoptosis were altered in Spib−/−Spic+/− B cells, flow cytometry was performed on WT, Spib−/−, and Spib−/−Spic+/− spleens. Cells were gated into T1, T2, MZ, and FO B cell populations based on B220, CD93, IgM, and CD23 staining, and apoptotic cells were gated for Annexin V+7-AAD− (Supplemental Fig. 1A). No differences were detected in apoptotic frequency in total B cells (Supplemental Fig. 1B) or gated T1, T2, MZ, or FO B cells (Supplemental Fig. 1C) among WT, Spib−/−, and Spib−/−Spic+/− mice. These results suggest that reduced Spi-C expression does not significantly alter steady-state levels of apoptosis in Spib−/− mice.

To determine the source of rescued numbers of FO B cells in Spib−/−Spic+/− spleens compared with Spib−/− spleens, immature transitional B cell populations were examined. Immature B cells were gated on expression of both B220 and CD93 (Fig. 3A). There were increased frequencies of B220+CD93+ immature B cells in both Spib−/− and Spib−/−Spic+/− spleens compared with WT spleens, but there was no significant difference in the frequency of these cells between Spib−/− and Spib−/−Spic+/− spleens. In contrast, absolute numbers of immature B cells were nearly doubled in Spib−/−Spic+/− spleens compared with WT or Spib−/− spleens (Fig. 3B). To determine which immature B cell subset was accountable for overall increased numbers, analysis of T1, T2, and transitional type 3 (T3) B cell populations was performed by gating immature B220+CD93+ B cells for differential expression of IgM and CD23 (Fig. 3C). No differences in T3 B cells were observed among WT, Spib−/−, and Spib−/−Spic+/− spleens. In contrast, Spib−/− and Spib−/ Spic+/− spleens contained elevated frequencies and absolute numbers of T1 B cells compared with WT spleens, although there was no quantitative difference in the T1 population between Spib−/− and Spib−/−Spic+/− mice. For T2 populations, Spib−/− spleens contained fewer cells than did WT spleens, and both the frequency and absolute numbers of T2 cells were rescued in Spib−/−Spic+/− spleens (Fig. 3D). To confirm that T2 B cell numbers were rescued in Spib−/−Spic+/− spleens compared with Spib−/− spleens, splenic B cells were examined using an alternative staining scheme based on CD21 and IgM expression (Supplemental Fig. 2A). Absolute numbers of T2 B cells were significantly reduced in Spib−/− spleens compared with WT spleens and were rescued in Spib−/−Spic+/− spleens using the alternative staining scheme (Supplemental Fig. 2B). In summary, these results showed that reduced Spi-C in Spib−/−Spic+/− mice resulted in a substantial rescue of the frequency of T2 cells in the spleen of Spib−/− mice.

FIGURE 3.
FIGURE 3.

Spib−/−Spic+/− mice have elevated transitional B cells and restored T2 B cells compared with Spib−/− mice. (A) Total transitional B cell populations were quantified in WT, Spib−/−, and Spib−/−Spic+/− mice. The dashed box represents total transitional B cells. Splenocytes were stained with CD93 and B220 and analyzed by flow cytometry. (B) Increased frequencies and absolute number of total transitional B cells in spleens of Spib−/− and Spib−/−Spic+/− mice. (C) Transitional B cell subsets T1 (upper left quadrant, IgMhighCD23−), T2 (upper right quadrant, IgMhighCD23+), and T3 (lower right quadrant, IgMlowCD23+) were quantified in WT, Spib−/−, and Spib−/−Spic+/− mice. Cells were gated on B220 and CD93 and analyzed for IgM and CD23 staining by flow cytometry. (D) Restored T2 subset in Spib−/−Spic+/− mice compared with Spib−/− mice. Quantitation of results shown in (C). Data are mean frequency and absolute numbers of gated B220+CD93+ splenocytes. Data in (B) and (D) are mean and SD of nine individual mice. *p < 0.05, **p < 0.01, ***p < 0.001.

