B cell receptor-induced IL-10 production from neonatal mouse CD19+CD43- cells depends on STAT5-mediated IL-6 secretion
- Laboratory of Bacterial Polysaccharides, Division of Bacterial Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, The US Food and Drug Administration, United States
- Facility for Biotechnology Resources, Center for Biologics Evaluation and Research, United States Food and Drug Administration, United States
Figures
Figure 1 with 9 supplements
BCR-induced IL-10 production in adult and neonatal splenic B cell subsets.
(A and B) Splenic B cells were isolated from adult and neonatal mice. Isolated cells were incubated in the absence (unstimulated) or presence of 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 20 h and intracellular IL-10 production was measured by flow cytometry in gated B cell subsets (technical triplicate per experiment). Gating is dependent on fluorescence-minus-one (FMO) control. All experiments were repeated twice for reproducibility, and data are shown as the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (**p<0.01, ***p<0.001, and ****p<0.0001). (C and D) Isolated adult and neonatal CD19+CD43- B cells were incubated at 3 million cells/mL in the absence or presence of 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for indicated duration and culture supernatant IL-10 levels (C) and mRNA expression (D) were measured in ELISA and q-PCR, respectively. Data are shown as the mean ± s.d. of three biological replicates. P values (Adult anti-IgM vs Neonate anti-IgM) were calculated using two-way ANOVA (***p<0.001 and ****p<0.0001). (E) Representative flow cytometry dot plots and the mean of CD5+CD1d+ cell-frequency (Upper) and B10 cell (CD5+CD1dhi)-frequency (Lower) among CD19+CD43- B cells are plotted (technical triplicate per experiment). The experiments were repeated once for reproducibility, and data are shown as the mean ± s.d. of two biological replicates. P values were calculated using two-tailed Student’s t-test (***p<0.001). (F) Isolated CD19+CD43- B cells were incubated in the absence (unstim) or presence of 10 µg/mL F(ab’)2 fragments of anti-IgM antibodies for 17 hr, and then analyzed for intracellular IL-10 production in flow cytometry (technical triplicate per experiment). The experiments were repeated twice for reproducibility. Representative flow cytometry dot plots of intracellular IL-10 in B10 cells are shown. The other subsets are shown in Figure 1—figure supplement 8. The mean ± s.d. of frequency (Left) and MFI (Right) of IL-10-expressing subsets from three biological replicates are shown. P values versus adult counterparts were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05) and two-tailed Student’s t-test (†p<0.05).
Figure 1—figure supplement 1
Isolation of splenic CD19+ B cells.
Splenic CD19+ B cells were isolated using CD19 MicroBeads.
Figure 1—figure supplement 2
Staining of splenic B cell subsets for IL-10 expression.
(A) Flow cytometry gating strategy for identification of splenic CD19+CD43+ B cells and CD19+CD43- B cells. (B) Adult and neonatal splenocytes were analyzed for CD19+CD43+ B cells and CD19+CD43- B cells (technical duplicate per experiment). Experiments were repeated once for reproducibility, and data are shown as the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (****p<0.0001).
Figure 1—figure supplement 3
BCR-induced IL-10 production in adult and neonatal splenic B cell subsets.
IL-10 MFI of each subset are shown (technical triplicates per experiment). Experiments were repeated twice for reproducibility, and data are shown as the mean ± s.d. of the three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (**p<0.01, ***p<0.001 and ****p<0.0001).
Figure 1—figure supplement 4
Isolation of splenic CD19+CD43 B cells.
Splenic CD19+CD43- B cells were isolated using B Cell Isolation Kit (130-090-862, Miltenyi Biotec) which contains anti-CD43 antibody as well as anti-CD4 and anti-Ter119 antibody. (A) Representative flow cytometry plots before and after isolation are shown. (B) The mean ± s.d. MFI of CD43 in purified CD19+ B cells (biological triplicate) are shown. P values versus adult counterparts were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (NS: no significance, *p<0.05). (C) The mean ± s.d. reads per kilobase of exon per million reads mapped (RPKM) of Spn gene coding for CD43 in isolated CD19+CD43- B cells (biological triplicates) are shown.
Figure 1—figure supplement 5
Neonatal splenic CD19+CD43 cells express higher levels of IgM.
