Unlike conventional B cells, regulatory B cells exhibit immunosuppressive functions to downregulate inflammation via IL-10 production. However, the molecular mechanism regulating the production of IL-10 is not fully understood. In this study, we report the finding that activation-induced cytidine deaminase (AID) is highly upregulated in the IL-10–competent B cell (B10) cell from Innp5dfl/flAicdaCre/+ mice, whereas the 5′ inositol phosphatase SHIP-1 is downregulated. Notably, SHIP-1 deficiency in AID+ B cells leads to a reduction in cell count and impaired IL-10 production by B10 cells. Furthermore, the Innp5dfl/flAicdaCre/+ mouse model shows B cell–dependent autoimmune lupus-like phenotypes, such as elevated IgG serum Abs, formation of spontaneous germinal centers, production of anti-dsDNA and anti-nuclear Abs, and the obvious deposition of IgG immune complexes in the kidney with age. We observe that these lupus-like phenotypes can be reversed by the adoptive transfer of B10 cells from control Innp5dfl/fl mice, but not from the Innp5dfl/flAicdaCre/+ mice. This finding highlights the importance of defective B10 cells in Innp5dfl/flAicdaCre/+ mice. Whereas p-Akt is significantly upregulated, MAPK and AP-1 activation is impaired in B10 cells from Innp5dfl/flAicdaCre/+ mice, resulting in the reduced production of IL-10. These results show that SHIP-1 is required for the maintenance of B10 cells and production of IL-10, and collectively suggests that SHIP-1 could be a new potential therapeutic target for the treatment of autoimmune diseases.

B lymphocytes use their surface-expressed BCRs to sense and acquire Ags, which subsequently trigger the activation, proliferation, and differentiation of B cells for the production of Abs (13). However, the suppressive function of B cells was also suggested when B cell–deficient mice exhibited chronic inflammation (4, 5). These observations led to the identification of regulatory B cells (Bregs), a subset of B cells characterized by their key ability to produce IL-10 (59). Bregs have been shown to suppress inflammation to prevent immunopathology and to support immunological tolerance (10, 11). Of the different Breg subsets identified thus far, transitional 2 marginal-zone precursors (T2-MZPs) Bregs and IL-10–competent B cells (B10 cells) are the two most important subsets (6, 12, 13). Additionally, B1 cells may also play an immunosuppressive function by producing IL-10 (14, 15). In MRL lpr/lpr systemic lupus erythematosus (SLE)–prone mice, the deficiency of Bregs favored the expansion of autoreactive B cells and the production of autoantibodies against small nuclear ribonucleoproteins, which were also termed anti-Sm Abs, one of the characteristic Abs produced in SLE patients (11, 1618). In different autoimmune disease models, B10 cells were able to control the initiation of experimental autoimmune encephalomyelitis (19), whereas T2-MZP cells could prevent and even ameliorate the experimental arthritis (12). B10 cells also exhibited an important protective role in the systemic autoimmune disease (20). In human PBMCs, a population of CD24highCD27+ Bregs has been identified which parallels B10 cells in mice (21). Clinically, it has been reported that the IL-10–producing capability of Bregs is severely compromised in both SLE (22) and rheumatoid arthritis (2325) patients. This compromised capability of Bregs to produce IL-10 is likely linked to the exacerbated clinical manifestation, especially in patients who have had rheumatoid arthritis for <5 y. These results demonstrate the crucial role of B10 cells in suppressing autoimmune disease.

Despite all these reports demonstrating the function of Bregs in the suppression of autoimmunity in both experimental mice models and clinical studies (11), the molecular mechanism regulating the production of IL-10 is not fully understood. This question is especially intriguing when considering the published clinical data that illustrates that the number of Bregs did not drop, but rather increased, in some SLE patients; however, the IL-10–producing capability of Bregs drastically dropped in SLE patients (22). In this article, we provide a model to show that SHIP-1 is required for IL-10 production in B10 cells through a mechanism of releasing the inhibitory function of AKT on the MAPK and AP-1 activation.

The generation of Innp5dfl/fl mice has been described previously (26). Innp5dfl/fl mice were the gift from Dr. W. Song (University of Maryland, College Park, MD) and Dr. S. Bolland (Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health). AicdaCre/+ mice were purchased from Taconic (La Verne, CA). Mice were housed in a specific pathogen-free barrier facility, and all mice were used according to governmental and institutional guidelines for animal welfare.

Single-cell leukocyte suspensions from the bone marrow, spleen, and peritoneal cavity were generated by gentle dissection. CD16/CD32 (93; BioLegend) was used for blocking of Fc receptors. The following Abs (conjugated to eFluor 450, FITC, PE, PE-Cy5, PerCP/Cy5.5, PE-Cy7, or allophycocyanin) were used: anti-CD1d (1B1; BioLegend), anti-CD3e (145-2C11; eBioscience), anti-CD5 (53–7.3; BD Biosciences), anti-CD19 (1D3; BD Biosciences), anti-CD21 (7G6; BD Biosciences), anti-CD23 (B3B4; eBioscience), anti-CD43 (S7; BD Biosciences), anti-CD93 (AA4.1; eBioscience), anti-CD95 (15A7; eBioscience), anti-B220 (RA3-6B2; BD Biosciences), anti-F4/80 (BM8; eBioscience), anti-GL7 (GL-7; eBioscience), anti-IgD (11-26c.2a; BD Biosciences), anti-IgM (II/41; eBioscience), and anti–IL-10 (JES5-16E3; BD Biosciences). Detection of cell surface marker expression was performed with an LSRFortessa cytometer (BD Biosciences) and cell sorting was performed with the FACSAria (BD Biosciences). Typically, living lymphocytes, judged by forward and side-scatter parameters, were gated for analysis. The data were further analyzed with FlowJo (Tree Star). For IL-10 intracellular staining, splenocytes were resuspended with LPS (10 μg/ml), PMA and ionomycin (50 ng/ml and 1 μg/ml, respectively; Multi Sciences), and monensin (2 μM; BD Biosciences) for 5 h.