LPS- and anti-IgM–mediated proliferation is rescued in Spib−/−Spic+/− B cells compared with Spib−/− B cells

B cells lacking Spi-B were reported to have impaired proliferative responses to either LPS or anti-IgM (4, 5). To determine whether Spic heterozygosity could rescue functional defects in Spib-knockout B cells, proliferation of Spib−/− and Spib−/−Spic+/− splenic B cells in response to LPS or anti-IgM was assessed using an MTT proliferation assay. Compared with WT B cells, Spib−/− B cells proliferated poorly in response to LPS or anti-IgM (Fig. 4A). In contrast, Spib−/−Spic+/− B cells were substantially rescued for LPS- or anti-IgM–mediated proliferation compared with Spib−/− B cells (Fig. 4A, 4B). To confirm the proliferation rescue, LPS-mediated proliferation was measured by flow cytometry following the staining B cells with CFSE (Fig. 4C). Fewer cells underwent cell division in Spib−/− B cells stimulated with LPS compared with WT B cells. However, more proliferating cells were detected in Spib−/−Spic+/− B cells compared with Spib−/− B cells (Fig. 4D). Therefore, these results suggest that B cell proliferation in response to LPS or anti-IgM is positively regulated by Spi-B but is negatively regulated by Spi-C.

FIGURE 4.
FIGURE 4.

B cell proliferation is rescued in Spib−/−Spic+/− mice compared with Spib−/− mice. (A) Spib−/−Spic+/− B cells have a rescue in proliferation compared with Spib−/− B cells. Proliferation was measured using an MTT proliferation assay. Data are mean and SD of triplicate wells and are a representative of five experiments. (B) Quantitation of five experiments from (A) showing proliferation index, which was calculated by normalizing corrected OD570 values to the untreated WT control. Data are mean and SEM. (C) Proliferation assessed by flow cytometry following CFSE staining on WT, Spib−/−, and Spib−/−Spic+/− B cells. Gate shown represents the frequency of proliferating cells. Data shown are a representative of four individual experiments. (D) Quantitation of CFSE experiments, as performed in (C), showing frequency of cells that underwent cell division. (E) Cell counts based on total cells isolated from the spleens of Spib−/−Spic+/− and Eμ–Spi-C Spib−/−Spic+/− mice. (F) Quantitation of the frequency and absolute number of MZ and FO mature B cells (B220+CD93−) in the spleens of Spib−/−Spic+/− and Eμ–Spi-C Spib−/−Spic+/− mice. FO and MZ B cell populations were quantified based on CD23 and IgM surface expression measured by flow cytometry. Data in (E) and (F) are mean and SD of five individual mice. (G) Proliferation assessed by flow cytometry following CFSE staining on Spib−/−Spic+/− and Eμ–Spi-C Spib−/−Spic+/− B cells. Data are mean and SD for four individual experiments. (H) Phospho-Syk levels were assessed in WT, Spib−/−, and Spib−/−Spic+/− B cells using flow cytometry. Splenocytes were stained with B220, stimulated with anti-IgM for 2 min or left untreated, and stained with phospho-Syk prior to analysis. (I) Quantitation of phospho-Syk levels in B220+ splenocytes following anti-IgM stimulation. Phospho-Syk levels are shown as normalized phospho-Syk mean fluorescence intensity (MFI) of anti-IgM to untreated. Data are mean and SD of five individual experiments. (J) Syk protein expression measured in WT, Spib−/−, and Spib−/−Spic+/− B cell lysates. Immunoblot was performed using anti-Syk Ab and anti–β-actin as a loading control. Data are a representative of three individual experiments. For (A)–(D) and (G), total B cells were enriched by magnetic separation and stimulated with LPS or anti-IgM for 72 h. *p < 0.05, **p < 0.01, ***p < 0.001.

To determine whether the Spib−/−Spic+/− B cell phenotype could be reversed by ectopic expression of Spi-C, Spib−/−Spic+/− mice were crossed to Eμ–Spi-C–transgenic mice (14). Spleens from Eμ–Spi-C Spib−/−Spic+/− mice contained fewer cells than did Spib−/−Spic+/− spleens (Fig. 4E). Flow cytometry analysis was performed on Eμ–Spi-C Spib−/−Spic+/− spleens to assess for differences in B cell populations. Frequencies of MZ and FO B cells in Eμ–Spi-C Spib−/−Spic+/− spleens were unchanged compared with Spib−/−Spic+/− spleens; however, there was a significant reduction in the absolute number of MZ and FO B cells in Eμ–Spi-C Spib−/−Spic+/− mice (Fig. 4F). Proliferation of Eμ–Spi-CSpib−/−Spic+/− B cells following LPS stimulation was assessed by CFSE staining. Compared with Spib−/−Spic+/− B cells, frequencies of proliferating Eμ–Spi-CSpib−/−Spic+/− B cells decreased (Fig. 4G). These data suggest that reintroducing Spi-C using a lymphocyte-specific transgene reverses the Spib−/−Spic+/− B cell–proliferation phenotype.

