TLR8 deficiency leads to autoimmunity in mice

Abstract

TLRs play an essential role in the induction of immune responses by detecting conserved molecular products of microorganisms. However, the function of TLR8 is largely unknown. In the current study, we investigated the role of TLR8 signaling in immunity in mice. We found that Tlr8(-/-) DCs overexpressed TLR7, were hyperresponsive to various TLR7 ligands, and showed stronger and faster NF-κB activation upon stimulation with the TLR7 ligand R848. Tlr8(-/-) mice showed splenomegaly, defective development of marginal zone (MZ) and B1 B cells, and increased serum levels of IgM and IgG2a. Furthermore, Tlr8(-/-) mice exhibited increased serum levels of autoantibodies against small nuclear ribonucleoproteins, ribonucleoprotein, and dsDNA and developed glomerulonephritis, whereas neither Tlr7(-/-) nor Tlr8(-/-)Tlr7(-/-) mice showed any of the phenotypes observed in Tlr8(-/-) mice. These data provide evidence for a pivotal role for mouse TLR8 in the regulation of mouse TLR7 expression and prevention of spontaneous autoimmunity.

Figure 1. Targeted disruption of the mouse Tlr8 gene.

(A) PCR analysis of mouse genomic DNA with specific primers gave a 240-bp band for Tlr8^+/+ mice, a 589-bp band for Tlr8^–/– mice, and both bands for Tlr8^+/– mice. M, molecular weight marker (100-bp DNA ladder; Invitrogen). (B) Expression of Tlr8 and Hprt mRNA by WT and Tlr8^–/– BMDCs either untreated or stimulated with 50 nM R848 or 1 ng/ml LPS for 4 hours, as determined by RT-PCR. (C) Northern blot analysis of RNA from untreated, 1 ng/ml LPS–, or 50 nM R848–stimulated WT and Tlr8^–/– BMDCs. Ethidium bromide staining after RNA transfer to the membrane is included as control (bottom). (D) Reporter gene expression (LacZ reporter, blue) in spleen and mesenteric lymph node sections from Tlr8^–/– mice. (A–D) Data are representative of 2–4 independent experiments.

Figure 2. Enhanced responses to TLR7 ligands by Tlr8^–/– DCs.

(A) BMDCs from WT and Tlr8^–/– mice were stimulated with the indicated amounts of R848, polyA:U, CpG, polyI:C, and LPS. After 20 hours, the concentrations of IL-6, IL-12p40, and TNF in the culture supernatants were assessed by ELISA. (B) WT and Tlr8^–/– BMDCs were left untreated or stimulated for the indicated times with 50 nM R848. Total RNA was extracted from the cells, and expression of Ifnb was assessed by Q-PCR. (C) WT and Tlr8^–/– BMDCs were left untreated or stimulated with the indicated amounts of R848 for 16 hours, and cell surface expression of MHCII and CD86 was analyzed by flow cytometry on gated CD11c⁺ cells. (D) WT and Tlr8^–/– CD11c⁺ splenic cells were isolated by magnetic-activated cell sorting, then left unstimulated or stimulated for 16 hours with 30 nM R848, 10 ng/ml LPS, or 30 nM CpG. The production of IL-12p40 in the culture supernatants was assessed by ELISA. *P < 0.05. (E) WT and Tlr8^–/– mice (8 weeks old) were injected i.p. with 100 μl of 10 μM R848. Sera were collected 2 or 6 hours later, and serum levels of IL-6 and IL-12p40 were determined by ELISA. (A, D, and E) Data are mean ± SD of 3–4 (A and D) or 8–10 (E) mice per group and are representative of 2–5 independent experiments. (B) Data are mean ± SD of duplicates and are representative of 2 independent experiments.

Figure 3. Increased TLR7 expression and NF-κB activation in Tlr8^–/– BMDCs.

