Little effect of Zbtb24-deficiency on the development and phenotype of B cells in mice

Given that the germline knockout of Zbtb24 is embryonic lethal in mice [8], we generated mice harboring a conditional Zbtb24loxp/+ allele with two loxp sites flanking the exon 2, which contains the translation starting site (Fig. 1A). The N-terminal 316 amino acids, including the BTB domain, the AT-hook domain and the first C2H2 Zinc finger motif, of ZBTB24 protein would be specifically removed in Cre-expressing cells/tissues, which results in a frame shift in the protein coding sequence and thereby generates a premature stop codon. To address cell-intrinsic roles of Zbtb24 in the development and function of B cells in vivo, we crossed the Zbtb24loxp/+ mice with the Cd19-driven Cre-expressing mouse strain (Cd19Cre/+) to generate B cell specific Zbtb24 deficient (Zbtb24B−CKO, Cd19Cre/+Zbtb24loxp/loxp) and the littermate control (Cd19Cre/+, Cd19Cre/+Zbtb24+/+) mice. Zbtb24B−CKO mice grew normally, and were phenotypically indistinguishable from the control Cd19Cre/+ mice.

In Zbtb24B−CKO mice, Zbtb24 was specifically and efficiently deleted from CD19-expressing pre-B and pro-B cells onward in the BM and periphery, while its expressions in CD19-negative BM pre-pro B cells, splenocytes and thymocytes were intact as analyzed by Q-PCR and/or western blot (Fig. 1B–D). Notably, lack of Zbtb24 affected neither the percentages/absolute numbers of different developing stages of B cells in the BM (Fig. 1E), nor the numbers of splenic FOB/MZB cells as well as the peritoneal B2, B1a/B1b cells in the periphery (Fig. S1A and B). Moreover, the percentages of GCB in the spleens, mesenteric lymph nodes (MLN) and Peyer’s Patches (PP), and serum total IgM, IgG, and IgA levels were all normal in Zbtb24B−CKO mice (Fig. S1C and Fig. 1F). Thus, depletion of B cell Zbtb24 has little impact on the early BM B cell development, the phenotype and maintenance of peripheral B cells, and serum antibody levels in naive mice.

Largely normal humoral responses to TD-Ags in Zbtb24 B−CKO mice

The major immunological features of patients with ICF2 are hypogammaglobulinemia and lack of circulating Bm cells, possibly owing to the disrupted germinal center reactions [3, 19, 20, 44]. We thus first characterized TD-Ag-induced humoral responses in mice with B cell specific Zbtb24 deficiency. Then, 1 week after immunization with NP19-OVA emulsified in IFA (NP-OVA/IFA), serum levels of NP-specific IgM, IgG1, and IgG2c were significantly reduced in Zbtb24B−CKO mice. Surprisingly, on day 14 only NP-specific IgG2c was still slightly decreased, while the other two subtypes were reverted to normal levels in Zbtb24B−CKO mice (Fig. 2A, B). Moreover, the percentages and numbers of CD19+CD95highCD38low GCB as well as CD19lowCD138++ PC in spleens were all comparable between control and Zbtb24B−CKO mice (Fig. 2C, D). Within gated GCB cells, percents of CD86lowCXCR4high dark zone centroblasts and CD86highCXCR4low light zone centrocytes were comparable between the two genotypes of mice as well (Fig. 2C). Consistent with our previous findings in human cells, lack of ZBTB24 did not affect the expressions of BCL6, the key driving TF for GC reactions, in gated either GCB (defined as CD19+CD95highCD38low) compartment or total CD19+ B cells (Fig. S2A, B). In addition, the numbers of splenic GCB and PC cells were comparable in Cd19Cre/+ and Zbtb24B−CKO mice immunized with sheep red blood cells (SRBC) as well (Fig. S2C), further corroborating the dispensability of Zbtb24 on their differentiations in vivo. Notably, ZBTB24 was efficiently depleted in purified GCB cells (Fig. S2D), thus it is unlikely that ZBTB24-expressing cells outcompete ZBTB24-null ones to fill the GCB compartment in TD-Ag-immunized Zbtb24B−CKO mice.

