Abstract
Intracellular calcium (Ca2+) mobilization after engagement of the BCR has been proposed to play an important role in B cell development and function. BCR activation causes an initial Ca2+ release from the endoplasmic reticulum that is mediated by inositol 1,4,5-trisphosphate receptor (IP3R) and then triggers store-operated Ca2+ entry once endoplasmic reticulum Ca2+ store is depleted. Store-operated Ca2+ entry has been shown to regulate B cell function but is dispensable for B cell development. By contrast, the function of IP3R-mediated Ca2+ release in B cells remains to be determined. In this study, we generated a B cell–specific IP3R triple-knockout (IP3R-TKO) mouse model and revealed that loss of IP3Rs increased transitional B cell numbers and reduced recirculating mature B cell numbers in bone marrow. In the peripheral tissues, the numbers of conventional B2 B cells and B1 B cells were both significantly decreased in IP3R-TKO mice. Ablation of IP3Rs also dramatically reduced BCR-mediated B cell proliferation and survival. Furthermore, T cell–dependent and T cell–independent Ab responses were altered in IP3R-TKO mice. In addition, deletion of IP3Rs reduced IL-10–producing regulatory B cell numbers and led to defects in NFAT activation, which together resulted in decreased IL-10 secretion. Taken together, our study demonstrated for the first time, to our knowledge, that IP3R-mediated Ca2+ release plays an essential role in regulating B cell development, proliferation, Ab production, and B cell regulatory function in vivo.
Introduction
B lymphopoiesis originates in the fetal liver and switches to bone marrow after birth. In the bone marrow, B cells progress through a highly ordered series of developmental stages: progenitor B cell, precursor B (pre-B) cell, and immature B cell stages (1). To prevent any further development of self-reactive cells, immature B cells undergo negative selection with survivors developing into transitional B cells, the majority of which then migrate to the spleen, where they undergo further maturation and differentiation (2). Within the spleen, transitional B cells give rise to two types of mature conventional B2 B cells: follicular (FO) B cells and marginal zone (MZ) B cells (2). Naive FO B cells, which constitute the majority of mature splenic B cells, reside in the primary follicles of B cell zones and gain the ability to recirculate through second lymphoid tissues (3). With the assistance of T cells, naive FO B cells can be further activated and play a major role in T cell–dependent (TD) Ab response (3). Unlike FO B cells, MZ B cells are noncirculating mature B cells located at the white pule of the spleen and are responsible for T cell–independent (TI) Ab response (4). Besides conventional B2 B cells, there is another subclass of B cells, B1 B cells, which predominate in body cavities and provide “innate-like” protection against pathogens by producing natural Abs (5). In addition to these Ab-producing B cells, there is a special subset of B cells termed regulatory B cells, which negatively regulate autoimmunity by secreting anti-inflammatory cytokines such as IL-10 (6).
Signals from BCRs are essential for B cell development, survival, and activation. One of the key signals in response to BCR stimulation is intracellular calcium (Ca2+) elevation, which leads to activation of many transcription factors to control the gene expression that mediates diverse genetic programs (7). In B lymphocytes, BCR-mediated Ca2+ elevation occurs via two main mechanisms: Ca2+ release from endoplasmic reticulum (ER) stores and Ca2+ influx from the extracellular space across the plasma membrane (8). Cross-linking of BCRs activates several protein tyrosine kinases and eventually results in activation of phospholipase C (PLC)-γ2. Activated PLC-γ2 hydrolyzes the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate, into two intracellular second messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 binds to IP3 receptors (IP3Rs) in the ER membrane, leading to a transient release of Ca2+ from ER stores, which stands for the first phase of BCR-induced Ca2+ signal. Subsequently, the decrease of ER luminal Ca2+ concentration is detected by stromal interaction molecule 1 (STIM1), which further triggers the opening of Orai1 Ca2+ channels, to induce a sustained Ca2+ influx across the plasma membrane. These processes are called store-operated Ca2+ entry (SOCE), which represents the second phase of BCR-induced Ca2+ signal (9).
Evidence showing the importance of Ca2+ signaling in B cells has mainly originated from studies of mutant mice in which SOCE was genetically abrogated. BCR-induced Ca2+ influx was almost completely blocked in STIM1 and STIM2 double-knockout (DKO) B cells, whereas residual SOCE was present in STIM1- and Orai1-deficient B cells (10, 11). Accordingly, BCR-mediated proliferation is most severely inhibited in STIM1/STIM2 DKO B cells and only partially decreased in B cells lacking either STIM1 or Orai1 (10, 11). Furthermore, loss of both STIM1 and STIM2 in B cells dramatically impairs IL-10 production and exacerbates the severity of experimental autoimmune encephalomyelitis in vivo (11). However, B cell populations in the bone marrow and secondary lymphoid organs were shown to be normal in Orai1−/−, Orai1R93W knock-in, and Stim1f/fStim2f/fMb1-Cre mice (10–12). Human patients with mutations in ORAI1 and STIM1 were also found to have normal numbers of B cells (13, 14). Collectively, these data suggest that SOCE plays an important role in regulating B cell function but is dispensable for B cell development.
Compared with the role of SOCE in B cells, the role of IP3Rs in B cell development and function is not well understood. In mammals, there are three different IP3R subtypes (IP3R1, IP3R2, and IP3R3) encoded by three genes, Itpr1, Itpr2, and Itpr3, respectively (15). Various studies utilizing IP3R knockout mice have demonstrated that IP3Rs regulate brain function (16), exocrine secretion (17), taste perception (18), embryonic development (19, 20), vascular smooth muscle contractility (21), and T cell development (22). On the other hand, IP3R subtypes have also been shown to play a redundant role in regulating BCR-induced Ca2+ mobilization and cell apoptosis in DT40 cells, a chicken pre-B cell line (23). However, the in vivo role of IP3Rs in B cell development and physiological function remains unclear. In this study, we generated a B cell–specific IP3R triple-knockout (IP3R-TKO) mouse model and found that loss of IP3R-mediated Ca2+ release reduced conventional B2 and B1 B cell population. Deletion of all IP3Rs also impaired BCR-mediated cell proliferation in vitro and altered TD and TI Ab responses in vivo. Furthermore, we found that loss of IP3Rs reduced regulatory IL-10–producing B cell (B10) numbers and caused defective activation of the calcineurin-NFAT signaling pathway, which together resulted in decreased BCR-mediated IL-10 production. In summary, we provide evidence for the first time, to our knowledge, that IP3R-mediated Ca2+ release plays an essential role in regulating B cell development and function.
Materials and Methods
Mice
The generation of floxed alleles of the genes encoding IP3R1, IP3R2, and IP3R3 has been described previously (22, 24). We crossed IP3R triple-floxed (Itpr1f/fItpr2f/fItpr3f/f) mice with Cd19tm1(cre)Cgn/J (CD19-Cre) mice (The Jackson Laboratory). CD19-Cre mice were maintained on the pure C57BL/6 background. IP3R triple-floxed mice were originally generated on a mixed genetic background of Black Swiss, 129, and C57BL/6, and had been backcrossed with C57BL/6 mice for more than seven generations. CD19-Cre is constitutively expressed and has been frequently used as a B cell–specific Cre (25). Itpr1f/fItpr2f/fItpr3f/f CD19-Cre+ mice were used as B cell–specific IP3R-TKO mice. Itpr1f/fItpr2f/fItpr3f/fCD19-Cre− mice were used as control mice. All mice used in this study were at the age of 8–10 wk. All mice were housed under specific pathogen-free conditions with a 12/12 h day/night cycle. All animal care and use procedures in this study were approved by the Institutional Animal Care and Use Committee.
