B cell-intrinsic TLR9 expression is protective in murine lupus

Abstract

Toll-like receptor 9 (TLR9) is a regulator of disease pathogenesis in systemic lupus erythematosus (SLE). Why TLR9 represses disease while TLR7 and MyD88 have the opposite effect remains undefined. To begin to address this question, we created 2 alleles to manipulate TLR9 expression, allowing for either selective deletion or overexpression. We used these to test cell type-specific effects of Tlr9 expression on the regulation of SLE pathogenesis. Notably, Tlr9 deficiency in B cells was sufficient to exacerbate nephritis while extinguishing anti-nucleosome antibodies, whereas Tlr9 deficiency in dendritic cells (DCs), plasmacytoid DCs, and neutrophils had no discernable effect on disease. Thus, B cell-specific Tlr9 deficiency unlinked disease from autoantibody production. Critically, B cell-specific Tlr9 overexpression resulted in ameliorated nephritis, opposite of the effect of deleting Tlr9. Our findings highlight the nonredundant role of B cell-expressed TLR9 in regulating lupus and suggest therapeutic potential in modulating and perhaps even enhancing TLR9 signals in B cells.

Keywords: Autoimmunity; B cells.

Figure 1. TLR9 is expressed on B cell, DC, and myeloid populations.

Quantification of TLR9 MFI is shown for each of the defined populations representing TLR9^–/– BALB/c (n = 5), BALB/c (n = 7), 5-week-old prediseased MRL/lpr (n = 5), and diseased 21-week-old MRL/lpr mice (n = 5). Scatter plots represent individual mice, with bars identifying mean and error bars representing standard deviations. **P < 0.01, as determined by ANOVA with Tukey’s multiple comparison test evaluating for differences within each cell type.

Figure 2. B cell–specific TLR9 deficiency results in exacerbated renal disease.

(A) Schematic representation of Tlr9 floxed allele generation. Exons (open rectangles), LoxP sites (black triangles), FLP recombinase target sites (block open arrowheads) are also shown. (B) Sorted B cells from control or CD19-Cre Tlr9^fl/fl mice were stimulated with CpG ODN 1826 (5 μg/mL) for 3 days and IgM secretion was quantified by ELISA (n = 2 per group). Scatter plots display data from individual mice with black lines showing means. **P < 0.01 using the 2-tailed Student’s t test. Phenotypic markers were assessed in 16-week-old MRL/lpr mice from each indicated genotype, including (C) proteinuria, (D) glomerular renal disease, (E) interstitial and perivascular renal infiltrates, with (F) representative images of H&E kidney sections from mice of indicated genotype, where black arrowheads indicate interstitial inflammation and white arrows show glomeruli. Additional phenotypic endpoints were assessed for each noted genotype including (G) dermatitis, (H) spleen weight, and (I) lymph node weight. Controls: n = 24–31; CD19-Cre Tlr9^fl/fl: n = 19–24. Scatter plots display data from individual mice with black lines showing median values. *P < 0.05, 2-tailed Mann-Whitney U test.

Figure 3. B cell–intrinsic deletion of TLR9 alters autoantibody patterns.

(A) Representative HEp-2 ANA staining patterns from serum of control or CD19-Cre Tlr9^fl/fl mice. Arrows indicate mitotic chromatin staining. Original magnification ×200. (B) Frequency of ANA staining patterns produced by sera from control and CD19-Cre Tlr9^fl/fl mice with numbers in the circles indicating the number of mice analyzed. Patterns were compared using χ² analysis. (C) Serum concentrations of anti–nucleosome, anti–Sm, and anti–RNA IgG of control (n = 27) and CD19-cre Tlr9^fl/fl (n = 19), as measured by ELISA. Scatter plots display data from individual mice with black lines showing median values. ****P < 0.0001, 2-tailed Mann-Whitney U test.

Figure 4. Myeloid-specific TLR9 deficiency does not alter clinical parameters of SLE pathogenesis.

Evaluation of phenotypic markers of disease including proteinuria, glomerulonephritis, interstitial and perivascular renal infiltrates, dermatitis, spleen weight, and lymph node weight for the indicated Cre lineages. (A) Tlr9^fl/fl versus LysM-Cre Tlr9^fl/fl, n = 37–38 and n = 36 per group, respectively. (B) Tlr9^fl/fl versus MRP8-Cre Tlr9^fl/fl, n = 40 per group, and (C) Tlr9^fl/fl versus CD11c-Cre Tlr9^fl/fl, n = 26 and n = 32 per group, respectively, for phenotypic markers and n = 22 and n = 29 for histologic scoring. Scatter plots display data from individual mice, with black lines representing median values. No comparisons were statistically significant by 2-tailed Mann-Whitney U test.

Figure 5. Alterations in autoantibody production after Tlr9 deletion in myeloid cell lineages.

