Monoallelic IRF5 deficiency in B cells prevents murine lupus

Abstract

Gain-of-function polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. We now show that monoallelic IRF5 deficiency in B cells markedly reduced disease in a murine lupus model. In contrast, similar reduction of IRF5 expression in macrophages, monocytes, and neutrophils did not reduce disease severity. B cell receptor and TLR7 signaling synergized to promote IRF5 phosphorylation and increase IRF5 protein expression, with these processes being independently regulated. This synergy increased B cell-intrinsic IL-6 and TNF-α production, both key requirements for germinal center (GC) responses, with IL-6 and TNF-α production in vitro and in vivo being substantially lower with loss of 1 allele of IRF5. Mechanistically, TLR7-dependent IRF5 nuclear translocation was reduced in B cells from IRF5-heterozygous mice. In addition, we show in multiple lupus models that IRF5 expression was dynamically regulated in vivo with increased expression in GC B cells compared with non-GC B cells and with further sequential increases during progression to plasmablasts and long-lived plasma cells. Overall, a critical threshold level of IRF5 in B cells was required to promote disease in murine lupus.

Keywords: Autoimmune diseases; Autoimmunity.

Figure 1. B cell–specific reduction of IRF5 expression in IRF5^ΔB mice.

All analyses were done in 8- to 10-week-old FcγRIIB^−/−Yaa mice. (A) Representative Western blot of IRF5 protein expression in sorted splenic B cells (CD19⁺) and myeloid cells (CD11b⁺Ly6G^–) from IRF5^F/+ and IRF5^ΔB mice. All lanes were run on the same gel but were noncontiguous. (B) Expression of IRF5 in B cells and myeloid cells from IRF5^ΔB mice normalized to IRF5^F/+ (n = 4). Data were analyzed using 2-tailed, unpaired Welch’s t test; **P < 0.01. (C) Representative flow cytometry plots of intracellular IRF5 expression in CD19⁺ B cells, CD11b⁺Ly6C⁺ monocytes, CD11b⁺Ly6G⁺ neutrophils, and CD11b^–PDCA1⁺Ly6C⁺ pDCs from IRF5^F/+, IRF5ΔB, and IRF5^–/– global knockout mice. (D) MFI values of IRF5 in B cells, monocytes, neutrophils, and pDCs from IRF5^F/+ and IRF5^ΔB mice (representative experiment of 3 individual experiments, n = 2 for each genotype). (E) IRF5 expression in B cells, monocytes, neutrophils, and pDCs from IRF5^ΔB mice normalized to the IRF5^F/+ littermate control in each experiment (n = 6). Data are shown as mean ± SEM and were analyzed using 1-way ANOVA with Tukey’s post hoc test; ****P < 0.0001. IRF5, IFN regulatory factor 5; pDCs, plasmacytoid DCs.

Figure 2. Splenomegaly and T cell activation are reduced in IRF5^ΔB mice.

All analyses were done in 5-month-old FcγRIIB^−/−Yaa mice. (A) Spleen weights from WT (n = 10), mb1cre (n = 13), IRF5^F/+ (n = 16), IRF5^ΔB (n = 8), and IRF5^+/– (global heterozygous deletion, n = 5) mice. (B) Splenic cell counts from WT (n = 5), mb1cre (n = 5), IRF5^F/+ (n = 14), and IRF5^ΔB (n = 8) mice. (C) Representative flow cytometry plots of CD4⁺CD62L^–CD44⁺ (effector/memory) and CD62L⁺CD44^– (naive) T cells from spleen of IRF5^F/+ and IRF5^ΔB mice. (D and E) Percentage and number of CD62L^–CD44⁺ CD4⁺ T cells from WT (n = 5), mb1cre (n = 5), IRF5^F/+ (n = 15), and IRF5^ΔB (n = 8) mice. (F and G) Percentage and number of CD62L⁺CD44^–CD4⁺ T cells. (H) Representative flow cytometry plots of CD8⁺CD62L^–CD44⁺ (effector/memory) and CD62L⁺CD44^– (naive) T cells from spleen of IRF5^F/+ and IRF5^ΔB mice. (I and J) Percentage and number of CD62L^–CD44⁺CD8⁺ T cells from WT (n = 5), mb1cre (n = 5), IRF5^F/+ (n = 15), and IRF5^ΔB (n = 8) mice. (K and L) Percentage and number of CD62L⁺CD44^–CD8⁺ T cells. Data are shown as mean ± SEM and were analyzed using 1-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IRF5, IFN regulatory factor 5.

Figure 3. Serum IgG, autoantibodies, and plasma cells are reduced in IRF5^ΔB mice.

All analyses were done in 5-month-old FcγRIIB^−/−Yaa mice. (A–D) IRF5^F/+ (n = 11) and IRF5^ΔB mice (n = 5–7) were analyzed. (A) Serum IgG isotype concentrations. (B) Serum antinuclear autoantibody titers. (C) Serum anti-nucleosome IgG concentration. (D) Serum anti-Sm/RNP IgG concentrations. (E) Representative flow cytometry plots and total numbers of splenic plasmablasts in IRF5^F/+ (n = 15) and IRF5^ΔB (n = 8) mice. (F) Representative flow cytometry plots and percentages of bone marrow plasma cells in IRF5^F/+ (n = 15) and IRF5^ΔB (n = 8) mice. Data are shown as mean ± SEM and were analyzed using 2-tailed, unpaired Welch’s t test; **P < 0.01, ***P < 0.001, ****P < 0.0001. IRF5, IFN regulatory factor 5.

