Synergism of Toll-like receptor-induced interleukin-12p70 secretion by monocyte-derived dendritic cells is mediated through p38 MAPK and lowers the threshold of T-helper cell type 1 responses

Toll-like receptors (TLRs) recognise specific molecular signatures of pathogens and trigger antimicrobial defence responses. Thereby, two independent signalling pathways can be distinguished: The inflammatory signalling pathway acting via the adapter molecule MyD88, leading to the activation of nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) such as SAPK/JNK and p38 MAPK and the interferon (IFN) dependent pathway that signals via TRIF and results in the production of IFN-α/β. Several evolutionarily conserved molecular patterns are expressed by pathogens, leading to the question if concerted targeting of different TLRs may induce exaggerated immune responses by signalling via both TLR pathways. Here we report that monocyte-derived dendritic cells (MoDCs) combine and integrate signals received via the IFN-dependent pathway by engagement of TLR3 (poly I:C) and activation of TRIF with the MyD88-dependent pathway by ligation of TLR2 (PGN), TLR2/TLR6 (zymosan) and TLR5 (flagellin). The generally low IL-12p70 inducers resulted in combination of both pathways in cytokine levels similar to LPS, which acts via TLR4 and induces recruitment of MyD88/Tirap and TRIF/TRAM adapter proteins. The combination of TLR3 (poly I:C) or TLR4 (LPS) engagement with TLR8 (R848) ligation induced synergistic effects on cytokine production with a boost especially in IL-12p70 secretion. SB203580, a specific p38 MAPK inhibitor, completely blocked TLR ligand mediated IL-12p70 secretion, whereby specific inhibitors for SAPK/JNK (SP600125) and NF-κB (PDTC) only repressed partially the IL-12p70 secretion. Enhanced phosphorylation in poly I:C and R848 activated MoDCs revealed the critical contribution of p38 MAPK in synergistically induced IL-12p70 induction. Further investigation of primary and recall CD8⁺ T cell responses to the MUC_12-20 M1.2 peptide LLLLTVLTV and the influenza A virus matrix_58-66 peptide GILGFVFTL proved that synergistically activated MoDCs were superior compared with LPS or R848 alone. The results indicate that dendritic cells process, combine and integrate signals delivered by pathogens to launch effective adaptive immune responses.     

Blockade of TLR9 agonist-induced type I interferons promotes inflammatory cytokine IFN-gamma and IL-17 secretion by activated human PBMC

Type I interferons (IFN) (IFN-alpha/beta) are recognized as both inhibitors and effectors of autoimmune disease. In multiple sclerosis, IFN-beta therapy appears beneficial, in part, due to its suppression of autoimmune inflammatory Th cell responses. In contrast, in systemic lupus erythematosus (SLE) triggering of plasmacytoid DC (pDC) Toll-like receptors (TLRs) by autoimmune complexes (autoICs) results in circulating type I IFN that appear to promote disease by driving autoantigen presentation and autoantibody production. To investigate how pDC-derived type I IFN might regulate Th cells in SLE, we examined a model in which sustained pDC stimulation by autoICs is mimicked by pretreating normal human PBMC with TLR9 agonist, CpG-A. Subsequently, PBMC Th cells are activated with superantigen, and APC are activated with CD40L. The role of CpG-A/TLR9-induced type I IFN in regulating PBMC is determined by blocking with virus-derived soluble type I IFN receptor, B18R. In summary, pretreatment with either rhIFN-alpha/beta or CpG-A inhibits PBMC secretion of superantigen-induced IFN-gamma and IL-17, and CD40L-induced IL-12p70 and IL-23. B18R prevents these effects. Data indicate that CpG-A-induced type I IFN inhibit IL-12p70-dependent PBMC IFN-gamma secretion by enhancing IL-10. Our results suggest that in SLE, circulating type I IFN may potentially act to inhibit inflammatory cytokine secretion.     

