Abstract
22 min readBacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-κB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins. Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-κB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins. Group A streptococcus (GAS 4The abbreviations used are: GAS, group A streptococcus; BMDM, bone marrow-derived macrophages; IFN, interferon; IRF, interferon regulatory factor; MAPK, mitogen-activated protein kinase; PRRs, pattern-recognition receptors; TLR, Toll-like receptor; TNFα, tumor necrosis factor α; IL, interleukin; MOI, multiplicity of infection; ELISA, enzyme-linked immunosorbent assay; qRT, quantitative reverse transcription. ; Streptococcus pyogenes) is an important human Gram-positive pathogen responsible for a wide spectrum of infections, ranging from mild diseases (e.g. tonsillitis) to serious illness (e.g. necrotizing fasciitis, sepsis, or severe poststreptococcal sequelae) (1Carapetis J.R. Steer A.C. Mulholland E.K. Weber M. Lancet. 2005; 5: 685-694Abstract Full Text Full Text PDF PubMed Scopus (2012) Google Scholar). The persistence of GAS in the human population and the severity of some GAS diseases are the result of activities of a number of virulence factors that enable the pathogen to escape immune surveillance or, on contrary, induce an overreaction of the immune system (2Bisno A.L. Brito M.O. Collins C.M. Lancet. 2003; 3: 191-200Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 3Chhatwal G.S. McMillan D.J. Trends Mol. Med. 2005; 11: 152-155Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Although GAS is generally regarded as an extracellular pathogen, recent findings suggest that GAS can survive (although not multiply) within various host cells, such as neutrophils, macrophages, epithelial cells, and fibroblasts (4Staali L. Morgelin M. Bjorck L. Tapper H. Cell. Microbiol. 2003; 5: 253-265Crossref PubMed Scopus (71) Google Scholar, 5Nakagawa I. Amano A. Mizushima N. Yamamoto A. Yamaguchi H. Kamimoto T. Nara A. Funao J. Nakata M. Tsuda K. Hamada S. 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Typically, pathogen-associated molecular patterns are components of the bacterial cell wall (e.g. lipopolysaccharide and lipoteichoic acid), but they may also be derived from the inside of bacteria (e.g. DNA). The primary function of PRRs is to trigger signaling cascades that activate antimicrobial defense programs. The best studied class of PRRs is the Toll-like receptor (TLR) family, which consists of 13 transmembrane glycoproteins in mammals (8Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8899) Google Scholar). Virtually all pathogenic bacteria are recognized by one or more TLRs, with TLR2 being the receptor for lipoteichoic acid of Gram-positive bacteria, TLR4 the receptor for lipopolysaccharide of Gram-negative bacteria, and TLR9 the receptor for bacterial DNA containing unmethylated CpG sequences. Ligand binding to TLR3, TLR4, TLR7, and TLR9 but not to TLR2 launches two distinct signaling pathways that result in the production of proinflammatory cytokines (e.g. TNFα and IL-1) and type I IFNs, respectively. Five TIR domain-containing adaptor molecules are involved in signaling downstream of TLRs: MyD88, TIRAP (or MAL), TRIF (or TICAM1), TRAM (or TICAM2), and SARM, a negative regulator of TRIF (10O'Neill L.A. Bowie A.G. Nat. Rev. 2007; 7: 353-364Google Scholar). MyD88, an essential signaling component of all TLRs except TLR3, is required for activation of MAPKs and NF-κB that cause production of proinflammatory cytokines. Signaling components that are required for the production of type I IFNs are less uniform, and they are partially TLR- and cell type-specific. Recently, much attention has been paid to the elucidation of pathways leading to TLR-independent production of type I IFN in response to intracellular bacteria and/or intracellular DNA (11Stockinger S. Reutterer B. 