Cells were lysed in 2× lysis buffer diluted to 1× with PBS. The 2× lysis buffer consisted of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, and 4% NP-40. Proteins were separated by 10% Bis-Tris PAGE (Life technologies), transferred to polyvinylidene difluoride film, and probed with the Ab of interest and appropriate HRP-conjugated secondary Abs (Tiangen). The targeted signal was normalized to the internal reference. For the phospho-specific signal, it was normalized to the total value, and the internal reference was also shown as reference. Abs specific for SHIP-1, β-actin, phospho-Akt, phospho-JNK, JNK, phospho-ERK, ERK, phospho-p38, p38, c-Jun, phospho-GSK3α/β, and GSK3α/β were purchased from Cell Signaling Technology. Tubulin was from Abcam. GAPDH was from Goodhere. Akt was from Millipore.

Mice were injected with 25 μg of NP33-KLH and 75 μl CFA at day 0, and recalled with 20 μg NP33-KLH in PBS at day 42. Sera were collected at day 7, 14, 21, 42, 49, 56, 70, and 119. The levels of Ag-specific Abs of different isotypes were determined by ELISA using 96-well plates coated with NP-BSA (NP8-BSA or NP30-BSA). The plates were further blocked with 0.3% gelatin in PBS buffer (2 h at 37°C), followed by the addition of serially diluted serum. After an incubation at 37°C for 1 h, 1:10,000 diluted HRP-conjugated goat anti-mouse IgM or IgG (Boster) was used to detect Ab. After washing, O-phenylenediaminde dihydrochloride peroxide solution (Amresco) was added, and the OD was measured with a microtiter plate reader at 490 nm (Bio-Rad) after terminating by 2M H2SO4.

To determine the autoantibody and serum total Ig, serum was obtained at the indicated time. The anti-dsDNA Ab was measured with kits from EUROIMMUN. To determine anti-nuclear Ab (ANA) positivity, serum was diluted to the predetermined optimal concentration and added to fixed HEp-2 cell ANA slides (EUROIMMUN) with Alexa Fluor 488–conjugated goat anti-mouse IgG (Fcγ fragment) (Jackson ImmunoResearch) used as the indirect immunofluorescence detection reagent, and nuclei were detected using ProLong Gold Antifade Mountant with DAPI (Life technologies).

For the serum IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3; goat anti-mouse Igκ-UNLB (Southern Biotech) was used as coating Ag. Serum was diluted to the indicated concentration and incubated for 1 h at room temperature. Bound Abs were revealed by HRP-conjugated goat anti-mouse IgM or IgG (Boster).

For cryosections, kidneys were fixed in lysine-paraformaldehyde fixative for 12 h and then dehydrated in 30% sucrose overnight. OCT-embedded, 8-μm cryostat sections were incubated in 0.1 M Tris⋅HCl buffer containing 0.3% Triton X-100 and 2% FBS before staining. Alexa Fluor 488–conjugated goat anti-mouse IgG (Fcγ fragment) (Jackson ImmunoResearch) was followed for 1 h at room temperature. Slides were washed for 15 min in 0.1 M Tris⋅HCl before mounting in ProLong Gold Antifade Mountant with DAPI (Life technologies). Fluorescence images were acquired using a confocal fluorescence microscope (LSM710; Zeiss) and further analyzed by Bitplane Imaris (BITPLANE scientific software). The fluorescence intensity of glomerular IgG staining was quantified using ImageJ software (National Institutes of Health) and expressed as density per unit area.

Bregs were sorted with the indicated marker. Total RNA was isolated following the instructions of the user’s manual (Life Technologies). Total RNA was reverse transcribed to cDNA using a Reverse Transcription System (Thermo Scientific). Quantitative RT-PCR was performed using the SYBR Green Master Mix (Takara) with analysis using a Bio-Rad CFX96 Touch according to the manufacturer’s instructions. The primer sequences were as follows: for mouse IL-10, 5′-TCC TTG CTG GAG GAC TTT AAG GGT-3′ (forward) and 5′-TGT CTG GGT CTT GGT TCT CAG CTT-3′ (reverse); for mouse TLR4, 5′-TGG CTG GTT TAC ACA TCC ATC GGT-3′ (forward) and 5′-TGG CAC CAT TGA AGC TGA GGT CTA-3′(reverse); for mouse activation-induced cytidine deaminase (AID), 5′-ACC TTC GCA ACA AGT CTG GCT-3′ (forward) and 5′-AGC CTT GCG GTC TTC ACA GAA-3′(reverse); for mouse c-FOS, 5′-CCG CGA ACG AGC AGT GAC CG-3′ (forward) and 5′-AAA GCT CGG CGA GGG GTC CA-3′ (reverse); for mouse c-Jun, 5′-CGC GGG AGC CAA CCA ACG TG-3′ (forward) and 5′-GCG TCC CCG CTT CAG TAA CAA AGT-3′(reverse); and for mouse GAPDH, 5′-TGT GTC CGT CGT GGA TCT GA-3′ (forward) and 5′-TTG CTG TTG AAG TCG CAG GAG-3′ (reverse). Relative expression of the quantitative RT-PCR products was determined using the ΔΔ threshold cycle (CT) technique. Cells from Innp5dfl/fl mice were used as the calibrator. Briefly, each set of samples was normalized using the difference in CT between the target gene and housekeeping gene (GAPDH): ΔCT = (CT target gene − CT GAPDH). Relative mRNA levels were calculated by the expression 2−ΔΔCT, where ΔΔCT = ΔCT sample − ΔCT calibrator.