The host type I interferon response to viral and bacterial infections

Type I interferons (IFN) are well studied cytokines with anti-viral and immune-modulating functions. Type I IFNs are produced following viral infections, but until recently, the mechanisms of viral recognition leading to IFN production were largely unknown. Toll like receptors (TLRs) have emerged as key transducers of type I IFN during viral infections by recognizing various viral components. Furthermore, much progress has been made in defining the signaling pathways downstream of TLRs for type I IFN production. TLR7 and TLR9 have become apparent as universally important in inducing type I IFN during infection with most viruses, particularly by plasmacytoid dendritic cells. New intracellular viral pattern recognition receptors leading to type I IFN production have been identified. Many bacteria can also induce the up-regulation of these cytokines. Interestingly, recent studies have found a detrimental effect on host cells if type Ⅰ IFN is produced during infection with the intracellular gram-positive bacterial pathogen, Listeria monocytogenes. This review will discuss the recent advances made in defining the signaling pathways leading to type I IFN production.     

Regulation of TLR7/9 signaling in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs), also known as type I interferon (IFN)-producing cells, are specialized immune cells characterized by their extraordinary capabilities of mounting rapid and massive type I IFN response to nucleic acids derived from virus, bacteria or dead cells. PDCs selectively express endosomal Toll-like receptor (TLR) 7 and TLR9, which sense viral RNA and DNA respectively. Following type I IFN and cytokine responses, pDCs differentiate into antigen presenting cells and acquire the ability to regulate T cell-mediated adaptive immunity. The functions of pDCs have been implicated not only in antiviral innate immunity but also in immune tolerance, inflammation and tumor microenvironments. In this review, we will focus on TLR7/9 signaling and their regulation by pDC-specific receptors.     

Differential effects of CpG DNA on IFN-beta induction and STAT1 activation in murine macrophages versus dendritic cells: alternatively activated STAT1 negatively regulates TLR signaling in macrophages

Classical STAT1 activation in response to TLR agonists occurs by phosphorylation of the Y701 and S727 residues through autocrine type I IFN signaling and p38 MAPK signaling, respectively. In this study, we report that the TLR9 agonist CpG DNA induced Ifn-beta mRNA, as well as downstream type I IFN-dependent genes, in a MyD88-dependent manner in mouse myeloid dendritic cells. This pathway was required for maximal TNF and IL-6 secretion, as well as expression of cell surface costimulatory molecules. By contrast, neither A- nor B-type CpG-containing oligonucleotides induced Ifn-beta in mouse bone marrow-derived macrophages (BMM) and a CpG-B oligonucleotide did not induce IFn-beta in the macrophage-like cell line, J774. In BMM, STAT1 was alternatively activated (phosphorylated on S727, but not Y701), and was retained in the cytoplasm in response to CpG DNA. CpG DNA responses were altered in BMM from STAT1(S727A) mice; Il-12p40 and Cox-2 mRNAs were more highly induced, whereas Tlr4 and Tlr9 mRNAs were more repressed. The data suggest a novel inhibitory function for cytoplasmic STAT1 in response to TLR agonists that activate p38 MAPK but do not elicit type I IFN production. Indeed, the TLR7 agonist, R837, failed to induce Ifn-beta mRNA and consequently triggered STAT1 phosphorylation on S727, but not Y701, in human monocyte-derived macrophages. The differential activation of Ifn-beta and STAT1 by CpG DNA in mouse macrophages vs dendritic cells provides a likely mechanism for their divergent roles in priming the adaptive immune response.     

Raftlin is involved in the nucleocapture complex to induce poly(I:C)-mediated TLR3 activation

The double-stranded RNA analog, poly(I:C), extracellularly activates both the endosomal Toll-like receptor (TLR) 3 and the cytoplasmic RNA helicase, melanoma differentiation-associated gene 5, leading to the production of type I interferons (IFNs) and inflammatory cytokines. The mechanism by which extracellular poly(I:C) is delivered to TLR3-positive organelles and the cytoplasm remains to be elucidated. Here, we show that the cytoplasmic lipid raft protein, Raftlin, is essential for poly(I:C) cellular uptake in human myeloid dendritic cells and epithelial cells. When Raftlin was silenced, poly(I:C) failed to enter cells and induction of IFN-β production was inhibited. In addition, cellular uptake of B-type oligodeoxynucleotide that shares its uptake receptor with poly(I:C) was suppressed in Raftlin knockdown cells. Upon poly(I:C) stimulation, Raftlin was translocated from the cytoplasm to the plasma membrane where it colocalized with poly(I:C), and thereafter moved to TLR3-positive endosomes. Thus, Raftlin cooperates with the uptake receptor to mediate cell entry of poly(I:C), which is critical for activation of TLR3.     

