B cell-intrinsic TLR7 expression drives severe lupus in TLR9-deficient mice

Abstract

The endosomal Toll-like receptor 7 (TLR7) is a major driver of murine and human systemic lupus erythematosus (SLE). The role of TLR7 in lupus pathogenesis is enhanced when the regulatory role of TLR9 is absent. TLR7 signaling in plasmacytoid DCs (pDC) is generally thought to be a major driver of the IFN response and disease pathology; however, the cell types in which TLR7 acts to mediate disease have not been distinguished. To address this, we selectively deleted TLR7 in either CD11c+ cells or CD19+ cells; using a TLR7-floxed allele, we created on the lupus-prone MRL/lpr background, along with a BM chimera strategy. Unexpectedly, TLR7 deficiency in CD11c+ cells had no impact on disease, while TLR7 deficiency in CD19+ B cells yielded mild suppression of proteinuria and a trend toward reduced glomerular disease. However, in TLR9-deficient MRL/lpr mice with accelerated SLE, B cell-specific TLR7 deficiency greatly improved disease. These results support revision of the mechanism by which TLR7 drives lupus and highlight a cis regulatory interaction between the protective TLR9 and the pathogenic TLR7 within the B cell compartment. They suggest B cell-directed, dual TLR7 antagonism/TLR9 agonism or dual TLR7/9 antagonism as a potential future therapeutic strategy to treat SLE.

Keywords: Autoimmune diseases; Autoimmunity; Immunology; Innate immunity; Lupus.

Figure 1. TLR7 is expressed in B cells, DCs, and myeloid cells and is deleted in targeted subsets using Cre-lox approaches.

(A and B) Representative histograms and quantification in the change in anti-TLR7 PE MFI between WT and Tlr7^–/y for each of the defined populations in 6-week-old male, prediseased MRL/lpr mice. (C) Representative histograms showing TLR7 expression in a littermate control (Tlr7^fl/y) mouse (blue line), a CD11c-Tlr7^Δ (CD11c-Cre^+/– Tlr7^fl/y) mouse (red line), and a Tlr7^–/y mouse (black dotted line) in relevant cell subsets. (D) Representative histograms showing TLR7 expression in a littermate control (Tlr7^fl/y) (blue line), a B-Tlr7^Δ (CD19-Cre^+/– Tlr7^fl/y) mouse (red line), and a Tlr7^–/y mouse (black dotted line) in relevant cell subsets.

Figure 2. CD11c⁺ cell–specific TLR7 deficiency mildly suppresses the anti-RNA–associated autoantibody response but does not affect clinical parameters of SLE.

Control (Tlr7^fl/fl and Tlr7^fl/y) and CD11c-Tlr7^Δ (CD11c-Cre^+/– Tlr7^fl/fl and CD11c-Cre^+/– Tlr7^fl/y) mice were aged until 19 weeks (female) or 22 weeks (male). (A) Serum concentrations of anti–RNA IgG2a, anti–Sm IgG, and anti–nucleosome IgG in control and CD11c-Tlr7^Δ mice (n = 24 and n = 28, respectively). (B and C) Evaluation of phenotypic disease markers in control and CD11c-Tlr7^Δ mice including proteinuria (n = 18 and n = 24), glomerulonephritis, and interstitial/perivascular infiltrates (n = 24 and n = 28) (B) and spleen weight, lymph node weight (n = 24 and n = 28), and dermatitis (n = 18 and n = 24) (C). Scatterplots display data from individual mice, with black lines showing median values and dotted lines indicating the lower limit of detection. *P < 0.05, **P < 0.01 by 2-tailed Mann-Whitney U test.

Figure 3. B cell–intrinsic TLR7 deficiency improves proteinuria and suppresses the anti-RNA–associated autoantibody responses.

Control (Tlr7^fl/fl and Tlr7^fl/y) and B-Tlr7^Δ (CD19-Cre^+/– Tlr7^fl/fl and CD19-Cre^+/– Tlr7^fl/y) mice were aged until 19 weeks (female) or 22 weeks (male). (A) Serum concentrations of anti–RNA IgG2a, anti–Sm IgG, and anti–nucleosome IgG in control and B-Tlr7^Δ mice (n = 37 and n = 31, respectively). (B and C) Evaluation of phenotypic disease markers in control and B-Tlr7^Δ mice including proteinuria, glomerulonephritis, and interstitial and perivascular infiltrates (B) and spleen and lymph node weight and dermatitis (n = 37 control and n = 31 B-Tlr7^Δ) (C). Scatterplots display data from individual mice, with black lines showing median values and dotted lines indicating the lower limit of detection. **P < 0.01, ****P < 0.0001 by 2-tailed Mann-Whitney U test.

