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

Koichi Tabeta*†, Philippe Georgel*†, Edith Janssen‡, Xin Du*, Kasper Hoebe*, Karine Crozat*, Suzanne Mudd*, Louis Shamel*, Sosathya Sovath*, Jason Goode*, Lena Alexopoulou§, Richard A. Flavell§¶ʈ, and Bruce Beutler*ʈ

*Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; ‡Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121; and §Section of Immunobiology and ¶The Howard Hughes Medical Institute, Yale University, New Haven, CT 06520

Contributed by Richard A. Flavell, January 22, 2004 Several subsets of dendritic cells have been shown to produce type I cascades leading to the synthesis of proinflammatory molecules. IFN in response to viral infections, thereby assisting the natural killer TLR3, which recognizes double-stranded RNA (dsRNA) (14), cell-dependent response that eliminates the pathogen. Type I IFN and TLR9, which recognizes double-stranded DNA unmethyl- production can be induced both by unmethylated CpG-oligode- ated at CpG motifs (15, 16), are likely to play a key role in viral oxynucleotide and by double-stranded RNA. Here, we describe a infections (17). Indeed, two groups have reported that herpes codominant CpG-ODN unresponsive phenotype that results from an simplex virus (HSV)-1- and HSV-2-induced IFN-␤ production is N-ethyl-N-nitrosourea-induced missense mutation in the Tlr9 gene abrogated in cells harvested from TLR9 knockout mice (11, 12). (Tlr9CpG1). Mice homozygous for the Tlr9CpG1 allele are highly suscep- A role for TLR3 in viral detection has been suggested by in vivo tible to mouse cytomegalovirus infection and show impaired infec- and ex vivo analyses that showed that this receptor is involved in tion-induced secretion of IFN-␣͞␤ and natural killer cell activation. We the recognition of poly I:C and dsRNA from Lang reovirus (14). also demonstrate that both the Toll-like receptor (TLR) 9 3 MyD88 However, none of these studies has demonstrated that these and TLR3 3 Trif signaling pathways are activated in vivo on viral TLRs are crucial for an efficient antiviral response in vivo. inoculation, and that each pathway contributes to innate defense We previously described the phenotype of Lps2 mice, which against systemic viral infection. Whereas both pathways lead to type carry a nonsense mutation in the Trif͞Ticam-1 gene (18, 19). The I IFN production, neither pathway offers full protection against mouse Trif gene encodes an essential adaptor molecule for MyD88- cytomegalovirus infection in the absence of the other. The Tlr9CpG1 independent signaling downstream from TLRs 3 and 4 (18, 20). mutation alters a leucine-rich repeat motif and lies within a receptor In macrophages harvested from homozygous Lps2 mutant mice, domain that is conserved within the evolutionary cluster encompass- neither LPS nor dsRNA is capable of triggering phosphorylation ing TLRs 7, 8, and 9. In other TLRs, including three mouse-specific TLRs or dimerization of IFN response factor (IRF)-3. Moreover, the described in this paper, the affected region is not represented. The animals display impaired responses to MCMV, fail to produce phenotypic effect of the Tlr9CpG1 allele thus points to a critical role for type I IFN when infected, and exhibit an abnormally elevated TLR9 in viral sensing and identifies a vulnerable amino acid within the splenic viral titer during infection. ectodomain of three TLR proteins, essential for a ligand response. In the present study, we have identified a codominant N-ethyl- N-nitrosourea (ENU)-induced phenotype in which homozygous atural killer cells (NK) make an essential contribution to animals fail to respond to CpG-oligodeoxynucleotide (ODN) Nmammalian innate immune defense against viral infections and also show severely impaired responses to MCMV infection (1). NK can directly sense viral pathogens through a specific and as evidenced by viral titers measurement and survival analysis. complex set of inhibitory and activating receptors (2–4). The This immunodeficiency phenotype is caused by a hypomorphic molecular interactions that are required for viral sensing and Tlr9 allele (Tlr9CpG1), encoding a structurally aberrant receptor. response have been best documented in the case of mouse cyto- Tlr9CpG1 homozygotes display a low level of cytokine induction megalovirus (MCMV) infection. The MCMV-encoded protein and NK activation on viral infection. Mice homozygous for a null m157 interacts directly with the NK membrane protein LY49H, allele of MyD88 show a similar phenotype, indicating that the which contains an immunoreceptor tyrosine-based activation motif TLR9 3 MyD88 axis is essential for effective innate antiviral (ITAM) (5, 6). On ligand binding, LY49H causes NK activation, defense. We also tested the response to MCMV infection in mice manifested in part by IFN-␥ and perforin production. homozygous for a null allele of Tlr3. Our results indicate that However, an effective NK response also depends on extrinsic TLR3 plays a significant role, although a less crucial role than signals. Once MCMV infection is established, NK are activated by TLR9, in defense against systemic MCMV infection. cytokines and chemokines, including type I IFN and IL-12, which are secreted by dendritic cells (DC). These molecules are known to Materials and Methods Ϫ Ϫ Ϫ Ϫ influence the outcome of MCMV infection (7–9). Indeed, during Mice. C57BL͞6, BALB͞c, Tlr3 / mice (14), MyD88 / mice (pro- MCMV infection, bidirectional communication between DC and vided by S. Akira, Osaka University, Osaka), and TLR9CpG1/CpG1 NK is established. Both DC-derived cytokines and direct pathogen recognition are essential for NK activation. In turn, NK participate in the maintenance of the DC population (10). However, little is Abbreviations: DC, dendritic cells; dsRNA, double-stranded RNA; ENU, N-ethyl-N- nitrosourea; LRR, leucine-rich repeat; MCMV, mouse cytomegalovirus; NK, natural killer known about the earliest events that lead to type I IFN synthesis, cells; ODN, oligodeoxynucleotide; pfu, plaque-forming units; TLR, Toll-like receptor; HSV, which is known to occur during viral infection. herpes simplex virus; TNF, tumor necrosis factor; TCR, T cell antigen receptor. The host sensors that initially detect viral pathogens and cause Data deposition: The sequences reported in this paper have been deposited in the GenBank cytokine production by myeloid cells have been investigated by database (accession nos. AY510704–AY510706). several groups, some of which have pointed to possible involve- †K.T. and P.G. contributed equally to this work. ment of Toll-like receptors (TLRs) (11, 12). The TLRs constitute ʈTo whom correspondence should be addressed. E-mail: richard.fl[email protected] or a family of ligand-binding molecules that engage a variety of [email protected]. microbial products (13). On binding, they activate signaling © 2004 by The National Academy of Sciences of the USA