Abs and reagents
For flow cytometry, FITC-conjugated anti-CD43 (catalog number [no.] 11-0431-81), anti-CD93 (catalog no. 11-5892-81), anti-GL7 (catalog no. 53-5902-80), Alexa Fluor 488–conjugated anti-CD1d (catalog no. 53-0011-80), PE-conjugated anti-IgM (catalog no. 12-5790), anti-CD21/CD35 (catalog no. 12-0211-81), anti-Fas (catalog no. 12-0951-81), PE-cyanine5.5–conjugated anti-CD19 (catalog no. 35-0193-80), PE-cyanine7–conjugated anti-CD23 (catalog no. 25-0232-81), allophycocyanin-conjugated anti-B220 (catalog no. 17-0452), allophycocyanin-eFluor 780–conjugated anti-CD21/CD35 (catalog no. 47-0211-80), and streptavidin PE (catalog no. 12-4317) were purchased from eBioscience. Biotin-conjugated anti-CD5 (catalog no. 553019) and PE-conjugated anti-CD93 (catalog no. 558039) were purchased from BD Biosciences. For immunoblotting, the primary Abs against JNK (catalog no. 9258), ERK1/2 (catalog no. 9102), p-JNK (catalog no. 9251), and p-ERK (catalog no. 9101), and NFAT1 (catalog no. 4389) were purchased from Cell Signaling Technology. The Ab against β-actin was purchased from Santa Cruz Biotechnology (catalog no. SC-47778). The Ab against IP3R3 was purchased from BD Biosciences (catalog no. 610312). The Abs against IP3R1 and IP3R2 were raised in our laboratory as previously described (22). For cell stimulation, anti-mouse IgM F(ab′)2 and anti-CD40 were purchased from Jackson Immunoresearch (catalog no. 115-006-075) and BD Biosciences (catalog no. 550285), respectively. LPS (catalog no. L2630), cyclosporin A (CsA; catalog no. C3662), and FK506 (catalog no. F4679) were purchased from Sigma-Aldrich.
Sample preparation and flow cytometry
Single-cell suspensions were prepared from the bone marrow, spleen, inguinal lymph node, peritoneal cavity, and peripheral blood by passing through a cell strainer to remove clumps and debris. Cells were treated with RBC lysis buffer (Beyotime) and washed with ice-cold PBS containing 0.1% BSA. Cells were then incubated with Abs for 30 min, washed twice, and resuspended in an appropriate volume of PBS with 0.1% BSA. Data were acquired on FACSCalibur and FACSCanto II flow cytometers (BD Biosciences) and analyzed with FlowJo (Tree Star).
B cell isolation and calcium measurement
Single-cell suspensions were obtained from spleens of control and IP3R-TKO mice. After RBCs were depleted by hypotonic lysis, splenic B cells were purified by the negative selection of CD43+ cells with anti-CD43 magnetic beads (Invitrogen). The purity of the B cells (B220+) was >95% determined by FACS analysis.
For Ca2+ imaging, purified splenic B cells were planted on glass coverslips coated with poly-d-lysine (Sigma), washed with regular physiological saline solution (PSS; in mM: NaCl 137, KCl 5.4, MgSO4 1.0, glucose 10, CaCl2 1.8, and HEPES 10 [pH 7.4]), and then incubated with 5 μM Fluo4-AM (Invitrogen) for 30 min at 37°C. Cells were imaged with a Zeiss LSM510 inverted confocal microscope. To separate BCR-induced Ca2+ release from SOCE, we perfused cells with a Ca2+-free PSS (zero [Ca2+] plus 1.0 mM EGTA) for a short time before the administration of anti-mouse IgM F(ab′)2 (10 μg/ml). To measure IP3-induced Ca2+ release, we incubated splenic B cells with a membrane-permeant caged derivative of IP3, caged-IP3/PM (catalog no. cag-iso-2-145-100; SiChem), together with Fluo4-AM for 30 min at 37°C. Photolysis of caged-IP3 in selected B cells was evoked by a 20-ms exposure of 405 nm UV laser. To measure thapsigargin (TG)-induced Ca2+ signal, we also perfused cells with a Ca2+-free PSS for a short time before the administration of 2 μM TG (Sigma). Image processing and data analysis were performed as previously described (26).
In vitro proliferation and survival
Purified splenic B cells were incubated with 5 μM CFSE (Sigma) at a density of 1 × 107 cells/ml in PBS containing 0.1% BSA for 5 min at 37°C. The staining was quenched by the addition of 10 vol of ice-cold PBS containing 5% FBS. After three washes with PBS containing 0.1% BSA, CFSE-labeled B cells were resuspended in RPMI 1640 with 10% FBS and plated in 96-well flat-bottom dishes at a density of 1 × 106 cells/ml. Thereafter, cells were treated with vehicle (RPMI 1640 with 10% FBS), 10 μg/ml anti-IgM F(ab′)2, 10 μg/ml anti-CD40, 10 μg/ml LPS, or a combination of 10 μg/ml anti-IgM with 10 μg/ml anti-CD40 for 48 h, respectively. Cells were stained with anti-B220 to indicate B cells and propidium iodide (PI; eBioscience) for cell viability analysis. In some experiments, cells were stained with anti-B220, anti-CD93, anti-CD21, and anti-CD23 to separate different B cell subsets. The percentages of PI− and CFSE-diluted B cell subsets were assessed by FACSCalibur (BD Biosciences).
Immunoblot analysis
Purified splenic naive B cells or LPS-activated B cells (pretreated with 10 μg/ml LPS for 48 h) were stimulated with 10 μg/ml anti-IgM F(ab′)2 in serum-free RPMI 1640 medium for indicated time periods and then lysed with lysis buffer (8 M urea, 2 M thiourea, 3% SDS, 75 mM DTT, 0.05 M Tris-HCl [pH 6.8], and 0.03% bromophenol blue). Cell lysates were separated by standard SDS-PAGE and analyzed by Western blotting using the Abs against IP3Rs, NFAT1, β-actin, ERK, p-ERK, JNK, and p-JNK.
p-ERK analysis
Pervanadate treatment and flow cytometric analysis of p-ERK were performed as previously described (27). In brief, freshly isolated single-cell suspension from bone marrow of control and IP3R-TKO mice was placed on ice for 1 h in dPBS with Ca2+ and Mg2+ containing 1% FBS. Cells were treated with 60 μM sodium pervanadate for 5 min at 37°C, fixed in 4% paraformaldehyde, and permeabilized in PBS containing 0.2% Triton-X 100, 5% BSA, and 2% horse serum. Cells were then washed with PBS and stained with primary Abs against ERK1/2 (catalog no. 9102; Cell Signaling Technology) and p-ERK1/2 (catalog no. 9101; Cell Signaling Technology) for 45 min at room temperature, respectively. Thereafter, cells were washed again and further stained with the Abs recognizing surface markers, as well as the secondary Ab FITC-conjugated anti-rabbit IgG (catalog no. 406403; BioLegend).