Splenocytes were isolated from adult and neonatal mice. CD19+ B cells were identified by flow cytometry. (A) Representative flow cytometry dot plots and the frequency of IgMhi-expressing cells and IgM MFI for each subset are shown (technical triplicates per experiment). (B) Representative flow cytometry dot plots and the frequency of IgDhi-expressing cells and IgD MFI for each subset are shown (technical triplicates per experiment). Experiments were repeated once for reproducibility. Data shown are the mean ± s.d. of a representative experiment. P values versus adult counterparts were calculated using two-tailed unpaired t-tests (NS: no significance, **p<0.01, ***p<0.001 and ****p<0.0001).
Figure 1—figure supplement 6
CD19+CD43low B cells do not contribute to IL-10 production.
Splenic CD19 lowB cells were isolated from adult and neonatal mice. Isolated cells were incubated in the absence (unstimulated) or presence of 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 20 hr and intracellular IL-10 production was measured by flow cytometry in stringently gated CD19+CD43- B cells from adult (A) and neonatal (B) mice to assess the contribution of CD19+CD43low cells (technical triplicates per experiment). Experiments were repeated twice for reproducibility. (C) The mean ± s.d. frequencies of IL-10-expressing CD19+CD43- B cells from a representative experiment are shown. P values versus adult counterparts were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05, ****p<0.0001).
Figure 1—figure supplement 7
Identification of splenic Breg subsets in adult and neonatal spleens.
(A) Representative counter plots depicting transitional-2 marginal zone precursor cells (T2-MZP; CD19+CD21hiCD23hiCD24hi), (B) Marginal zone B cells (MZB; CD19+CD21hiCD23-), and (C) Tim-1 B cells (CD19+Tim-1+) in adult and neonatal spleens are shown. Data shown are the mean ± s.d. of biological triplicates. P values versus adult counterparts were calculated using two-tailed unpaired t-tests (*p<0.05 and ****p<0.0001).
Figure 1—figure supplement 8
CD1d and CD5 expression on adult and neonatal splenic CD19+CD43 B cells.
Isolated CD19+CD43- B cells were analyzed for CD1d and CD5 expression separately (technical triplicates per experiment). Data shown are the mean ± s.d. of three biological replicates. P values were calculated using two-tailed Student’s t-test (**p<0.01 and ***p<0.001).
Figure 1—figure supplement 9
BCR-induced IL-10 production in each subset of adult and neonatal splenic CD19+CD43 B cells.
Isolated CD19+CD43- B cells were incubated in the absence (unstim) or presence of 10 µg/mL F(ab’)2 fragments of anti-IgM antibodies for 17 hr, and then analyzed for intracellular IL-10 production in flow cytometry (technical triplicates per experiment). Representative dot plots of each CD19+CD43- subset from adult (A) and neonatal (B) mice are shown. Mean ± s.d. frequency (C) and MFI (D) of IL-10-expressing subsets of CD19+CD43- B cells are shown. P values versus adult counterparts were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (****p<0.0001) and two-tailed Student’s t-test (††p<0.01).
Figure 2 with 3 supplements
STAT3 and STAT5 are uniquely activated in neonatal B cells following BCR cross-linking.
In all experiments, splenic CD19+CD43- B cells were isolated and stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies to engage BCR under different conditions. RNA-seq analysis was performed on CD19+CD43- cells stimulated with anti-IgM antibodies for 7 hr (biological triplicates). Total RNA was isolated for regular RNA sequencing. (A) Gene set enrichment analysis (GSEA) was performed using the hallmark gene sets in C5 gene ontology (GO). Normalized enrichment scores (NES) for representative gene sets from the C5 GO molecular signature databases that are correlated with the anti-IgM-stimulated phenotype (positive values) or unstimulated phenotype (negative values) following GSEA. Gene sets significantly enriched (FDR q-val <0.25) are ordered by the increasing NES. Bars in red indicate gene sets related to cytokine receptor and STAT signaling. (B) GSEA was performed using the hallmark gene sets in C3 transcription factor targets (TFT). Heat maps show top 10 genes in selected TFs. AU: adult unstimulated; AS: adult stimulated; NU: neonate unstimulated; NS: neonate stimulated. (C and D) CD19+CD43- cells were stimulated with anti-IgM antibodies for the indicated duration, and whole cell extracts were collected for immunoblot analysis of STAT1, STAT3, and STAT5. All experiments were repeated twice for reproducibility, and data are shown as the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001).