3516–3521 ͉ PNAS ͉ March 9, 2004 ͉ vol. 101 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400525101 Downloaded by guest on September 30, 2021 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.

mice were maintained under pathogen-free conditions in The measuring the protection conferred by serially diluted serum Scripps Research Institute animal care facility. All mice used in the against the cytopathic effect of the vesicular stomatitis virus in experiments were 6–9 weeks in age. All experimental procedures L929 cells (22). Purified IFN-␣ (R & D Systems) was used as a were conducted in accordance with institutional guidelines for standard. animal care and use. Fluorescence-Activated Cell Sorting Assay. Splenocytes were har- ENU Mutagenesis and in Vitro Stimulation of Thioglycolate-Elicited vested and passed through a cell strainer 36 h postinfection, into Macrophages. Germ-line mutagenesis was performed in mice by Dulbecco’s modified Eagle’s medium containing 10% FBS and using ENU (19). Ex vivo stimulation of peritoneal macrophages ͞

2% penicillin streptomycin solution (GIBCO). After fixation IMMUNOLOGY with multiple microbial inducers and measurement of secreted and permeabilization, cells were stained for the surface markers ␣ tumor necrosis factor (TNF)- activity were conducted as de- NK1.1 and T cell antigen receptor (TCR) ␤ as well as intracel- CpG1/CpG1 scribed in refs. 18 and 19. For analysis of the Tlr9 lular IFN-␥ by using antibodies from BD Bioscience. Flow phenotype, CpG-ODN (5Ј-tccatgacgttcctgatgct-3Ј) was used at ␣ cytometry was performed by using a FACSCalibur cell sorter the stated concentrations. Secreted TNF- activity was mea- and analyzed with CELLQUEST software. sured 4 hours after induction by using the L-929 cell bioassay.