WT and Tlr8^–/– BMDCs were left untreated or stimulated with 50 nM (A and B) or 100 nM (C and H) R848 for the indicated times. Total mRNA was extracted from the cells, and the expression of (A) Tlr8 or (B) Tlr7 was assessed by Q-PCR. Total protein lysates were prepared, and (C) the expression of Tlr7 and β-actin were assessed at 0, 4, and 8 hours after stimulation, or (H) phosphorylation of NF-κBp65 was determined by Western blot. Total NF-κBp65 and β-actin were used as loading controls. (D–F) WT and Tlr8^–/– BMMs were left untreated or stimulated with 50 nM R848. At the indicated time points, either total mRNA was extracted from the cells and the expression of (D) Tlr8 and (E) Tlr7 was assessed by Q-PCR, or (F) total protein lysates were prepared, and the expression of Tlr7 and β-actin were assessed by Western blot. (G) Tlr8^–/– BMDCs were transfected with 50 pmol Tlr8-FLAG or GFP mRNA. 5 hours later, cells were harvested, total protein lysates were prepared, and the expression of Tlr7, Tlr8-FLAG, and β-actin were assessed by Western blot. The Tlr7/β-actin ratio is shown at right. (A, B, D, and E) Data are mean ± SD of duplicates and are representative of 2–3 independent experiments. (C, F, G, and H) Data are representative of 3–5 (C, F, and H) or 2 (G) independent experiments.

Figure 4. Defect of MZ B cells in Tlr8^–/– mice.

(A) Spleen weight and size of 8-week-old male WT and Tlr8^–/– mice. Each point represents the value obtained from 1 mouse; horizontal bars denote mean values. ***P < 0.001. (B) Erythrocyte-depleted splenocytes from 6-month-old WT, Tlr8^–/–, and Tlr8^–/–Tlr7^–/– mice were analyzed by flow cytometry for the expression of CD19, CD21, and CD23. Profiles using anti-CD21 and anti-CD23 were performed on CD19⁺ gated cells. Numbers denote the percentage of immature B cells (CD21^–CD23^–), follicular B cells (CD21^intCD23^hi) and MZ B cells (CD21^hiCD23^lo/–) in the indicated circles. (C) Spleen sections from WT or Tlr8^–/– mice stained for MOMA1 (red) and B220 (green), or for B220 (red) and CD3 (blue). Scale bars: 200 μm. (D) BM cells (2 × 10⁶ cells) from WT or Tlr8^–/– mice were transferred i.v. into lethally irradiated WT or Tlr8^–/– mice. After 6–8 weeks, splenocytes were analyzed by flow cytometry for the presence of MZ B cells. (A) Data are representative of 3 independent experiments with 5–9 mice per group. (B) Data are representative of 5 independent experiments with 3–4 mice per group. (C and D) Data are representative of 2 independent experiments with 3 mice per group.

Figure 5. Reduced numbers of B1 B cells in Tlr8^–/– mice.

Peritoneal cavity cells from (A) 11-week-old WT and Tlr8^–/– mice or (B) lethally irradiated WT and Tlr8^–/– mice reconstituted i.v. with 2 × 10⁶ WT or Tlr8^–/– BM cells were analyzed by flow cytometry for expression of CD19, B220, and CD5. Shown are percent B1a (B220^loCD5^int), B1b (B220^loCD5^lo), and B2 (B220^hiCD5^–) B cells on CD19⁺ gated cells of the peritoneal cavity. Data are representative of 2–3 independent experiments with 3 mice per group.

Figure 6. Serum levels of IgM and IgGs in WT, Tlr8^–/–, and Tlr7^–/– mice.

Levels of IgM, IgG, IgG1, IgG2a, IgG2b, and IgG3 in the sera of 3-month-old male WT, Tlr8^–/–, and Tlr7^–/– mice were evaluated by ELISA. Each point represents the value obtained from 1 mouse; horizontal bars and error bars denote mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Data are representative of 3 independent experiments.

Figure 7. Tlr8^–/– mice develop glomerulonephritis.

(A) smRNP-, RNP-, and dsDNA-specific autoantibodies were quantified in sera from WT and Tlr8^–/– mice aged 10–13 months. Each point represents the value obtained from 1 mouse; horizontal bars and error bars denote mean ± SD. *P < 0.05; **P < 0.01. (B) ANA staining patterns on Hep2 human epithelial cells for serum derived from WT, Tlr8^–/–, Tlr7^–/–, and Tlr8^–/–Tlr7^–/– mice at 1:160 dilution. (C) IgM, IgG, and C3 immunofluorescence staining of kidney sections from 4-month-old WT, Tlr8^–/–, and Tlr7^–/– mice. (A–C) Data are representative of 2–3 independent experiments with at least 3 mice per group. Scale bars: 50 μm.