Fig. 2

No significant defects in antibody responses in Zbtb24B−CKO mice post primary/secondary NP-OVA/IFA immunization. Mice (10-week old males in A-D; and 8-week old females in E–G) were intraperitoneally (i.p.) immunized with NP19-OVA emulsified in IFA (1:1, 50 μg/100 μl/mouse) on D0, followed by i.p. rechallenging with NP19-OVA/IFA (3:1, 20 μg/100 μl/mouse) on D53 (only in E–G). Sera were collected on indicated days and NP-specific antibodies were detected by ELISA. Cells in spleens on day (D) 14 (C, D) or D70 (G) were stained with antibodies, in combination with 7-AAD to exclude dead cells, to visualize the percents of CD19+CD38lowCD95high germinal center B (GCB) cells or CD19−/lowCD138high plasma cells (PC) by flow cytometry. A, E, schematic diagrams illustrating the immunization and bleeding schedules of mice. B, F, bar graphs showing the optical density (OD) values of NP-specific antibodies in sera of Cd19Cre/+ and Zbtb24B−CKO mice on indicated days. C representative pseudo-plots showing the percentages of GCB (within gated 7-AAD−CD19+ B cells) and frequencies of dark zone centroblasts (DZ, CXCR4highCD86low) and light zone centrocytes (LZ, CXCR4lowCD86high) within gated GCB cells in control Cd19Cre/+ and Zbtb24B−CKO mice. D, G Representative pseudo-plots showing the percentages of PCs in spleens of mice on D14 (D) or D70 (G). Cumulative data on the percentages or absolute numbers of indicated B cell subsets were shown in the right of C, D or bottom of G. Each symbol represents a single mouse of the indicated genotype, and numbers below horizontal lines indicate P values while those in brackets denote percentages

Given that the generation of specific antibodies appeared to be delayed in Zbtb24B−CKO mice upon immunization, we immunized mice with NP-OVA/IFA on day 0, and boosted the mice with the same antigen intraperitoneally once again on day 53 to further determine the impact of B cell Zbtb24 on humoral responses elicited by TD-Ags in vivo (Fig. 2E). Akin to previous results, control mice produced significantly more NP-specific IgG1 and IgG2c on day 10, and the differences went down greatly on day 20 post the primary immunization (Fig. 2F). Of note, comparable levels of NP-specific antibodies were detected between the two groups thereafter, even on day 7 and 17 post the secondary challenge (Fig. 2F). The numbers of PCs were normal in spleens of Zbtb24B−CKO as well (Fig. 2G).

We also immunized mice with a high dose of NP19-OVA (100 μg/mouse) adsorbed on alum, which predisposes to Th2 immune responses instead of Th1 triggered by IFA in vivo. Again, reduced antigen-specific IgG1 were only observed early (day 7) post the primary immunization (Fig. S3A, B). Moreover, specific deletion of Zbtb24 in B cell compartment did not affect the ratios of high-affinity to low-affinity (against NP2-BSA and NP25-BSA, respectively) IgG1 and IgG2c before (i.e., day 70) and two weeks (i.e., day 84) post the secondary immunization (Figure S3C, D). Given that ZBTB proteins may act redundantly to maintain the integrity and function of immune cells [16], we conducted RNA sequencing (RNA-Seq) analysis on purified splenic B cells before and after LPS stimulation to determine if any ZBTB proteins were evidently upregulated to compensate for the loss of Zbtb24. Levels of all ZBTB proteins, except ZBTB24, did not differ significantly between time-matched control and Zbtb24B−CKO splenic B cells (Table S2).

To exclude any potential off-target deletions of Zbtb24 in Zbtb24B−CKO mice, we next adoptively transferred purified splenic control or Zbtb24-deficient CD19+ B cells, together with WT CD4+ Th cells, into Rag2−/− recipients before immunization (Fig. S4A). Although the total IgM and IgG levels were reduced in Rag2−/− mice reconstituted with Zbtb24-depleted B cells, serum levels of antigen-specific IgG, and total numbers of splenic CD19+ B cells or CD19lowCD138++ PCs did not differ in Rag2−/− recipients implanted with control versus Zbtb24-deficient B cells (Fig. S4B–D). Decreased total polyreactive IgM might underlie the reduced levels of NP-specific IgM in Zbtb24B−CKO mice (Fig. S4B, C), as ablation of Zbtb24 did not profoundly affect the CSR ability of murine splenic B cells in vitro (Fig. S5).

