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Regulation of age-associated B cells by IRF5 in systemic autoimmunity

Nature Immunology volume 19, pages 407–419 (2018)Cite this article

Abstract

Age-associated B cells (ABCs) are a subset of B cells dependent on the transcription factor T-bet that accumulate prematurely in autoimmune settings. The pathways that regulate ABCs in autoimmunity are largely unknown. SWAP-70 and DEF6 (also known as IBP or SLAT) are the only two members of the SWEF family, a unique family of Rho GTPase–regulatory proteins that control both cytoskeletal dynamics and the activity of the transcription factor IRF4. Notably, DEF6 is a newly identified human risk variant for systemic lupus erythematosus. Here we found that the lupus syndrome that developed in SWEF-deficient mice was accompanied by the accumulation of ABCs that produced autoantibodies after stimulation. ABCs from SWEF-deficient mice exhibited a distinctive transcriptome and a unique chromatin landscape characterized by enrichment for motifs bound by transcription factors of the IRF and AP-1 families and the transcription factor T-bet. Enhanced ABC formation in SWEF-deficient mice was controlled by the cytokine IL-21 and IRF5, whose variants are strongly associated with lupus. The lack of SWEF proteins led to dysregulated activity of IRF5 in response to stimulation with IL-21. These studies thus elucidate a previously unknown signaling pathway that controls ABCs in autoimmunity.

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Fig. 1: Spontaneous expansion of the ABC population in DKO mice.
Fig. 2: IL-21 regulates the generation of DKO ABCs in vitro and in vivo
Fig. 3: DKO ABCs exhibit a distinctive transcriptome.
Fig. 4: The chromatin landscape of DKO ABCs shows enrichment for IRF and AP-1-BATF motifs.
Fig. 5: Wild-type and DKO ABCs exhibit distinct transcriptional and chromatin profiles.
Fig. 6: IRF5 regulates the IL-21-mediated formation of DKO ABCs.
Fig. 7: Enhanced binding of IRF5 to ABC regulatory regions in the absence of SWEF proteins.
Fig. 8: Monoallelic deletion of Irf5 abolishes the accumulation of ABCs and lupus development in DKO mice.

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Acknowledgements

We thank I. Rogatsky (Hospital for Special Surgery) for expression constructs encoding full-length wild-type human IRF5 in the plasmid pcDNA3P, and the late P. Pitha-Rowe (Johns Hopkins University) for Irf5fl/fl mice. Supported by the Barbara Volcker Center and a gift made in honor of Anne Kennedy O’Neil (M.M.), the US National Institutes of Health (NIH F31 Ruth L. Kirschstein National Service Award to E.R.; AR064883 and AR070146 to A.B.P; and AI044938 to L.B.I.), the Peter Jay Sharp Foundation, the Tow Foundation (support for the David Z. Rosensweig Genomics Research Center), Giammaria Giuliani, the Ambrose Monell Foundation and the Epigenomics Core and the Flow Cytometry Core Facility of Weill Cornell Medical College (Office of the Director of the National Institutes of Health under Award Number S10OD019986 to Hospital for Special Surgery; technical support).

Author information

Authors and Affiliations

  1. Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, NY, USA

    Michela Manni, Sanjay Gupta, Man Shi & Alessandra B. Pernis

  2. Graduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA

    Edd Ricker, Lionel B. Ivashkiv & Alessandra B. Pernis

  3. Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, NY, USA

    Yurii Chinenov, Sung Ho Park & Lionel B. Ivashkiv

  4. David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, NY, USA

    Yurii Chinenov, Sung Ho Park, Lionel B. Ivashkiv & Alessandra B. Pernis

  5. Research Division and Precision Medicine Laboratory, Hospital for Special Surgery, New York, NY, USA

    Tania Pannellini

  6. Institute of Physiological Chemistry, Technische Universität, Dresden, Germany

    Rolf Jessberger

  7. Department of Medicine, Weill Cornell Medical College, Cornell University, New York, NY, USA

    Lionel B. Ivashkiv & Alessandra B. Pernis

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Contributions

M.M. designed and performed the experiments, interpreted the experiments and wrote the manuscript; S.G., E.R. and M.S. performed the experiments; Y.C. conducted the RNA-seq bioinformatics analysis; S.H.P. conducted the ATAC-seq bioinformatics analysis; T.P. assisted with the histological analysis; R.J. generated the Swap70−/− mice and helped write the manuscript; L.B.I. supervised the bioinformatics analysis and helped write the manuscript; and A.B.P. designed and supervised the study, interpreted the experiments, and wrote the manuscript.