Oral administration of poly-gamma-glutamate induces TLR4- and dendritic cell-dependent antitumor effect

Previously, we reported that the oral administration of high molecular mass poly-γ-glutamate (γ-PGA) induced antitumor immunity but the mechanism underlying this antitumor activity was not understood. In the present study, we found that application of high molecular mass γ-PGA induced secretion of tumor necrosis factor (TNF)-α from the bone-marrow-derived macrophages of wild type (C57BL/6 and C3H/HeN) and Toll-like receptor 2 knockout (TLR2^−/−) mice, but not those of myeloid differentiation factor 88 knockout (MyD88^−/−) and TLR4-defective mice (C3H/HeJ). Production of interferon (IFN)-γ-inducible protein 10 (IP-10) in response to treatment with γ-PGA was almost abolished in C3H/HeJ mice. In contrast to LPS, γ-PGA induced productions of TNF-α and IP-10 could not be blocked by polymyxin B. Furthermore, γ-PGA-induced interleukin-12 production was also impaired in immature dendritic cells (iDCs) from MyD88^−/− and C3H/HeJ mice. Downregulation of MyD88 and TLR4 expression using small interfering RNA (siRNA) significantly inhibited γ-PGA-induced TNF-α secretion from the RAW264.7 cells. γ-PGA-mediated intracellular signaling was markedly inhibited in C3H/HeJ cells. The antitumor effect of γ-PGA was completely abrogated in C3H/HeJ mice compared with control mice (C3H/HeN) but significant antitumor effect was generated by the intratumoral administration of C3H/HeN mice-derived iDCs followed by 2,000 kDa γ-PGA in C3H/HeJ. These findings strongly suggest that the antitumor activity of γ-PGA is mediated by TLR4. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Essential role for IKKβ in production of type 1 interferons by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are characterized by their ability to produce high levels of type 1 interferons in response to ligands that activate TLR7 and TLR9, but the signaling pathways required for IFN production are incompletely understood. Here we exploit the human pDC cell line Gen2.2 and improved pharmacological inhibitors of protein kinases to address this issue. We demonstrate that ligands that activate TLR7 and TLR9 require the TAK1-IKKβ signaling pathway to induce the production of IFNβ via a pathway that is independent of the degradation of IκBα. We also show that IKKβ activity, as well as the subsequent IFNβ-stimulated activation of the JAK-STAT1/2 signaling pathway, are essential for the production of IFNα by TLR9 ligands. We further show that TLR7 ligands CL097 and R848 fail to produce significant amounts of IFNα because the activation of IKKβ is not sustained for a sufficient length of time. The TLR7/9-stimulated production of type 1 IFNs is inhibited by much lower concentrations of IKKβ inhibitors than those needed to suppress the production of NFκB-dependent proinflammatory cytokines, such as IL-6, suggesting that drugs that inhibit IKKβ may have a potential for the treatment of forms of lupus that are driven by self-RNA and self-DNA-induced activation of TLR7 and TLR9, respectively.     