Virus Culture and Assay. MCMV (Smith strain) was prepared by in Transfection of HEK 293 Cells and Assay of TLR9 Signaling Activity. ͞ vivo propagation in 3-week-old BALB͞c mice infected with 1 ϫ Full-length muTLR9 cDNAs (amplified from C57BL6 J and CpG1/CpG1 104 plaque-forming units (pfu) by an i.p. route. Salivary gland Tlr9 mutant peritoneal macrophage mRNA) were homogenates were prepared at day 14 postinfection, and viral cloned into pFlagCMV3 mammalian expression vector (Sigma). titer was determined by plaque assay (21). Splenic viral titers Plasmids were isolated by using the Endo-free Maxi prep kit were determined by plaque assay 4 days after MCMV infection. (Qiagen, Valencia, CA). HEK293 cells (2 ϫ 105) were trans- fected by using Lipofectamine (Invitrogen) with 0.4 ␮g of each Cytokine Measurements. IL-12 and IFN-␥ were measured in serum expression vector and endothelial leukocyte adhesion molecule harvested 36 h after MCMV infection by using an ELISA (R & 1 (ELAM-1) luciferase reporter vector (23). After 24 h, cells D Systems). Type I IFN titers were determined with a bioassay, were stimulated with 0.1 and 1.0 ␮M CpG-ODN for 5 h.

Tabeta et al. PNAS ͉ March 9, 2004 ͉ vol. 101 ͉ no. 10 ͉ 3517 Downloaded by guest on September 30, 2021 Luciferase assays were performed by using the Steady-Glo luciferase assay system (Promega).

