TLR8 on dendritic cells and TLR9 on B cells restrain TLR7-mediated spontaneous autoimmunity in C57BL/6 mice

Abstract

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with diverse clinical presentations characterized by the presence of autoantibodies to nuclear components. Toll-like receptor (TLR)7, TLR8, and TLR9 sense microbial or endogenous nucleic acids and are implicated in the development of SLE. In mice TLR7-deficiency ameliorates SLE, but TLR8- or TLR9-deficiency exacerbates the disease because of increased TLR7 response. Thus, both TLR8 and TLR9 control TLR7 function, but whether TLR8 and TLR9 act in parallel or in series in the same or different cell types in controlling TLR7-mediated lupus remains unknown. Here, we reveal that double TLR8/9-deficient (TLR8/9(-/-)) mice on the C57BL/6 background showed increased abnormalities characteristic of SLE, including splenomegaly, autoantibody production, frequencies of marginal zone and B1 B cells, and renal pathology compared with single TLR8(-/-) or TLR9(-/-) mice. On the cellular level, TLR8(-/-) and TLR8/9(-/-) dendritic cells were hyperesponsive to TLR7 ligand R848, but TLR9(-/-) cells responded normally. Moreover, B cells from TLR9(-/-) and TLR8/9(-/-) mice were hyperesponsive to R848, but TLR8(-/-) B cells were not. These results reveal that TLR8 and TLR9 have an additive effect on controlling TLR7 function and TLR7-mediated lupus; however, they act on different cell types. TLR8 controls TLR7 function on dendritic cells, and TLR9 restrains TLR7 response on B cells.

Keywords: endosomal TLRs; innate immunity; knockout mice.

Fig. 1.

TLR8/9^−/− mice show increased splenomegaly and more severe defect on MZ and B1 B cells compared with TLR8^−/− or TLR9^−/− mice. (A) Spleen weight of 4- to 6-mo-old female WT, TLR8^−/−, TLR8/9^−/−, and TLR9^−/− mice. Each point represents one mouse (n = 7–8 mice per genotype) and horizontal bars denote the median. (B) Splenocytes from 12-wk-old female WT, TLR8^−/−, TLR8/9^−/−, and TLR9^−/− mice were analyzed by flow cytometry for the expression of CD19, CD21, and CD23. Numbers denote the percentage of MZ (CD21^hiCD23^lo/-), follicular (CD21^intCD23^hi), and immature (CD21⁻CD23⁻) B cells in the indicated circles. (C) FACS analysis of B220 and CD5 expression on CD19⁺ gated cells of the peritoneal cavity shows the percentage of B1a (B220^loCD5^int), B1b (B220^loCD5^lo), and B2 (B220^hiCD5⁻) B cells. Data in B and C are representative of two to three independent experiments (n = 3–4 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Increased autoantibodies, plasmablasts, and germinal centers in TLR8/9^−/− vs. TLR8^−/− or TLR9^−/− mice. Sera from 10- to 13-wk-old female WT, TLR8^−/−, TLR8/9^−/−, and TLR9^−/− mice were used for the evaluation of (A) serum levels of IgM and IgG2a and (B) dsDNA-, RNA-, smRNP-, and RNP-specific autoantibody production by ELISA. Each point represents value from one mouse and horizontal bars denote the median. Representative flow cytometry plots of (C) B220^loCD138⁺ plasmablasts and (D) B220⁺GL7⁺CD38⁻ germinal center B cells in 5 mo old male WT, TLR8^−/−, TLR8/9^−/−, and TLR9^−/− mice. Data in C and D are representative of two independent experiments (n= 3–4 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Increased renal pathology in TLR8/9^−/− mice compared with TLR8^−/− or TLR9^−/− mice. (A) ANA staining patterns on Hep2 human epithelial cells for sera derived from 9-wk-old mice (Original Magnification 400×). Kidney sections from 5- to 6-mo-old female WT, TLR8^−/−, TLR8/9^−/−, and TLR9^−/− mice were stained with (B) H&E (scale bars, 50 uM) or (C) immunofluorescence anti-IgG (Original Magnification 100×). Data in A and C are representative of two independent experiments (n = 3–4 per group).

Fig. 4.

Enhanced responses of TLR8^−/− and TLR8/9^−/− DCs and pDCs to TLR7 ligand. (A–C) BM-DCs from WT, TLR8^−/−, TLR8/9^−/−, and TLR9^−/− mice were stimulated with R848, LPS, or poly I:C. (A) After 16 h, the concentration of IL-6 in the culture supernatants was assessed by ELISA. (B and C) BM-DCs were left untreated or stimulated with 50 nM R848 for the indicated time points. Total RNA was extracted from the cells and the expression of (B) IFN-β or (C) TLR7 was assessed by quantitative PCR. (D and E) BM-pDCs from WT, TLR8^−/−, TLR8/9^−/−, and TLR9^−/− mice were stimulated with R848, Pam₃CKS₄, poly I:C, or CpG for 16 h. (D) The concentration of IL-6 in culture supernatants was assessed by ELISA. (E) The surface expression of CD86 and MHC class II was analyzed by flow cytometry on B220⁺CD11c^intCD11b⁻ cells from WT (black line), TLR8^−/− (blue line), TLR8/9^−/− (red line), and TLR9^−/− (green line) mice. Data in all panels are representative of two to four independent experiments (n = 3–4 per group). *P < 0.05.

Fig. 5.

Increased activated memory T cells in TLR8^−/− and TLR8/9^−/− mice. Flow cytometric histograms (Upper) and graphical analysis (Lower) on splenocytes from 7-mo-old female WT, TLR8^−/−, TLR8/9^−/−, and TLR9^−/− mice, analyzed for the expression of CD3, CD4, CD8, and CD44. CD44 staining profiles of gated (A) CD4⁺ and (B) CD8⁺ T cells to identify activated memory (CD44^hi) subpopulations. Data are representative of three independent experiments (n = 4 per group). *P < 0.05.

Fig. 6.

Enhanced response of TLR9^−/− and TLR8/9^−/− B cells to TLR7 stimulation. Total splenocytes (A and C) or isolated B cells (B and D) from WT (black line), TLR8^−/− (blue line), TLR8/9^−/− (red line), and TLR9^−/− (green line) mice were left untreated or stimulated with R848 or LPS for 16 h. Representative flow cytometry plots of CD69 on (A) CD119⁺B220⁺ splenocytes or (B) isolated B cells. IL-6 production on culture supernatants of (C) splenocytes or (D) isolated B cells. Data are representative of two to four independent experiments (n = 3 per group). *P < 0.05.