Figure 4. Decreased renal disease in IRF5^ΔB mice.

All analyses were done in 5-month-old FcγRIIB^−/−Yaa mice. (A) Representative renal histology of IRF5^F/+ and IRF5^ΔB mice. Red arrow depicts necrotic cells; black arrow depicts cellular crescent (original magnification, ×20). (B–D) Quantification of renal disease by (B) percentage of glomeruli with crescents or necrosis, (C) glomerular injury score, and (D) interstitial disease. IRF5^F/+ (n = 11) and IRF5B (n = 8). (E) Representative examples and (F) quantitation of glomerular IgG and complement C3 deposition measured by fluorescence intensity in 11–14 glomeruli per mouse from 5 mice per group. All scored glomeruli are shown (original magnification, ×20). Data are shown as mean ± SEM and were analyzed using 2-tailed, unpaired Welch’s t test; ***P < 0.001, ****P < 0.0001. IRF5, IFN regulatory factor 5.

Figure 5. LysMcre-mediated deletion of IRF5 does not reduce disease in FcγRIIB^−/−Yaa mice.

All analyses were done in FcγRIIB^−/−Yaa littermates that either did not express LysMcre (IRF5^fl/fl) or did express LysMcre (IRF5^ΔM). (A) Flow cytometry quantitation of intracellular IRF5 expression (MFI values) in neutrophils (CD11b⁺Ly6G⁺), monocytes (CD11b⁺Ly6C⁺), and B cells (CD19⁺) from spleen, and in peritoneal macrophages (F/480⁺CD11b⁺), in 8- to 10-week-old FcγRIIB^−/−Yaa littermates (n = 4 or n = 5). Data are shown as mean ± SEM and were analyzed using 2-tailed, unpaired Welch’s t test; ***P < 0.001, ****P < 0.0001. (B–D) All analyses were done in 5-month-old FcγRIIB^−/−Yaa littermates. (B) Spleen weight and cell counts from IRF5^fl/fl (n = 10–12) and IRF5^ΔM mice (n = 10–12). (C) Serum antinuclear autoantibody titers, serum anti-nucleosome IgG concentration, and serum anti-Sm/RNP IgG concentration (n = 5–10). (D) Quantification of renal disease by glomerular injury score, percentage of glomeruli with crescents or necrosis, and interstitial disease (n = 10). Data are shown as mean ± SEM and were analyzed using 2-tailed, unpaired Welch’s t test. IRF5, IFN regulatory factor 5.

Figure 6. GC B cells, Tfhs, and T-bet⁺ B cells are reduced in the spleens of IRF5^ΔB mice.

Spleen cells from 8- to 10-week-old FcγRIIB^−/−Yaa WT (n = 6), IRF5^F/+ (n = 10), IRF5^ΔB (n = 8), IRF5^+/– (global heterozygous deletion, n = 7), and IRF5^–/– (global homozygous deletion, n = 7) mice were analyzed. (A and B) Representative flow cytometry plots and total numbers of CD95⁺CD38^– GC B cells (gated on CD19⁺B220⁺ cells). (C and D) Representative flow cytometry plots and total numbers of CXCR5⁺PD-1⁺ Tfhs (gated on CD3⁺CD4⁺). (E) Upper panel indicates CD23^–CD21^– B cells (gated on B220⁺CD19⁺CD43^–CD93 cells); lower panel indicates T-bet⁺CD11c⁺ ABCs gated on the CD23^–CD21^– B cells shown in the upper panel. A representative example is shown. (F) Total number of T-bet⁺CD11c⁺ ABCs. Data are shown as mean ± SEM and were analyzed using 1-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001. GC, germinal center; Tfhs, T follicular helper cells; IRF5, IFN regulatory factor 5; ABCs, age-associated B cells.

Figure 7. Reduced IRF5 expression in B cells decreases IL-6 and TNF-α production in vitro, and serum IL-6 and TNF-α is reduced in IRF5^ΔB mice.

(A–C) B cells were isolated from the spleens of FcγRIIB^−/−Yaa mice at 8–10 weeks of age and stimulated for 24 hours with anti-IgM, anti-CD40, R848, and CpG-B alone or in combination. (A) Representative experiments showing mean IL-6 and TNF-α production by B cells from WT, IRF5^+/–, and IRF5^–/– mice (n = 2 for each genotype). (B and C) IL-6 (n = 8) and TNF-α (n = 3) production after R848 stimulation (B) and CpG-B stimulation (C) by B cells from WT, IRF5^+/–, and IRF5^–/– mice normalized to the WT control in each experiment. (D and E) IL-6 and TNF-α production by B cells from IRF5^ΔB mice normalized to the littermate IRF5^F/+ control in each experiment (n = 4 for each genotype). Data are shown as mean ± SEM and were analyzed using 2-way ANOVA with Tukey’s post hoc test; **P < 0.01, ***P < 0.001, ****P < 0.0001. (F) Mean serum IL-6 and TNF-α levels from 5-month-old IRF5^ΔB (n = 8) and littermate IRF5^F/+ (n = 8) mice. Data are shown as mean ± SEM and were analyzed using 2-tailed, unpaired Welch’s t test; *P < 0.05, ***P < 0.001. IRF5, IFN regulatory factor 5.