Quantitative real-time PCR analysis
Total RNA was extracted using TRIzol reagent (Invitrogen) or RNeasy MiniKit (Qiagen), and cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech). Quantitative real-time PCR (RT-PCR) was performed using TransStart Tip Green qPCR SuperMix (TransGen Biotech) according to the manufacturer’s instructions. The sequences for primers of Itpr1-3 and Gapdh were used as previously described (21). The primer sequences for Il-10, Ccnd2, Bip, GRP94, Calr, Baffr, Mef2c, Foxo1, Rag1, and Rag2 are presented as follows: Il-10 (forward: 5′-TGCCTTCAGTCAAGTGAAGACT-3′; reverse: 5′-AAACTCATTCATGGCCTTGTA-3′), Ccnd2 (forward: 5′-CAGAGCTTCGATTTGCTCCT-3′; reverse: 5′-ACACACTCACGTGTGATGCC-3′), Bip (forward: 5′-ACATGGACCTGTTCCGCTCTA-3′; reverse: 5′-TGGCTCCTTGCCATTGAAGA-3′), GRP94 (forward: 5′-GGGAGGTCACCTTCAAGTCG-3′; reverse: 5′-CTCGAGGTGCAGATGTGG G-3′), Calr (forward: 5′-AGCAGTTCTTGGACGGAGATG-3′; reverse: 5′-TTCTCCAGGTCCCCGTAAAAT-3′), Baffr (forward: 5′-CCTCCGCTCAAAGAAGATGCA-3′; reverse: 5′-GTGGAGCCCAGTTCTGT-3′), Mef2c (forward: 5′-AGATCTGACATCCGGTGCAG-3′; reverse: 5′-TCTTGTTCAGGTTACCAGGTG-3′), Foxo1 (forward: 5′-TGCTGTGAAGGGACAGATTG-3′; reverse: 5′-GAGTGGATGGTGAAGAGCGT-3′), Rag1 (forward: 5′-CTGAAGCTCAGGGTAGACGG-3′; reverse: 5′-CA ACCAAGCTGCAGACATTC-3′), Rag2 (forward: 5′-CTTCCTGCTTGTGGATGTGA-3′; reverse: 5′-AGTGACTCTTCCCCAAGTGC-3′). Relative transcript abundance was normalized to Gapdh. Each sample was run at least in duplicate.
ELISA
To determine basal Ig titers, we prepared serum from unimmunized control and IP3R-TKO mice, and titers were measured using HRP-conjugated goat anti-mouse IgM, IgG1, IgG2b, IgG3, and IgA (Southern Biotech).
To determine TD responses, we immunized control and IP3R-TKO mice i.p. with 100 μg of 4-hydroxy-3-nitrophenyl (NP)-chicken γ-globulin (CGG) (NP-CGG; Biosearch Technologies) in alum (Thermo Scientific). To determine TI responses, we immunized mice i.p. with NP-LPS (TI-1 Ag; Biosearch Technologies) or NP-Ficoll (TI-2 Ag; Biosearch Technologies). Blood was collected at days 0, 7, 14, and 21 by orbital sinus blood sampling. For detection of NP-specific Abs, ELISA plates were precoated with 2 μg/ml NP29-BSA (Biosearch Technologies). Serum samples were serially diluted and relative anti-NP titers were measured using isotype-specific HRP-conjugated secondary Abs (Southern Biotech).
For IL-10 production detection, LPS-activated splenic B cells were stimulated with 10 μg/ml anti-IgM F(ab′)2 for 24 h. IL-10 in the cell culture medium was detected by ELISA according to the manufacturer’s protocol (Invitrogen).
Results
Deletion of all three IP3R subtypes abolished BCR-induced Ca2+ mobilization
We first sorted B cells from control mouse bone marrow and examined expression of all three IP3R subtypes in pro/pre-B cells (B220+CD19+IgM−), immature B cells (B220lowCD19+IgMint), transitional B cells (B220intCD19+IgMhigh), and recirculating mature B cells (B220highCD19+IgMint) using quantitative RT-PCR. Consistent with expression in DT40 cells (23), all three IP3R subtypes were detected at all B cell developmental stages (Supplemental Fig. 1A). However, the expression level of each subtype varied between different stages. Interestingly, all three IP3R subtypes were highly expressed at the transitional stage compared with mature B cell stage (Supplemental Fig. 1A), which suggests that IP3R-mediated Ca2+ signaling could play a role in B cell development from the transitional to mature stage.
To further explore the role of IP3Rs in B cell development and function, we generated a B cell–specific IP3R-TKO mouse model using a CD19-Cre mouse line that has been shown to be constitutively expressed from the early progenitor B cell stage and throughout all the later B cell developmental stages (25). Once IP3R-TKO mice were generated, we first examined whether CD19-Cre could efficiently delete IP3Rs in B cells. In purified IP3R-TKO splenic B cells, both mRNA and protein levels of all three IP3R subtypes were dramatically reduced compared with control B cells (Fig. 1A, 1B). In addition, we found that mRNA levels of all three IP3R subtypes were dramatically decreased in immature, transitional, and recirculating mature B cells purified from the bone marrow of IP3R-TKO mice compared with control mice (Supplemental Fig. 1B). We also found that deletion of IP3Rs was less efficient in pro/pre-B cells of IP3R-TKO mice (Supplemental Fig. 1B), which is consistent with weaker expression of CD19-Cre in pro/pre-B cells (28). Furthermore, we checked whether deletion of IP3Rs could efficiently abrogate IP3-induced Ca2+ release. Splenic B cells were isolated and incubated with a membrane-permeant caged derivative of IP3, and a UV laser was applied to selected cells to release IP3 from the caged compound, which can thus trigger the opening of IP3Rs. In control B cells, photolysis of caged-IP3 was able to induce a rapid Ca2+ transient, which was totally absent in IP3R-TKO B cells (Supplemental Fig. 1C, 1D), further indicating that all IP3R subtypes were successfully deleted in these IP3R-TKO B cells. We next investigated whether deletion of all three IP3Rs could alter ER Ca2+ content and induce ER stress and the unfolded protein response. We depleted ER Ca2+ store with TG, an ER Ca2+ ATPase blocker, in the absence of extracellular Ca2+, and measured TG-induced Ca2+ signals in the cytosol. We found that deletion of all three IP3R subtypes did not significantly increase the amplitude of TG-induced Ca2+ signals (Supplemental Fig. 1E, 1F), suggesting that the filling state of ER Ca2+ stores between control and IP3R-TKO B cells was comparable. Consistently, we found that expression of prototypical unfolded protein response target genes Bip, GRP94, and Calr was also not significantly changed in IP3R-TKO B cells compared with control B cells (Supplemental Fig. 1G).
B cell–specific deletion of IP3Rs abrogated BCR-mediated Ca2+ mobilization. Splenic B cells were isolated from control and IP3R-TKO mice by negative selection of CD43+ cells with anti-CD43 magnetic beads. Quantitative RT-PCR (A) and Western blot analysis (B) were used to measure the RNA and protein levels of each IP3R subtype in control and IP3R-TKO B cells, respectively. n = 3–5 mice per group. Data represent mean ± SEM. (C) Sequential confocal images of splenic B cells at the time point of t0, t1, t2, and t3 as indicated by the arrows in (D). Splenic B cells were loaded with Ca2+ indicator Fluo4 and imaged using confocal microscopy. Intracellular Ca2+ mobilization was first elicited by anti-IgM (10 μg/ml) in Ca2+-free (zero [Ca2+]o) buffer with subsequent reintroduction of regular buffer with normal extracellular Ca2+ concentration (2 mM [Ca2+]o). Scale bar, 100 μm. (D) Representative traces of averaged Ca2+ signals in control (black) and IP3R-TKO (red) B cells. Data are representative of at least three independent experiments. Significance was determined by the two-tailed, unpaired Student t test. ***p < 0.001 versus control B cells.
B cell–specific deletion of IP3Rs abrogated BCR-mediated Ca2+ mobilization. Splenic B cells were isolated from control and IP3R-TKO mice by negative selection of CD43+ cells with anti-CD43 magnetic beads. Quantitative RT-PCR (A) and Western blot analysis (B) were used to measure the RNA and protein levels of each IP3R subtype in control and IP3R-TKO B cells, respectively. n = 3–5 mice per group. Data represent mean ± SEM. (C) Sequential confocal images of splenic B cells at the time point of t0, t1, t2, and t3 as indicated by the arrows in (D). Splenic B cells were loaded with Ca2+ indicator Fluo4 and imaged using confocal microscopy. Intracellular Ca2+ mobilization was first elicited by anti-IgM (10 μg/ml) in Ca2+-free (zero [Ca2+]o) buffer with subsequent reintroduction of regular buffer with normal extracellular Ca2+ concentration (2 mM [Ca2+]o). Scale bar, 100 μm. (D) Representative traces of averaged Ca2+ signals in control (black) and IP3R-TKO (red) B cells. Data are representative of at least three independent experiments. Significance was determined by the two-tailed, unpaired Student t test. ***p < 0.001 versus control B cells.
