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International Reviews of Immunology, 30:16–34, 2011 C Informa Healthcare USA, Inc. Copyright ISSN: 0883-0185 print / 1563-5244 online DOI: 10.3109/08830185.2010.529976 Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. Pathogen Recognition by the Innate Immune System Himanshu Kumar,1 Taro Kawai,2 and Shizuo Akira2 1 Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka, Japan, and Laboratory of Immunology, Indian Institute of Science Education and Research (IISER), Bhopal, India; 2 Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka, Japan, and Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Microbial infection initiates complex interactions between the pathogen and the host. Pathogens express several signature molecules, known as pathogen-associated molecular patterns (PAMPs), which are essential for survival and pathogenicity. PAMPs are sensed by evolutionarily conserved, germline-encoded host sensors known as pathogen recognition receptors (PRRs). Recognition of PAMPs by PRRs rapidly triggers an array of anti-microbial immune responses through the induction of various inflammatory cytokines, chemokines and type I interferons. These responses also initiate the development of pathogen-specific, long-lasting adaptive immunity through B and T lymphocytes. Several families of PRRs, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and DNA receptors (cytosolic sensors for DNA), are known to play a crucial role in host defense. In this review, we comprehensively review the recent progress in the field of PAMP recognition by PRRs and the signaling pathways activated by PRRs. Keywords Innate immunity, Toll-like receptors, RIG-I-like receptors, NOD-like receptors INTRODUCTION Invasion of a host by pathogenic infectious agents triggers a battery of immune responses through interactions between a diverse array of pathogen-borne virulence factors and the immune surveillance mechanisms of the host. Host–pathogen interactions are generally initiated via host recognition of conserved molecular structures known as pathogen-associated molecular patterns (PAMPs) [1] that are essential for the life-cycle of the pathogen. However, these PAMPs are either absent or compartmentalized inside the host cell, and are sensed by the host’s germline encoded pattern recognition receptors (PRRs), which are expressed on innate immune cells such as dendritic cells, macrophages and neutrophils [2–5]. Effective sensing of PAMPs rapidly induces host immune responses via the activation of complex signaling pathways that culminate in the induction of inflammatory responses mediated by various cytokines and chemokines, which subsequently facilitate the eradication of the pathogen [2–6]. The innate immune system is the primary, or early, barrier to infectious agents and acts immediately. Furthermore, the innate immune system also mounts an effective defense against infectious agents through the initiation of adaptive immunity, which Address correspondence to Himanshu Kumar, Laboratory of Immunology, Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Bhopal, Transit campus: ITI (Gas Rahat) building, Govindpura, Bhopal 460 023, India. E-mail: [email protected] Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. Pathogen Recognition by Innate Immunity is long-lasting and has immunological memory. Adaptive immunity is mediated via the generation of pathogen (antigen)-specific B and T lymphocytes through a process of gene rearrangement [7, 8]. To date, several classes of PRRs, such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and DNA receptors (cytosolic sensors for DNA), have been discovered and characterized. These PRRs are at the forefront of both extracellular and intracellular pathogen recognition and sense various classes of molecules including proteins, lipids, carbohydrates and nucleic acids [2–6]. In this review, we discuss recent progress in our understanding of PAMP recognition and signaling through the various PRRs. TLRs TLRs are the most widely studied PRRs and are considered to be the primary sensors of pathogens. The field of TLR immunobiology expanded rapidly after the discovery of toll proteins in flies [9]. In humans, 10 TLR family members have been identified (there are 12 in mice). TLR1 to 9 are conserved in both humans and mice. TLR10 is expressed in humans but not in mice (because of a stop codon in the murine TLR10 gene), whereas TLR11 is expressed in mice, but not in humans. TLR10 in humans and TLR12 and TLR13 in mice are not well characterized and their function remains unclear [2]. TLR1, 2, 4, 5 and 6 are primarily expressed on the cell surface and recognize PAMPs derived from bacteria, fungi and protozoa, whereas TLR3, 7, 8 and 9 are exclusively expressed within endocytic compartments and primarily recognize nucleic acid PAMPs derived from various viruses and bacteria [2, 3, 6]. TLRs are type I membrane glycoproteins and consist of extracellular leucine rich repeats (LRRs) that are required for PAMP recognition, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain, required for downstream signaling. The crystal structure of the extracellular recognition