Collectively, these data indicate that B cell intrinsic Zbtb24 does not play an essential role in the generation and maintenance of GCB cells, their affinity maturation as well as later differentiation toward Bm or PCs after immunization, albeit that Zbtb24B−CKO mice show delayed/mitigated early antibody productions induced by TD-Ag. Notably, mice with a specific depletion of Zbtb24 in T cells (Cd4-Cre) or the whole hematopoietic lineages (Vav1-Cre) exhibit largely intact antibody responses in vivo before and after TD-Ag immunizations (Fig. S6A–D), implying that Zbtb24 may modulate TD-Ag-elicited GC reactions and antibody responses outside of the hematopoietic system.

Significantly reduced humoral responses to TID-Ags in Zbtb24 B−CKO mice

The reduced total serum IgM and IgG in Rag2−/− mice reconstituted with Zbtb24-deficient total splenic B cells, most of which likely react against autoantigens and commensal gut bacteria, indicate that lack of Zbtb24 hampers antibody responses elicited by TID-Ags (Figure S4B). Indeed, Zbtb24B−CKO mice produced significantly decreased amounts of NP-specific IgM, IgG1, and IgG3, three major antibody subtypes against soluble protein or carbohydrate antigens in mice [39], in the first 2 weeks after immunization with the type II TID-Ag NP-Ficoll. On day 21 and 35, levels of IgG1 were still reduced, and the other two antibody subtypes tended to decrease as well (Fig. 3A). Likewise, significantly less NP-specific IgM, IgG1, IgG3, and IgG2b were detected in Zbtb24B−CKO mice after immunization with the type I TID-Ag NP-LPS (Fig. 3F). As expected, reduced percentages/numbers of CD19lowCD138++ PCs were detected in spleens and/or peritoneal cavities (PeC) of Zbtb24B−CKO mice (Fig. 3B–E and G–H). Moreover, depletion of Zbtb24 in hematopoietic cells resulted in diminished antibody productions induced NP-Ficoll as well (Fig. S6E). These data strongly indicate that B cell intrinsic Zbtb24 promotes the differentiation of PC and production of antibodies against TID-Ags in vivo.

Fig. 3

Significantly reduced antigen-specific antibody levels in Zbtb24B−CKO mice after TID-Ag immunization. Mice were intraperitoneally immunized with the type II TID-Ag NP-Ficoll (10 μg/200 μl PBS/mouse, A–E) or type I TID-Ag NP-LPS (20 μg/200 μl PBS/mouse, F–H) on D0. Blood was taken at indicated times and NP-specific antibodies in sera were determined by ELISA in plates coated with NP25-BSA. A, F Bar graphs showing the optical density (OD) values of NP-specific antibodies in diluted sera of Cd19Cre/+ versus Zbtb24B−CKO mice on indicated days post immunization with NP-Ficoll (A) or NP-LPS (F). B, C Representative pseudo/zebra-plots illustrating the gating strategies for CD19+CD138+ plasma blasts (PB)/CD19lowCD138high plasma cells (PC) and percents of NP+ cells within gated PB (PB-NP+) and PC (PC-NP+) cells in peritoneal cavities (PeC, B) or spleens (SPL, C) of mice on D35 post NP-Ficoll immunization. D, E, G, H, bar graphs showing the percentages and absolute numbers of PB and PC cells as well as the NP+ cells within gated PB/PC cells (PB-NP+/PC-NP+, respectively) in PeC (D, G) and SPL (E, H) of mice on D35 post NP-Ficoll (D, E) or on D21 post NP-LPS (G, H) immunization. Each symbol represents a single mouse of the indicated genotype (female, 10 weeks of age in A–E; and male, 9 weeks of age in F–H), and numbers below horizonal lines in bar graphs indicate P values. Data in A, F are representative of two independent experiments