We further investigated the effects of IP3R deletion on BCR-induced Ca2+ mobilization in B cells. To characterize the phase of Ca2+ release and the following phase of SOCE separately, we first applied anti-IgM in the absence of extracellular Ca2+ (zero [Ca2+]o) to induce Ca2+ release from the ER. When ER Ca2+ store was depleted, regular extracellular solution with 2 mM [Ca2+]o was added to induce SOCE. In control cells, application of anti-IgM was able to elicit a transient Ca2+ elevation, and subsequent reintroduction of extracellular Ca2+ was able to induce a second phase of Ca2+ elevation. However, both phases of Ca2+ increase were completely abolished in IP3R-TKO B cells (Fig. 1C, 1D), suggesting that IP3Rs are essential for BCR-induced Ca2+ release and subsequent activation of SOCE.
IP3R-mediated Ca2+ release regulates B2 and B1 B cell development
Next, we investigated the role of IP3R-mediated Ca2+ signal in B cell development by characterizing distinct B cell subsets using FACS analysis. B cells were isolated from bone marrow and peripheral secondary lymphoid tissues. First, total numbers of CD19+B220+ B cells in bone marrow of IP3R-TKO mice were not significantly changed compared with control mice (3.71 ± 0.42 × 106 cells in control mice versus 3.45 ± 0.33 × 106 cells in IP3R-TKO mice, n = 11 mice for both groups). The numbers of pro/pre-B and immature B cells were also not significantly changed in IP3R-TKO mice (Fig. 2A, 2B). By contrast, the number of transitional B cells was significantly increased, whereas the number of recirculating mature B cells was significantly reduced, in bone marrow of IP3R-TKO mice compared with control mice (Fig. 2A, 2B).
Deletion of IP3Rs partially blocked late conventional B2 B cell development. (A) Surface CD19 and B220 expression of lymphocytes in the bone marrow of control and IP3R-TKO mice. Lymphocytes were first gated based on FSC and SSC. CD19+B220+ cells were subgated, and surface expression of IgM was examined. (B) Cell numbers of pro/pre-B (B220+IgM−), immature (B220lowIgMint), transitional (B220intIgMhigh), and recirculating mature (B220highIgMint) B cells. n = 11 mice per group. Data represent mean ± SEM. (C) B cell subsets in the spleen of control and IP3R-TKO mice. Immature (B220+CD93+) and mature (B220+CD93−) B cells are depicted within the lymphocyte gate. Gated on immature B cells, T1 (CD23−IgMhigh), T2 (CD23+IgMhigh), and T3 (CD23+IgMlnt) were distinguished based on differential expression of CD23 and IgM. Gated on mature B cells, FO (CD21intCD23high) and MZ (CD21highCD23−) B cells were shown based on CD21 and CD23 expression. (D) Cell numbers of T1, T2, T3, FO, and MZ B cells in spleen of control and IP3R-TKO mice. n = 4 mice per group. Data represent mean ± SEM. (E and F) Percentages of B220+ cells within the lymphocyte gate in lymph node, peripheral blood, and peritoneal cavity of control and IP3R-TKO mice. n = 5–7 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.
Deletion of IP3Rs partially blocked late conventional B2 B cell development. (A) Surface CD19 and B220 expression of lymphocytes in the bone marrow of control and IP3R-TKO mice. Lymphocytes were first gated based on FSC and SSC. CD19+B220+ cells were subgated, and surface expression of IgM was examined. (B) Cell numbers of pro/pre-B (B220+IgM−), immature (B220lowIgMint), transitional (B220intIgMhigh), and recirculating mature (B220highIgMint) B cells. n = 11 mice per group. Data represent mean ± SEM. (C) B cell subsets in the spleen of control and IP3R-TKO mice. Immature (B220+CD93+) and mature (B220+CD93−) B cells are depicted within the lymphocyte gate. Gated on immature B cells, T1 (CD23−IgMhigh), T2 (CD23+IgMhigh), and T3 (CD23+IgMlnt) were distinguished based on differential expression of CD23 and IgM. Gated on mature B cells, FO (CD21intCD23high) and MZ (CD21highCD23−) B cells were shown based on CD21 and CD23 expression. (D) Cell numbers of T1, T2, T3, FO, and MZ B cells in spleen of control and IP3R-TKO mice. n = 4 mice per group. Data represent mean ± SEM. (E and F) Percentages of B220+ cells within the lymphocyte gate in lymph node, peripheral blood, and peritoneal cavity of control and IP3R-TKO mice. n = 5–7 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.
To determine whether expression of CD19-Cre could affect B cell development, we further examined B cell populations in bone marrow of Itpr1f/+Itpr2f/+Itpr3f/+CD19-Cre+ and Itpr1f/+Itpr2f/+Itpr3f/+CD19-Cre− mice, and found that the percentages of total CD19+B220+, pro/pre-B, immature, transitional, and recirculating B cells were not significantly changed in Cre+ mice compared with Cre− mice (Supplemental Fig. 2A), suggesting that expression of CD19-Cre alone did not significantly alter B cell development. Furthermore, we observed no changes in the percentages of total CD19+B220+, pro/pre-B, immature, transitional, and recirculating B cells in bone marrow of Itpr1f/fItpr2f/fItpr3f/+CD19-Cre+ (IP3R1/R2 DKO), Itpr1f/fItpr2f/+Itpr3f/fCD19-Cre+ (IP3R1/R3 DKO), and Itpr1f/+Itpr2f/fItpr3f/f CD19-Cre+ (IP3R2/R3 DKO) mice compared with control mice (Supplemental Fig. 2B). Consistently, we found that BCR-induced Ca2+ signals in IP3R1/R2-DKO, IP3R1/R3-DKO, or IP3R2/R3-DKO B cells were also comparable with that in control B cells (Supplemental Fig. 2C), suggesting that different IP3R subtypes could play a redundant role in regulating both BCR-mediated Ca2+ release and B cell development.
We further examined B cell maturation in the spleen. Once exiting bone marrow, transitional B cells migrate to the spleen and undergo further maturation (2). In the spleen, CD93-expressing transitional B cells can be subdivided into three subsets: transitional type 1 (T1), transitional type 2 (T2), and transitional type 3 (T3) B cells, based on differential expression of CD23 and IgM (2). Among these transitional B cell types, T2 B cells are thought to give rise to mature FO B cells and MZ B cells (2). Compared with control mice, we found that both the number and the percentage of T2 B cells (25.6 ± 1.1% in control mice versus 38.0 ± 1.6% in IP3R-TKO mice within B220+CD93+ B cells gate, n = 4 mice for both groups) were significantly increased in the spleen of IP3R-TKO mice (Fig. 2C, 2D). Consistent with what we found in the bone marrow, the number of FO B cells was also significantly decreased in IP3R-TKO spleens (Fig. 2C, 2D). However, we did not find significant changes in the number of MZ B cells in IP3R-TKO spleens (Fig. 2C, 2D). FO B cells can freely recirculate among the peripheral secondary lymphoid tissues. Therefore, we further investigated B cell population in the lymph node, peripheral blood, and peritoneal cavity of control and IP3R-TKO mice. Consistently, the percentage of B220+ B cells was significantly decreased in all peripheral secondary lymphoid tissues of IP3R-TKO mice compared with control mice (Fig. 2E, 2F). In summary, these data suggest that IP3R-mediated Ca2+ signaling may play an important role in late B cell development, especially from T2 to FO B cell development.
