B Cell αv Integrins Regulate TLR-Driven Autoimmunity

Abstract

Systemic lupus erythematosus (SLE) is defined by loss of B cell tolerance, resulting in production of autoantibodies against nucleic acids and other cellular Ags. Aberrant activation of TLRs by self-derived RNA and DNA is strongly associated with SLE in patients and in mouse models, but the mechanism by which TLR signaling to self-ligands is regulated remains poorly understood. In this study, we show that αv integrin plays a critical role in regulating B cell TLR signaling to self-antigens in mice. We show that deletion of αv from B cells accelerates autoantibody production and autoimmune kidney disease in the Tlr7.1 transgenic mouse model of SLE. Increased autoimmunity was associated with specific expansion of transitional B cells, extrafollicular IgG2c-producing plasma cells, and activation of CD4 and CD8 T cells. Our data show that αv-mediated regulation of TLR signaling in B cells is critical for preventing autoimmunity and indicate that loss of αv promotes escape from tolerance. Thus, we identify a new regulatory pathway in autoimmunity and elucidate upstream signals that adjust B cell activation to prevent development of autoimmunity in a mouse model.

FIGURE 1.

αv deletion promotes expansion of plasma cells. (A) Survival of female and male Tlr7.tg control and αv-CD19.Tlr7 mice (n ≥ 17 mice per group). (B) Spleen weight from control mice (con), αv-CD19 (αv), control Tlr7.tg (con-Tlr7.tg), and αv-CD19.Tlr7 mice (αv Tlr7.tg). Groups analyzed are 7–8-wk-old males (n ≥ 6 per group) and females (n ≥ 10 per group) and 10–12-wk-old females (n ≥ 7 mice per group). (C) Spleen B cell frequency in female mice at 7–8 and 10–12 wk of age (n ≥ 4 mice per group). (D–G) Splenocytes were gated on CD19⁺ cells, and the frequencies of immature/transitional (Imm/Trans), T1 and T2, MZ, Fo, and CD24-negative B cells determined by flow cytometry as shown. Analysis of non-tg control and αv-CD19 mice are included for comparison (n = 3–6 per group of non-tg and 6–10 per group of tg mice). (H–J) Spleen plasma cells were identified based on CD138 staining and analyzed for intracellular IgG2c and quantified by percentage of parent population (n = 5 for non-tg mice and n ≥ 13 per group for tg mice) (I) or total number of cells per spleen (n = 8 mice per group) (J). Data are presented as data points from individual mice. Shown are p values <0.05 for comparisons between control Tlr7.tg and αv-CD19.Tlr7 mice. *p < 0.05, **p < 0.01, calculated using log-rank test [survival curves (A)] or Mann–Whitney U test.

FIGURE 2.

Increased TLR signaling in αv-deficient Tlr7.1 tg B cells. (A) Sorted transitional (T1), MZ, and Fo B cells from Tlr7.tg control and αv-CD19.Tlr7 mice were cultured with 5 μg/ml TLR7 ligand R848 for 0–120 min. Nuclear NF-κB (p65) localization was measured by Western blot, and LSD1 is shown as nuclear loading control. (B and C) Cytokine (B) and IgG2c (C) levels in supernatants of cultured B cell subpopulations after stimulation with R848 for 24 h [cytokines (B)] or 4 d [IgG2c (C)]. Data shown are mean ± SD of replicate cultures from one experiment. The p values <0.05 (Student t test) for comparisons between control Tlr7.tg and αv-CD19.Tlr7 mice are shown (*p < 0.05). Similar results were seen in at least three independent experiments.

FIGURE 3.

Increased T cell activation in αv-CD19.Tlr7.1 tg mice. (A and B) Spleens from 7- to 8-wk-old wild-type control mice (con), Tlr7.tg controls (con-Tlr7.tg), and αv-CD19.Tlr7 mice (αv-Tlr7.tg) were analyzed for CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T cells as a frequency of total splenocytes by FACS (A). CD4 and CD8 were further analyzed for expression of CD44 and CD62L cells as shown (B). Each data point represents a single mouse. The p values <0.05 (Mann–Whitney U test) for comparisons between control Tlr7.1 tg and αv-CD19.Tlr7.1 tg mice are shown (n = 6 non-tg and n ≥ 13 per group tg mice). *p < 0.05, **p < 0.01. (C–E) B cells from TLR7.tg controls and αv-CD19.Tlr7 mice were cultured with CD4⁺ T cells from control mice in the presence of anti-CD3. T cell activation (based on CD25 and CD69) (C and D) and cell proliferation measured by [³H]thymidine incorporation (E) were measured after 3 d. (F) Sorted transitional, MZ, and Fo B cells from control TLR7.1 tgs (con-Tlr7.1) and αv-CD19.Tlr7.1 tg mice (αv-Tlr7.1) were cultured with control CD4⁺ T cells in the presence of anti-CD3, and cell proliferation was measured after 48 h. All data shown are mean ± SD of replicate cultures from one experiment. Similar results were seen in at least three independent experiments. The p values <0.05 (Student t test) for comparisons between control Tlr7.1 tg and αv-CD19.Tlr7.1 tg mice are shown. *p < 0.05, **p < 0.01.

FIGURE 4.

αv deletion from B cells increases autoantibody production (A). (A and B) Serum titers of anti-RNA and anti-snRNP Abs from 7 to 8-wk-old wild-type control, control Tlr7.1 tg, and αv-CD19.Tlr7.1 tg mice (n ≥ 11 per group). The p values <0.05 (Student t test) for comparisons between control Tlr7.1 tg and αv-CD19.Tlr7.1 tg mice are shown (*p < 0.05, **p < 0.01). (C) HEp-2 cell staining patterns of serum Abs from 7- to 8-wk-old control Tlr7.1 tg and αv-CD19.Tlr7.1 tg mice. Two representative images are shown per genotype. Original magnification ×40. Pie charts show summary of patterns for 5–6 mice per genotype. (D) Serum IgM and IgG autoantibodies from 7- to 8-wk-old control Tlr7.1 tg (n = 4) and αv-CD19.Tlr7.1 tg (n = 3) mice measured using an autoantibody array chip containing 88 specific autoantigens. Data are shown as a heat map of z-scores. Autoantigen class is shown, along with control Abs (anti-KLH and anti-LPS).

FIGURE 5.

αv deletion increases kidney Ab deposition and glomerulonephritis. (A and B) Glomerular immune complex deposits determined by immunofluorescence staining for IgG (A) and complement C3 (B). Left panels show representative images from 7- to 8-wk-old control Tlr7.1 tg and αv-CD19.Tlr7.1 tg mice. Original magnification ×60. Right panels show intensity and area of staining. (C) Histological analysis of H&E-stained representative sections. Original magnification ×40. Graph represents quantification of glomerular size. Data are mean ± SD for at least n = 3 mice per group. The p values <0.05 (Mann–Whitney U test) for comparisons between control Tlr7.1 tg and αv-CD19.Tlr7.1 tg mice are shown. *p < 0.05, **p < 0.01.