Serum concentrations of anti–nucleosome, anti–Sm, and anti–RNA IgG from (A) Tlr9^fl/fl versus LysM-Cre Tlr9^fl/fl, n = 37 and n = 36 per group, (B) Tlr9^fl/fl versus MRP8-Cre Tlr9^fl/fl, n = 40 and n = 39, respectively, per group, and (C) Tlr9^fl/fl versus CD11c-Cre Tlr9^fl/fl, n = 22 and n = 29 per group respectively. Scatter plots display data from individual mice with black lines representing median values. *P < 0.05; ****P < 0.0001, 2-tailed Mann-Whitney U test. (D) Frequency of ANA staining patterns produced by sera from control (Tlr9^fl/fl) and CD11c-Cre Tlr9^fl/fl mice, with numbers in the circles indicating the number of mice analyzed. *P < 0.05; ****P < 0.0001, using χ² analysis.

Figure 6. Generation and validation of a conditional TLR9 overexpression allele.

(A) Diagram of the TLR9 overexpression plasmid and insertion into the rosa26 locus. The plasmid contains 2 rosa26 homology arms flanking the expression vector. The vector is composed of a floxed region (demarcated by black triangles) containing eGFP, a Neo cassette, and a transcriptional stop sequence. This floxed sequence is followed by an HA-tagged Tlr9. PGK-DTA was used as a negative selection marker for ES cells; bPA represents the bovine growth hormone polyadenylation site. The top panels show the targeting plasmid and rosa26 locus, and the bottom 2 panels show the Rosa^Tlr9 locus before and after Cre-mediated excision. (B) qPCR analysis of TLR9 expression in sorted B cells from control (n = 7) and CD19-Cre Rosa^Tlr9 mice (n = 4) mice. (C) Representative Western blot showing TLR9-HA expression in CD19-Cre Rosa^Tlr9 mice but not control mice. Sorted B cells were immunoprecipitated (IP) with isotype control antibody (rat IgG1) or anti–HA antibody and immunoblotted with anti–HA antibody. Arrows depict the full-length (FL-TLR9) and cleaved (C-TLR9) forms of TLR9. (D) Sorted B cells from control and CD19-Cre Rosa^Tlr9 mice were stimulated with CpG ODN 1826 (at indicated concentrations) for 3 days and IgM secretion was measured by ELISA. Scatter plots display data from individual mice, with black lines showing means. (E) Left shows representative FACS plots showing GFP expression in CD19⁺ (red) and TCRβ⁺ cells derived from CD19-Cre Rosa^Tlr9 mice, with the right panel showing summary data from CD19-Cre⁺ and Cre-negative Rosa^Tlr9 mice (n = 42 and n = 46, respectively). For tabulated data, each dot denotes an individual mouse and horizontal lines represent the mean and standard deviation. *P < 0.05; **P < 0.01; ****P < 0.0001 using Student’s t test.

Figure 7. B cell–specific overexpression of Tlr9 results in ameliorated renal disease and altered antibody profile in MRL/lpr mice.

CD19-Cre Rosa^Tlr9 MRL/lpr and Cre-negative Rosa^Tlr9 MRL/lpr controls were aged until 19 weeks (female) and 21 weeks (male). Phenotypic markers were assessed, including (A) proteinuria, (B) dermatitis, (C) glomerular renal disease, and (D) interstitial and perivascular renal infiltrates, with (E) representative images of H&E kidney sections from mice of indicated genotypes, where black arrowheads denote interstitial inflammation and white arrows show glomeruli. Original magnification ×200. Additionally, (F) spleen weight and (G) lymph node weight were assessed as markers of lymphoproliferation. Serum concentrations of (H) anti–nucleosome, (I) anti–RNA antibody formation, and (J) ratio of anti–nucleosome/anti–RNA antibodies of Rosa^Tlr9 controls (n = 45) and CD19-Cre Rosa^Tlr9 (n = 46). Scatter plots display data from individual mice with black lines showing median values. *P < 0.05; **P < 0.01; ***P < 0.001, 1-tailed Mann-Whitney U test.

Figure 8. B cell–specific overexpression of Tlr9 results in ameliorated renal disease and altered antibody profile in Fcgr2b^–/–.Yaa mice.

CD19-Cre Rosa^Tlr9 Fcgr2b^–/–.Yaa and Cre-negative Rosa^Tlr9 Fcgr2b^–/–.Yaa male controls were aged until 24 weeks. Phenotypic markers assessed included (A) proteinuria, (B) glomerular renal disease, and (C) interstitial and perivascular renal infiltrates, with (D) representative images of H&E kidney sections from mice of indicated genotype, where black arrowheads indicate interstitial inflammation and white arrows show glomeruli. Original magnifications ×40 and ×200; scale bar: 200 µm. Additionally, (E) spleen weight and (F) lymph node weight were measured as markers of lymphoproliferation. Scatter plots display data from individual mice, with lines showing median values. *P < 0.05; ****P < 0.0001, 2-tailed Mann-Whitney U test.