B1 and MZB cells are considered the main responders to TID-Ags [45]. We thus tried to delineate which cell type was responsible for the diminished antibody levels in TID-Ag-immunized Zbt24B−CKO mice via detailed flow cytometry analyses. At 5 weeks post NP-Ficoll immunization, the numbers of NP-specific cells in gated different B cell compartments were all comparable in the spleens and PeC between Cd19Cre/+ and Zbtb24B−CKO mice (Fig. S7), possibly owing to the late analyzing time point. Of note, significantly less NP+ B1, but not FOB or MZB, cells were observed in the spleens of Zbtb24B−CKO mice 3 weeks post NP-LPS inoculation, and NP+ B1 cells tended to decrease in their PeC as well (Fig. S8), indicating that B1 was the target B cell subset through which Zbtb24 promoted TID-Ag-induced antibody responses in vivo.

Zbtb24-deficiency impairs the differentiation and antibody-producing ability of B1 cells in vivo

To unambiguously reveal the roles of Zbtb24 in B1 cells in vivo, we adoptively transferred peritoneal B1 cells, purified from Cd19Cre/+ or Zbtb24B−CKO mice by FACS-sorting, into Rag2−/− mice with no mature T/B cells and serum antibodies (Fig. 4A). Total IgM levels were reduced by ~ 70% in Rag2−/− recipients received Zbtb24-deficient B1 cells (Fig. 4B). Moreover, 7 days after NP-LPS immunization (i.e., day 27 post transfer), Zbtb24-deficient B1-reconstituted Rag2−/− recipients produced significantly less NP-specific IgM than those harboring control Cd19Cre/+ B1 cells (Fig. 4C). Total or NP-specific IgG was barely detectable in recipients implanted with Zbtb24-deficient or sufficient B1 cells (data not shown).

Fig. 4

Impaired function of Zbtb24-deficient B1 cells in vivo. FACS-sorted peritoneal cavity CD19+B220lowCD23− B1 cells from control Cd19Cre/+ and Zbtb24B−CKO mice (female, 12 weeks of age) were adoptively transferred into Rag2−/− recipient mice (female, 8 weeks of age) intraperitoneally (2 × 105 cells/200 μl PBS/mouse). On D20 post injection, Rag2−/− recipient mice were immunized with NP-LPS (20 μg/200 μl PBS/mouse) intraperitoneally. Blood was taken at indicated times and cells in peritoneal cavities (PeC) and spleens (SPL) were analyzed by flow cytometry on D27. A A schematic flow-chart showing the experiment setup. B, C Bar graphs showing levels of total (B and tIgM in C) and NP-specific (NP-IgM in C) IgM in sera of recipient mice on D10 and D20 (B) or D27 (C). D, E Representative offset histograms/bar graphs showing the percentages of CD19+ B cells and CD19+CD138+ plasma cells (PC) within gated B cells in the spleens (SPL, D) and peritoneal cavities (PeC, E) of recipients implanted with Cd19Cre/+ or Zbtb24B−CKO B1 cells. Each dot represents a single recipient mouse, and numbers below horizontal lines indicate P values. Data are representative of two experiments

Upon activation, some peritoneal B1 cells transit to the spleen to divide and differentiate into PCs, while others stay in PeC and rapidly differentiate into PCs even without division [46]. We thus compared the number of B and PCs in the spleens and PeC of recipients. The percentages of Zbtb24-deficient CD19+ B cells and CD138+ PCs were significantly reduced in spleens, while only the latter was decreased in the PeC of recipients (Fig. 4D, E).

In sum, our data (depicted in Figs. 3, 4, and S6E, S7, S8) indicate that lack of ZBTB24 protein suppresses the differentiation of B1 cells toward PCs and thereby reduces the antibody levels against TID-Ags in vivo. Given that surface levels of CD11b, which couples with CD18 to form MAC-1/CR3 and governs the migration of B1 cells from the PeC into the spleens [46, 47], and surface IgM were comparable on Zbtb24-deficient and -sufficient peritoneal B1 cells (Fig. S9A, B), it is unlikely that reduced PC differentiation of B1 cells was attributable to impaired migration or BCR-triggering.