domain of several TLRs bound to their agonist or antagonist PAMPs has been characterized. TLRs have a unique horseshoe, or “m” shaped architecture [10, 11]. TLR1 and TLR2 form stable heterodimers and the addition of Pam3CSK4 (a ligand for TLR1TLR2) activates downstream signaling. The TLR1-TLR2-Pam3CSK4 crystal shows that two of the three hydrophobic lipid chains on Pam3CSK4 are submerged in the hydrophobic spaces within TLR2, with the remaining hydrophobic lipid chain penetrating into the hydrophobic space within TLR1 [12]. The crystal structure of TLR4 bound to Lipid A (an agonist PAMP) or Eritoran (an antagonist PAMP) has also been reported. Lipid A is composed of phosphorylated diglucosamine and six acyl chains, whereas Eritoran consists of phosphorylated diglucosamine and four acyl chains. Crystal structure studies suggest that five acyl chains of lipid A and four acyl chains of Eritoran are submerged within the hydrophobic space of MD2 (myeloid differentiation factor-2), a co-receptor for TLR4, and the remaining acyl chain of lipid A is submerged within the hydrophobic space of TLR4. This facilitates the interaction between MD2 and TLR4 that is required for downstream signaling, which is not the case for Eritoran. Therefore, lipid A activates the TLR4 signaling pathway, but Eritoran does not [13, 14]. Furthermore, co-crystallization of TLR3 with dsRNA shows that the recognition domain of TLR3 has a horseshoe-shaped solenoid structure and that dsRNA binds to the lateral convex surface of the TLR3 ectodomain [15]. PAMPs RECOGNIZED BY TLRs Rapid progress has been made not only in our understanding of the structure of TLRs but also in revealing the complexity of TLR-mediated signaling and in the identification of PAMPs derived from microbial pathogens such as mycobacteria, bacteria, viruses, fungi and parasites. The PAMPs recognized by the various TLRs are shown in C Informa Healthcare USA, Inc. Copyright H. Kumar et al. Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. TABLE I TLR Ligands and Cellular Location TLR and (co-receptors) Cellular localization TLR1/2 TLR2 (Dectin-1, C-type lectin) Cell surface Cell surface TLR3 Endosome TLR4 (MD2, CD14, LBP) Cell surface TLR5 TLR6/2 (CD36) TLR7 Cell surface Cell surface Endolysosome TLR8 (only in human) TLR9 Endolysosome TLR11 (only in mouse) Cell surface Endolysosome TLR ligands Triacyl lipopeptides Peptidoglycan, lipoarabinomannan, hemagglutinin, phospholipomannan, glycosylphosphophatidyl inositol mucin, zymosan ssRNA virus, dsRNA virus, respiratory syncytial virus, murine cytomegalovirus Lipopolysaccride, mannan, glycoinositolphospholipids, envelope and fusion proteins from mammary tumor virus and respiratory syncytial virus, respectively, endogenous oxidized phospholipids produced after H5N1 avian influenza virus infection, pneumolysin from streptococcus pneumonia, paclitaxel. Flagellin from flagellated bacteria Diacyl lipopeptides from mycoplasma), lipoteichoic acid ssRNA viruses, purine analog compounds (imidazoquinolines). RNA from bacteria from group B streptococcus ssRNA from RNA virus, purine analog compounds (imidazoquinolines). dsDNA viruses herpes simplex virus and murine cytomegalovirus, CpG motifs from bacteria and viruses, hemozoin malaria parasite Uropathogenic bacteria, profillin-like molecule from Toxoplasma gondii Table I and have been discussed in great detail in several recent reviews [2, 3, 6]. Several of the bacterial, viral, fungal and parasite PAMPs sensed by TLRs are described below. BACTERIAL PAMPs SENSED BY TLRs Of the TLRs, TLR1, 2, 4, 5, 6, 7, and 9 are primarily dedicated to the recognition of various bacterial components. LPS is a major cell wall component of gram-negative bacteria and is primarily sensed by TLR4 complexed with another molecule known as MD2 [16, 17]. Another essential major component of gram-positive bacteria is peptidoglycan, which is sensed by TLR2 [18]. Mycobacteria, another class of bacteria rich in lipoarabinomannan (LAM), are also sensed by TLR2 [19]. TLR2 (in conjugation with TLR1 or TLR6) senses diacyl or triacyl lipopeptides on bacteria, mycobacteria and mycoplasma [6, 20]. TLR5 and TLR9 sense the flagellin protein expressed by flagellated bacteria and bacterial/viral genomic DNA rich in unmethylated CpG, respectively [6, 20].Group B streptococci, which reside in the phagosome, are recognized by TLR7 [21]. Also, bacterial RNA produced in the lysosomal compartment is likely to act as a PAMP for TLR7 [21]. Recognition of PAMPs by TLR1, TLR2, TLR4, TLR5 and TLR6 primarily induces the production of inflammatory cytokines, whereas TLR7 and TLR9 induce type I interferons. VIRAL PAMPs SENSED BY TLRs Nucleic acids (single stranded (ss)/double stranded (ds) RNA or ss/dsDNA) derived from viruses are recognized by several TLRs. DNA from herpes simplex virus (HSV), murine cytomegalovirus (MCMV), as well as CpG motifs containing synthetic oligonucleotides that contain unmethylated CpG DNA, are sensed by TLR9, which induces the production of type I interferons, most likely through plasmacytoid dendritic cells International Reviews of Immunology Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. Pathogen Recognition