Because anti-IgM–mediated proliferation was restored in Spib−/−Spic+/− B cells compared with Spib−/− B cells, it was possible that Spib−/−Spic+/− B cells could have restored BCR signaling. To test for this, phosphorylated levels of Syk were measured in B cells using flow cytometry (Fig. 4H). There was a detectable increase in phospho-Syk levels following anti-IgM stimulation; however, there was no difference in the increase among WT, Spib−/−, and Spib−/−Spic+/− B cells (Fig. 4I). Next, total Syk was measured in B cells using immunoblot analysis. There was a noticeable increase in total Syk protein in Spib−/− and Spib−/−Spic+/− B cells compared with WT B cells (Fig. 4J). Therefore, the restoration of BCR signaling in Spib−/−Spic+/− B cells is likely downstream of Syk.

It was reported previously that Spib−/− mice had normal basal levels of all Ig isotypes (4). To determine whether reduced Spi-C had an effect on isotype switching in Spib−/−Spic+/− mice, basal serum levels of different Igs were measured by ELISA. No significant differences in basal levels of IgM (Supplemental Fig. 3A) or IgG2b (Supplemental Fig. 3B) were measured among WT, Spib−/−, and Spib−/−Spic+/− mice. Therefore, the data suggest that Spi-C does not alter basal levels of Ig isotypes in Spib−/−Spic+/- mice.

Spi-C represses Nfkb1 promoter activation by Spi-B

Finally, a potential mechanism for Spi-C’s regulation of B cell proliferation was investigated. Stimulation of B cells with LPS or anti-IgM results in NF-κB transcription factor activation (21). P50 (encoded by Nfkb1) is required for murine B cell proliferation in response to LPS or anti-IgM (22). We recently showed that PU.1 and Spi-B are required for Nfkb1 transcription in B cells, and retroviral transduction with p50 substantially rescues LPS-induced proliferation of Spi1+/−Spib−/− B cells (23). Therefore, it was determined whether restored proliferation in Spib−/−Spic+/− B cells was associated with increased mRNA transcript levels of Nfkb1 compared with Spib−/− B cells. Reverse transcription–qPCR was performed on cDNA synthesized from MZ and FO B cells sorted from Spib−/− and Spib−/−Spic+/− spleens. Steady-state Nfkb1 mRNA transcript levels were increased in MZ and FO B cells from Spib−/−Spic+/− mice compared with Spib−/− mice (Fig. 5A), suggesting that Spi-C may negatively regulate Nfkb1 in B cells.

FIGURE 5.
FIGURE 5.