It is known that TLR4 ligand stimulation induces the upregulation of AID in B cells (27), which mediates the molecular processes leading to both class-switched recombination and somatic hypermutation (28). It is also known that the TLR4 ligand, LPS, potently drives the production of IL-10 in B10 cells. We thus speculated that B10 cells may upregulate the expression of AID upon LPS stimulation. To validate this hypothesis, we first compared the AID expression level in B10 cells versus mature naive B cells. The AID expression measured via quantitative RT-PCR was more highly upregulated in B10 cells than in the follicular B cells, both under the naive condition and LPS induction in vivo (2931). The results also showed that LPS induction did not alter the AID mRNA expression level in the follicular B cells, but drastically enhanced the AID mRNA expression level in the B10 cells (Fig. 1A). Considering the expression level of SHIP-1 is drastically lower in B cells from SLE patients than in healthy controls (32), we crossed Innp5dfl/fl mice that contain the loxP-flanked SHIP-1 allele with Aicda-Cre transgenic mice for the purpose of generating mice lacking SHIP-1 in AID+ cells. We anticipated that the upregulation of AID would induce the downregulation of SHIP-1 in the B10 cells from Innp5dfl/flAicdaCre/+ but not in the control Innp5dfl/fl mice. Thus, it would be of interest to examine the IL-10 production in B10 cells lacking the expression of SHIP-1. Because the activation of TLR4R is required to drive the production of IL-10 in B10 cells, we first examined the TLR4 expression level in B10 cells by comparing it to that in B1a cells, which are known to highly express TLR4 (33). Indeed, B10 cells (gated as in the previous study showing that B10 cells also highly express CD21, CD24, and IgM [6]) showed higher expression of TLR4 than B1a cells (Supplemental Fig. 1A). These results strongly validated that B10 cells could serve as good responder cells to LPS stimulation. Additionally, we found comparable TLR4 expression levels in splenic B220+CD5+IgMhighCD24high B10 cells from both Innp5dfl/flAicdaCre/+ and the Innp5dfl/fl control mice (Supplemental Fig. 1B). More importantly, the expression level of SHIP-1 was significantly reduced in the LPS-induced Innp5dfl/flAicdaCre/+ B10 cells in vivo compared with those from the control Innp5dfl/fl mice (Fig. 1B). These results showed that AID is upregulated and SHIP-1 is downregulated in B10 cells in Innp5dfl/flAicdaCre/+ mice.

FIGURE 1.

AID was upregulated and SHIP-1 was downregulated in B10 cells from Innp5dfl/flAicdaCre/+ mice. (A) AID transcript expression analyses in spleen B10 cells. RNA was isolated from B10 cell subsets (CD19+CD5+/−CD1dhigh, as shown later in representative flow data) and follicular B cells (B220+IgM+IgD+) from 16-wk-old Innp5dfl/flAicdaCre/+ or Innp5dfl/fl mice. For the LPS induction in vivo, 20 μg LPS was injected per mice at day 0 and day 3, and splenic B10 cells and follicular B cells were sorted out at day 6. AID mRNA levels were determined by quantitative RT-PCR assays, and normalized relative to internal control GAPDH transcripts. **p < 0.01, ***p < 0.001. (B) SHIP-1 was determined by Western blot assays, and GAPDH was used as internal control. Results shown are representative of three independent experiments.

FIGURE 1.

AID was upregulated and SHIP-1 was downregulated in B10 cells from Innp5dfl/flAicdaCre/+ mice. (A) AID transcript expression analyses in spleen B10 cells. RNA was isolated from B10 cell subsets (CD19+CD5+/−CD1dhigh, as shown later in representative flow data) and follicular B cells (B220+IgM+IgD+) from 16-wk-old Innp5dfl/flAicdaCre/+ or Innp5dfl/fl mice. For the LPS induction in vivo, 20 μg LPS was injected per mice at day 0 and day 3, and splenic B10 cells and follicular B cells were sorted out at day 6. AID mRNA levels were determined by quantitative RT-PCR assays, and normalized relative to internal control GAPDH transcripts. **p < 0.01, ***p < 0.001. (B) SHIP-1 was determined by Western blot assays, and GAPDH was used as internal control. Results shown are representative of three independent experiments.

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Next, we tested whether the deletion of SHIP-1 in AID+ B cells would influence B cell development. We observed comparable percentages of B cells in the bone marrow, spleen, and peritoneal cavity in Innp5dfl/flAicdaCre/+ mice and the control Innp5dfl/fl mice. There were no obvious differences in terms of the percentages of pro-B cells (B220+IgDIgMCD43+), pre-B cells (B220+IgDIgMCD43), immature B cells (B220+IgDIgM+CD43), and mature B cells (B220+IgD+) in the bone marrow of these two types of mice. Similarly, we observed comparable proportions of transitional B cells (B220+CD93+), follicular B cells (B220+CD93CD23+CD21+), and marginal zone B cells (B220+CD93CD23CD21high) in the spleen in Innp5dfl/flAicdaCre/+ mice versus the control Innp5dfl/fl mice. However, we found a significantly reduction of B1a cells (CD19+CD3eF4/80CD5+CD23) and B1b cells (CD19+CD3eF4/80CD5CD23) in the peritoneal cavity of Innp5dfl/flAicdaCre/+ mice. In marked contrast, B2 B cells (CD19+CD3eF4/80CD23+) were elevated in the peritoneal cavity of Innp5dfl/flAicdaCre/+ mice (Fig. 2A–D). As AID is usually highly expressed in germinal-center (GC) B cells in T cell–dependent Ab responses, we checked the SHIP-1 expression in GC B cells, which were sorted (as shown later) from mice immunized with SRBCs. Immunoblotting indicated that SHIP-1 was efficiently knocked out in the sorted GC B cells of Innp5dfl/flAicdaCre/+ mice in comparison with those from the control Innp5dfl/fl mice (Fig. 2E). Collectively, Innp5dfl/flAicdaCre/+ mice showed normal B cell development, except for a reduction of B1 cells and an increase of B2 B cells in the peritoneal cavity.

FIGURE 2.

Innp5dfl/flAicdaCre/+ mice showed a normal B cell development except for reduction of peritoneal B1 cells. (A) Bone marrow cells, (B) peritoneal cavity cells, or (C) spleen cells were analyzed by the indicated markers and percentages are given for the individual populations in bar diagrams. (D) B cell percentages in bone marrow, peritoneal cavity, and spleen. **p < 0.01, ***p < 0.001. (E) Western blot analysis of SHIP-1 expression in GC B cells (B220+CD95+GL-7+ cells were sorted at day 6 after SRBC immunization, the gating strategy for FACS analysis is shown later). Results shown are representative of three independent experiments. BM, bone marrow; FO, follicular B cells; MZ, marginal zone; PC, peritoneal cavity; SP, spleen.

FIGURE 2.