We next investigated whether increased numbers of transitional B cells in IP3R-TKO mice resulted from altered positive or negative selection of immature B cells. It has been shown that activation of Ras could break B cell tolerance to promote autoreactive B cell differentiation and inhibit receptor editing via ERK and PI3K pathways (27). Therefore, we measured ERK1/2 phosphorylation and expression levels of Foxo1, Rag1, and Rag2, all of which were suggested to be regulated by PI3K and essential for receptor editing, in bone marrow immature B cells of control and IP3R-TKO mice. We found that ERK1/2 phosphorylation is slightly decreased in IP3R-TKO immature B cells (Supplemental Fig. 3A). In addition, we did not find any significant changes of Foxo1, Rag1, and Rag2 mRNA levels between control and IP3R-TKO immature B cells (Supplemental Fig. 3B). Taken together, these data suggested that increased numbers of transitional B cells in IP3R-TKO mice might not be caused by altered positive or negative selection of immature B cells. Because BAFF-R has also been shown to play an important role in regulating the survival and maturation of transitional B cells (29), we further checked the expression of Baffr in bone marrow transitional B cells. However, we did not find any significant change of Baffr mRNA levels in bone marrow transitional B cells between control and IP3R-TKO mice (Supplemental Fig. 3C).
In addition to conventional B2 B cells, B1 B cells are another subclass of B cell lymphocytes, which constitute a minor fraction of B cells in the spleen but predominately reside in the peritoneal and pleural cavities (30). In mice, B1 B cells can be further divided into B1a (CD5+) and B1b (CD5−) subsets (31). A series of mutations that interfere with normal BCR signaling has been shown to reduce B-1 B cell numbers, suggesting a crucial requirement of BCR signals for B1 B cell development (31). Therefore, we next investigated the effect of IP3R deficiency in B1 B cell development. Compared with control mice, the proportion of B1a B cells in the peritoneal cavity was significantly reduced in IP3R-TKO mice (Fig. 3A, 3B).Similarly, the proportion of splenic B1a and B1b B cells was also decreased in IP3R-TKO mice (Fig. 3C). Taken together, these results suggest that IP3R-mediated Ca2+ signaling could regulate both conventional B2 B cell and B1 B cell development in vivo.
Loss of IP3Rs resulted in reduced B1 B cell numbers. (A) Surface expression of CD43 and CD5 on B220+-gated lymphocytes isolated from the peritoneal cavities of control and IP3R-TKO mice. Percentages of B1a (CD43+CD5+), B1b (CD43+CD5−), and B2 (CD43−CD5−) cells in the (B) peritoneal cavity and (C) spleen of control and IP3R-TKO mice. n = 5–6 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.
Loss of IP3Rs resulted in reduced B1 B cell numbers. (A) Surface expression of CD43 and CD5 on B220+-gated lymphocytes isolated from the peritoneal cavities of control and IP3R-TKO mice. Percentages of B1a (CD43+CD5+), B1b (CD43+CD5−), and B2 (CD43−CD5−) cells in the (B) peritoneal cavity and (C) spleen of control and IP3R-TKO mice. n = 5–6 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.
Loss of IP3Rs impairs BCR-mediated cell proliferation and survival
We next investigated the role of IP3Rs in regulating B cell proliferation and survival under various mitogenic stimuli including anti-IgM, LPS, and anti-CD40. Purified splenic B cells were labeled with CFSE, and cell proliferation was then measured by CFSE dilution. BCR stimulation induced several rounds of cell division in control B cells, whereas IP3R-TKO B cells did not undergo cell division in response to anti-IgM stimulation (Fig. 4A, 4B). By contrast, cell division induced by LPS or anti-CD40 was unchanged in IP3R-TKO B cells (Fig. 4A, 4B), suggesting that LPS-induced or anti-CD40–induced cell proliferation is independent of IP3Rs, which is also consistent with a previous report that anti-CD40 or LPS did not induce Ca2+ mobilization in B cells (11). However, we also found that costimulation of anti-IgM with anti-CD40 could significantly restore the proliferative ability of IP3R-TKO B cells (Fig. 4A, 4B). Furthermore, we examined the effects of IP3R deficiency on B cell survival in response to various mitogenic stimuli. The percentage of viable B cells was determined by PI exclusion. Survival rate was dramatically increased in control B cells with BCR stimulation, which was severely inhibited in IP3R-TKO B cells (Fig. 4C). By contrast, survival rates of B cells in response to LPS or anti-CD40 were comparable between control and IP3R-TKO mice (Fig. 4C), suggesting that IP3Rs could also be dispensable for LPS-induced or anti-CD40–induced B cell survival. Furthermore, costimulation of anti-IgM with anti-CD40 could partially restore the survival rate of IP3R-TKO B cells (Fig. 4C). Taken together, these results clearly demonstrate that IP3R-mediated Ca2+ signaling is specifically required for BCR-induced B cell proliferation and survival. In contrast, our data also suggest that signaling pathways evoked by anti-CD40 could compensate for IP3R deficiency after BCR stimulation, even though the effects of anti-CD40 on B cell proliferation and survival are not dependent on IP3Rs.
Deletion of IP3Rs impaired BCR-mediated proliferation and survival. (A) Proliferation as assessed by CFSE labeling. Splenic B cells were isolated from control and IP3R-TKO mice, and stimulated for 48 h with vehicle, anti-IgM (10 μg/ml), anti-CD40 (10 μg/ml), LPS (10 μg/ml), and a combination of anti-IgM (10 μg/ml) with anti-CD40 (10 μg/ml), respectively. The numbers within each histogram represent the percentage of divided cells as measured by CFSE dilution. (B) Percentages of dividing cells in response to different stimuli. n = 4 mice per group. Data represent mean ± SEM. (C) Percentages of PI− control and IP3R-TKO B220+ B cells. Cells were cultured and stimulated as described in (A). n = 4 mice per group. Data represent mean ± SEM. (D) Western blot analysis of whole-cell lysates of control and IP3R-TKO splenic B cells stimulated with anti-IgM (10 μg/ml) for various durations as depicted. β-Actin was used as loading control. Results represent one of three representative experiments. (E) Quantitative RT-PCR analysis of the expression of cyclin D2 (Ccnd2) in control and IP3R-TKO splenic B cells at basal levels or stimulated for 12 h with anti-IgM (10 μg/ml). n = 3 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.
Deletion of IP3Rs impaired BCR-mediated proliferation and survival. (A) Proliferation as assessed by CFSE labeling. Splenic B cells were isolated from control and IP3R-TKO mice, and stimulated for 48 h with vehicle, anti-IgM (10 μg/ml), anti-CD40 (10 μg/ml), LPS (10 μg/ml), and a combination of anti-IgM (10 μg/ml) with anti-CD40 (10 μg/ml), respectively. The numbers within each histogram represent the percentage of divided cells as measured by CFSE dilution. (B) Percentages of dividing cells in response to different stimuli. n = 4 mice per group. Data represent mean ± SEM. (C) Percentages of PI− control and IP3R-TKO B220+ B cells. Cells were cultured and stimulated as described in (A). n = 4 mice per group. Data represent mean ± SEM. (D) Western blot analysis of whole-cell lysates of control and IP3R-TKO splenic B cells stimulated with anti-IgM (10 μg/ml) for various durations as depicted. β-Actin was used as loading control. Results represent one of three representative experiments. (E) Quantitative RT-PCR analysis of the expression of cyclin D2 (Ccnd2) in control and IP3R-TKO splenic B cells at basal levels or stimulated for 12 h with anti-IgM (10 μg/ml). n = 3 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01, ***p < 0.001 versus control.