T cell-independent, TLR-induced IL-12p70 production in primary human monocytes

IL-12p70 is a key cytokine for the induction of Th1 immune responses. IL-12p70 production in myeloid cells is thought to be strictly controlled by T cell help. In this work we demonstrate that primary human monocytes can produce IL-12p70 in the absence of T cell help. We show that human monocytes express TLR4 and TLR8 but lack TLR3 and TLR7 even after preincubation with type I IFN. Simultaneous stimulation of TLR4 and TLR8 induced IL-12p70 in primary human monocytes. IL-12p70 production in peripheral blood myeloid dendritic cells required combined stimulation of TLR7/8 ligands together with TLR4 or with TLR3 ligands. In the presence of T cell-derived IL-4, but not IFN-gamma, stimulation with TLR7/8 ligands was sufficient to stimulate IL-12p70 production. In monocytes, type I IFN was required but not sufficient to costimulate IL-12p70 induction by TLR8 ligation. Furthermore, TLR8 ligation inhibited LPS-induced IL-10 in monocytes, and LPS alone gained the ability to stimulate IL-12p70 in monocytes when the IL-10 receptor was blocked. Together, these results demonstrate that monocytes are licensed to synthesize IL-12p70 through type I IFN provided via the Toll/IL-1R domain-containing adaptor inducing IFN-beta pathway and the inhibition of IL-10, both provided by combined stimulation with TLR4 and TLR8 ligands, triggering a potent Th1 response before T cell help is established.     

Toll-Like Receptor Expression and Induction of Type I and Type III Interferons in Primary Airway Epithelial Cells

Interferons (IFNs) are a critical component of the first line of antiviral defense. The activation of Toll-like receptors (TLRs) expressed by dendritic cells triggers different signaling cascades that result in the production of large amounts of IFNs. However, the functional consequences of TLR activation and differential IFN production in specific cell populations other than antigen-presenting cells have not yet been fully elucidated. In this study, we investigated TLR expression and polarization in airway epithelial cells (AECs) and the consequences of TLR agonist stimulation for the production of type I (IFN-α/β) and type III (IFN-λ) IFNs. Our results show that the pattern of expression and polarization of all TLRs in primary AEC cultures mirrors that of the human airways ex vivo and is receptor specific. The antiviral TLRs (TLR3, TLR7, and TLR9) are mostly expressed on the apical cell surfaces of epithelial cells in the human trachea and in primary polarized AECs. Type III IFN is the predominant IFN produced by the airway epithelium, and TLR3 is the only TLR that mediates IFN production by AECs, while all TLR agonists tested are capable of inducing AEC activation and interleukin-8 production. In response to influenza virus infection, AECs can produce IFN-λ in an IFNAR- and STAT1-independent manner. Our results emphasize the importance of using primary well-differentiated AECs to study TLR and antiviral responses and provide further insight into the regulation of IFN production during the antiviral response of the lung epithelium.     

Type I IFN Triggers RIG-I/TLR3/NLRP3-dependent Inflammasome Activation in Influenza A Virus Infected Cells

Influenza A virus (IAV) triggers a contagious and potentially lethal respiratory disease. A protective IL-1β response is mediated by innate receptors in macrophages and lung epithelial cells. NLRP3 is crucial in macrophages; however, which sensors elicit IL-1β secretion in lung epithelial cells remains undetermined. Here, we describe for the first time the relative roles of the host innate receptors RIG-I (DDX58), TLR3, and NLRP3 in the IL-1β response to IAV in primary lung epithelial cells. To activate IL-1β secretion, these cells employ partially redundant recognition mechanisms that differ from those described in macrophages. RIG-I had the strongest effect through a MAVS/TRIM25/Riplet–dependent type I IFN signaling pathway upstream of TLR3 and NLRP3. Notably, RIG-I also activated the inflammasome through interaction with caspase 1 and ASC in primary lung epithelial cells. Thus, NS1, an influenza virulence factor that inhibits the RIG-I/type I IFN pathway, strongly modulated the IL-1β response in lung epithelial cells and in ferrets. The NS1 protein derived from a highly pathogenic strain resulted in increased interaction with RIG-I and inhibited type I IFN and IL-1β responses compared to the least pathogenic virus strains. These findings demonstrate that in IAV-infected lung epithelial cells RIG-I activates the inflammasome both directly and through a type I IFN positive feedback loop.     