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Correspondence to Alessandra B. Pernis.

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Supplementary Figure 1 Spontaneous population expansion of ABCs in DKO mice

(a) Spleens of WT and DKO female mice (>20 weeks old) were evaluated by flow cytometry for the presence of plasmacytoid dendritic cells. Representative FACS plots for CD11c and PDCA-1 expression after gating on B220+ or B220+CD19+ cells. Graphs show the frequencies and numbers for individual mice and mean value of 3 independent experiments (n = 3 WT and 5 DKO). ns: not significant. (two-tailed Student-t test). (b) Representative FACS plots for CD11b and CD11c expression after gating on B220+CD19+ cells in the spleens of 10 weeks old WT and DKO female mice. Graphs show frequencies and numbers of individual mice and mean value of 2 independent experiments (n = 4 WT and 5 DKO). ** P = 0.0011; *** P = 0.0001. (two-tailed Student-t test). (c) Representative FACS plots for CD11b and CD11c expression after gating on B220+CD19+ cells in skin draining lymph nodes of WT, DKO, Def6–/– and Swap70–/– mice (18–24 weeks old). Graphs show frequencies and numbers of individual mice and mean value of 3 independent experiments (n = 3 WT, 5 DKO, 5 Def6–/–, 4 Swap70–/–). ** P = 0.0018 (WT vs. DKO), P = 0.0011 (DKO vs. Def6–/–), P = 0.0055 (DKO vs. Swap70–/–) (One-way ANOVA followed by Bonferroni’s multiple comparisons test). (d) Representative FACS plots for CD11c and T-bet expression gated on B220+ cells or B220+CD19+ cells. Graphs show frequencies and numbers for individual mice and mean values of 3 independent experiments (n = 3 WT, 5 DKO). * P = 0.0137 (B220+) and P = 0.0131 (B220+CD19+); ** P = 0.068 (B220+) and P = 0.0075 (B220+CD19+). (two-tailed Student-t test). (e) Representative FACS plots for IgG1 and IgG2c expression on B220+ CD19+CD11c+CD11b+ cells from WT and DKO female mice (>20 weeks old). Graphs show frequencies and numbers for individual mice and mean values of 16 independent experiments, (n = 17 WT and 30 DKO). ** P = 0.0062; *** P = 0.0006; **** P < 0.0001. (two-tailed Student-t test). (f) Gating strategy employed to sort ABC and FoB cells.

Supplementary Figure 2 IL-21 regulates the generation of DKO ABCs in vitro and in vivo

(a) Generation of ABCs (B220+CD11c+CD11b+) B cells from cultures of CD23+ B cells purified from WT and DKO female mice (8–10 weeks of age) stimulated with αIgM (5 μg/ml), αCD40 (5 μg/ml), IL-21 (50 ng/ml) or imiquimod (1 μg/ml) for 3 days as assessed by flow cytometry. Representative FACS plot is shown. Graph shows mean and individual value of 5 independent experiments (n = 5 cell culture). *** P = 0.0001. (One-way ANOVA followed by Bonferroni’s multiple comparisons test). (b) Generation of ABCs (B220+CD11c+T-bet+) B cells from cultures of CD23+ B cells purified from WT and DKO female mice (8–10 weeks of age) and stimulated with various combinations of αIgM (5 μg/ml), αCD40 (5 μg/ml), IL-21 (50 ng/ml), IL-4 (10 ng/ml), or IFN-γ (20 ng/ml) as indicated. Representative FACS plot is shown. Graph shows mean and individual values of 3 independent experiments (n = 3 cell cultures). **** P < 0.0001. (One-way ANOVA followed by Bonferroni’s multiple comparisons test). (c-e) Representative FACS plots showing the presence of TFH cells (CD4+CXCR5+PD1+Foxp3-), germinal center (GC) B cells (B220+FAS+GL-7+) and B220intCD138+ plasma cells (PC) in the spleens of WT, DKO, IL21–/– DKO, and SAP–/– DKO female mice (>24 weeks old). (f) Quantification of TFH, GC, and PC of c,d,e. Graphs show percentages and numbers of specific cells types in individual mice and mean of 4 independent experiments (n = 4 WT, 4–8 DKO, 7 IL21–/– DKO, 7 SAP–/– DKO). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. (One-way ANOVA followed by Bonferroni’s multiple comparisons test).