Spi-C inhibits activation of the Nfkb1 promoter by Spi-B. (A) Measurement of transcript levels of Nfkb1 in Spib−/−Spic+/− B cells compared with Spib−/− B cells. Reverse transcription–qPCR analysis was performed on RNA prepared from unstimulated sorted FO and MZ B cells. mRNA transcript levels of genes indicated on the x-axis were quantified after normalizing to β2 microglobulin (B2m). Data are mean and SD and are a representative of three individual mice. (B) Alignment of the of the annotated Nfkb1 promoter among multiple species, showing nt 105–130 of the conserved 213-bp region. Gray text represents conserved nucleotides, and boxes indicate predicted PU.1/Spi-B binding sites with similarity score and strand location (+/−) listed. Circled nucleotide represents known TSS in the human Nfkb1 promoter. Dots represent TSS upstream or downstream of the annotated sequence. (C) Schematic diagram of luciferase reporter vector. The Nfkb1 promoter was cloned by PCR and ligated into pGL3-basic. ETS binding sites (wt) were mutated (mut) using site-directed mutagenesis. (D) WEHI-279 B cell lymphoma cell lines were generated that overexpress 3XFLAG–Spi-B or Spi-C. Immunoblot analysis demonstrates the overexpression levels of Spi-B or Spi-C in these cell lines. (E) Overexpression of Spi-C inhibits activation of the Nfkb1 promoter. Mutation of the predicted ETS binding sites reduces promoter activity in WEHI-279 MIGR1 and WEHI-279 Spi-B cells. (F) Spi-C expression in WEHI-279 Spi-B cells decreases Nfkb1 activation. WEHI-279 Spi-B cells were cotransfected with a Spi-C expression vector (pcDNA3 Spi-C) along with the luciferase vectors. In (E) and (F), cells were transfected with the plasmids indicated on the x-axis. The y-axis indicates fold-induction of luciferase activity relative to pGL3-basic. Luciferase activity was normalized by transfection with Renilla luciferase expression vector. Data are mean and SEM of three independent experiments. (G) Spi-C directly binds to the Nfkb1 promoter. ChIP analysis was performed on WEHI-279 MIGR1 and WEHI-279 Spi-C cells immunoprecipitated with anti-FLAG or anti–Spi-C Ab. Data show percentage enrichment relative to input for Spi-C interaction with Nfkb1 and Gapdh promoters. Error bars represent SEM from six independent experiments. *p < 0.05, **p < 0.01, paired Student t test.

To assess whether Spi-B and Spi-C regulate Nfkb1 directly, the murine Nfkb1 promoter sequence was aligned with multiple species using a published transcription start site (TSS) for reference (24). The Nfkb1 5′ untranslated region contained two highly conserved ETS binding sites predicted among all species near the TSS using MatInspector software (Fig. 5B). Next, the mouse Nfkb1 proximal promoter was cloned, as described in Materials and Methods, and ligated into the pGL3-basic luciferase reporter vector. Site-directed mutagenesis was performed to mutate two predicted ETS binding sites closest to the TSS by substituting GGAA (TTCC on complement strand) with GGAC (GTCC), which was reported previously to abolish Spi-B binding (7, 19) (Fig. 5C). To determine whether Spi-B and Spi-C regulate Nfkb1 transcription in opposing fashion, luciferase assays were performed using transient transfection of WEHI-279 B lymphoma cells that ectopically express 3XFLAG-tagged Spi-B or Spi-C (7, 19). WEHI-279 cells infected with an empty MIGR1 virus were used as a control (WEHI-279 MIGR1). Expression of 3XFLAG Spi-B and Spi-C protein was confirmed by anti-FLAG immunoblot analysis in infected cell lines (Fig. 5D). The Nfkb1 promoter was active in WEHI-279 MIGR1 cells, and mutation of the ETS sites impaired its activation (Fig. 5E). WEHI-279 Spi-B cells displayed a higher level of activation compared with WEHI-279 MIGR1 cells, whereas mutation of the ETS sites reduced Nfkb1 activation. Interestingly, the activity of the Nfkb1 promoter in WEHI-279 Spi-C cells was lower than in both WEHI-279 MIGR1 and WEHI-279 Spi-B cells, and mutation of the ETS site resulted in no change of activity (Fig. 5E). To confirm that Spi-C expression decreased Nfkb1 transcriptional activation, luciferase activity was measured following cotransfection of a Spi-C expression vector (pcDNA3 Spi-C) and the pGL3-Nfkb1 vector into WEHI-279 Spi-B cells. Cotransfection with pcDNA3 Spi-C resulted in decreased Nfkb1 activation compared with cotransfection with the vector control (pcDNA3) (Fig. 5F). These results suggest that Spi-B transcriptionally activates Nfkb1, and Spi-C inhibits Nfkb1 transcription.

We recently showed that PU.1 and Spi-B directly interact with the Nfkb1 promoter to activate its transcription (23). To determine whether Spi-C directly interacts with the Nfkb1 promoter, chromatin immunoprecipitation (ChIP) analysis was performed using anti–Spi-C and anti-FLAG Ab on chromatin isolated from WEHI-279 Spi-C and WEHI-279 MIGR1 cells. Relative amounts of immunoprecipitated DNA were determined by qPCR from the Nfkb1 promoter region, as well as the Gapdh promoter as a negative control. ChIP analysis confirmed that Spi-C was significantly enriched at the Nfkb1 promoter compared with the Gapdh promoter in WEHI-279 Spi-C cells but not in WEHI-279 MIGR1 cells (Fig. 5G). Together, these data indicate that Spi-C directly interacts with the Nfkb1 promoter, suggesting that it opposes Spi-B–mediated activation of Nfkb1 transcription in B cells.