We further investigated BCR-induced cell survival and proliferation in different B cell subsets, including transitional, FO, and MZ B cells. We found that cell proliferation induced by anti-IgM was all reduced in transitional, FO, and MZ B cells of IP3R-TKO mice compared with control mice (Supplemental Fig. 4A–C). In contrast, we found that the percentages of viable PI− transitional and FO B cells after stimulation of anti-IgM were significantly reduced in IP3R-TKO mice compared with control mice, whereas the percentage of viable PI− MZ B cells was comparable between control and IP3R-TKO mice (Supplemental Fig. 4D).
To further understand the molecular mechanisms underlying the impaired BCR-induced B cell proliferation and survival after IP3R deletion, we examined the expression and activation of downstream effectors including NFAT, ERK, and JNK in isolated naive B cells after BCR ligation. First, BCR stimulation in control B cells resulted in apparent NFAT1 dephosphorylation, which was almost totally blocked in IP3R-TKO B cells (Fig. 4D). On the contrary, phosphorylation of ERK and JNK induced by BCR stimulation was increased in IP3R-deficient B cells compared with control B cells (Fig. 4D), consistent with a previous report using Mef2c-knockout B cells in which defective cell proliferation was accompanied by enhanced activation of ERK and JNK (32). Even though we did not find a significant change of Mef2c expression level between control and IP3R-TKO splenic B cells (Supplemental Fig. 4E), we found that expression of Ccnd2, a downstream target of Mef2c, was significantly reduced at both basal levels and in response to BCR stimulation (Fig. 4E). Because Mef2c has also been suggested as a target of calcineurin (32), these results implicate that activation of calcineurin after IP3R-mediated Ca2+ release could be a critical step underlying the regulation of BCR-mediated cell proliferation.
Deletion of IP3Rs affects in vivo immune responses
We next investigated whether deletion of IP3Rs could affect the ability of the immune system to produce Abs as a consequence of altered B cell development and function. First, we found that serum titers of IgM, IgG1, IgG2b, IgG3, and IgA were comparable between unimmunized IP3R-TKO and control mice (Fig. 5A), indicating that IP3R-mediated Ca2+ signaling is not necessary for maintaining basal serum Ig levels. To assess humoral immune responses, we first examined TD Ab responses by immunizing mice with NP-CGG, and we monitored the production of NP-specific IgM and IgG1 Abs for the next 3 consecutive wk. The production of both NP-specific IgM and NP-specific IgG1 was significantly reduced in IP3R-TKO mice at 1 wk postimmunization, but recovered to comparable levels within the following 2 wk when compared with control mice (Fig. 5B). During TD responses, TD Ags will lead to germinal center (GC) formation, where B cells undergo expansion and affinity maturation (33). Consistently, we also found that the percentage of splenic GC B cells (defined as B220+FashighGL7+) at 1 wk post NP-CGG immunization in IP3R-deficient mice was significantly decreased when compared with control mice (Fig. 5C). We then analyzed TI Ab responses by immunizing mice with NP-LPS (TI-1 Ag) or NP-Ficoll (TI-2 Ag), and we monitored the production of Ag-specific IgM and IgG3 Abs for the next 3 consecutive wk. We found that the level of NP-specific IgM in IP3R-TKO mice was slightly higher than that of control mice at 1 wk postimmunization of NP-LPS (Fig. 5D), and the level of NP-specific IgM in IP3R-TKO mice was slightly lower than that of control mice at 1 wk postimmunization with NP-Ficoll (Fig. 5E). However, we did not observe any significant difference in the levels of NP-specific IgG3 between control and IP3R-TKO within 3 wk postimmunization of NP-LPS or NP-Ficoll (Fig. 5D, 5E). Taken together, our results suggest that loss of IP3R-mediated Ca2+ release in B cells might have an acute, but not chronic, influence on humoral immune response.
TD and TI Ab responses in control and IP3R-TKO mice. (A) ELISA was used to measure basal Ig titers of IgM, IgG1, IgG2b, IgG3, and IgA in the sera of nonimmunized control and IP3R-TKO mice. n = 8 mice per group. Data represent mean ± SEM. (B) IgM and IgG1 NP-specific Ab responses of control and IP3R-TKO mice immunized with NP-CGG, as measured by NP-specific ELISA at each time point. n = 4–5 mice per group. Data represent mean ± SEM. (C) Percentages of splenic GC B cells (B220+FashighGL7+) within B220+ gate in control and IP3R-TKO mice at baseline (n = 3 mice for both control and IP3R-TKO groups) and 1 wk after NP-CGG immunization (n = 7 mice for both control and IP3R-TKO groups). Data represent mean ± SEM. (D and E) NP-specific Ab responses of control and IP3R-TKO mice immunized with NP-LPS (D) or NP-Ficoll (E), as measured by NP-specific ELISA at each time point. n = 3–4 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01 versus control.
TD and TI Ab responses in control and IP3R-TKO mice. (A) ELISA was used to measure basal Ig titers of IgM, IgG1, IgG2b, IgG3, and IgA in the sera of nonimmunized control and IP3R-TKO mice. n = 8 mice per group. Data represent mean ± SEM. (B) IgM and IgG1 NP-specific Ab responses of control and IP3R-TKO mice immunized with NP-CGG, as measured by NP-specific ELISA at each time point. n = 4–5 mice per group. Data represent mean ± SEM. (C) Percentages of splenic GC B cells (B220+FashighGL7+) within B220+ gate in control and IP3R-TKO mice at baseline (n = 3 mice for both control and IP3R-TKO groups) and 1 wk after NP-CGG immunization (n = 7 mice for both control and IP3R-TKO groups). Data represent mean ± SEM. (D and E) NP-specific Ab responses of control and IP3R-TKO mice immunized with NP-LPS (D) or NP-Ficoll (E), as measured by NP-specific ELISA at each time point. n = 3–4 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01 versus control.
Deletion of IP3Rs reduces IL-10 production
In addition to producing Abs to protect against infectious Ags, B cells possess a suppressive capability to negatively regulate inflammatory responses by secreting anti-inflammatory cytokines such as IL-10 (34). We next investigated whether deletion of IP3Rs could influence the regulatory function of B cells by measuring IL-10 production in isolated splenic B cells. It has been shown that BCR stimulation alone is not able to induce IL-10 secretion, but BCR stimulation could potentiate IL-10 secretion when B cells are first preactivated with the agonist of TLRs such as LPS (11, 35, 36). Therefore, we pretreated splenic naive B cells with LPS for 48 h and then applied BCR stimulation with anti-IgM to induce IL-10 secretion. Using quantitative RT-PCR analysis, we found that BCR stimulation could dramatically increase the expression of IL-10 in LPS-activated control B cells, which was significantly inhibited in LPS-activated IP3R-TKO B cells (Fig. 6A). Moreover, BCR stimulation also dramatically increased the level of secreted IL-10 in the medium of LPS-activated control B cells, which was also dramatically inhibited in LPS-activated IP3R-TKO B cells (Fig. 6B).