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Signaling downstream of this unknown PRR employs the serine threonine kinase TBK1 or, less frequently, its relative IKK (15Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2099) Google Scholar, 16Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1371) Google Scholar, 17Hemmi H. Takeuchi O. Sato S. Yamamoto M. Kaisho T. Sanjo H. Kawai T. Hoshino K. Takeda K. Akira S. J. Exp. Med. 2004; 199: 1641-1650Crossref PubMed Scopus (465) Google Scholar, 18Perry A.K. Chow E.K. Goodnough J.B. Yeh W.C. Cheng G. J. Exp. Med. 2004; 199: 1651-1658Crossref PubMed Scopus (313) Google Scholar), which phosphorylates primarily the transcription factor IRF3, causing the activation of the IFN-β gene, the first type I IFN to be expressed (19Levy D.E. J. Exp. Med. 2002; 195: F15-18Crossref PubMed Scopus (53) Google Scholar, 20Taniguchi T. Takaoka A. Curr. Opin. Immunol. 2002; 14: 111-116Crossref PubMed Scopus (406) Google Scholar). Other cytoplasmic PRRs, such as the NOD proteins, recognize bacterial products in the cytoplasm and activate MAPKs and NF-κB but not IFN production (21Strober W. Murray P.J. Kitani A. Watanabe T. Nat. Rev. 2006; 6: 9-20Crossref Google Scholar). Despite a considerable knowledge of GAS virulence factors, the PRRs that are responsible for recognition of this pathogen by the host are unknown. Remarkably little information is available also about the signaling pathways triggered by GAS in infected innate immune cells that are known to be required for defense against GAS (22Goldmann O. Rohde M. Chhatwal G.S. Medina E. Infect Immun. 2004; 72: 2956-2963Crossref PubMed Scopus (97) Google Scholar). Here we show that GAS activates p38 MAPK, NF-κB, TNFα, and IL-6 production in infected bone marrow-derived mouse macrophages (BMDMs). Similar to other bacteria, these responses depend on MyD88. However, in contrast to most other pathogenic bacteria, the MyD88-mediated signaling was independent of TLR2, the receptor for Gram-positive bacteria, and the other bacterial receptors TLR4 and TLR9. We also rule out the involvement of IL-1 receptor signaling. We further demonstrate that GAS also elicits MyD88-independent signaling that results in type I IFN production. The IFN production did not require the presence of the GAS-encoded cytolysins SLO and SLS. This finding is surprising, since during infections with other Gram-positive bacteria, either the cytolysin itself or cytolysin-mediated cytoplasmic escape of bacteria from phagocytic vesicles was implicated in triggering IFN production (12Stetson D.B. Medzhitov R. 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Our data implicate that GAS is recognized by a yet unknown receptor upstream of MyD88 and establish GAS as a Gram-positive pathogen capable of inducing type I IFN synthesis without molecules usually required for entry of bacterial products into the cytoplasm of infected cells. Bacterial Strains—Escherichia coli DH5-α and TOP10 were used as hosts for cloning. S. pyogenes serotype M1 (ATCC 700294) is a clinical strain originally isolated from an infected wound. The isogenic sagA- and slo-deficient mutants were constructed using a thermosensitive strategy described previously (26Mangold M. Siller M. Roppenser B. Vlaminckx B.J. Penfound T.A. Klein R. Novak R. Novick R.P. Charpentier E. Mol. Microbiol. 2004; 53: 1515-1527Crossref PubMed Scopus (96) Google Scholar). First, using wild-type genomic DNA as template and primers containing flanking restriction sites, a 1111-bp sagA upstream fragment (primers OLEC120/OLEC143), a 1200-bp sagA downstream fragment (primers OLEC123/OLEC144), a 1159-bp slo upstream fragment (primers OLEC248/OLEC249), and a 1033-bp slo downstream fragment (primers OLEC250/OLEC251) were amplified. After digestion with the respective restriction enzymes, the upstream and downstream fragments were cloned into thermo-sensitive plasmids pRDN18 (sagA-deficient mutant) and pEC84 (slo-deficient mutant) (26Mangold M. Siller M. Roppenser B. Vlaminckx B.J. Penfound T.A. Klein R. Novak R. Novick R.P. Charpentier E. Mol. Microbiol. 