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Figure 2—source data 1
Raw data of all western blots from Figure 2.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig2-data1-v2.zip
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Figure 2—source data 2
Complete and uncropped membranes of all western blots from Figure 2.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig2-data2-v2.pdf
Figure 2—figure supplement 1
STAT3 and STAT5 are uniquely activated in neonatal CD19+CD43 B cells following BCR cross-linking.
Isolated CD19+CD43- cells were stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 7 hr, and then total RNA was isolated for regular RNA sequencing. (A) Volcano plots of differential gene expression levels in adult and neonatal B cells following BCR cross-linking are shown. (B and C) Flow cytometry analyses of STAT5 phosphorylation (B) and STAT3 phosphorylation (C) were done on stimulated CD19+CD43- B cells. Isolated splenic CD19+CD43- B cells were left unstimulated or stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 15 min (B) or 4 hr (C) and phospho-STAT5 and phosphor-STAT3 were measured. Experiments were repeated twice for reproducibility. Data shown are the mean ± s.d. of three biological replicates. ***p<0.001 and ****p<0.0001. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test.
Figure 2—figure supplement 2
STAT1 is phosphorylated in adult and neonatal splenic CD19+CD43 B cells in response to IL-21 but not to BCR cross-linking.
Splenic CD19+CD43- cells were isolated from adult mice and were stimulated with 100 ng/mL recombinant IL-21 or 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for the indicated duration. Whole cell extracts from stimulated cells were collected for immunoblot analysis of STAT1. This experiment was repeated once for reproducibility.
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Figure 2—figure supplement 2—source data 1
Raw data of all western blots from Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig2-figsupp2-data1-v2.zip
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Figure 2—figure supplement 2—source data 2
Complete and uncropped membranes of all western blots from Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig2-figsupp2-data2-v2.pdf
Figure 2—figure supplement 3
Akt, p38, JNK, and ERK phosphorylations in adult and neonatal splenic CD19+CD43 B cells following BCR cross-linking.
Splenic CD19+CD43- cells were isolated from adult and neonatal mice and were stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for the indicated duration. Whole cell extracts from stimulated cells were collected for immunoblot analysis of Akt, p38, JNK, and ERK. Data are shown as the mean ± s.d. of three biological replicates. pP values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (**p<0.01, ***p<0.001 and ****p<0.0001).
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Figure 2—figure supplement 3—source data 1
CRaw data of all western blots from Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig2-figsupp3-data1-v2.zip
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Figure 2—figure supplement 3—source data 2
Complete and uncropped membrane of all western blots from Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig2-figsupp3-data2-v2.pdf
Figure 3 with 1 supplement
STAT3 is activated by autocrine IL-6 in a STAT5-dependent manner.
In all experiments splenic CD19+CD43- B cells were isolated and stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies to engage BCR under different conditions. (A and B) CD19+CD43- cells were pre-treated with DMSO, 20 μM Pimozide, or 100 μM S3I-201 for 1 hr and then incubated in the absence or presence of anti-IgM antibodies for 7 hr (biological triplicate). Il10 mRNA expression was determined by RT-qPCR (A) and the amount of secreted IL-10 was determined by ELISA (B). (C) CD19+CD43- cells were pre-treated with DMSO (DM) or 1 μM Pyridone 6 (P6) for 1 hr prior to stimulation with anti-IgM antibodies for the indicated duration and changes in STAT3 (Tyr705) and STAT5 (Tyr694/699) phosphorylations were detected in Western blot analysis (biological triplicate). (D) CD19+CD43- cells were pre-treated with DMSO, 20 μM Pimozide (Pim), or 1 μM Pyridone 6 (P6) for 1 hr and then incubated in the absence or presence of anti-IgM antibodies for 4 hr and changes in STAT3 (Tyr705) phosphorylation were detected in western blot analysis (biological triplicate). (E and F) CD19+CD43- cells were incubated with anti-IgM antibodies for the indicated duration and Il6 mRNA expression was determined by RT-qPCR (E) and the amount of secreted IL-6 was measured by ELISA (F) (biological triplicate). P values (Adult anti-IgM vs Neonate anti-IgM) were calculated using two-way ANOVA. (G and H) CD19+CD43- cells were pre-treated with DMSO, 20 μM Pimozide, or 1 μM Pyridone 6 for 1 hr and then incubated in the absence or presence of anti-IgM antibodies for 4 hr and Il6 mRNA expression was determined by RT-qPCR (G) and the amount of secreted IL-6 was determined by ELISA (H) (biological triplicate). (I) CD19+CD43- cells were transfected with siRNA targeting STAT5A (siSTAT5A) or non-targeting control siRNA (siControl) for 48 hr, and then incubated in the absence or presence of anti-IgM antibodies for 4 hr. The amount of STAT5A was examined by immunoblot analysis. The amount of secreted IL-6 was determined by ELISA (biological triplicate). Unless otherwise is indicated, P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). No significant difference; NS. In all experiments data are shown as the mean ± s.d. of three biological replicates.