Figure 4. B cell–intrinsic TLR7 deficiency in mixed BM chimeras suppresses features of SLE in female mice.

Mixed radiation BM chimeric mice were generated and were then analyzed 25 weeks after irradiation. (A) Theoretical reconstitution in the mixed BM chimeras indicating the expected genotype and composition of each listed cellular compartment. (B–D) Evaluation of clinical SLE parameters in control versus B-Tlr7^–/– female (n = 13 and n = 13, respectively) and male (n = 11 and n = 10, respectively) mice, including proteinuria (B), glomerulonephritis (C), and interstitial nephritis (D). (E and F) Total B cell (E) and follicular and marginal zone B cell (F) frequencies in male and female B-Tlr7^–/– mice compared with controls. Scatterplots display data from individual mice, with black lines showing median values. *P < 0.05, **P < 0.01 by 1-tailed Mann-Whitney U test.

Figure 5. B cell–intrinsic TLR7 drives severe renal disease, splenomegaly, and the anti-RNA–associated autoantibody responses in TLR9-deficient mice.

Control (Tlr7^fl/fl Tlr9^–/– and Tlr7^fl/y TLR9^–/–) and B-Tlr7^Δ Tlr9^–/– (CD19-Cre^+/– Tlr7^fl/fl Tlr9^–/– and CD19-Cre^+/– Tlr7^fl/y Tlr9^–/–) mice were aged until 16 weeks (female) or 19 weeks (male). (A) Serum concentrations of anti–RNA IgG2a, anti–Sm IgG, and anti–nucleosome IgG in control and B-Tlr7^Δ Tlr9^–/– (n = 23 and n = 37, respectively). (B) Evaluation of renal disease including proteinuria, glomerulonephritis, and interstitial and perivascular infiltrates in control versus B-Tlr7^Δ Tlr9^–/– mice (n = 23 and n = 37, respectively). (C) Representative images of H&E-stained kidney sections for indicated genotypes. Original magnification, 200×. (D) Quantification of spleen weight, lymph node weight, and dermatitis in control versus B-Tlr7^Δ Tlr9^–/– mice (n = 23 and n = 37, respectively). Scatterplots display data from individual mice, with black lines showing median values and dotted lines indicating the lower limit of detection. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-tailed Mann-Whitney U test.

Figure 6. B cell–intrinsic TLR7 drives naive T cell activation, double-negative T cell expansion, and B cell lymphopenia in TLR9-deficient mice.

Flow cytometric analysis was performed on spleens from control (Tlr7^fl/fl Tlr9^–/– and Tlr7^fl/y Tlr9^–/–, solid black circles) and B-Tlr7^Δ Tlr9^–/– (CD19-Cre^+/– Tlr7^fl/fl Tlr9^–/– and CD19-Cre^+/– Tlr7^fl/y Tlr9^–/–, open gray squares) mice. (A–D) Analysis of the B cell compartment in control versus B-Tlr7^Δ Tlr9^–/–, including proportions of B cells as percent of total live cells (A), follicular and marginal zone B cells as a percent of total B cells (B), CD11b⁺CD11c⁺ ABCs as a percent of total B cells (C), and plasmablasts as a percent of total TCRβ^– cells (D). (E–G) Analysis of the T cell compartment in control versus B-Tlr7^Δ TLR9^–/–, including proportions of T cells as a percent of total live cells (E); CD4⁺, CD8⁺, and double-negative (CD4^–CD8^–) T cells as a percent of total T cells (F); and percent naive CD4⁺ and naive CD8⁺ T cells as a percent of CD4⁺ and CD8⁺ T cells, respectively (G). (H–J) Analysis of selected myeloid populations, including proportions of macrophages, monocytes, and neutrophils as a percent of total CD19^–CD11b⁺ cells (H); cDCs as a percent of total CD19^– cells (I); and pDCs as a percent of total CD19^– cells (J). Scatterplots display data from individual mice, with black lines showing median values. *P < 0.05, **P < 0.01, ***P < 0.001, by 1-tailed Mann-Whitney U test.