Supplementary Figure 3 DKO ABCs exhibit a distinctive transcriptome

(a) Selected GSEA Hallmark pathways analysis of WT FoB compared to DKO FoB cells (n = 2 WT and 3 DKO/group). (b) Apoptotic rates as measured by caspase 3 cleavage in B220+CD11c–T-bet–B cells from WT and DKO mice (left panel) or in CD11c–T-bet– B cells and CD11c+T-bet+ (ABC) B cells from DKO mice (Right panel). Shown is a representative histogram. All data are representative of 3 independent experiments. (n = 4 mice/group). (c) Proliferation of B220+CD11c–T-bet–cells and ABCs (B220+CD11c+T-bet+) was assessed by evaluating dilution of cell trace violet (CTV) by flow cytometry. CD23+ B cells were purified from WT and DKO female mice (6–9 weeks old), labeled with CTV and cultured with αIgM (5 μg/ml), αCD40 (5 μg/ml), and IL-21 (50 ng/ml) for 3 days. Shown is a representative histogram of 5 independent experiments. (n = 5 cell cultures). (d) Cell viability of CD23+ B cells purified from WT and DKO female mice (6–9 weeks old) and stimulated with αIgM (5 μg/ml), αCD40 (5 μg/ml), IL-21 (50 ng/ml) or imiquimod (1 μg/ml) for 3 or 5 days was assessed by staining with CaspGlow or propidium iodide as indicated. Graphs show percentages of live cells +SEM in 3 independent experiments (n = 3 cell cultures). (One-way ANOVA followed by Bonferroni’s multiple comparisons test) (e) Viability was assessed as in d in CD23+B cells stimulated with αIgM (5 μg/ml), αCD40 (5 μg/ml) and IL-21 (50 ng/ml) and gated on CD11c+ or CD11c- cells. Graphs show percentages of live cells +SEM in 3 independent experiments (n = 3 cell cultures). (One-way ANOVA followed by Bonferroni’s multiple comparisons test) (f) Selected GSEA pathways analysis of DKO FoB compared to DKO ABC cells. (n = 3 mice/group).

Supplementary Figure 4 The chromatin landscape of DKO ABCs shows enrichment for IRF and AP-1–BATF motifs

(a) Expression (RNAseq) of total ABC-specific peaks associated genes (n = 2,482) from three independent experiments (n = 3) (b) Functionally enriched Gene Ontology (GO) categories of selected genes associated with ABC-specific peaks of ATAC-seq (n = 487). (c) qPCR analysis of the expression of Il6 mRNA in sorted FoB (B220+CD19+CD11c-CD11b-CD23+) cells from WT and DKO female mice and ABC (B220+CD19+CD11c+CD11b+) cells from DKO female mice as indicated. The data were normalized relative to Ppia mRNA expression. Mean of one representative experiment of 2 independent experiments is shown (n = 2 mice/group). SEM of technical replicates is shown. ** P = 0.0052.