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Figure 3—source data 1
Raw data of all western blots from Figure 3.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig3-data1-v2.zip
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Figure 3—source data 2
Complete and uncropped membrane of all western blots from Figure 3.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig3-data2-v2.pdf
Figure 3—figure supplement 1
The effect of inhibitors on STAT3 and STAT5 phosphorylations in splenic CD19+CD43 cells.
(A) Isolated splenic CD19+CD43- cells were pre-treated with DMSO (DM) or 1 μM Pyridone 6 (P6) for 1 hr prior to stimulation with 20 ng/mL recombinant IL-6 or 20 ng/mL recombinant IL-21 for the indicated duration and phosphorylation of STAT3 and STAT5 were assessed in western blot analysis. Data shown are the mean ± s.d. of biological triplicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (***p<0.001 and ****p<0.0001). (B) Splenic CD19+CD43- cells were isolated from neonatal mice and were pre-treated with DMSO, BTK inhibitor Ibrutinib, PKC inhibitor Staurosporine, pan-JAK inhibitor Pyridone 6, or JAK2 inhibitor AG90 for 1 hr prior to stimulation with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for the indicated duration. Phosphorylation of STAT5 was determined in Western blot analysis. Data shown are the mean ± s.d. of biological triplicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (NS: no significant difference, and ****p<0.0001). (C) Isolated splenic CD19+CD43- B cells were left unstimulated or stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 17 hr and intracellular IL-6 were measured in CD19+CD43- B cells. Experiments were repeated twice for reproducibility. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (**p<0.01 and ***p<0.001).
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Figure 3—figure supplement 1—source data 1
Raw data of all western blots from Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig3-figsupp1-data1-v2.zip
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Figure 3—figure supplement 1—source data 2
Complete and uncropped membrane of all western blots from Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig3-figsupp1-data2-v2.pdf
Figure 4 with 4 supplements
Autocrine IL-6 activates IL-10 expression in a STAT3-dependent manner.
In all experiments, splenic CD19+CD43- B cells were isolated and stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies to engage BCR under different conditions. (A) CD19+CD43- cells were stimulated with anti-IgM antibodies for the indicated duration and changes in STAT3 (Tyr705) and STAT5 (Tyr694/699) phosphorylations were detected in Western blot analysis (biological triplicate). (B) CD19+CD43- cells were pre-treated with 10 μg/mL isotype control antibodies (Rat IgG1/Rat IgG2b), anti-IL-6R antibody or anti-IL-10R antibody for 1 hr prior to stimulation with of anti-IgM antibodies for 7 h and Il10 mRNA expression was determined by RT-qPCR (biological triplicate). (C) CD19+CD43- cells, isolated from wild-type (WT) adult, WT neonate, and IL-6-deficient (IL6KO) neonate, were stimulated with anti-IgM antibodies for the indicated duration and changes in STAT3 (Tyr705) and STAT5 (Tyr694/699) phosphorylations were detected in Western blot analysis (biological triplicate). (D and E) CD19+CD43- cells were incubated with anti-IgM antibodies for the indicated duration. Il10 and Tnf mRNA expression were determined by RT-qPCR (D) and the amounts of secreted IL-10 and TNF-α were determined by ELISA (E) (biological triplicate). P values (Neonate WT anti-IgM vs Neonate IL6KO anti-IgM) were calculated using two-way ANOVA (****p<0.0001). Unless otherwise indicated, in other experiments P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). No significant difference; NS. In all experiments, data are shown as the mean ± s.d. of three biological replicates.
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Figure 4—source data 1
Raw data of all western blots from Figure 4.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig4-data1-v2.zip
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Figure 4—source data 2
Complete and uncropped membrane of all western blots from Figure 4.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig4-data2-v2.pdf
Figure 4—figure supplement 1
Inhibition of gp130 blocks STAT3 activation in BCR stimulated neonatal cells.