Identification and cDNA Cloning of TLRs 11, 12, and 13. All murine and human ESTs were downloaded from the National Center for Biotechnology Information database, translated in all six reading frames by using the program PEPDATA, and searched for Toll͞ IL-1R homology (TIR) domain homology (HMMERSEARCH, E Ͻ 10) by using a hidden Markov model based on representatives of all known animal TIR domains. Three hits were acquired among the mouse ESTs. The corresponding cDNAs were amplified from macrophage mRNA by using primers based on genomic sequence to capture complete coding regions. Alignment of all human and mouse TLR protein sequences was performed by using the CLUSTALW program (gap opening penalty ϭ 10; gap extension penalty ϭ 0.20; PAM series as the protein weight matrix). The TLR coding sequences were submitted to GenBank (accession nos. AY510704–AY510706). Results CpG1 ␣ Identification of an ENU-Induced CpG-ODN Non-Responder Phenotype Fig. 2. In vivo effects of the Tlr9 mutation. (A) TNF- secretion by macro- phages from controls C57BL͞6(Tlr9ϩ/ϩ, filled dots), heterozygotes (Tlr9CpG1/ϩ, and Determination of the Causative Mutation Within the Tlr9 Locus. CpG1/CpG1 Ͼ gray dots) and homozygotes (Tlr9 , open dots) animals after CpG-ODN Among 11,000 mice examined to date, we identified an F3 induction (0.1 ␮M). Each dot represents the result of a duplicate induction assay mutant male in which thioglycolate-elicited peritoneal macro- performed on cells from a single animal. (B) TNF-␣ production or (C) IL-12p40 phages were markedly hyporesponsive to stimulation with CpG- production by peritoneal macrophages at low (0.1 ␮M) CpG ODN concentration, ODN. Normal TNF-␣ production was observed when macro- as influenced by Tlr9 genotype. Error bars indicate SD; n ϭ 5 mice. (D) Kaplan– phages were stimulated with all other TLR agonists (data not Meier survival curves for Tlr9CpG1/CpG1 mice and Tlr9ϩ/ϩ mice, after sensitization shown). This finding suggested that the mutation, termed CpG1, with D-galactosamine and challenge with CpG-ODN. Mice were monitored for a specifically affected TLR9 signaling. 3-day period, at which time all survivors seemed healthy. On sequencing the Tlr9 coding region, a T 3 C base transition was observed at position 1496 (gi:13626029), predicting the amino ϩ ϩ ϩ Tlr9CpG1/ Tlr9 / B C acid substitution L499P (Fig. 1A). To examine the affected residue and genotypes (Fig. 2 and ). Hence, the codominant character of the Tlr9CpG1 allele is observed only at with reference to all other mammalian TLRs, a HMMERSEARCH of low CpG-ODN concentrations (Ͻ0.1 ␮M). all human and mouse ESTs was performed to generated an updated CpG1/CpG1 set of TIR domain proteins for alignment. Three mouse TLRs were The Tlr9 genotype was found to protect D- galactosamine-sensitized animals against the lethal effect of identified (TLRs 11, 12, and 13). They have not previously been ␣ reported and lack human orthologs. The total complement of CpG-ODN-induced TNF- in vivo. Homozygous mutants and mouse TLR proteins now numbers 12 because the mouse ortholog wild-type controls were sensitized with D-galactosamine (20 mg of human TLR10 is a degenerate pseudogene. per mouse) and then injected with CpG-ODN (20 nmol per All 10 human TLRs, and all 12 mouse TLRs, were optimally mouse). One hundred percent of the homozygous mutants survived the injection whereas 83% of wild-type C57BL͞6 mice aligned. Residue 499 of the mouse TLR9 protein lies within a ϭ part of the receptor ectodomain that, on comparison with all succumbed within 10 h (Fig. 2D; P 0.0025). other TLRs in the multiple alignment plot, is represented only 3 in TLRs 7, 8, and 9 [known to represent an evolutionary cluster The TLR9 MyD88 Signaling Axis Is Critical to Control MCMV CpG1/CpG1 (24)]. The leucine in question is located near the beginning of the Infection. Tlr9 mice were examined in a second screen, eleventh leucine-rich repeat (LRR) motif predicted by simple designed to identify mutations that impair MCMV resistance. In modular research tool (SMART) analysis. In TLR8, an isoleucine this screen, a systemic infection is induced by i.p. injection of ϫ 5 is represented at this position. MCMV, by using an inoculum (5 10 pfu) that is well tolerated ͞ When reconstructed and overexpressed in transfected HEK by normal C57BL 6 mice. This protocol has been established to ͞ 293 cells, the mutant TLR9 protein was insensitive to stimulation distinguish between sensitive strains (such as BALB c), which with CpG-ODN whereas the wild-type protein, expressed in the succumb 4–5 days after the inoculation, and resistant strains ͞ same system, was strongly CpG-ODN responsive (Fig. 1B). In (such as C57BL 6), which survive even 8 days after the infection. addition, homozygous mutant mice were bred to normal As shown in Fig. 3A, Tlr9CpG1/CpG1 mice accumulate exagger- C57BL͞6 mice and then backcrossed to the mutant stock so that ated viral loads 4 days after infection, with titers approaching CpG1 those observed in the BALB͞c strain. Because TLR9 signals Tlr9 homozygotes and heterozygotes were obtained. Cells Ϫ Ϫ from Tlr9CpG1/CpG1 mice were uniformly unresponsive to CpG- through MyD88 (25), we determined the viral load in MyD88 / ODN (0.1 ␮M concentration) stimulation whereas cells from mice and observed that these animals also show high viral titers. Tlr9ϩ/ϩ mice were uniformly responsive. Heterozygotes We have previously reported that the Lps2 allele of the Tlr9CpG1/ϩ exhibited an intermediate phenotype. These data Trif͞Ticam-1 gene impairs macrophage IFN-␤ production in establish that the CpG1 phenotype is linked to the observed response to dsRNA. As a result, these mice are also permissive mutation in Tlr9 (P Ͻ 0.0001) and also reveal that the mutant for MCMV infection (18). Because Trif is necessary for TLR3 allele is codominant, or alternatively, that the wild-type allele is signaling (18, 20), which is MyD88-independent (18), we rea- haploinsufficient (Fig. 2A). soned that either component of the Trif 3 TLR3 axis might Ϫ Ϫ The effect of heterozygosity for the Tlr9CpG1 allele on TNF-␣ impair the immune response to MCMV. Accordingly, Tlr3 / and IL-12 secretion was further examined by varying the con- mice were examined for MCMV susceptibility. The average viral centration of CpG-ODN used as a stimulus. A high CpG-ODN titer in the spleen of infected Tlr3Ϫ/Ϫ mice never reached that concentration (1 ␮M) allows a clear-cut distinction between observed in Tlr9CpG1/CpG1 or MyD88Ϫ/Ϫ mice but showed a highly Tlr9CpG1/ϩ and Tlr9CpG1/CpG1 genotypes but fails to resolve significant increase compared with wild-type controls (Fig. 3A),