Supplementary Figure 5 WT and DKO ABCs exhibit distinct transcriptional and chromatin profiles

(a) Expression of selected transcription factors in WT and DKO ABC cells as identified by RNA-seq analysis. (n = 2 mice/group). (b) Genomic distribution of WT ABC-specific and DKO ABC-specific peaks of ATAC-seq relative to annotated genomic features. (n = 2 mice/group) (c) Normalized ATAC-seq tag distributions tracks for representative genomic regions at Maf, Mafb and Znhit1 genes. Highlighted are WT ABC or DKO ABC-specific ATAC-seq peaks (n = 2 mice/group).

Supplementary Figure 6 IRF5 regulates the IL-21-mediated formation of DKO ABCs

(a) Flow cytometric analysis of B220+CD11c+CD11b+B cells in the spleens of WT, DKO and Cd11c-Cre Irf4fl/fl DKO female mice (>18 weeks-old). Graphs show frequencies and numbers for individual mice and mean values of 4 independent experiments (n = 4 WT, 5 DKO, 6 Cd11c-Cre Irf4fl/fl DKO). ns: not significant; * P = 0.0485; ** P = 0.0072. (One-way ANOVA followed by Bonferroni’s multiple comparisons test). (b) qPCR analysis of the expression of Irf5 mRNA in cultures of CD23+ B cells purified from WT, DKO and Cd21-Cre Irf5fl/– DKO female mice (8–10 weeks of age) stimulated with αIgM (5 μg/ml) and αCD40 (5 μg/ml). Data were normalized relative to Ppia mRNA expression. Mean of one representative experiment of 4 independent experiments is shown (n = 4 mice/group). SEM of technical replicates is shown. ns: not significant. (One-way ANOVA followed by Bonferroni’s multiple comparisons test).

Supplementary Figure 7 Enhanced binding of IRF5 to ABC regulatory regions in the absence of the SWEF proteins

(a) ChIP assays were performed with either an IRF5 antibody or a T-bet antibody on CD23+ B cells purified from WT, DKO, and Cd21-Cre Irf5fl/– DKO mice and stimulated with αIgM (5 μg/ml) and αCD40 (5 μg/ml) (top panel) or with αIgM (5 μg/ml), αCD40 (5 μg/ml) and IL-21 (50 ng/ml) (bottom panel) for 2 days. qPCR for the ABC specific ATAC-seq peak at the Il6 TSS or the Zeb2 Exon 8 locus was performed. Data are representative of 2 independent experiments. Mean ± SEM is shown (n = 2 mice/grup). ns: not significant; ** P = 0.0079. (One-way ANOVA followed by Bonferroni’s multiple comparisons test). (b) Nuclear extracts were prepared from CD23+ B cells purified from WT, DKO and Cd21-Cre Irf5fl/– DKO female mice (8–10 weeks of age) stimulated with αIgM (5 μg/ml), αCD40 (5 μg/ml) +/− IL-21 (50 ng/ml) or imiquimod (1 μg/ml) for 3 days. Extracts were analyzed by immunoblotting with pSTAT3, STAT3, IRF5, and HDAC1 antibodies. Data are representative of 2 independent experiments. (c) Quantification of immunoblot in (b) by densitometry. P-STAT3, STAT3 and IRF5 were normalized to HDAC1 density. Mean and individual values of 2 independent experiments is shown. (d) Nuclear extracts were prepared from cells stimulated as in a. for 2 days and subjected to ONP assay with a biotinylated oligonucleotide from the Il6 TSS. Precipitated proteins were analyzed by immunoblotting with IRF5 and T-bet antibodies. Data are representative of 2 independent experiments. (e) Binding of IRF5 to the ONP shown in Fig. 7d. 293T cells were transiently transfected with various IRF5 and T-bet constructs as indicated. Nuclear extracts were prepared and subjected to ONP assay with a biotinylated oligonucleotide from the Cxcl10 cluster. Precipitated proteins were analyzed by immunblotting with a FLAG antibody. Data are representative of 2 independent experiments. (f) and (g) 293T cells were transiently transfected with various DEF6, SWAP-70, and IRF5 constructs as indicated. Immunoprecipitations were performed using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting using an anti-IRF5 or anti-HA antibody. Data are representative of 2 independent experiments with similar results. (h), (i), and (j) 293T cells were transiently transfected with various constructs as indicated. Immunoprecipitations were performed using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting using an anti-FLAG, T-bet, DEF6 or HA antibodies. Data are representative of 2 independent experiments with similar results.