Isolated splenic CD19+CD43- cells were pre-treated with DMSO or 2 μM of the gp130 inhibitor SC144 for 1 hr prior to stimulation with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for the indicated duration. Phosphorylation of STAT3 was determined in Western blot analysis. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (NS: no significant difference, ****p<0.0001).
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Figure 4—figure supplement 1—source data 1
Raw data of all western blots from Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig4-figsupp1-data1-v2.zip
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Figure 4—figure supplement 1—source data 2
Complete and uncropped membrane of all western blots from Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig4-figsupp1-data2-v2.pdf
Figure 4—figure supplement 2
Autocrine IL-10 has a minor role in STAT3 activation following BCR cross-linking.
(A) Isolated splenic CD19+CD43- cells were stimulated with 10 nM recombinant IL-10 for the indicated duration and STAT3 phosphorylation was assessed in western blot analysis. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test. (B) Isolated CD19+CD43- B cells were pre-treated with 10 μg/mL isotype control antibodies (Rat IgG1 and Rat IgG2b), anti-IL-6R antibody and anti-IL-10R antibody for 1 hr prior to stimulation with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for the indicated duration and STAT3 phosphorylation was assessed in western blot analysis. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (NS: no significant difference, and ****p<0.0001). (C) Isolated CD19+CD43- B cells from wild-type (WT) adult, WT neonate and IL-6-deficient (IL6KO) neonates were stimulated with 20 ng/mL recombinant IL-6 for the indicated duration. STAT3 phosphorylation was determined in Western blot analysis. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (NS: no significant difference).
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Figure 4—figure supplement 2—source data 1
Raw data of all western blots from Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig4-figsupp2-data1-v2.zip
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Figure 4—figure supplement 2—source data 2
Complete and uncropped membrane of all western blots from Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig4-figsupp2-data2-v2.pdf
Figure 4—figure supplement 3
Autocrine/paracrine IL-1β is not involved in neonatal BCR-induced IL-10 production.
(A–C) Splenic CD19+CD43- cells were isolated from wild-type (WT) adult, WT neonate, and IL-6-deficient (IL6KO) neonate mice. Isolated cells were treated with 10 μg/mL isotype control antibody (American Hamster IgG) or anti-IL1β antibody for 1 hr prior to stimulation with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 18 hr. Il1b (A) and Il10 (B) mRNA expressions were determined by RT-qPCR and the secreted IL-10 (C) were measured by ELISA. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (****p<0.0001). (D) Splenic CD19+CD43- cells were isolated from adult and were treated with 10 μg/mL isotype control antibody (Armenian Hamster IgG) or anti-IL1β antibody for 1 h prior to stimulation with 20 ng/mL recombinant IL-1β for the indicated duration. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (***p<0.001). (E) Splenic CD19+CD43- cells were isolated from adult and neonatal mice. Isolated cells were stimulated with 20 ng/mL recombinant IL-6, 20 ng/mL recombinant IL-1β, 10 μg/mL LPS, or 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 8 hr. Culture supernatant IL-10 levels were determined by ELISA (n=3). Data shown are the mean ± s.d. of three biological replicates. P values versus adult counterparts were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05 and ****p<0.0001) and two-tailed unpaired t-tests (††††p<0.0001).
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Figure 4—figure supplement 3—source data 1
Raw data of all western blots from Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig4-figsupp3-data1-v2.zip
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Figure 4—figure supplement 3—source data 2
Complete and uncropped membrane of all western blots from Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig4-figsupp3-data2-v2.pdf
Figure 4—figure supplement 4
Autocrine/paracrine IL-35 is not involved in neonatal BCR-induced IL-10 production.
Splenic CD19+CD43- cells were isolated from adult and neonatal mice. Isolated cells were stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 5 hr and 24 hr. Culture supernatant IL-35, IL-6, and IL-10 levels were determined by ELISA (technical triplicates per experiment). Experiments were repeated once for reproducibility. Data shown are the mean ± s.d. of a representative experiment. P values versus adult counterparts were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (****p<0.0001).
Figure 5 with 3 supplements
Neonatal BCRs activate STAT5 in PKC/FAK/Rac1/Syk and Btk signaling axis.