3518 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400525101 Tabeta et al. Downloaded by guest on September 30, 2021 Fig. 3. Tlr9CpG1/CpG1, MyD88Ϫ/Ϫ, and Tlr3Ϫ/Ϫ 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 ϫ 105 pfu of MCMV. (B) Mice of the indicated genotype were infected i.p. with 5 ϫ 105 pfu of MCMV, and survival was monitored for a period of 7 days. P values indicate comparisons with the survival curve of C57BL͞6 control animals.

comparable to that previously reported in TrifLps2/Lps2 mutant Fig. 4. In vivo impairment of cytokine production after MCMV infection in mice 3 ␣͞␤ mice (19). In the course of our infection studies, we repeatedly lacking the TLR9 MyD88 signaling pathway. (A) IFN- activity was measured observed that, as in BALB͞c mice, MyD88Ϫ/Ϫ and Tlr9CpG1/CpG1 in the serum of noninfected (ni) controls and MCMV-infected animals 36 h postinoculation. Values are expressed as international units (IU)͞ml of serum. mice showed obvious signs of sickness 4 days after MCMV IL-12p40 (B) and IFN-␥ (C) concentrations were measured in the serum of the same inoculation. As shown in Fig. 3B, death follows viral infection of animals as those shown in A by ELISA and are expressed in ng͞ml. Data represent Ϫ Ϫ Tlr9CpG1/CpG1 and MyD88 / mice almost as rapidly as it does in mean values with SD (n ϭ 4 mice). Statistical analysis was performed by using the Ϫ Ϫ BALB͞c animals. Tlr3 / mutants showed no significant sur- ANOVA test with PRISM software (GraphPad, San Diego) (*, P Ͻ 0.05, **, P Ͻ 0.001, vival difference compared with C57BL͞6 controls although, at a with respect to the C57BL͞6 control values). subjective level, the Tlr3Ϫ/Ϫ animals seemed sicker than controls, and a trend toward higher mortality was observed. IMMUNOLOGY A clear link has been established between cytokine induction and Cytokine Induction and NK Activation Are Impaired in Tlr9CpG1͞CpG1 NK activation during MCMV infection (8–10). We analyzed splenic ؊/؊ NK and NKT cell populations 36 h postinfection by gating on and MyD88 Mice. Because the secretion of certain cytokines is a ϩ Ϫ ϩ ϩ NK1.1 ͞TCR␤ (for NK) and NK1.1 ͞TCR␤ (for NKT cells) well known and essential response for the clearance of viral ␥ ͞ and then measuring intracellular IFN- . Fig. 5 shows the results of infections, we infected C57BL 6 control mice and mutant mice with this analysis. The values (mean Ϯ SD, n ϭ 4 mice) indicate the an inoculum of virus that was sublethal during the term of the proportion of IFN-␥ϩ NK or NKT cells among the total population. ϫ 4 experiment (5 10 pfu per mouse) and monitored their cytokine These data illustrate the pronounced effect (a 4-fold reduction) of levels in the serum at a time (36 h postinfection) when this response both the Tlr9CpG1/CpG1 and the MyD88Ϫ/Ϫ genotypes on NK and is at its maximum (26). As shown in Fig. 4A, type I IFN secretion NKT cell activation that occurs in the course of MCMV infection. Ϫ/Ϫ CpG1/CpG1 is dramatically reduced in MyD88 mice as well as in Tlr9 The Tlr3Ϫ/Ϫ genotype was associated with a smaller, but still ␥ mice. IL-12 p40 levels (Fig. 4B) and IFN- levels (Fig. 4C) are also significant, decrease in the proportion of activated NK and NKT diminished in the homozygous mutants. We also note that IFN-␥ cells. production is strongly inhibited in MyD88Ϫ/Ϫ animals, which might be attributable to additional signaling defects in these mice (see Discussion Discussion). The decrease in serum cytokine levels is less pro- The L499P substitution specified by Tlr9CpG1 falls within a centrally nounced in Tlr3Ϫ/Ϫ mice but is statistically significant. placed LRR motif: 1 of 19 such motifs found in the TLR9