Zbtb24-deficiency impedes the PC differentiation of B1 cells without compromising their survival, activation and proliferation in vitro

Upon activation by LPS in vitro, the differentiation of purified peritoneal B1, but not B2, cells towards CD138+ PCs was significantly impaired by Zbtb24 depletion (Fig. 5A, B). Accordingly, levels of IgM and IgG3 in culture supernatants were reduced in LPS-cultivated Zbtb24-null B1 cells (Fig. 5D). Given that B1 cells are more sensitive to TLR agonists-induced PC differentiation than FOB cells [29], we further cultured splenic B cells with anti-IgM/CD40 or high amounts of LPS to mimic the T-cell dependent or independent stimulations, respectively. Again, deficiency of Zbtb24 did not suppress the PC differentiation of splenic B2 cells as well as their antibody producing abilities in these conditions (Fig. S9C–E), corroborating our in vivo findings depicted in Figs. 2D, G and S4D. Zbtb24-deficiency did not reduce the viabilities and cell numbers in these B1/B2 cultures (Figs. 5C, and S10A, D, E). Moreover, the ability of B1/B2 cells to upregulate surface activation makers (CD69 and CD86) were not impaired by Zbtb24 depletion (Fig. S10B, C, F), excluding a generally disrupted signaling transduction downstream of TLR4 in B cells deprived of Zbtb24.

Fig. 5

Zbtb24-deficiency specifically inhibits LPS-induced differentiation of peritoneal B1 cells toward PCs in vitro. CD19+B220lowCD23− B1 and CD19+B220highCD23+ B2 cells were FACS-sorted from peritoneal cavities of Cd19Cre/+ and Zbtb24B−CKO mice, and subsequently cultured (2–5 × 104 cells/well in 96-U bottom plate) in medium (M), 0.1/1 μg/ml LPS (L-0.1/L-1, respectively) for 3 days. A Representative contour-plots showing the percentages of CD19lowCD138+ PCs in differently cultured B1 or B2 cells on day 3. B, C Bar graphs showing the percentages/absolute numbers of PCs (B) or total living cells (C) 3 days post stimulation. D Bar graphs showing the IgM and IgG3 levels in culture supernatants of LPS-stimulated B1 or B2 cells on day 3. Antibody levels in supernatants of B cells cultured in medium were too low to be detected after 5 × dilution. Each dot represents a single mouse of the indicated genotype (male, 10 weeks of age). E Representative overlayed histograms showing the comparable division profiles (top panel) or contour-plots showing the proliferation (CFSE) versus differentiation (CD138, middle and bottom panels) of cultured peritoneal B1 cells isolated from Cd19Cre/+ versus Zbtb24B−CKO mice (male, 14 weeks old, n = 3 and 4 for Cd19Cre/+ and Zbtb24B−CKO, respectively). B1 cells were labeled with the cell division tracker CFSE (10 μM) and subsequently cultured in medium (M), LPS (0.1/0.5 μg/ml) or anti-IgM (αCD40, 1/5 μg/ml) for 3 days before flow cytometry analysis. Numbers below horizontal lines in B–D indicate P values. Data are representative of 2–4 independent experiments

As reported previously [48], B1 cells were anergic to BCR triggering and thus failed to undergo activation upon anti-IgM stimulation in vitro (Fig. S10B, C, F). To further determine the role of ZBTB24 in cell proliferation, we labeled B1 cells with cell division tracker CFSE before culture. Interestingly, LPS stimulation induced massive PC differentiation accompanied by minimal cell divisions, whereas anti-CD40 elicited robust proliferation concurrent with little CD138 upregulation (Fig. 5E), implying that B1 cells were able to uncouple PC differentiation from the cell division process. Of note, Zbtb24-depletion significantly repressed PC differentiation of B1 cells without impeding their mitoses induced by LPS or anti-CD40 in vitro (Fig. 5E).

On the basis of these data, we conclude that ZBTB24 promotes the PC differentiation of B1 cells independent of their survival, activation and proliferation in vitro.