2004; 53: 1515-1527Crossref PubMed Scopus (96) Google Scholar). After introduction of the recombinant plasmids into S. pyogenes ATCC 700294, a series of temperature shifts with appropriate antibiotic selection was performed, thus leading to the final deficient mutants (strain EC548 (sagA-deficient mutant) and EC997 (slo-deficient mutant)) in which the entire coding sequence of the gene was deleted in a nonpolar fashion. To create the sagA/slo-deficient strain (EC1142), the deletion of slo was performed as mentioned above in the background of the sagA-deficient strain. The correct deletion event in the mutants was checked by PCR, Southern blot, and sequencing analysis. SLS and SLO hemolysis assays further confirmed that the sagA-, slo-, and sagA/slo-deficient mutants were defective in SLS and/or SLO activity as described (27Ruiz N. Wang B. Pentland A. Caparon M. Mol. Microbiol. 1998; 27: 337-346Crossref PubMed Scopus (90) Google Scholar, 28Sierig G. Cywes C. Wessels M.R. Ashbaugh C.D. Infect. Immun. 2003; 71: 446-455Crossref PubMed Scopus (107) Google Scholar). DNA manipulations are described in the supplemental materials. Bacterial Culture—S. pyogenes strains were grown at 37 °C with 5% CO2 without agitation in Todd-Hewitt broth (BD Biosciences) supplemented with 0.2% yeast extract and on tryptic soy agar supplemented with 3% defibrinogated sheep blood. Transformation of E. coli and S. pyogenes was performed as previously described (29Caparon M.G. Stephens D.S. Olsen A. Scott J.R. Infect. Immun. 1991; 59: 1811-1817Crossref PubMed Google Scholar, 30Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, pp. 1.74–1.84, Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar). Whenever required, antibiotics were added to the medium at the following final concentrations: erythromycin, 300 μg/ml for E. coli and 3 μg/ml for S. pyogenes; spectinomycin, 100 μg/ml for both E. coli and S. pyogenes. Bacterial cell growth was turbidimetrically monitored at 620 nm with a microplate reader. Macrophage Cell Culture—Primary BMDMs were obtained from the femur bone marrow of 6–10-week-old mice. Cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in the presence of L cell-derived CSF-1, as described (31Baccarini M. Bistoni F. Lohmann-Matthes M.L. J. Immunol. 1985; 134: 2658-2665PubMed Google Scholar). MyD88–/–, TLR2–/–, TLR4–/–, TLR9–/–, TLR2/4/9–/–, IFNAR1–/–, IRF3–/–, IL1-RI–/–, and control WT mice, all on a C57Bl/6 background, were housed under specific pathogen-free conditions (32Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar, 33Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar, 34Takeuchi O. Hoshino K. Kawai T. 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After 30 min of incubation at 37 °C, nonadherent extracellular bacteria were eliminated by removing the culture medium, and adherent extracellular bacteria were subsequently killed by incubation with fresh medium (without fetal calf serum) containing 60 μg/ml penicillin. At specific time points after infection, supernatants were collected for ELISA, and whole cell extracts were prepared for Western blot analysis. At least three mice of each genotype were used in all infection experiments. Antibodies—Antibodies to Tyr701-phosphorylated Stat1 (pY701-S1) and phosphorylated p38 (pp38) were purchased from Cell Signaling (Frankfurt/Main, Germany). Antibodies to IκB-α and p38 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody to Stat1-α C terminus was previously described (37Kovarik P. Stoiber D. Novy M. Decker T. EMBO J. 1998; 17: 3660-3668Crossref PubMed Scopus (201) Google Scholar). ELISA and Western Blot Analysis—For ELISAs, supernatants of infected macrophages were collected and diluted 1:5 in reagent diluent. TNFα and IL-6 were assayed using DuoSET ELISA kits (R&D Systems, Minneapolis, MN). Western blot analysis was performed using fluorophore-linked secondary antibodies (Molecular Probes-Invitrogen (Lofer, Austria) and Rockland (Gilbertsville, PA)) and an Odyssey infrared imaging system (LI-COR Bioscience, Lincoln, NE). qRT-PCR—Total RNA was isolated using Trizol® LS reagent (Invitrogen). Reverse transcription of total RNA was performed using oligo(dT)18 as primer and Moloney murine leukemia virus reverse transcriptase (Fermentas, St. Leon-Rot, Germany). The