Innp5dfl/flAicdaCre/+ mice showed a normal B cell development except for reduction of peritoneal B1 cells. (A) Bone marrow cells, (B) peritoneal cavity cells, or (C) spleen cells were analyzed by the indicated markers and percentages are given for the individual populations in bar diagrams. (D) B cell percentages in bone marrow, peritoneal cavity, and spleen. **p < 0.01, ***p < 0.001. (E) Western blot analysis of SHIP-1 expression in GC B cells (B220+CD95+GL-7+ cells were sorted at day 6 after SRBC immunization, the gating strategy for FACS analysis is shown later). Results shown are representative of three independent experiments. BM, bone marrow; FO, follicular B cells; MZ, marginal zone; PC, peritoneal cavity; SP, spleen.

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Next, we investigated the percentage and probability of IL-10 production in B10 cells of Innp5dfl/flAicdaCre/+ mice. First, we observed a drastic drop in the percentage of IL-10+ B cells in Innp5dfl/flAicdaCre/+ mice compared with the control Innp5dfl/fl mice (Fig. 3A). Correspondingly, the percentage of B10 cells (CD19+CD5+/−CD1dhigh) were also dramatically decreased in Innp5dfl/flAicdaCre/+ mice compared with the control Innp5dfl/fl mice (Fig. 3B). It should be noted that the majority, but not entirety, of all cells were CD5+, due to the lack of a clear boundary distinguishing CD5+ and CD5 B cell populations, consistent with prior studies (34, 35). The decrease of IL-10 production in the Innp5dfl/flAicdaCre/+ mice was further confirmed by quantitative RT-PCR using the same numbers of B10 cells from these two types of mice (Fig. 3C). Results verified that both the percentage and IL-10–producing capability of B10 cells decreased in Innp5dfl/flAicdaCre/+ mice. We also examined the IL-10–producing capacity in CD19+CD5+CD24highIgMhigh B10 cells that were cultured with LPS, PMA, ionomyin, and monensin for 5 h (Supplemental Fig. 2). IL-10+ B cells expressed higher levels of CD5, CD24, and IgM than IL-10 B cells (Supplemental Fig. 2A). On the contrary, an average of 18.2% IL-10 expression was observed in the CD5+CD24highIgMhigh B cell subset, whereas CD5 or CD5+CD24lowIgMlow B cells were negative for IL-10 expression (Supplemental Fig. 2B). Subsequently, these markers were used for the investigation of B10 cell function in the Innp5dfl/flAicdaCre/+ mice. We used LPS to stimulate B10 cells, as TLR4 signaling is crucial for the stimulation of B10 cells (9). After 5 h of LPS induction in vitro, the percentage of B220+CD5+CD24highIgMhigh B10 cells in the purified splenic B cells were significantly decreased in the Innp5dfl/flAicdaCre/+ mice compared with the control Innp5dfl/fl mice (Supplemental Fig. 3A). Moreover, the IL-10 mRNA expression level was also decreased in the same number of B10 cells (CD5+CD24high IgMhigh B cells) from Innp5dfl/flAicdaCre/+ mice compared with those from Innp5dfl/fl mice (Supplemental Fig. 3B). These results further prove that both the percentage and IL-10–producing capability of B10 cells is decreased in the Innp5dfl/flAicdaCre/+ mice.

FIGURE 3.

Decreased B10 cells with dampened IL-10–producing capability in the Innp5dfl/flAicdaCre/+ mice. (A) Spleen lymphocytes were stimulated ex vivo with LPS, PMA, ionomycin, and monensin for 5 h and stained for cell surface CD19 and intracellular IL-10. Representative flow cytometry data show IL-10 expression by viable and single spleen CD19+ B cells. Numbers indicate the frequencies of cells within the indicated gates. Values in the bar graphs represent mean (16-wk-old, n = 6) frequencies of IL-10+ B cells. (B) The spleen B10 cells were indicated both in the representative flow cytometry data and bar graphs. The data are presented as in (A) (16-wk-old, n = 6). (C) IL-10 transcript expression by spleen B10 cells. RNA was isolated from B10 cell subset of 16-wk-old Innp5dfl/flAicdaCre/+ and Innp5dfl/fl mice. IL-10 mRNA levels were determined in quantitative RT-PCR assays and normalized relative to internal control GAPDH transcripts. *p < 0.05, **p < 0.01.

FIGURE 3.

Decreased B10 cells with dampened IL-10–producing capability in the Innp5dfl/flAicdaCre/+ mice. (A) Spleen lymphocytes were stimulated ex vivo with LPS, PMA, ionomycin, and monensin for 5 h and stained for cell surface CD19 and intracellular IL-10. Representative flow cytometry data show IL-10 expression by viable and single spleen CD19+ B cells. Numbers indicate the frequencies of cells within the indicated gates. Values in the bar graphs represent mean (16-wk-old, n = 6) frequencies of IL-10+ B cells. (B) The spleen B10 cells were indicated both in the representative flow cytometry data and bar graphs. The data are presented as in (A) (16-wk-old, n = 6). (C) IL-10 transcript expression by spleen B10 cells. RNA was isolated from B10 cell subset of 16-wk-old Innp5dfl/flAicdaCre/+ and Innp5dfl/fl mice. IL-10 mRNA levels were determined in quantitative RT-PCR assays and normalized relative to internal control GAPDH transcripts. *p < 0.05, **p < 0.01.