Tabeta et al. PNAS ͉ March 9, 2004 ͉ vol. 101 ͉ no. 10 ͉ 3519 Downloaded by guest on September 30, 2021 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 TCR␤ surface markers. (Left) IFN-␥ intracellular staining (and isotype antibody as control) for NK (NK1.1ϩTCR␤Ϫ). (Right) Results for NKT cells (NK1.1ϩTCR␤ϩ). Mean values Ϯ SD are indicated (n ϭ 4 mice).

ectodomain. Within this LRR, the mutation would be expected to sensing apparatus within the NK of the C57BL͞6 mouse is disrupt an alpha helix that normally contributes to the single loop inadequate to contain an MCMV infection. formed by all LRRs (27). The region of the receptor within which Although MyD88 serves most of the TIR domain receptors the mutation occurs is represented only in a subset of TLR proteins. (excluding TLR3), it is logical to suppose that most of the Neither the previously published TLRs (TLRs 1–6 and TLR10) nor protection that MyD88 affords during MCMV infection results the three mouse TLRs reported herein (TLRs 11, 12, and 13) from its interaction with TLR9. Consistent with this conclusion, exhibit a homologous region. It might therefore be imagined that TLR2 and TLR4 deficiencies have no influence on the course of the residue in question has a specialized function, related to the MCMV infection (P.G., unpublished results). On the other hand, types of ligands that are engaged by TLRs 7, 8, and 9 (nucleotide- the viral resistance phenocopy observed in MyD88Ϫ/Ϫ and based molecules). Tlr9CpG1/CpG1 is imperfect. The exceptionally low level of serum The Tlr9CpG1 allele was identified in two independent screen- IFN-␥ observed in infected MyD88Ϫ/Ϫ animals (Fig. 4C) might ing procedures: an ex vivo assay designed to identify new reflect a requirement for MyD88 in certain TLR-independent components of the TLR signaling pathways, and an in vivo signaling pathways for NK activation. IL-18, which signals protocol used to recover mutations that affect the innate anti- through MyD88 (28) but not Trif (P.G., E.J., and K.H., unpub- viral response. The mutation markedly impairs the antiviral lished results) and is essential for NK expansion after mCMV response, and the magnitude of the impairment is very similar to infection (10), may account for the discrepancy. that associated with the MyD88Ϫ/Ϫ genotype, and indeed, to that Because the DNA of herpesviruses such as MCMV is G͞C-rich associated with the BALB͞c genotype. Hence, absent input from and has immunostimulatory activities (29), the MCMV-sensitivity the TLR9 3 MyD88 signaling axis, the normal m157 3 LY49H phenotype that we have observed is likely due to a lack of