Figure 8. TLR7 signaling is required for IRF5 phosphorylation, and IRF5 nuclear translocation is reduced in B cells from FcγRIIB^−/−Yaa IRF5^+/– mice.

(A and B) B cells were isolated from the spleens of FcγRIIB^−/−Yaa mice at 8–10 weeks of age. (A) B cells were stimulated with anti-IgM, anti-CD40, and R848 alone or in combination for 2 hours and the protein lysate analyzed using phospho-Tag gel (upper panel) or standard gel (lower panels). B cells isolated from an IRF5-deficient (IRF5^–/–) mouse are shown in the first lane. p-IRF5 denotes phosphorylated IRF5. A representative example of 7 individual experiments is shown. (B) Ratio of p-IRF5 to unphosphorylated IRF5 (u-IRF5). Intensity of p-IRF5 was normalized to the intensity of unphosphorylated IRF5 (lowest band of IRF5 on p-Tag gel as shown in A) (n = 7). (C and D) B cells from FcγRIIB^−/−Yaa (WT) and FcγRIIB^−/−Yaa IRF5^+/– mice were stimulated for 2 hours with R848, or not stimulated (untreated), and IRF5 was probed in the nuclear and cytoplasmic fractions. (C) A representative experiment of 4 individual experiments is shown. (D) Ratio of IRF5 expression in nucleus relative to the WT after R848 stimulation; nuclear IRF5 intensity in each sample was first normalized to its own loading control (histone; n = 5). Data are shown as mean ± SEM and were analyzed using 1-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001. IRF5, IFN regulatory factor 5.

Figure 9. IRF5 expression is increased in activated B cells in vitro.

(A–E) Splenic B cells were isolated from 8- to 10-week-old FcγRIIB^−/−Yaa or C57BL/6 mice and were either not stimulated or stimulated with anti-IgM, anti-CD40, and R848 alone or in combination for 24 hours. (A) Intracellular IRF5 levels were measured using flow cytometry. A representative experiment of 6 individual experiments using B cells from FcγRIIB^−/−Yaa mice is shown. (B and D) MFI of IRF5 with and without stimulation in B cells from FcγRIIB^−/−Yaa mice (B) and C57BL/6 mice (D) (n = 2 per strain). (C and E) Fold change of IRF5 expression normalized to unstimulated control in B cells from FcγRIIB^−/−Yaa mice (C) and C57BL/6 mice (E) (n = 6 per strain). Data are shown as mean ± SEM and were analyzed using 1-way ANOVA with Tukey’s post hoc test; **P < 0.01, ****P < 0.0001. IRF5, IFN regulatory factor 5.

Figure 10. IRF5 expression is increased in GC B cells, splenic PBs, and BM PCs in vivo.

(A–G) Flow cytometry performed on splenocytes and bone marrow from FcγRIIB^−/−Yaa mice at 8–10 weeks of age. (A) Representative examples of IRF5 expression in GC B cells (CD38^–CD95⁺CD19⁺) and non-GC B cells (CD38⁺CD95^–; upper panel); PBs (CD44⁺CD138⁺) and CD19⁺CD138^– B cells (non-PBs; middle panel); and BM PCs (CD44⁺ CD138⁺) and non-PB B cells from spleens (lower panel). (B) Fold change in IRF5 expression in GC B cells normalized to non-GC B cells (n = 9). (C) Fold change in IRF5 expression in PBs normalized to non-PBs (n = 8). (D) Fold change in IRF5 expression in PCs normalized to splenic non-PBs (n = 8). (E–G) IRF5 expression in NZB/W mice (n = 6), MRL/lpr mice (n = 6), and C57BL/6 (n = 6) mice immunized with 4-hydroxy-3-nitrophenylacetyl coupled to chicken γ-globulin. (E) Fold change of IRF5 expression in GC B cells. (F) Fold change of IRF5 expression in PBs. (G) Fold change of IRF5 expression in plasma cells. Black histogram shows isotype control in non-GC B cells, non-PBs, or BM PCs; gray-tinted histogram shows isotype control in GC B cells, PBs, or BM PCs; blue histogram shows IRF5 expression in non-GC B cells and non-PBs; red histogram shows IRF5 expression in GC B cells, PBs, or BM PCs. Data are shown as mean ± SEM and were analyzed using 2-tailed, unpaired Welch’s t test; *P < 0.05, **P < 0.01. IRF5, IFN regulatory factor 5; GC, germinal center; PBs, plasmablasts; BM PCs, bone marrow plasma cells.