In all experiments, isolated splenic CD19+CD43- B cells were used. (A) CD19+CD43- cells were analyzed for surface levels of IL-6Rα. Data are shown as the mean ± s.d. of three biological replicates. P values versus adult counterparts were calculated using two-tailed unpaired t-tests (*p<0.05). (B) CD19+CD43- cells were stimulated with recombinant IL-6 at indicated concentrations for 30 min. Changes in STAT3 (Tyr705) phosphorylation were detected in western blot analysis. Data are representative of three biological replicates. Data are shown as the mean ± s.d. of normalized ratio of P-STAT3 to total STAT3 in three biological replicates. P values versus adult counterparts were calculated using two-way ANOVA with a Sidak’s multiple comparisons test for the dose-response curve (***p<0.001 and ****p<0.0001) and two-tailed Student’s t-test for the IL-6 EC50 comparison (*p<0.05). (C) CD19+CD43- cells were stimulated with increasing concentrations of recombinant IL-6 for 48 hr. The amount of secreted IL-10 was determined by ELISA. Data are shown as the mean ± s.d. of two biological replicates. P values versus adult counterparts were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (****p<0.0001). (D) Schematic representation of cellular events leading to enhanced IL-10 production from neonatal CD19+CD43- B cells following BCR cross-linking. Larger population of neonatal CD19+CD43- B cells activate STAT5 through highly expressed IgM, and STAT5 causes IL-6. Large amount of IL-6 and highly expressed IL-6Rα on neonatal CD19+CD43- B cells synergistically activate STAT3 in an autocrine/paracrine fashion, leading to IL-10 production.
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Figure 5—source data 1
Raw data of all western blots from Figure 5.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig5-data1-v2.zip
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Figure 5—source data 2
Complete and uncropped membrane of all western blots from Figure 5.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig5-data2-v2.pdf
Figure 5—figure supplement 1
Comparable expression of gp130 on adult and neonatal B cells.
Il6ra gene expression was not statistically higher in neonatal B cells than in adult counterparts.Isolated CD19 + CD43 B cells were analyzed for Il6ra mRNA expressions by RT-qPCR. Data shown are the mean ±s.d. of three biological replicates. P values versus adult counterparts were calculated using two-tailed unpaired t-tests (NS, p=0.0520).
Figure 5—figure supplement 2
Comparable expression of gp130 on adult and neonatal B cells.
Isolated CD19+CD43- B cells were analyzed for surface levels of gp130. Data shown are the mean ± s.d. of three biological replicates. P values versus adult counterparts were calculated using two-tailed unpaired t-tests (NS: no significance).
Figure 5—figure supplement 3
TLR-induced IL-10 production is not dependent on IL-6.
Splenic CD19+CD43- cells were isolated from wild-type (WT) adult, WT neonate, and IL-6-deficient neonate (IL6KO). Isolated cells were stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies, 10 μg/mL CpG 1826, or 10 μg/mL lipopolysaccharide (LPS) for 18 h. Culture supernatant IL-10 levels were determined by ELISA. Data shown are the mean ± s.d. of two biological replicates. P values versus adult counterparts were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (**p<0.01 and ****p<0.0001).
Figure 6 with 3 supplements
In all experiments, splenic CD19+CD43- B cells were isolated and stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies to engage BCR under different conditions.
(A) CD19+CD43- cells were stimulated with anti-IgM antibodies for the indicated duration and changes in PKC (Ser660), FAK (Tyr397), and FAK (Tyr567) phosphorylations were detected in western blot analysis (biological triplicate). (B) CD19+CD43- cells were pre-treated with DMSO, 20 nM PKC inhibitor Staurosporine (Staur), or 10 μM FAK inhibitor 14 (F-14) for 1 hr and then stimulated with anti-IgM antibodies for the indicated duration. Changes in FAK (Tyr397) and STAT5 (Tyr694/699) phosphorylations were detected in Western blot analysis (biological triplicate). (C) Isolated CD19+CD43- B cells were pre-treated with DMSO, 20 nM Staurosporine, or 10 μM F-14, or 20 μM Pimozide for 1 hr prior to incubation in the absence or presence of anti-IgM antibodies for 4 hr and Il6 mRNA expression was determined by RT-qPCR (biological triplicate). (D) CD19+CD43- cells were pre-treated with Rac1 inhibitor NSC23766 for 1 hr and then stimulated with anti-IgM antibodies for 15 min. Changes in STAT5 (Tyr694/699) phosphorylation was detected in western blot analysis (biological triplicate). (E) Isolated CD19+CD43- B cells were pre-treated with NSC23766 for 1 hr prior to incubation in the absence or presence of anti-IgM antibodies for 20 hr and Il6 mRNA expression was determined by RT-qPCR (biological triplicate). In all experiments, data shown as the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001).