Zbtb24-deficiency inhibits the PC differentiation of B1 cells via attenuating heme synthesis

We next performed RNA-Seq analysis to explore how ZBTB24 regulates the differentiation of B1 cells. Upon activation by LPS, Zbtb24-deficient peritoneal B1, but not splenic B2 cells, expressed significantly lower levels of hallmark genes involved in PC differentiation, unfolded protein response (UPR), and protein secretion, such as Prdm1 (the driving force of PC differentiation), Sdc1 (encoding CD138), Il10, Irf4, and Xbp1 (Fig. 6A, B, E, and Table S2). Of note, expressions of Bach2 and Bcl6, two major TFs repressing Prdm1, were not influenced by Zbtb24 depletion in B1 cells (Fig. 6B). Therefore, it is unlikely that the B1-specific effect of ZBTB24 on PC differentiation is attributable to its direct binding and regulation of these key regulators.

Fig. 6

Zbtb24-deficiency blunts the differentiation of B1 cells into PCs through repressing the biosynthesis of heme. FACS-sorted peritoneal CD19+B220lowCD23− B1 cells were stimulated with 0.1 μg/ml LPS in 96-U bottom plate for 24 h (RNA-seq/Q-PCR, A-D) or 2–3 days without/with additional L-AC (250 μM)/Hemin (25 μM) to induce PC differentiation (E–J). A, GSEA plots of genes involved in PC differentiation, protein secretion, unfolded protein response (UPR), and heme metabolism in LPS-stimulated Zbtb24B−CKO versus Cd19Cre/+ B1 cells. B, C, heatmaps showing the z-score normalized on the raw expression counts of dysregulated genes regulating the differentiation of PC cells (B) or heme metabolism in cells (C) identified by RNA-seq analysis. The complete lists of enriched genes regulating UPR and heme metabolism were provided in Fig. S11C. D mRNA levels of CPOX and ALAD in Cd19Cre/+ versus Zbtb24B−CKO B1 cells determined by Q-PCR. E Representative half-offset histograms showing the levels of intracellular PRDM1 in LPS-stimulated B1 cells on D2. F Representative overlayed histograms (left) and cumulative data (right) showing the levels of intracellular PPIX in resting control versus Zbtb24B−CKO B1 cells (D0) or gated CD138− non-PC and CD138+ PC cells in LPS-stimulated cultures on D2. G, I Representative contour-plots (G) and bar graphs (I) showing the percentages of CD19lowCD138+ PCs in differentially cultured Cd19Cre/+ versus Zbtb24B−CKO B1 cells on D3. H Representative overlayed contour-plots showing the intracellular ROS levels (visualized by DCFH-DA) and mitochondrial mass/membrane potentials (detected by MitoTracker Orange CMTMRos) in gated CD138− non-PC (black) versus CD138+ PC (red) cells in cultured B1 cells on D3. J Bar graphs showing the ROS levels (MFI of DCFH-DA, top) or mitochondrial mass/membrane potential (MFI of MitoTracker, bottom) in gated non-PC/PC cells in Cd19Cre/+ versus Zbtb24B−CKO B1 cultures. Gates for CD138− non-PC and CD138+ PC were illustrated in G. Each dot represents a single mouse of the indicated genotype in D, F and I (male, 12–14 weeks of age), while results in J are expressed as mean ± SEM (n = 3). Numbers in F, I and J indicate P values determined by student t-test. Blue numbers in F denote percents of reduction. Data in E–J are representative of two experiments

Enormous studies recently showed that the activation and function of lymphocytes are intertwined with metabolic reprogramming and adaption [31,32,33]. In association with the altered function, multiple genes relating to nutrients uptake and metabolism, such as Slc2a1/Glut1, Slc16a3/Mct3, Ak4, Pdk1, Pfkl, and Tpi1, were significantly upregulated in Zbtb24B−CKO B1 cells (Fig. S11A). Accordingly, GSEA identified a positive enrichment of gene sets regulating the glycolysis and pyruvate metabolism in Zbtb24-null B1 cells (Fig. S11B). By contrast, a panel of genes involved in heme metabolism were downregulated in Zbtb24B−CKO cells, albeit that the differences did not reach the cutoff value and thus were much milder compared with Zbtb24 direct targets, such as Cdca7 and Ostc (Figs. 6A, C, and S11C). Expressions of Alad (5-aminolevulinic acid dehydratase) and Cpox (Coproporphyrinogen oxidase), two enzymes that directly catalyze reactions along the heme synthesis pathway in cytosol and mitochondrial intermembrane space, respectively [49], were consistently reduced in Zbtb24-null B1 but not B2 cells (Fig. 6C, D, S11C, and Table S2). Moreover, the level of Hmox1, a heme-induced oxygenase that mediates its degradation and serves as a readout for intracellular heme content [38], was mildly decreased in Zbtb24B−CKO B1 cells as well (Fig. 6C). Given that heme promotes PRDM1 expression and PC differentiation of B cells by inactivating BACH2 [38], we reasoned that heme biosynthesis may be attenuated in Zbtb24-depleted B1 cells, thereby leading to impaired PC differentiation.