Discussion

The purpose of these experiments was to understand how Spi-C contributes to B cell development and function. Spi-C heterozygosity restored many aspects of the Spib−/− phenotype. Spib−/−Spic+/− mice had restored numbers of FO and T2 B cells in their spleens relative to Spib−/− mice. Furthermore, proliferation in response to LPS- and anti-IgM–mediated stimulation was restored in Spib−/−Spic+/− B cells compared with Spib−/− B cells. Investigation of a potential mechanism for the Spib−/−Spic+/− phenotypic rescue revealed that steady-state levels of Nfkb1 were elevated in sorted FO and MZ B cells from Spib−/−Spic+/− spleens compared with Spib−/− spleens. Ectopic expression of Spi-B in B cells resulted in increased Nfkb1 promoter activity, which was dependent on the ETS binding site. Conversely, overexpression of Spi-C inhibited Nfkb1 activation by Spi-B. ChIP and ChIP-sequencing analysis demonstrated that both Spi-B and Spi-C are capable of directly binding to the Nfkb1 promoter. In summary, these results suggest that Spi-C opposes Spi-B in B cell transcriptional regulation.

Intercrosses of Spib−/−Spic+/− mice generated Spib−/−Spic−/− mice at a frequency < 1% of live births. Similar findings were reported where intercrosses of Spic+/− mice generated only 9% Spic−/− mice (15). Previous studies demonstrated that injecting embryos with Spic small interfering RNA resulted in a reduced rate of blastocyst development (25). It is possible that Spi-B and Spi-C may have overlapping roles in prenatal development; therefore, embryonic death may be occurring at the blastocyst stage in Spib−/−Spic−/− mice. Therefore, the low birth frequency of Spib−/−Spic−/− mice is likely due to reduced embryonic or fetal viability.

Few target genes for Spi-C have been identified in B cells, and Spi-C was reported to function either as an activator or as a repressor of gene transcription. It is possible that altered expression of other unidentified target genes may contribute to the Spib−/−Spic+/− B cell phenotype. Spi-C ectopically expressed in pro-B cells can directly bind and oppose transcription of the Fcgr2b gene mediated by PU.1 (13). In contrast, transcription of the gene Fcer2a, encoding CD23, was reported to be directly activated by Spi-C in WEHI-279 Spi-C cells (7). Earlier reports showed that Spi-C cooperated with STAT6 to directly induce transcription of IgE under the control of IL-4 (12). In macrophages, Spi-C was reported to transcriptionally activate Vcam1 expression directly (15). Our data demonstrated that Spi-C directly opposes transcriptional activation of Nfkb1 by preventing Spi-B, and possibly PU.1, from binding to the Nfkb1 promoter in B cells. In summary, Spi-C is capable of both activation and repression of its target genes, and it also can transcriptionally regulate target genes by preventing other transcription factors from binding.

Previous studies demonstrated that the transcription factors PU.1 and Spi-B are required to maintain proper levels of Nfkb1 in B cells (23). Spi-B directly binds to the Nfkb1 promoter and activates transcription. In contrast, Spi-C directly binds to the Nfkb1 promoter, but it does not activate transcription. Therefore, decreased Spi-C expression may reduce its occupancy of the Nkfb1 promoter in Spib−/−Spic+/− B cells, permitting increased activation of Nfkb1 by PU.1. Elevated Nfkb1 transcript levels in Spib−/−Spic+/− B cells compared with Spib−/− B cells suggest that Spi-C and Spi-B oppose each other to regulate appropriate Nfkb1 levels. Spib−/− and Nfkb1−/− B cells proliferate similarly to LPS and anti-IgM. LPS-stimulated Nfkb1−/− B cells fail to proliferate, and anti-IgM stimulation results in reduced proliferation (22). Overall, elevated Nfkb1 expression in Spib−/−Spic+/− B cells compared with Spib−/− B cells may sufficiently explain the rescue in proliferation.