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Figure 6—source data 1
Raw data of all western blots from Figure 6.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig6-data1-v2.zip
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Figure 6—source data 2
Complete and uncropped membrane of all western blots from Figure 6.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig6-data2-v2.pdf
Figure 6—figure supplement 1
Neonatal BCRs activate FAK.
Isolated splenic CD19+CD43- B cells were left unstimulated or stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 30 s and intracellular phospho-FAK was measured in CD19+CD43- B cells. Experiments were repeated twice for reproducibility. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05 and **p<0.01).
Figure 6—figure supplement 2
BCR-induced STAT5 activation is dependent on Syk, Btk, and PKC.
(A) Isolated CD19+CD43- B cells were stimulated with 10 μg/mL PMA for the indicated duration and phosphorylation of PKC, p65, IκBα, FAK, and STAT5 were determined in western blot analysis. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (****p<0.0001). (B) Isolated CD19+CD43- cells from neonatal spleen were pre-treated with DMSO, 10 μM Syk inhibitor (Syk-i), 5 μM Btk inhibitor Ibrutinib (Ibr), or 20 nM Staurosporine for 1 hr and then stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for the indicated duration and phosphorylation of STAT5 was determined in western blot analysis. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (****p<0.0001).
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Figure 6—figure supplement 2—source data 1
Raw data of all western blots from Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig6-figsupp2-data1-v2.zip
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Figure 6—figure supplement 2—source data 2
Complete and uncropped membrane of all western blots from Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/83561/elife-83561-fig6-figsupp2-data2-v2.pdf
Figure 6—figure supplement 3
IL-6 is not responsible for IL-10 production by CD43-expressing B1 subset in neonatal mouse.
Isolated CD19+ cells were incubated in the absence or presence of 10 µg/mL F(ab’)2 fragments of anti-IgM for 20 hr (technical triplicates per experiment). Experiments were repeated twice for reproducibility. Data shown are the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (**p<0.01, NS: no significance) and two-tailed unpaired t-tests (††p<0.01, †††p<0.001 and ††††p<0.0001).
Figure 7 with 2 supplements
IL-10 secreted from neonatal CD19+CD43 B cells suppress TNF-α secretion from macrophages.
(A and B) Isolated splenic CD19+CD43- B cells were stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 24 hr. IL-10 (A) and TNF-α (B) levels in conditioned medium (CM) were determined by ELISA. (C) Peritoneal macrophages isolated from adult mice were treated with 10 μg/mL anti-IL10R or isotype (Rat IgG1) control antibodies for 1 hr and then cultured in CM for 24 hr. Levels of TNF-α secreted by macrophages cultured in CM were determined by ELISA. Data are shown as the mean ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05, **p<0.01, and ****p<0.0001).
Figure 7—figure supplement 1
Schematic description of experimental design to test the suppressive effect of IL-10 secreted from neonatal CD19+CD43 B cells.
Isolated splenic CD19+CD43- B cells from adult and neonatal mice were plated at 3.0 million cells per mL and stimulated with 10 μg/mL F(ab’)2 fragments of anti-IgM antibodies for 24 hr. Conditioned media were collected and filtered through 0.2 µm membrane. Peritoneal macrophages isolated from adult mice were treated with 10 μg/mL anti-IL10R (1B1.3A) or isotype control antibodies (Rat IgG1) for 1 hr prior to culture in conditioned media. After 24 hr incubation, supernatants were collected for the assessments.
Figure 7—figure supplement 2
Schematic representation of IL-10 production in BCR stimulated neonatal CD19+CD43 cells.
Recognition of antigen by BCR induces IL-6 secretion from CD19+CD43- B cells through a pathway involving Syk, Btk, PKC, FAK, Rac1, and STAT5. The secreted IL-6 binds to its receptors IL-6Rα and gp130 on CD19+CD43- B cells to stimulate the production of IL-10 in a JAK- and STAT3-dependent manner.
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B cell receptor-induced IL-10 production from neonatal mouse CD19+CD43- cells depends on STAT5-mediated IL-6 secretion
eLife 12:e83561.
https://doi.org/10.7554/eLife.83561