Indeed, Zbtb24-depletion significantly mitigated LPS-induced upregulation of PPIX, the final substrate of heme biosynthesis [49], in B1 cells, albeit no differences were observed in resting cells (Fig. 6F). Notably, ablation of Zbtb24 resulted in a more pronounced PPIX reduction in CD138− non-PC cells than that in CD138+ PC cells (26.2% versus 10.1%, Fig. 6F). Moreover, addition of exogenous hemin in cultures almost completely abrogated the differentiation defects of Zbtb24-deficient B1 cells (Fig. 6G, I), while its supplementation similarly promoted/suppressed PC differentiation/CSR in control versus Zbtb24B−CKO splenic B cells (Fig. S12A, B). Together, these data indicate that ZBTB24 specifically promotes the PC differentiation of B1 cells via augmenting heme synthesis. Exogenous hemin only slightly potentiated the PC differentiation of control B1 cells (Fig. 6G, I), implying that activated peritoneal B1 cells may contain abundant intracellular heme to facilitate their accelerated and heightened PC differentiation.

Zbtb24-depletion represses the heme synthesis partially through mTORC1 in B1 cells

The rather mildly downregulated genes along heme synthesis pathway, compared with Zbtb24-direct targets, such as Cdca7, implied that ZBTB24 exerts the modulation of former genes indirectly. It has been recently shown that endogenous ROS inhibits heme synthesis, and activated splenic B cells with intermediate mitochondrial mass/membrane potential are predisposed to become PCs [37]. We thus wondered whether ZBTB24 promoted heme synthesis in B1 cells via regulating intracellular ROS and/or mitochondrial function. Compared with activated splenic B cells, B1 cells had significantly higher levels of ROS, which were further increased by Zbtb24-depletion (Figs. 6H, J, and S12C, D). However, addition of the antioxidant L-ascorbic acid (L-AC) failed to promote PC differentiation in peritoneal B1 cells, albeit that it did reduce ROS levels in B1 cell and augment the PC differentiation of splenic B2 cells (Figs. 6G–J, and S12). The mitochondrial mass/membrane potential was markedly lower in splenic B2-derived PCs as previously reported [37], but no such differences were observed in B1 cultures, and no significant impact of Zbtb24-deficiency was observed (Figs. 6H, J, and S12C, D). Thus, the metabolic reprogramming and mitochondrial function differ significantly between B1 versus B2 cells along their differentiation toward PCs. Because ROS represses the final step of heme synthesis by inhibiting the addition of ferrous ions to PPIX [37], it is unlikely that disturbed ROS levels were responsible for the attenuated PPIX accumulation in Zbtb24-deficient B1 cells.

Expressions of Slc3a2 and Slc7a5, encoding the respective heavy and light chain of CD98, positively correlate with mTORC1 activity in B cells [35]. RNA-Seq data showed that levels of Slc3a2 and Slc7a5 were decreased, coinciding with the disturbed mTORC1 signaling in activated Zbtb24-null B1 cells as revealed by GSEA (Fig. S11A, B). mTORC1 activity regulates the protein synthesis and metabolism of mammalian cells, and promotes PC differentiation of murine splenic B cells partially via augmenting heme synthesis [35]. In keeping with these findings obtained in B2 cells, inclusion of an mTORC1 specific antagonist, rapamycin, significantly suppressed the PC differentiation and PPIX accumulation in peritoneal B1 cells (Fig. 7A, B), demonstrating that mTORC1 promotes heme synthesis and PC differentiation of B1 cells as well. In line with this notion, Zbtb24-depletion significantly repressed the expression of phosphorylated ribosomal protein S6 (p-S6), an event controlled by mTORC1 activity [35], in LPS-stimulated B1 cells, albeit that no differences were observed in the upstream AKT activity (Fig. 7C, D). Notably, the inhibition of p-S6 was more pronounced, akin to PPIX contents, in CD138− cells as compared with that in CD138+ PCs (Figs. 6F, and 7C, D), indicating that the impaired mTORC1 activity underlies, at least partially, the attenuated heme synthesis and PC differentiation of Zbtb24-null B1 cells.