Supplementary Figure 8 Monoallelic deletion of Irf5 abolishes the accumulation of ABCs and lupus development in DKO mice

(a,b) Percentage and numbers of B220+CD19+CD11c+T-bet+ (a) and of B220+CD19+CD21–CD23–CD11c+CD11b+ (b) B cells in the spleens of WT, Irf5fl/fl DKO, Irf5fl/– DKO, Cd11c-Cre Irf5fl/– DKO and Cd21-Cre Irf5fl/– DKO female mice (>20 weeks-old). Graphs show the frequencies and numbers for individual mice and mean value of 6 independent experiments (n = 7 WT, 5 Irf5fl/fl DKO, 6 Irf5fl/– DKO, 5 Cd11c-Cre Irf5fl/– DKO and 6 Cd21-Cre Irf5fl/– DKO). *** P = 0.0001; **** P < 0.0001. (One-way ANOVA followed by Bonferroni’s multiple comparisons test). (c) Flow cytometric analysis of CD11c+CD11b+ B cells in the spleens of WT, Irf5fl/fl DKO and Cd11c-Cre Irf5fl/– DKO female mice (>20 weeks-old). Representative FACS plots of 10 independent experiments is shown. (d) Spleen size of indicated mice. Graph shows numbers of individual mice and mean value of 10 independent experiments. (n = 9 WT, 10 Irf5fl/fl DKO, 6 Irf5fl/– DKO, 10 Cd11c-Cre Irf5fl/– DKO and 5 Cd21-Cre Irf5fl/– DKO female mice >20 weeks of age). * P = 0.0254; *** P = 0.0004; **** P < 0.0001. (One-way ANOVA followed by Bonferroni’s multiple comparison test). (e) Quantification of flow cytometric analysis of TFH (CD4+CXCR5+PD1+Foxp3–), GC (B220+FAS+GL-7+), and PC (B220intCD138+) in spleens of WT, Irf5fl/fl DKO, Irf5fl/– DKO, Cd11c-Cre Irf5fl/– DKO and Cd21-Cre Irf5fl/– DKO female mice (>20 weeks). Graphs show percentages and numbers of specific cells types in individual mice and mean value of 10 independent experiments (n = 10 WT, 10 Irf5fl/fl DKO, 6 Irf5fl/– DKO, 10 Cd11c-Cre Irf5fl/– DKO and 5 Cd21-Cre Irf5fl/– DKO female mice). ** P < 0.01; *** P < 0.001; **** P < 0.0001. (One-way ANOVA followed by Bonferroni’s multiple comparisons test). (f) Quantification of flow cytometric analysis of Treg (CD4+Foxp3+), activated Treg (CD4+Foxp3+CD44+), and activated T cells (CD4+Foxp3–CD44+) in spleens of WT, Irf5fl/fl DKO, Irf5fl/– DKO, Cd11c-Cre Irf5fl/– DKO and Cd21-Cre Irf5fl/– DKO female mice (>20 weeks). Graphs show frequencies and numbers of individual mice and mean value of 10 independent experiments (n = 9 WT, 10 Irf5fl/fl DKO, 6 Irf5fl/– DKO, 10 Cd11c-Cre Irf5fl/– DKO and 5 Cd21-Cre Irf5fl/– DKO female mice). ** P < 0.01; *** P < 0.001; **** P < 0.0001. (One-way ANOVA followed by Bonferroni’s multiple comparisons test).

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Supplementary Table 1

Differentially expressed genes in WT and DKO ABC

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Manni, M., Gupta, S., Ricker, E. et al. Regulation of age-associated B cells by IRF5 in systemic autoimmunity. Nat Immunol 19, 407–419 (2018). https://doi.org/10.1038/s41590-018-0056-8

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