HIV and HCV Activate the Inflammasome in Monocytes and Macrophages via Endosomal Toll-Like Receptors without Induction of Type 1 Interferon

Innate immune sensing of viral infection results in type I interferon (IFN) production and inflammasome activation. Type I IFNs, primarily IFN-α and IFN-β, are produced by all cell types upon virus infection and promote an antiviral state in surrounding cells by inducing the expression of IFN-stimulated genes. Type I IFN production is mediated by Toll-like receptor (TLR) 3 in HCV infected hepatocytes. Type I IFNs are also produced by plasmacytoid dendritic cells (pDC) after sensing of HIV and HCV through TLR7 in the absence of productive pDC infection. Inflammasomes are multi-protein cytosolic complexes that integrate several pathogen-triggered signaling cascades ultimately leading to caspase-1 activation and generation pro-inflammatory cytokines including interleukin (IL)-18 and IL-1β. Here, we demonstrate that HIV and HCV activate the inflammasome, but not Type I IFN production, in monocytes and macrophages in an infection-independent process that requires clathrin-mediated endocytosis and recognition of the virus by distinct endosomal TLRs. Knockdown of each endosomal TLR in primary monocytes by RNA interference reveals that inflammasome activation in these cells results from HIV sensing by TLR8 and HCV recognition by TLR7. Despite its critical role in type I IFN production by pDCs stimulated with HIV, TLR7 is not required for inflammasome activation by HIV. Similarly, HCV activation of the inflammasome in monocytes does not require TLR3 or its downstream signaling adaptor TICAM-1, while this pathway leads to type I IFN in infected hepatocytes. Monocytes and macrophages do not produce type I IFN upon TLR8 or TLR7 sensing of HIV or HCV, respectively. These findings reveal a novel infection-independent mechanism for chronic viral induction of key anti-viral programs and demonstrate distinct TLR utilization by different cell types for activation of the type I IFN vs. inflammasome pathways of inflammation.     

Fever range temperature promotes TLR4 expression and signaling in dendritic cells

Fever improves survival and shortens disease duration in microbial infections. However, the mechanisms of these beneficial responses still remain elusive. Toll-like receptors (TLRs) play important roles in sensing microbes invading and therefore we hypothesized that fever range temperature may enhance responsiveness of dendritic cells (DCs) to lipopolysaccharide (LPS) by promoting TLR4 expression and signaling. In this study, we found that pretreatment of DCs with 39.5 degrees C temperature can up-regulate TLR4 expression in DCs and enhances LPS-induced DC production of interleukins (IL) IL-6, IL-10 and IL-12 but not tumor necrosis factor alpha (TNF-alpha). Blockade of the autocrine action of IL-10 could increase LPS-induced TNF-alpha and IL-12 production in DCs. Further experiments confirmed that TLR4 ligation activates extracellular signal-regulated kinase (ERK), p38, and nuclear factor-kappaB pathways more potently in DCs pretreated with 39.5 degrees C. We conclude that fever range temperature can promote TLR4 expression and signaling in DCs, leading to enhancement of immune responses to inflammatory stimuli. These results might reveal a possible mechanistic explanation for the significance of fever in activating innate immune responses.     

ITAM-coupled receptors inhibit IFNAR signaling and alter macrophage responses to TLR4 and Listeria monocytogenes

ITAM-coupled receptors play an essential role in regulating macrophage activation and function by cross-regulating signaling from heterologous receptors. We investigated mechanisms by which ITAM-associated receptors inhibit type I IFN (IFN-α/β) signaling in primary human macrophages and tested the effects of simultaneous ligation of ITAM-associated receptors and TLR4 on TLR4-induced Jak-STAT signaling that is mediated by autocrine IFN-β. Preligation of ITAM-coupled β2 integrins and FcγRs inhibited proximal signaling by the type I IFN receptor IFNAR. Cross-inhibition of IFNAR signaling by β2 integrins resulted in decreased Jak1 activation and was mediated by partial downregulation of the IFNAR1 subunit and MAPK-dependent induction of USP18, which blocks the association of Jak1 with IFNAR2. Simultaneous engagement of ITAM-coupled β2 integrins or Dectin-1 with TLR4 did not affect TLR4-induced direct activation of inflammatory target genes such as TNF or IL6 but abrogated subsequent induction of IFN response genes that is mediated by autocrine IFN-β signaling. Type I IFNs promote macrophage death postinfection by Listeria monocytogenes. Consequently, attenuation of IFN responses by β2 integrins protected primary human macrophages from L. monocytogenes-induced apoptosis. These results provide a mechanism for cross-inhibition of type I IFN signaling by ITAM-coupled β2 integrins and demonstrate that ITAM signaling qualitatively modulates macrophage responses to pathogen-associated molecular patterns and pathogens by selectively suppressing IFN responses.     