cDNA of the IFN-β and Mx2 genes was subsequently analyzed by qRT-PCR as described in the supplemental materials. GAS Activates Inflammatory Signaling Independently of TLR2, TLR4, and TLR9—To investigate the role of bacteria-recognizing TLRs in responses to GAS infection, we examined the activation of the transcription factor NF-κB by assaying the degradation of its inhibitor (IκB) and the phosphorylation of p38 MAPK in infected BMDMs. Activation of both NF-κB and p38 MAPK was shown in numerous studies to be essential for production of proinflammatory cytokines (38Alcamo E. Mizgerd J.P. Horwitz B.H. Bronson R. Beg A.A. Scott M. Doerschuk C.M. Hynes R.O. Baltimore D. J. Immunol. 2001; 167: 1592-1600Crossref PubMed Scopus (227) Google Scholar, 39Senftleben U. Li Z.W. Baud V. Karin M. Immunity. 2001; 14: 217-230Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 40Kim D.H. Feinbaum R. Alloing G. Emerson F.E. Garsin D.A. Inoue H. Tanaka-Hino M. Hisamoto N. Matsumoto K. Tan M.W. Ausubel F.M. Science. 2002; 297: 623-626Crossref PubMed Scopus (602) Google Scholar, 41Suzuki N. Suzuki S. 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IκB gradually reappeared at later time points but did not reach the original level during the time frame of observation (up to 4 h). The internalization of GAS (monitored by fluorescence microscopy) was equally efficient in cells of both genotypes (data not shown). These data demonstrate that TLR2 was not required for GAS-induced signaling. Other TLRs frequently engaged by bacteria are TLR4 and TLR9. TLR4 is the receptor for lipopolysaccharide of Gram-negative bacteria, but it has been shown to recognize also cell wall components of mycobacteria and several cytolysins of Gram-positive bacteria (25Park J.M. Ng V.H. Maeda S. Rest R.F. Karin M. J. Exp. Med. 2004; 200: 1647-1655Crossref PubMed Scopus (182) Google Scholar, 43Abel B. Thieblemont N. Quesniaux V.J. Brown N. Mpagi J. Miyake K. Bihl F. Ryffel B. J. Immunol. 2002; 169: 3155-3162Crossref PubMed Scopus (310) Google Scholar, 44Ferwerda G. Girardin S.E. Kullberg B.J. Le Bourhis L. de Jong D.J. Langenberg D.M. van Crevel R. 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To examine whether a combination of TLR2, TLR4, and TLR9 was used for GAS recognition, we analyzed GAS-induced signaling in TLR2/4/9 triple-deficient BMDMs. As depicted in Fig. 1D, the signaling events in triple-deficient BMDMs were comparable with WT cells. These data exclude a redundant function of TLR2, TLR4, and TLR9 in GAS recognition and demonstrate that macrophages sense GAS using a PRR other than the most common bacterial receptors. GAS-induced Inflammatory Signaling Depends on MyD88—All TLRs except for the double-stranded RNA receptor TLR3 require MyD88 for activation of MAPKs, NF-κB, and the subsequent proinflammatory cytokine production. To investigate whether MyD88 was involved in GAS-induced inflammatory signaling, we examined the activation of p38 MAPK and degradation of IκB in infected MyD88–/– BMDMs. The experiment revealed that GAS-induced activation of p38 MAPK was almost completely abolished, and the degradation of IκB was strongly diminished in MyD88–/– cells when compared with WT cells (Fig. 1E). A weak p38 MAPK activation and IκB degradation were observed in MyD88–/– cells at later time points of infection. Thus, the GAS-induced inflammatory signaling was markedly reduced and delayed in MyD88–/– cells. The activation of MAPKs and NF-κB plays a fundamental role in induction of proinflammatory cytokines, such as TNFα or IL-6. To estimate the effect of reduced GAS-induced inflammatory signaling in MyD88–/– BMDMs, we determined the amounts of secreted TNFα and IL-6 in supernatants of GAS-infected MyD88–/– and control WT cells. In parallel, we also measured TNFα and IL-6 production by infected TLR2/4/9 triple-deficient cells. Infection of WT BMDMs with GAS resulted in a robust cytokine production that was only slightly reduced (by less than 20%) in TLR2/4/9 triple-deficient cells (Fig. 1F). In marked contrast, TNFα and IL-6 production was completely abolished in