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A striking observation with the Innp5dfl/flAicdaCre/+ mice is that these mice exhibit spontaneous autoimmune syndrome with age. Such a phenotype was initially brought to our attention when we tried to quantify the levels of the total serum Abs in these two types of mice (Fig. 4A). We observed that 16-wk-old Innp5dfl/flAicdaCre/+ mice showed more IgG total serum Abs than the age-matched control Innp5dfl/fl mice, but exhibited similar IgM total serum Abs compared with the age-matched control Innp5dfl/fl mice. The enhanced levels of IgG2b and IgG2c were correlated with the spontaneously increased GC B cells in the spleen of Innp5dfl/flAicdaCre/+ mice compared with those of the control Innp5dfl/fl mice (Fig. 4B). However, the IgG1 and IgG3 Ab levels were not significantly different between the Innp5dfl/flAicdaCre/+ and control Innp5dfl/fl mice. The increase in GC B cells was much more obvious in the aged mice (16-wk-old mice) than in the young mice (6-wk-old mice). The aged Innp5dfl/flAicdaCre/+ mice (16-wk-old mice) consistently showed enlarged spleens compared with the corresponding aged control Innp5dfl/fl mice (Fig. 4C). These results suggest that Innp5dfl/flAicdaCre/+ mice might show a spontaneous autoimmune phenotype with age. Indeed, our speculation was supported by the detection of anti-dsDNA–specific Abs in aged Innp5dfl/flAicdaCre/+ mice, but not in their young counterparts. As a negative control, aged-matched control Innp5dfl/fl mice did not show anti-dsDNA Abs (Fig. 4D). More importantly, aged Innp5dfl/flAicdaCre/+ mice (16-wk-old mice) showed ANA (Fig. 4E) and exhibited an obvious deposition of IgG immune complexes in the kidney (Fig. 4F) in comparison with the age-matched control Innp5dfl/fl mice. These results demonstrate that Innp5dfl/flAicdaCre/+ mice exhibit a spontaneous lupus-like autoimmune disease phenotype with age.

FIGURE 4.

Innp5dfl/flAicdaCre/+ mice exhibited spontaneous lupus-like autoimmune phenotype with age. (A) Total serum Ig concentration of indicated isotypes was measured by ELISA in mice (16-wk-old). The data were expressed as mean ± SEM (n = 6 per group), assessed by two-way ANOVA. (B) Shown are the representative flow cytometric gating and the percentage of spontaneous GC B cells from 6-wk-old and 16-wk-old mice. (C) Spleen from Innp5dfl/flAicdaCre/+ mice and its control Innp5dfl/fl mice (16-wk-old). (D) Quantification of anti-dsDNA–specific IgG in the serum of Innp5dfl/flAicdaCre/+ mice and its control Innp5dfl/fl mice at 7–10, 12, and 16 wk of age. (E) Representative images (above) and quantification of mean fluorescence intensity (mFI) (below) of serum ANA IgG detected with fixed Hep-2 slides. Alexa Fluor 488–conjugated goat anti-mouse IgG was used as the indirect immunofluorescence detection reagent, and nuclei were detected using ProLong Gold Antifade Mountant with DAPI. Scale bar, 70 μm. (F) Immunofluorescence in kidney glomerulus sections showing IgG deposits. Sections were stained with Alexa Fluor 488–conjugated goat anti-mouse IgG, and nuclei were detected using ProLong Gold Antifade Mountant with DAPI. Scale bar, 10 μm. Results shown are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

Innp5dfl/flAicdaCre/+ mice exhibited spontaneous lupus-like autoimmune phenotype with age. (A) Total serum Ig concentration of indicated isotypes was measured by ELISA in mice (16-wk-old). The data were expressed as mean ± SEM (n = 6 per group), assessed by two-way ANOVA. (B) Shown are the representative flow cytometric gating and the percentage of spontaneous GC B cells from 6-wk-old and 16-wk-old mice. (C) Spleen from Innp5dfl/flAicdaCre/+ mice and its control Innp5dfl/fl mice (16-wk-old). (D) Quantification of anti-dsDNA–specific IgG in the serum of Innp5dfl/flAicdaCre/+ mice and its control Innp5dfl/fl mice at 7–10, 12, and 16 wk of age. (E) Representative images (above) and quantification of mean fluorescence intensity (mFI) (below) of serum ANA IgG detected with fixed Hep-2 slides. Alexa Fluor 488–conjugated goat anti-mouse IgG was used as the indirect immunofluorescence detection reagent, and nuclei were detected using ProLong Gold Antifade Mountant with DAPI. Scale bar, 70 μm. (F) Immunofluorescence in kidney glomerulus sections showing IgG deposits. Sections were stained with Alexa Fluor 488–conjugated goat anti-mouse IgG, and nuclei were detected using ProLong Gold Antifade Mountant with DAPI. Scale bar, 10 μm. Results shown are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

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Next, we further compared the functional capability of SHIP-1–deficient and normal B10 cells by measuring the extent of suppression of the autoimmune syndrome in the aged Innp5dfl/flAicdaCre/+ mice upon adoptive transfer of B10 cells (CD19+CD5+/−CD1dhigh) from either Innp5dfl/fl or Innp5dfl/flAicdaCre/+ mice into the 16-wk-old Innp5dfl/flAicdaCre/+ mice, once a week for 2 wk. Then, we evaluated the autoimmune phenotype in the host mice by quantifying anti-dsDNA Abs and ANA. The levels of anti-dsDNA Abs (Fig. 5A) and ANA (Fig. 5B, 5D) decreased after the transfer of B10 cells, shown as a ratio to Ab levels the day of transfer. Wild-type B10 cells from Innp5dfl/fl mice more significantly reduced the levels of anti-dsDNA Abs and ANA in Innp5dfl/flAicdaCre/+ mice than B10 cells from Innp5dfl/flAicdaCre/+ mice. In addition, the transfer of Innp5dfl/fl, instead of SHIP-1–deficient B10 cells, prevented the increase of the total serum IgG in serum. In contrast, the transfer of either the wild-type or SHIP-1–deficient B10 cells inhibits the increase in the serum IgM (Fig. 5C). As a system control, the Innp5dfl/flAicdaCre/+ mice that were transferred with PBS showed further increases in the levels of anti-dsDNA Abs, ANA, serum IgM, and IgG, reflecting an age-dependent exacerbation of the autoimmune disease. We also found that the adoptive transfer of wild-type Innp5dfl/fl B10 cells ameliorated the IgG deposition in the kidney compared with that of SHIP-1–deficient B10 cells (Fig. 5E). Collectively, SHIP-1–deficient B10 cells are functionally poor at rescuing autoimmune syndrome in Innp5dfl/flAicdaCre/+ mice in comparison with wild-type B10 cells.

FIGURE 5.