IL-10 production was impaired in IP3R-TKO B cells because of decreased CD1dhighCD5+ regulatory B cell numbers and defective activation of calcineurin-NFAT signaling. (A) Control and IP3R-TKO B cells were cultured with LPS (10 μg/ml) for 48 h (LPS-activated B cells) followed by stimulation with vehicle or anti-IgM for 3 h. Quantitative RT-PCR analysis was used to assess the mRNA levels of IL-10. n = 6 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. **p < 0.01 versus control. (B) ELISA analysis of IL-10 production in LPS-activated control and IP3R-TKO B cells stimulated with vehicle or anti-IgM for 24 h. n = 6 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01 versus control. (C) Expression of surface CD1d and CD5 on B220+-gated B cells isolated from control (n = 6) and IP3R-TKO (n = 5) mice. Histograms show the averaged percentages of CD1dhigh/CD5+ B cells in control and IP3R-TKO mice. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. ***p < 0.001 versus control. (D) Quantitative RT-PCR analysis of IL-10 mRNA levels in LPS-activated control and IP3R-TKO B cells treated with vehicle (DMSO), CsA (1 μM), or FK506 (1.25 μM) for 1 h followed by stimulation with anti-IgM for 3 h. n = 4 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. ***p < 0.001 versus DMSO. (E) Western blot analysis of NFAT1 in whole-cell lysates of LPS-activated control and IP3R-TKO B cells stimulated with anti-IgM (10 μg/ml) for various durations as depicted. β-Actin was used as loading control. Results represent one of three representative experiments.
IL-10 production was impaired in IP3R-TKO B cells because of decreased CD1dhighCD5+ regulatory B cell numbers and defective activation of calcineurin-NFAT signaling. (A) Control and IP3R-TKO B cells were cultured with LPS (10 μg/ml) for 48 h (LPS-activated B cells) followed by stimulation with vehicle or anti-IgM for 3 h. Quantitative RT-PCR analysis was used to assess the mRNA levels of IL-10. n = 6 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. **p < 0.01 versus control. (B) ELISA analysis of IL-10 production in LPS-activated control and IP3R-TKO B cells stimulated with vehicle or anti-IgM for 24 h. n = 6 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. *p < 0.05, **p < 0.01 versus control. (C) Expression of surface CD1d and CD5 on B220+-gated B cells isolated from control (n = 6) and IP3R-TKO (n = 5) mice. Histograms show the averaged percentages of CD1dhigh/CD5+ B cells in control and IP3R-TKO mice. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. ***p < 0.001 versus control. (D) Quantitative RT-PCR analysis of IL-10 mRNA levels in LPS-activated control and IP3R-TKO B cells treated with vehicle (DMSO), CsA (1 μM), or FK506 (1.25 μM) for 1 h followed by stimulation with anti-IgM for 3 h. n = 4 mice per group. Data represent mean ± SEM. Significance was determined by the two-tailed, unpaired Student t test. ***p < 0.001 versus DMSO. (E) Western blot analysis of NFAT1 in whole-cell lysates of LPS-activated control and IP3R-TKO B cells stimulated with anti-IgM (10 μg/ml) for various durations as depicted. β-Actin was used as loading control. Results represent one of three representative experiments.
We next investigated the potential cellular and molecular mechanisms underlying the regulation of IL-10 production by IP3R-mediated Ca2+ signaling. It has been shown that a regulatory B10 subset identified as CD1dhighCD5+ B cell was the major cell type responsible for producing IL-10 (11, 37). Therefore, we first examined whether deletion of IP3Rs could cause aberrant development of B10 cells. Compared with control mice, the percentage of splenic CD1dhighCD5+ B cells within the B220+ B cells gate in IP3R-TKO mice was significantly decreased (Fig. 6C), suggesting that IP3R-Ca2+ signaling plays an important role in regulatory B10 cell development or maintenance. Because the calcineurin-NFAT signaling pathway has been shown to play a critical role in regulating BCR-mediated IL-10 production (11, 38), we next examined whether deletion of IP3Rs could disrupt the calcineurin-NFAT signaling pathway in B cells. First, calcineurin inhibitors CsA and FK506 could completely abolish the expression of IL-10 induced by BCR stimulation in both LPS-activated control and IP3R-TKO B cells (Fig. 6D), indicating that IL-10 expression in both cell populations relies on the calcineurin-NFAT signaling pathway. Furthermore, we also found that deletion of IP3Rs diminished the activation of NFAT1 in LPS-activated B cells. In LPS-activated control cells, BCR stimulation could induce quick and sustained dephosphorylation of NFAT1. However, the dephosphorylation of NFAT1 was dramatically suppressed in LPS-activated IP3R-TKO cells (Fig. 6E). Taken together, these results suggest that deletion of IP3Rs in B cells resulted in a decreased percentage of CD1dhighCD5+ B cells and defective NFAT activation, both of which together caused reduced IL-10 expression and secretion.
Discussion
In this study, we demonstrated that IP3R-mediated Ca2+ release plays an essential role in regulating B cell development and function using B cell–specific IP3R knockout mouse models. In B cells, the strength of BCR signaling has been proposed to guide the positive selection of B cells and negative selection to eliminate autoreactive B cells, thereby regulating the development of B cells. A central response to BCR cross-linking is the elevation of intracellular Ca2+ concentration (39), which is mediated by Ca2+ release from the ER via IP3Rs and the subsequent SOCE. Mutations in molecules that positively or negatively regulate Ca2+ signaling have been reported to regulate B cell development and function. The absence of positive regulators such as Syk, Btk, BLNK, or PLC-γ2 results in impaired Ca2+ signaling and blocked B cell development at the early stages in bone marrow (7, 40–43). By contrast, loss of negative modulators such as SHP-1 or CD22 leads to enhanced Ca2+ increase, hyperresponsive B cells, and autoimmunity (44, 45). All of these studies implicate that Ca2+ signals in B cells may participate in regulating both development and function. Consistently, the physiological role of Ca2+ signals mediated by SOCE in regulating B cell function has been well studied in human patients carrying ORAI1 and STIM1 mutations (13, 14) or mice with deleted Orai1 and STIM proteins (10–12). On the contrary, endogenous Orai1, STIM1, and STIM2 were shown to be dispensable for B cell development (10–12). However, whether endogenous Ca2+ signals can influence B cell development and the functional difference between Ca2+ release from ER and SOCE remain unclear. Therefore, our study provided evidence for the first time, to our knowledge, that endogenous Ca2+ signals mediated by IP3Rs are essential for B cell development. Loss of all three IP3R subtypes in B cells could totally abrogate BCR-induced Ca2+ release and the subsequent SOCE, and impair development of conventional B2, B1, and regulatory B10 cells. It is intriguing that the initial intracellular Ca2+ increase mediated by IP3Rs alone was sufficient to drive normal B cell development, whereas sustained intracellular Ca2+ increase mediated by both IP3Rs and SOCE was required for normal B cell function.
Our results demonstrated that IP3R-mediated Ca2+ signals are specifically important for T2 B cell to FO B cell maturation. Deletion of IP3Rs increased transitional B cell numbers and decreased mature B cell numbers in the bone marrow. Consistently, the numbers of T2 B cells were significantly increased, whereas FO B cell numbers were decreased in the spleen. In addition, all peripheral lymphoid tissues including the lymph node, peripheral blood, and peritoneal cavity showed reduced proportions of B cells. A series of transcription factors such as Aiolos, Notch-2, c-Myb, Pax-5, and NF-κB have been reported to be involved in peripheral B cell development (46). Notably, the coordination of NF-κB family members, RelB and c-Rel, has been demonstrated to regulate late B cell development from transitional to mature B cells (47). Whether the defect in transitional to mature B cell development in the IP3R-deficient mice was caused by impaired activation of NF-κB or other transcription factors remains to be investigated. In contrast, IP3R-mediated Ca2+ signaling is essential for B1 B cell development. Previous studies have shown that B1 B cell development or maintenance is regulated by the calcineurin-NFAT signaling pathway, given that both calcineurin b1–deficient mice and NFAT2-deficient mice exhibited reduced B1 B cell numbers (48, 49). In IP3R-TKO B cells, the activation of NFAT1, another member of NFAT proteins, was defective in response to BCR stimulation, implicating that abnormal B1 B cell development in IP3R-TKO mice might result from defective calcineurin-NFAT signaling.