Spib−/−Spic+/− mice have restored FO and T2 B cell numbers compared with Spib−/− mice, indicating that Spi-B and Spi-C regulate peripheral B cell differentiation. FO and T2 cell differentiation both require NF-κB signaling and BCR signaling. Transitioning from a T1 to a T2 B cell requires noncanonical NF-κB signals and basal BCR signals, and maturation of a T2 B cell to an FO B cell requires canonical NF-κB signals and strong BCR signaling (1). Nfkb1−/−Nfkb2−/− mice have a developmental block at the T2 stage and fail to generate any mature B cells (26). Therefore, elevated Nfkb1 expression in developing Spib−/−Spic+/− mice could be sufficient to explain the restored FO B cell population. It is also conceivable that Spi-C may be a negative regulator of c-Rel expression, because PU.1 and Spi-B were reported to directly activate Rel transcription (27). Alternatively, it is possible that FO and T2 B cells in Spib−/−Spic+/− mice have a longer lifespan than in Spib−/− B cells, which could contribute to restoration of FO and T2 B cells. Spib−/− B cells have impaired BCR signaling and, thus, proliferate poorly in response to anti-IgM stimulation (4, 5). Transgenic Spi-C expression in B cells results in reduced transcript levels of the BCR signaling genes Btk and Blnk in FO B cells (14). Restored proliferative responses to anti-IgM in Spib−/−Spic+/− B cells compared with Spib−/− B cells suggest that BCR signaling is possibly restored as a result of reduced Spi-C expression. Because WT, Spib−/−, and Spib−/−Spic+/− B cells are capable of phosphorylating Syk at equivalent levels following anti-IgM stimulation, the rescue in BCR signaling is likely downstream of Syk. Therefore, rescued FO and T2 B cells in Spib−/−Spic+/− spleens might be caused by a combination of restored NF-κB and BCR signaling.

It is possible that aspects of the B cell phenotype of Spib−/−Spic+/− mice are not cell intrinsic. However, evidence was provided for a cell-intrinsic effect of Spi-C on B cell proliferation using the Eμ–Spi-C transgenic mouse. In this mouse model, the Spi-C transgene is under the control of a lymphocyte-specific Eμ intronic enhancer. We found that crossing this transgenic mouse to Spib−/−Spic+/− mice (Eμ–Spi-C Spib−/−Spic+/−) resulted in a reduction in proliferation from isolated B cells, suggesting that Spi-C has a cell-intrinsic effect on B cell function. Furthermore, overexpression of Spi-C in Eμ–Spi-C Spib−/−Spic+/− mice reduced the number of FO and MZ B cells, suggesting a cell-intrinsic role for Spi-C in B cell development.

In summary, our results demonstrate a novel mechanism for Spi-C as a negative regulator of B cell development and function. Understanding transcriptional regulation in B cells has human health implications, because dysfunctional B cell development can lead to leukemia of the B cell lineage (8, 19). Furthermore, Ab formation in B cells is transcriptionally regulated, so understanding the potential activators and repressors of this process can improve interventions for vaccine development. Further analysis on mice lacking Spi-B and reduced Spi-C can provide additional insight into other genes involved in B cell development, function, and disease.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Kenneth M. Murphy (Washington University School of Medicine) for the gift of Spic−/− mice. We acknowledge the London Regional Genomics core facility for assisting with DNA sequencing and Kristin Chadwick from the London Regional Flow Cytometry for performing cell sorting.

Footnotes

  • This work was supported by grants from the Canadian Institutes of Health Research (MOP-106581) and the National Sciences and Engineering Research Council (Grant 386046) (to R.P.D.). S.K.H.L. was the recipient of an Ontario Graduate Scholarship.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ChIP
    chromatin immunoprecipitation
    ETS
    E26 transformation specific
    FO
    follicular
    MZ
    marginal zone
    pro-B
    progenitor B
    qPCR
    quantitative PCR
    T1
    transitional type 1
    T2
    transitional type 2
    T3
    transitional type 3
    TSS
    transcription start site
    WT
    wild-type.

  • Received September 23, 2014.
  • Accepted February 9, 2015.

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