Adoptive transfer of wild-type, but not Innp5dfl/flAicdaCre/+, B10 cells rescued the autoimmune syndrome in Innp5dfl/flAicdaCre/+ mice. B10 cells from Innp5dfl/f mice and Innp5dfl/flAicdaCre/+ mice were FACS sorted and 5 × 105 cells were transferred once a week for 2- to 16-wk-old Innp5dfl/flAicdaCre/+ mice. Control mice received PBS injections. Shown are the ratio of anti-dsDNA–IgG (A), the ratio of ANA IgG (B), and the ratio of serum IgM and IgG (C), which were calculated by normalizing each measured ELISA value after the transfer of B10 cells to the corresponding value before the transfer of B10 cells. (D) Shown are representative immunofluorescence images for the quantification of ANA IgG detected with fixed Hep-2 slides. Alexa Fluor 488–conjugated goat anti-mouse IgG was used as the indirect immunofluorescence detection reagent. Nuclei were detected using ProLong Gold Antifade Mountant with DAPI. Scale bar, 70 μm. (E) Shown in the upper panel are representative immunofluorescence images showing the glomerular IgG deposits in the kidney, which was stained with Alexa Fluor 488–conjugated goat anti-mouse IgG, and nuclei were detected using ProLong Gold Antifade Mountant with DAPI. Scale bar, 20 μm. Given in the bottom panel was the statistical comparison for the mean fluorescence intensity (mFI) of IgG deposits as quantified in kidney glomerulus sections after the transfer of B10 cells from Innp5dfl/fl mice versus Innp5dfl/fl AicdaCre/+ mice. Fluorescence intensity of glomerular IgG staining was quantified using ImageJ software and expressed as density per unit area. Each bar represents data from 30 to 50 glomeruli of all the mice. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Adoptive transfer of wild-type, but not Innp5dfl/flAicdaCre/+, B10 cells rescued the autoimmune syndrome in Innp5dfl/flAicdaCre/+ mice. B10 cells from Innp5dfl/f mice and Innp5dfl/flAicdaCre/+ mice were FACS sorted and 5 × 105 cells were transferred once a week for 2- to 16-wk-old Innp5dfl/flAicdaCre/+ mice. Control mice received PBS injections. Shown are the ratio of anti-dsDNA–IgG (A), the ratio of ANA IgG (B), and the ratio of serum IgM and IgG (C), which were calculated by normalizing each measured ELISA value after the transfer of B10 cells to the corresponding value before the transfer of B10 cells. (D) Shown are representative immunofluorescence images for the quantification of ANA IgG detected with fixed Hep-2 slides. Alexa Fluor 488–conjugated goat anti-mouse IgG was used as the indirect immunofluorescence detection reagent. Nuclei were detected using ProLong Gold Antifade Mountant with DAPI. Scale bar, 70 μm. (E) Shown in the upper panel are representative immunofluorescence images showing the glomerular IgG deposits in the kidney, which was stained with Alexa Fluor 488–conjugated goat anti-mouse IgG, and nuclei were detected using ProLong Gold Antifade Mountant with DAPI. Scale bar, 20 μm. Given in the bottom panel was the statistical comparison for the mean fluorescence intensity (mFI) of IgG deposits as quantified in kidney glomerulus sections after the transfer of B10 cells from Innp5dfl/fl mice versus Innp5dfl/fl AicdaCre/+ mice. Fluorescence intensity of glomerular IgG staining was quantified using ImageJ software and expressed as density per unit area. Each bar represents data from 30 to 50 glomeruli of all the mice. *p < 0.05, **p < 0.01, ***p < 0.001.

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During T cell–dependent Ab responses, the generation of high-affinity memory B cells and plasma cells occurs within GCs, accounting for highly effective humoral immunity (3638). To examine the function of SHIP-1 in GC-mediated Ab responses, we immunized mice with SRBCs on day 0 and quantified the formation of primary GCs in the Innp5dfl/flAicdaCre/+ and control Innp5dfl/fl mice on day 6. We consistently observed a moderate increase in the percentage of GC B cells among total B cells in Innp5dfl/flAicdaCre/+ mice compared to control Innp5dfl/fl mice (Fig. 6A, 6B). To examine the formation of secondary GCs in Ag-recall responses, we immunized these mice again with SRBCs on day 21 and observed the drastically elevated percentage of GC B cells among total B cells in Innp5dfl/flAicdaCre/+ mice (Fig. 6C, 6D). To further assess the specific Ab titers upon T cell–dependent Ag immunization, we immunized both Innp5dfl/flAicdaCre/+ mice and control Innp5dfl/fl mice with a model T cell–dependent Ag, NP33-KLH, and CFA on day 0 with an Ag-recall immunization on day 42 (Fig. 6E). ELISA experiments indicated the comparable NP-specific IgM induction, high affinity NP-specific IgG (examined by NP8-BSA), and low affinity NP-specific IgG (examined by NP30-BSA) Abs in these two types of mice 7, 14, 21, and 42 d after the primary immunization. However, for Ag-recall responses, Innp5dfl/flAicdaCre/+ mice produced significantly higher levels of NP-specific IgM and IgG Abs than the control mice, which is consistent with the above observation of the significantly enhanced formation of GC B cells in the secondary, but not primary, responses to SRBC immunization in Innp5dfl/flAicdaCre/+ mice. When the extent of affinity maturation of NP-specific IgG Abs was measured as a ratio of anti–NP8-BSA IgG/anti–NP30-BSA IgG titers, we consistently observed an impaired affinity maturation in Innp5dfl/flAicdaCre/+ mice, but not in the control Innp5dfl/fl mice. Thus, all of these results confirm the negatively regulated function of SHIP-1 in GC responses, and particularly highlight the key role of SHIP-1 in promoting stringent selection of high-affinity GC B cells in GC responses.

FIGURE 6.