3520 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400525101 Tabeta et al. Downloaded by guest on September 30, 2021 TLR9-mediated viral DNA recognition and resulting impairment It is possible that this relatively modest superadditivity results of IFN-␣͞␤ secretion. Two independent studies have recently from signal transducer and activator of transcription (STAT)-1- identified plasmacytoid dendritic cells (pDC) and͞or IFN produc- mediated induction of additional type I IFN in response to IFN that ing cells (IPC) as the source of type I IFN production during HSV-1 is produced (33). However, when an infectious endpoint is exam- and HSV-2 infections (11, 12) and have shown that IFN production ined, the effect of mutational inactivation of either the TLR3 or is TLR9-dependent. Our data do not permit discrimination be- TLR9 pathway is even more pronounced, suggesting that numerous tween these two categories of DCs as principal sources of type I functional defects (rather than the relatively modest observed IFNs in MCMV infection. We have examined the splenic pDC defect of type I IFN production) contribute to immunocompro- ϩ ϩ Ϫ (CD8 ͞CD11c ͞CD11b ) population in MCMV-infected mise. MCMV titer is elevated by three orders of magnitude as the Ϫ Ϫ MyD88 / mice and observed that they undergo a reduction in result of Tlr3 mutation, and by four orders of magnitude as the numbers at day 1.5 after infection, and an expansion at day 4 after result of Tlr9 mutation. Whereas it might have been supposed that infection: a pattern also observed in wild-type control animals (data either pathway would complement the loss of the other, it seems, not shown). This finding, however, does not rule out their involve- to the contrary, that both pathways are essential for containment of ment in viral DNA recognition because this pattern of response is infection. Are these the only two pathways that allow DC to sense itself dependent on NK activation (10). It has also been suggested MCMV infection? It is possible that dsRNA sensing may also occur that IFN-␣͞␤ produced by IPC on viral challenge can activate by means of protein kinase R (PKR), which may prompt the immature DC (30), which in turn secrete type I IFN. Alternatively, production of type I IFN (34). The relative importance of this both types of DC, as well as macrophages [which also express TLR9 pathway in DC remains to be established. Compound homozygous (31)], may be activated by MCMV in vivo and synergize in resisting mutants (Tlr3Ϫ/Ϫ;Tlr9CpG1/CpG1) are currently being established and the viral pathogen. should clarify whether residual awareness of infection exists, absent Our data also implicate TLR3 as a key participant in the antiviral both receptors for viral sensing. response. Approximately 1,000-fold augmentation of viral load in In the past few years, a large volume of data has demonstrated Ϫ/Ϫ the spleen was observed in Tlr3 mice inoculated with MCMV, the involvement of TLRs in the defense against bacterial infections, a finding consistent with earlier studies that showed a comparable but their role in viral infection has remained unclear. In contrast to Lps2/Lps2 increase in Trif mice (18). TLR3 is known to serve as a previous reports (11, 12), our in vivo infectious model establishes a ligand for dsRNA, and we presume that dsRNA may be produced prominent and essential function for TLR9 in MCMV resistance as a consequence of bidirectional transcription from the MCMV because mice devoid of CpG-mediated signaling quickly die after genome in the course of infection (32). viral inoculation. Furthermore, our data suggest that both MyD88- The fact that impairment of either TLR3 or TLR9 signaling dependent and -independent signals are essential for cytokine pathways has a dramatic effect on the course of disease is responses. TLR3- and TLR9-dependent type I IFN production somewhat surprising. The TLR3 3 Trif signaling axis is believed 3 activates NK, which in turn confine the viral pathogen and prevent to be independent of the TLR9 MyD88 signaling axis. Yet its rapid spread during the critical interval before activation of an both signals lead to the induction of type I IFN (12, 18, 20). adaptive immune response. Abrogation of TLR3 signaling causes a Ͼ60% decrement, and Ͼ abrogation of TLR9 signaling causes a 90% decrement in the We thank Marc Dalod (Centre d’Immunologie de Marseille-Luminy, amount of type I IFN that is measured in serum after infection. Marseille, France) for helpful advice. This work was supported by Hence, the two pathways seem to elicit the production of type I National Institutes of Health Grant U54A154523 and by the Fondation IFN in a superadditive or codependent manner. Philippe (to P.G.).

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