We also demonstrated that IP3Rs are required for B cell proliferation and survival in response to BCR ligation. Calcineurin has been shown to play an important role in B cell proliferation (49). In IP3R-deficient B cells, dephosphorylation of NFAT1 in response to BCR stimulation was dramatically decreased. In addition, Mef2c, another calcineurin-regulated transcription factor, was also essential for BCR-mediated B cell proliferation and survival through regulating gene expression of cyclin D2 (32). IP3R-TKO B cells also showed decreased expression of cyclin D2 at the mRNA level when stimulated with anti-IgM. In Mef2c-deficient B cells, BCR stimulation resulted in increased phosphorylation levels of JNK and ERK1/2, which is consistent with what we found in IP3R-TKO B cells. These data together implicated that a defective calcineurin-Mef2c pathway might be one of the molecular mechanisms accounting for the reduced proliferation of IP3R-TKO B cells. In contrast, IP3R-TKO B cells proliferated normally in response to LPS and anti-CD40 stimulation compared with control B cells, suggesting that LPS-induced and anti-CD40–induced B cell proliferation is independent of intracellular Ca2+ signaling, which is also consistent with the fact that both LPS and anti-CD40 were not able to induce intracellular Ca2+ increase in B cells (11). Furthermore, restoration of BCR-induced cell proliferation in IP3R-deficient B cells by costimulation with anti-CD40 might be because of the fact that both BCR- and CD40-induced B cell proliferation can share several common molecular mechanisms, one of which has been identified as cyclin D2 upregulation (50).
In IP3R-TKO mice, TD Ab response was impaired after deletion of IP3Rs. It has been shown that FO B cells are the main mediator responsible for TD Ab response, in which B cells undergo clonal expansion by receiving cognate T cell help through cytokine stimulation and CD40 engagement in GCs (3). Therefore, reduced GC B cell numbers and defective proliferation of splenic B cells might account for the impaired TD Ab response in IP3R-TKO mice. However, it remains unclear whether reduced Ab production and GC formation by deletion of IP3Rs was due to impaired B cell development or cell-autonomous defects in mature B cells. Future experiments using an inducible CreERT2 system instead of a constitutive CD19-Cre system would be helpful to address this question. In addition to TD Ab response, we found that the level of NP-specific IgM in IP3R-TKO mice was slightly higher than that of control mice at 1 wk postimmunization of NP-LPS. B1 and MZ B cells have been proposed to be responsible for TI-2 Ab responses (4, 51). It is likely that the reduced B1 B cell numbers in IP3R-TKO mice contribute to the lower NP-specific IgM production at the first wk after immunization. Furthermore, we found that the level of NP-specific IgM was slightly increased in IP3R-TKO mice in response to TI-1 Ags, NP-LPS. Because TI-1 Ag is thought to bind both BCR and TLRs to transduce a strong signal for B cell activation (52), one possible explanation could be that TLR signaling in IP3R-TKO mice might compensate B cell activation and function in response to TI-1 Ags. Taken together, our data suggest that IP3R-mediated Ca2+ signaling contributes to Ab response in vivo, which is also partially consistent with a previous study of calcineurin b1–deficient mice (11). However, the mechanism underlying why loss of IP3Rs had only a short-term effect in Ab production requires further investigation. Because Ab production by B cells is critical for protecting from infection in vivo, deletion of IP3Rs in mice may potentially lead to difficulty in fighting infectious diseases, which would be worthy to be further examined using different mouse models of infection.
In addition, B cells are known to play a regulatory role in the immune system by secreting inflammatory cytokines, one of which is IL-10, that can function to prevent inflammatory and autoimmune pathologies (53). The signaling pathways mediated by TLRs, combined with BCR and CD40, are critical for B cells to suppress autoimmunity through IL-10 production in vivo (35, 53). In IP3R-TKO B cells that are preactivated by LPS, IL-10 production induced by BCR stimulation was severely impaired. In STIM1/STIM2 double-deficient B cells, IL-10 production is also reduced, which could result from the defective activation of the calcineurin-NFAT signaling pathway after BCR stimulation (11). Consistently, we also found that dephosphorylation of NFAT1 in response to BCR stimulation was decreased in LPS-activated IP3R-deficient B cells, suggesting that BCR-mediated IL-10 production requires both IP3R-mediated Ca2+ release and the subsequent SOCE. Intriguingly, we also found that the number of B10 cells is decreased in IP3R-defecient mice. Although there is no specific cell surface marker unique to all B10 cells, they have been proposed to be enriched within the splenic CD1dhighCD5+ subset (54). BCR signaling is thought to play a role in B10 cell development, given that human CD19-overexpressing mice showed increased BCR signaling and elevated B10 cell numbers (36), whereas CD19-deficient mice have impaired BCR signaling and fewer B10 cell numbers (37). Moreover, multiple strains of transgenic mice with a fixed BCR repertoire have reduced B10 cell numbers (55). IP3R-mediated Ca2+ signaling is critical to maintain CD1dhighCD5+ B cell numbers. In IP3R-TKO mice, the number of CD1dhighCD5+ B cells was decreased, whereas the numbers of this population is not altered by loss of both STIM1 and STIM2, which is also consistent with the finding that impaired B2 and B1 cell development was only found in IP3R-deficient mice and not in STIM1/STIM2 DKO mice. The role of regulatory B10 cells has been extensively examined in mouse models of experimental autoimmune encephalomyelitis, collagen-induced arthritis, and contact hypersensitivity (55). Further studies using these mouse models may help to determine whether reduced production of IL-10 and B10 cell numbers in IP3R-deficient mice could exacerbate disease severity.
In summary, we demonstrated that IP3R-mediated Ca2+ release is important for maintaining normal numbers of conventional B2, B1, and regulatory B10 B cells, as well as regulating the humoral immune response in vivo. We also found that IP3R-deficient B cells exhibited reduced BCR-mediated proliferation and survival, defective calcineurin-NFAT signaling, and impaired IL-10 production. Therefore, our study revealed a dual role of IP3R-mediated Ca2+ signals in B cell development and function.
Acknowledgements
We thank Xing Chang (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) for critical suggestions about experimental design, Ju Chen (University of California, San Diego) and Jennifer Veevers for critical reading of the manuscript, and Xiaodi Chen (Peking University Shenzhen Hospital) and Wugen Zhan (Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences) for technical support of blood collection and flow cytometry.
Footnotes
This work was supported by the National Key Basic Research Program of China (Grant 2013CB531200), the National Science Foundation of China (Grants 31370823 and 91439130), the Shenzhen Basic Research Foundation (Grants JCYJ20140509093817680 and JCYJ20160428154108239), and the Guangdong Province Basic Research Foundation (Grant 2016A020216003).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- B10
IL-10–producing B cell
- Ca2+
intracellular calcium
- CGG
chicken γ-globulin
- CsA
cyclosporin A
- DKO
double-knockout
- ER
endoplasmic reticulum
- FO
follicular
- GC
germinal center
- IP3
inositol 1,4,5-trisphosphate
- IP3R
IP3 receptor
- IP3R-TKO
IP3R triple-knockout
- MZ
marginal zone
- no.
number
- NP
4-hydroxy-3-nitrophenyl
- PI
propidium iodide
- PLC
phospholipase C
- pre-B
precursor B
- PSS
physiological saline solution
- RT-PCR
real-time PCR
- SOCE
store-operated Ca2+ entry
- STIM1
stromal interaction molecule 1
- T1
transitional type 1
- T2
transitional type 2
- T3
transitional type 3
- TD
T cell–dependent
- TG
thapsigargin
- TI
T cell–independent.
References
Disclosures
The authors have no financial conflicts of interest.