Innp5dfl/flAicdaCre/+ mice showed enhanced Ab recall responses, but impaired Ab affinity maturation. (A and B) Representative flow cytometric gating and GC B cell percentages in the primary Ab response. GC B cells were identified by gating B220+CD95+GL7+ splenocytes 6 d after SRBC immunization. (C and D) Representative flow cytometric gating and GC B cell percentages in the secondary GC. GC B cells were identified 6 d after day 21 SRBC recall immunization. (E) Sequential serum ELISA analysis to detect NP-specific Abs. Innp5dfl/flAicdaCre/+ mice and control Innp5dfl/fl mice were immunized with 25 μg NP33-KLH and CFA at day 0, and recalled with 20 μg NP33-KLH alone at day 42 (n = 5 samples per group). NP30-BSA for IgM and either NP30-BSA or NP8-BSA for IgG were used as the coating Ag. At day 7, 14, 21, 42, 49, 56, 70, and 119, the affinity of the NP-specific IgG was determined by the ratio NP8/NP30. *p < 0.05.

FIGURE 6.

Innp5dfl/flAicdaCre/+ mice showed enhanced Ab recall responses, but impaired Ab affinity maturation. (A and B) Representative flow cytometric gating and GC B cell percentages in the primary Ab response. GC B cells were identified by gating B220+CD95+GL7+ splenocytes 6 d after SRBC immunization. (C and D) Representative flow cytometric gating and GC B cell percentages in the secondary GC. GC B cells were identified 6 d after day 21 SRBC recall immunization. (E) Sequential serum ELISA analysis to detect NP-specific Abs. Innp5dfl/flAicdaCre/+ mice and control Innp5dfl/fl mice were immunized with 25 μg NP33-KLH and CFA at day 0, and recalled with 20 μg NP33-KLH alone at day 42 (n = 5 samples per group). NP30-BSA for IgM and either NP30-BSA or NP8-BSA for IgG were used as the coating Ag. At day 7, 14, 21, 42, 49, 56, 70, and 119, the affinity of the NP-specific IgG was determined by the ratio NP8/NP30. *p < 0.05.

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Lastly, we investigated why SHIP-1 reduction leads to the drastically reduced production of IL-10 in B10 cells. Because SHIP-1 is a well-characterized negative regulator of Akt activation by opposing the function of PI3K, we first examined the activation of Akt by detecting the phosphorylation of the Ser473 amino acid residue in purified B10 cells after in vitro stimulation with LPS for 5 h. As expected, we observed a significant enhancement in Akt activation in LPS-stimulated B10 cells from Innp5dfl/flAicdaCre/+ mice compared with the B10 cells from the control Innp5dfl/fl mice (Fig. 7A). In contrast, the phosphorylation of Jnk, Erk, and p38, as well as the expression of c-Jun were drastically impaired in B10 cells from Innp5dfl/flAicdaCre/+ mice (Fig. 7A). We also used quantitative RT-PCR to further confirm that both c-Jun and c-Fos were decreased in B10 cells from Innp5dfl/flAicdaCre/+ mice compared with the B10 cells from the control Innp5dfl/fl mice (Fig. 7B). Because it is well documented that AP-1 is one of the main transcription factors accounting for the transcription of the IL-10 gene (39, 40), the drastically reduced activation of MAPK pathway molecules, including Erk and Jnk (both of which are key molecules driving the activation of AP-1), explained the lack of IL-10 transcription in B10 cells from Innp5dfl/flAicdaCre/+ mice, as shown above. The transcription of IL-10 is also recognized to be regulated by GSK3α and GSK3β (41), but we could not observe obvious differences in terms of activation of these two molecules in the LPS-induced B10 cells from Innp5dfl/flAicdaCre/+ mice and the control Innp5dfl/fl mice (Fig. 7A). Thus, these data suggest that the impaired activation of the MAPK–AP-1 pathway accounts for the dampened IL-10 production in B10 cells in Innp5dfl/fl AicdaCre/+ mice.

FIGURE 7.

p-Akt was enhanced whereas MAPK–AP-1 pathway was impaired in B10 cells from Innp5dfl/fl AicdaCre/+ mice. (A) After 5 h LPS stimulation, phospho-Akt, phospho-Jnk, phospho-Erk, phospho-p38, phospho- GSK3, and c-Jun were evaluated in the sorted B10 cells from 16-wk-old Innp5dfl/flAicdaCre/+ and Innp5dfl/fl mice by Western blot assays. β-Actin and tubulin were used as internal controls. Results shown are representative of three independent experiments. (B) c-Jun and c-Fos mRNA levels were tested in B10 cells in 16-wk-old Innp5dfl/flAicdaCre/+ and Innp5dfl/fl mice by quantitative RT-PCR assays. *p < 0.05.

FIGURE 7.

p-Akt was enhanced whereas MAPK–AP-1 pathway was impaired in B10 cells from Innp5dfl/fl AicdaCre/+ mice. (A) After 5 h LPS stimulation, phospho-Akt, phospho-Jnk, phospho-Erk, phospho-p38, phospho- GSK3, and c-Jun were evaluated in the sorted B10 cells from 16-wk-old Innp5dfl/flAicdaCre/+ and Innp5dfl/fl mice by Western blot assays. β-Actin and tubulin were used as internal controls. Results shown are representative of three independent experiments. (B) c-Jun and c-Fos mRNA levels were tested in B10 cells in 16-wk-old Innp5dfl/flAicdaCre/+ and Innp5dfl/fl mice by quantitative RT-PCR assays. *p < 0.05.

