Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection

Abstract

Several subsets of dendritic cells have been shown to produce type I IFN in response to viral infections, thereby assisting the natural killer cell-dependent response that eliminates the pathogen. Type I IFN production can be induced both by unmethylated CpG-oligodeoxynucleotide and by double-stranded RNA. Here, we describe a codominant CpG-ODN unresponsive phenotype that results from an N-ethyl-N-nitrosourea-induced missense mutation in the Tlr9 gene (Tlr9(CpG1)). Mice homozygous for the Tlr9(CpG1) allele are highly susceptible to mouse cytomegalovirus infection and show impaired infection-induced secretion of IFN-alpha/beta and natural killer cell activation. We also demonstrate that both the Toll-like receptor (TLR) 9 --> MyD88 and TLR3 --> Trif signaling pathways are activated in vivo on viral inoculation, and that each pathway contributes to innate defense against systemic viral infection. Whereas both pathways lead to type I IFN production, neither pathway offers full protection against mouse cytomegalovirus infection in the absence of the other. The Tlr9(CpG1) mutation alters a leucine-rich repeat motif and lies within a receptor domain that is conserved within the evolutionary cluster encompassing TLRs 7, 8, and 9. In other TLRs, including three mouse-specific TLRs described in this paper, the affected region is not represented. The phenotypic effect of the Tlr9(CpG1) allele thus points to a critical role for TLR9 in viral sensing and identifies a vulnerable amino acid within the ectodomain of three TLR proteins, essential for a ligand response.

Fig. 1.

Isolation and identification of CpG1, an ENU-induced point mutation in TLR9. (A) Trace file of amplified genomic DNA from homozygous mutant mice (upper chromatogram) and normal animals (lower chromatogram). (Left) Location of the mutation within the 11th LRR in the TLR9 ectodomain. (Lower) Multiple alignment of all human and mouse TLR protein sequences reveals that the mutation resides in a region shared only by TLRs 7, 8, and 9. At left, the identity of each sequence is indicated. H, human paralog; M, mouse paralog. Note that TLRs 11 and 12 are nearest homologs, and TLR13 is most closely related to TLR3. (B) Transfection-based assay of TLR9 signaling activity. Error bars indicate the SD of duplicate transfections.

Fig. 2.

In vivo effects of the Tlr9^CpG1 mutation. (A) TNF-α secretion by macrophages from controls C57BL/6 (Tlr9^+/+, filled dots), heterozygotes (Tlr9^CpG1/+, gray dots) and homozygotes (Tlr9^CpG1/CpG1, open dots) animals after CpG-ODN induction (0.1 μM). Each dot represents the result of a duplicate induction assay performed on cells from a single animal. (B) TNF-α production or (C) IL-12p40 production by peritoneal macrophages at low (0.1 μM) CpG ODN concentration, as influenced by Tlr9 genotype. Error bars indicate SD; n = 5 mice. (D) Kaplan–Meier survival curves for Tlr9^CpG1/CpG1 mice and Tlr9^+/+ mice, after sensitization with d-galactosamine and challenge with CpG-ODN. Mice were monitored for a 3-day period, at which time all survivors seemed healthy.

Fig. 3.

Tlr9^CpG1/CpG1, MyD88^-/-, and Tlr3^-/- mice are hypersusceptible to viral infections. (A) Viral titers, expressed as log pfu per spleen, were determined in mice 4 days after i.p. inoculation with 5 × 10⁵ pfu of MCMV. (B) Mice of the indicated genotype were infected i.p. with 5 × 10⁵ pfu of MCMV, and survival was monitored for a period of 7 days. P values indicate comparisons with the survival curve of C57BL/6 control animals.

Fig. 4.

In vivo impairment of cytokine production after MCMV infection in mice lacking the TLR9 → MyD88 signaling pathway. (A) IFN-α/β activity was measured in the serum of noninfected (ni) controls and MCMV-infected animals 36 h postinoculation. Values are expressed as international units (IU)/ml of serum. IL-12p40 (B) and IFN-γ (C) concentrations were measured in the serum of the same animals as those shown in A by ELISA and are expressed in ng/ml. Data represent mean values with SD (n = 4 mice). Statistical analysis was performed by using the ANOVA test with prism software (GraphPad, San Diego) (*, P < 0.05, **, P < 0.001, with respect to the C57BL/6 control values).

Fig. 5.

Impairment of NK and NKT cell activation after MCMV inoculation in TLR9 and MyD88 mutant mice. Purified splenic cells from uninfected or MCMV-infected animals were recovered 36 h postinoculation and gated against NK1.1 and TCRβ surface markers. (Left) IFN-γ intracellular staining (and isotype antibody as control) for NK (NK1.1⁺TCRβ^-). (Right) Results for NKT cells (NK1.1⁺TCRβ⁺). Mean values ± SD are indicated (n = 4 mice).