Erythromycin differentially inhibits lipopolysaccharide- or poly(I:C)-induced but not peptidoglycan-induced activation of human monocyte-derived dendritic cells

Erythromycin (EM) has attracted attention because of its anti-inflammatory effect. Because dendritic cells (DCs) are the most potent APCs involved in numerous pathologic processes including innate immunity, we examined effects of EM on the activation of human DCs by pathogen-derived stimuli. Monocyte-derived DCs were pretreated with EM and subsequently stimulated with peptidoglycan, polyriboinosinic:polyribocytidylic acid (poly(I:C)), or LPS. The activation of DCs was assessed by surface molecule expression and cytokine production. To reveal the signaling pathways affected by EM, TLR expression, NF-kappaB, IFN regulatory factor-3, and AP-1 activation were examined. EM inhibited costimulatory molecule expression and cytokine production that was induced by poly(I:C) and LPS but not by peptidoglycan. EM pretreatment down- and up-regulated mRNA levels of TLR3 and TLR2, respectively, but did not affect that of TLR4. EM suppressed IFN regulatory factor-3 activation and IFN-beta production but not AP-1 activation induced by poly(I:C) and LPS. The inhibitory effect of EM on NF-kappaB activation was observed only in poly(I:C)-stimulated DCs. EM selectively suppressed activation of DCs induced by LPS and poly(I:C) in different ways, suggesting that the immuno-modulating effects of EM depend on the nature of pathogens. These results might explain why EM prevents the virus-induced exacerbation in the chronic inflammatory respiratory diseases and give us the clue to design new drugs to treat these diseases.     

Toll-like receptors 2 and 4 regulate the frequency of IFNγ-producing CD4+ T-cells during pulmonary infection with Chlamydia pneumoniae

TLR2 and TLR4 are crucial for recognition of Chlamydia pneumoniae in vivo, since infected TLR2/4 double-deficient mice are unable to control the infection as evidenced by severe loss of body weight and progressive lethal pneumonia. Unexpectedly, these mice display higher pulmonary levels of the protective cytokine IFNγ than wild type mice. We show here, that antigen-specific CD4(+) T-cells are responsible for the observed IFNγ-secretion in vivo and their frequency is higher in TLR2/4 double-deficient than in wild type mice. The capacity of TLR2/4 double-deficient dendritic cells to re-stimulate CD4(+) T-cells did not differ from wild type dendritic cells. However, the frequency of CD4(+)CD25(+)Foxp3(+) T-cells was considerably higher in wild type compared to TLR2/4 double-deficient mice and was inversely related to the number of IFNγ-secreting CD4(+) effector T-cells. Despite increased IFNγ-levels, at least one IFNγ-mediated response, protective NO-secretion, could not be induced in the absence of TLR2 and 4. In summary, CD4(+)CD25(+)Foxp3(+) regulatory T-cells fail to expand in the absence of TLR2 and TLR4 during pulmonary infection with C. pneumoniae, which in turn enhances the frequency of CD4(+)IFNγ(+) effector T-cells. Failure of IFNγ to induce NO in TLR2/4 double-deficient cells represents one possible mechanism why TLR2/4 double-deficient mice are unable to control pneumonia caused by C. pneumoniae and succumb to the infection.     

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.     

Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9

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-kappaB, 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.