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Bregs produce IL-10 for the purpose of suppressing inflammation (59). However, the molecular mechanism regulating the production of IL-10 is not fully understood. In this report, we demonstrate the regulatory function of SHIP-1 in the IL-10–producing capability of B10 cells. One of our critical observations is that AID was highly expressed in B10 cells in comparison with follicular B cells, especially upon the stimulation by the TLR4 ligand LPS. Moreover, we found that B10 cells also expressed a much higher level of TLR4 than B1a cells, which were known to highly express TLR4 (33). These observations enable us to investigate the function of SHIP-1 in B10 cells from Innp5dfl/flAicdacre/+ mice as SHIP-1 could be drastically eliminated in B10 cells in this strain of mice. AID was shown to be expressed even in early developing B cells, such as in pre-B and immature B cells, and was downregulated in developing B cells from Myd88−/− mice (42), suggesting that TLR4-mediated signaling is responsible for the AID expression in these cells. In our system, we found that the deletion of SHIP-1 in AID+ cells did not influence B cell development except for the reduction in B1 cells and the increase in B2 cells in the peritoneal cavity. We propose that there could be two models to explain the drop of B1 cells in Innp5dfl/flAicdacre/+ mice compared with the Innp5dfl/fl mice: 1) Previous reports showed the increase in B1 cells in SHIP−/− mice, which was apparently in contrast with our results that B1 cells reduced in the Innp5dfl/flAicdacre/+ mice. However, we hypothesize that SHIP-1–deficient mice would select B1 cells which have weakly autoreactive BCRs due to enhanced signaling compared with wild-type mice, resulting in selection of more B1 cells that could not be selected in the wild-type mice (4345). In Innp5dfl/flAicdaCre/+ mice, SHIP is conditionally knocked out only in highly autoreactive B1 cells because AID is induced by strong BCR signaling, which might lead to the narrowing of the positive selection window of B1 cells or negative selection of B1 cells with very high BCR signaling. 2) B10 cells appear to be heterogeneous, and some B10 cells might traverse from B1 cells (46). Thus, our second hypothesis is that the downregulation of B1 cells might be linked to the drop of B10 cells in Innp5dfl/flAicdaCre/+ mice.

Most importantly, we observed that the SHIP-1 deficiency in AID+ B cells leads to a deficiency in the cell count and IL-10–producing capability of B10 cells. As a consequence, we found severe autoimmune phenotypes in 16-wk-old Innp5dfl/flAicdaCre/+ mice, which included the presence of anti-dsDNA and anti-nuclear Abs, marked deposition of IgG immune complexes in the kidney, increased serum levels of IgG2b and IgG2c Abs, and spontaneous GC formation with increased GC B cells in the spleen. In this study, we investigate whether the autoimmune phenotypes result from the numerical and functional defects of B10 cells. The importance of B10 cells in the autoimmune pathogenesis could be confirmed by the adoptive transfer experiments with B10 cells. Adoptive transfer of Innp5dfl/fl wild-type B10 cells, but not SHIP-1–deficient Innp5dfl/flAicdaCre/+ B10 cells, could rescue the autoimmune phenotypes in 16-wk-old Innp5dfl/flAicdaCre/+ mice, which highlights the B10 cell’s function in regulating autoimmunity. These results are in accordance with a previous report showing the decreased expression of SHIP-1 in B cells in human SLE patients (32). In this report, we also demonstrate that the T cell–dependent Ab response is stronger in the Innp5dfl/flAicdaCre/+ mice, especially in the recall response, than that in the control mice. Recently, it was shown that B cell signal transduction in GC B cells was short-circuited by the significantly enhanced phosphatase function in comparison with that in follicular B cells, suggesting the role of phosphatase-including SHIP-1 and SHP-1 in the GC B cells (47). Therefore, the autoimmune phenotypes in the Innp5dfl/flAicdaCre/+ mice may be caused by multiple mechanisms including the numerical and functional defects of B10 cells and the disturbance of GC B cell signaling leading to persistence of autoreactive GC B cells. Another possible mechanism is the loss of anergy, which was observed in the Innp5dfl/flCD79a-cre mice (48). It was also well addressed that SHIP-1 was required for the establishment of anergy of B cells with high-affinity BCRs against proteinaceous autoantigens (49) as well as for the maintenance of unresponsiveness of anergic B cells (50). Thus, the newly arising GC-autoreactive B cells in Innp5dfl/flAicdaCre/+ mice may survive avoiding negative selection, as a result of lacking SHIP-1 (5153). As IgD was reported to attenuate the IgM-induced anergy response (54), we tested the levels of IgD and IgM in the blood B cells from Innp5dfl/flAicdaCre/+ and the littermate control mice, but did not find any differences between them (Supplemental Fig. 4).

Why does SHIP-1 deficiency in B10 cells lead to reduced production of IL-10? We show that the defective MAPK–AP-1 pathway is responsible for the impaired IL-10 production in Innp5dfl/flAicdaCre/+ mice. Previously, the requirement of STIM-dependent calcium influx and subsequent activation of calcineurin–NFAT was required for IL-10 production (55). Interestingly, the calcineurin–NFAT pathway was not required for the Ag-specific Ab response or the development of most of B cells. In our report, we illustrated that B10 cells in Innp5dfl/flAicdaCre/+ mice showed enhanced phosphorylation of Akt, the impaired MAPK function, and the reduced AP-1 expression. Our data is consistent with the report showing that SHIP-1 could positively regulate IL-10 production in LPS-induced macrophages via the MAPK–AP-1 pathway (56). In addition, PI3K could also regulate IL-10 production by the phosphorylation of GSK3 (α/β) (39, 40). In our experiment, the levels of phosphorylation of GSK3 (α/β) were not significantly different between B10 cells from the Innp5dfl/flAicdaCre/+ and littermate control mice.

In summary, we selectively downregulate SHIP-1 in B10 cells by generating Innp5dfl/flAicdaCre/+ mice as AID is highly upregulated in B10 cells and observe a deficiency in the cell count and IL-10–producing capability of B10 cells in the Innp5dfl/flAicdaCre/+ mice. The defect of B10 cells leads to autoimmunity, which could be rescued by the adoptive transfer of wild-type B10 cells. We also show that SHIP-1 positively modulates IL-10 production via the MAPK–AP-1 pathway. Based on our observations, we propose that SHIP-1 can be exploited to regulate B10 cells and autoimmunity.

Innp5dfl/fl mice were gifts from Dr. Wenxia Song (University of Maryland, College Park, MD) and Dr. Salvia Bolland (Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health).

This work was supported by funds from the National Science Foundation China (81730043, 81422020, 81621002, and 81671604) and the Ministry of Science and Technology of China (2014CB542500-03).

The online version of this article contains supplemental material.

Abbreviations used in this article:

AID

activation-induced cytidine deaminase

ANA

anti-nuclear Ab

B10

IL-10–competent B cell

Breg

regulatory B cell

CT

threshold cycle

GC

germinal center

RT-PCR

real-time PCR

SLE

systemic lupus erythematosus

T2-MZPs

transitional 2 marginal-zone precursor.

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The authors have no financial conflicts of interest.

Supplementary data