Fig. 7

Zbtb24-ablation represses the mTORC1 activity, which promotes heme synthesis and PC differentiation of B1 cells. Peritoneal CD19+B220lowCD23− B1 cells were stimulated with 0.1 μg/ml LPS in the absence/presence of rapamycin (Rapa, 10 nM) for ~ 2 days (A–D). A, B Representative overlayed histograms (A) and bar graphs (B) showing the PC differentiation (left panel) and PPIX expression (right panel) in B1 cells cultured with LPS ± Rapa on day 2. C, D representative FACS-plots/overlayed histograms (C) and bar graphs (D) showing the expression of phosphorylated AKT (p-AKT) or ribosomal protein S6 (p-S6) in gated CD138+ PC or CD138− non-PC cells derived from Cd19Cre/+/Zbtb24B−CKO B1 cells after stimulation with LPS for 30 h. tCD19 denotes total CD19+ B cells and blue numbers in D indicate percent of reduction in gated Zbtb24B−CKO populations compared with the corresponding control counterparts. E, F Representative histograms (E) and bar graphs (F) showing the PC differentiation of B1 cells cultured in medium (M), LPS (L), and LPS plus Hemin (25 μM, L + H), rapamycin (10 nM, L + R) or both (L + R + H) for 2 days. Each dot represents a single mouse of the indicated genotype (12-week males in A–D, and 9-week females in E, F). Black/blue numbers below horizontal lines indicate P values determined by Mann–Whitney test or paired t-test, respectively. Data are representative of two experiments

We noticed that PC differentiation and intracellular PPIX levels were still considerably reduced in mTORC1 repressed (i.e., cultured with LPS plus rapamycin) Zbtb24-null B1 cells (Fig. 7A, B). To determine the extent to which the two factors (mTORC1 activity versus heme accumulation) contribute to the defected PC generation of Zbtb24-deficient B1 cells, we differentiated B1 cells in the presence of rapamycin/hemin alone or both (Fig. 7E, F). Supplementation of hemin not only completely reverted the defects caused by Zbtb24-depletion as previously observed in Fig. 6G, I, but also partially relieved the inhibitory effect of rapamycin on PC differentiation of B1 cells. Of note, control and Zbtb24-null B1 cells exhibited comparable PC differentiation in the presence of both rapamycin and hemin (Fig. 7E, F), implying that attenuated intracellular heme synthesis is mainly responsible for the differentiation defects of Zbtb24-deficient B1 cells.

Collectively, our data show that ZBTB24 promotes the PC differentiation of B1 cells mainly through augmenting heme synthesis, and the attenuated heme biosynthesis in Zbtb24-depleted B1 cells is partially attributed to their impaired mTORC1 activity.

No effect of Zbtb24-deficiency on PC differentiation of MZB cells

Akin to B1 cells, MZB cells are specialized in responses to TID-Ags and are more sensitive, compared to conventional FOB cells, to TLR agonists-induced proliferation (Fig. 8A) and PC differentiation [29]. We thus questioned whether Zbtb24 exerts a similar function in this innate-like B cell subset as well. LPS stimulation induced robust cell division and PC differentiation of MZB cells, while BCR-crosslinking elicited massive proliferation accompanied by mild PC generation (Fig. 8A–C). Notably, neither the proliferation nor the PC differentiation of LPS-cultivated MZB cells were affected by Zbtb24 depletion, and the addition of exogenous hemin enhanced PC differentiation of Zbtb24 deficient or sufficient MZB cells to the same extent (Fig. 8A–D). Hence, depletion of Zbtb24 does not impact the PC differentiation of MZB cells.

Fig. 8