GAS-infected MyD88–/– BMDMs (Fig. 1E). These data show that GAS-induced proinflammatory cytokine production is absolutely dependent on MyD88. The slight but reproducible reduction of cytokine production by TLR2/4/9 triple-deficient BMDMs indicates that one of these TLRs or a combination of them plays a minor role in GAS-induced proinflammatory cytokine production. However, the largest part of GAS-induced inflammatory cytokine production is mediated by a receptor (or receptors) different from the most common bacteria-specific TLRs. Importantly, this receptor signals via MyD88. The only other known pathways that use the adaptor MyD88 for signaling are the IL-1/IL-18 pathways (32Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar). Although IL-18 is most relevant for the activation and IFN-γ production by Th1 cells (46Wei X.Q. Leung B.P. Niedbala W. Piedrafita D. Feng G.J. Sweet M. Dobbie L. Smith A.J. Liew F.Y. J. Immunol. 1999; 163: 2821-2828PubMed Google Scholar), IL-1 has been reported be released by innate immune cells upon infection with various pathogens that are sensed in the cytoplasm by components of the inflammasome (47Gavrilin M.A. Bouakl I.J. Knatz N.L. Duncan M.D. Hall M.W. Gunn J.S. Wewers M.D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 141-146Crossref PubMed Scopus (165) Google Scholar, 48Mariathasan S. Weiss D.S. Newton K. McBride J. O'Rourke K. Roose-Girma M. Lee W.P. Weinrauch Y. Monack D.M. Dixit V.M. 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GAS Induces Type I Interferon—Gram-negative and intracellular bacteria are known to elicit MyD88-independent signaling that causes activation of the transcription factor IRF3 and subsequent transcription of the type I IFN-β. The mechanisms of IFN production induced by these two types of bacteria differ upstream of IRF3. Whereas Gram-negative bacteria use TLR4 to stimulate IFN production, intracellular bacteria are recognized by a still unknown cytoplasmic receptor. Engagement of TLR9 can also trigger IRF3/IRF7-mediated type I IFN expression. GAS has been previously reported to induce IFN production in primary human macrophages (51Miettinen M. Lehtonen A. Julkunen I. Matikainen S. J. Immunol. 2000; 164: 3733-3740Crossref PubMed Scopus (184) Google Scholar). We asked whether GAS can exert similar effects in mouse macrophages and what the role of MyD88 or the bacteria-specific TLRs therein is. First, tyrosine phosphorylation of the crucial IFN-activated transcription factor Stat1 was analyzed. Tyrosine phosphorylation of Stat1 is a good indicator for autocrine IFN-β production, since this cytokine is the first IFN to be synthetized upon challenge of macrophages with bacteria or their products (52Gao J.J. Filla M.B. Fultz M.J. Vogel S.N. Russell S.W. Murphy W.J. J. Immunol. 1998; 161: 4803-4810PubMed Google Scholar, 53Toshchakov V. Jones B.W. Perera P.Y. Thomas K. Cody M.J. Zhang S. Williams B.R. Major J. Hamilton T.A. Fenton M.J. Vogel S.N. Nat. Immunol. 2002; 3: 392-398Crossref PubMed Scopus (682) Google Scholar, 54Decker T. Muller M. Stockinger S. Nat. Rev. 2005; 5: 675-687Crossref Scopus (843) Google Scholar). Infection of BMDMs with GAS revealed induction of Stat1 tyrosine phosphorylation after 4 h of infection of WT BMDMs (Fig. 3, A and B). This late tyrosine phosphorylation is similar to challenges with other bacteria (e.g. Listeria monocytogenes) that do not induce IFN synthesis through direct engagement of an IFN-inducing TLR (23Stockinger S. Materna T. Stoiber D. Bayr L. Steinborn R. Kolbe T. Unger H. Chakraborty T. Levy D.E. Muller M. Decker T. J. Immunol. 2002; 169: 6522-6529Crossref PubMed Scopus (137) Google Scholar). Consistently, Stat1 activation proceeded independently of MyD88 and TLR2, TLR4, and TLR9 signaling, as shown by GAS infection of BMDMs from MyD88–/– (Fig. 3A), TLR2–/–, TLR4–/–, TLR9–/– (all in Fig. S1), or TLR2/4/9 triple-deficient (Fig. 3B) mice. In fact, Stat1 tyrosine phosphorylation was slightly but reproducibly increased in MyD88–/– and TLR2/4/9 triple-deficient BMDMs (Fig. 3, A and
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