by Innate Immunity (pDCs) [2, 3, 6, 20]. RNA from RNA viruses is sensed by TLR7 and TLR8 (the function of murine TLR8 is not known). In addition, several synthetic antiviral compounds, such as R848, imiquimod and loxoribine, are also sensed by TLR7 and TLR8. Furthermore, the synthetic analog of dsRNA, known as poly IC, is sensed by TLR3 which also activate adaptive immunity when used as an vaccine adjuvant [22–24]. Another PAMP derived from viruses is a coat protein. The coat proteins of respiratory syncytial virus (RSV) and mouse mammary tumor virus (MMTV) are sensed by TLR4. However, in vivo studies show that TLR2 and TLR6 play an essential role in controlling RSV infection. The coat proteins of other viruses, such as the Measles virus hemagglutinin protein, are also sensed by TLR2, as is Vaccinia virus. Notably, this recognition induces the production of type I interferons by inflammatory monocytes [2, 3, 6, 20]. FUNGAL PAMPs SENSED BY TLRs Several fungi, such as Candida albicans and Aspergillus fumigatus, are sensed by several TLRs and induce inflammatory responses. However, this recognition requires additional receptors such as dectins, CD14, mannose receptors, and DC-SIGN. βglucans are the primary component of the majority of fungal cell walls, including those of baker’s yeast and some pathogenic fungi such as Candida albicans. These β-glucans are recognized by TLR2 in association with dectin-1. Glucuronoxylomannans, another fungal component sensed by CD14 and TLR4, also induce inflammatory responses [2, 3, 6, 20, 25]. PROTOZOAL PAMPs SENSED BY TLRs Protozoal infections are a serious problem in developing countries and cause diseases such as Toxoplasmosis (Toxoplasma gondii), malaria (Plasmodium species), leishmaniasis (Leishmania species), and sleeping sickness (Trypanosoma brucei). Unsaturated alkylacylglycerol and lipophosphoglycan (LPG) from Trypanosoma species and Leishmania species, respectively, are recognized by TLR2, and glycoinositolphospholipids and glycosylphosphatidylinositol anchors from Trypanosoma species, P. falciparum and T. gondii are recognized by both TLR2 and TLR4. The profilin-like protein of T. gondii is sensed by murine TLR11. The genomic DNA and hematin crystals of Trypanosoma and Plasmodium species, respectively, are sensed by TLR9 [2, 3, 6, 20]. TLR SIGNALING TLR signaling is primarily meditated via the recruitment of different TIR domaincontaining adaptor molecules such as MyD88, TRIF (TICAM-1), TIRAP (Mal), and TRAM to the TIR domains of the different TLRs [2, 3, 6, 20]. Recruitment of these adaptor molecules activates various transcription factors such as NF-κB, IRF3/7, and MAP kinases to induce the production of pro-inflammatory cytokines and type I interferons (Fig. 1). All TLRs, except for TLR3, recruit MyD88 and initiate MyD88-dependent signaling to activate NF-κB and MAP kinases to induce proinflammatory cytokines in macrophages and cDCs. In addition to MyD88, TLR1, TLR2, TLR4 and TLR6 recruit TIRAP to initiate MyD88-dependent signaling. TLR3 and TLR4 recruit TRIF and initiate TRIF-dependent signaling to activate NF-κB and IRF3 to induce production of pro-inflammatory cytokines and type I interferons. TLR4 recruits TRIF via an additional adaptor molecule, TRAM. TLR4 activates both MyD88- and TRIF-dependent signaling via recruitment of all four adaptors. TLR4 first recruits TIRAP, which facilitates the recruitment of MyD88 to initiate the first phase of NF-κB and MAPK C Informa Healthcare USA, Inc. Copyright Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. H. Kumar et al. FIGURE 1 PRRs-mediated signaling. Toll-like receptor (TLR) signaling. Recognition of PAMPs by plasma membrane-localized TLRs, such as TLR4, TLR5, TLR11, and TLR2 (TLR2 forms a heterodimer with TLR1 or TLR6 to form a functional receptor complex) and endosomal-localized TLRs, such as TLR3, TLR7, and TLR9, activates TLR signaling pathways. Endosomal-localized TLRs are transported to the endosomal compartment via the ER-localized protein, UNC93B. All TLRs, except TLR3, recruit MyD88 and activate MyD88-dependent signaling. TLR1, 2, 4, and 6 recruit the additional adaptor molecule, TIRAP, for the recruitment of MyD88. TLR3 recruits TRIF and activates TRIF-dependent signaling. TLR4 also activates TRIF-dependent signaling through an additional adaptor molecule, TRAM. In cDCs, MyD88-dependent signaling is initiated through the recruitment and activation of various signaling molecules, such as IRAK family proteins, TRAF6, and TAK1 which, in turn, activate the IKK complex. The active IKK complex activates NF-κB subunits to initiate the transcription of inflammatory cytokine genes. In pDCs, the TLR7 and TLR9-mediated signaling pathways activate NF-κB via an MyD88-dependent signaling pathway in the same manner as for cDCs. In addition, stimulation with TLR7 and TLR9 ligands induces MyD88-dependent type I interferon production through a direct interaction between MyD88 and IRF7 via IRAK family proteins and phosphorylated IRF7. Phosphorylated IRF7 translocates to the nucleus and initiates the transcription of type I interferons. cDCs stimulated with TLR3 PAMPs activate the TRIF-dependent signaling pathway through recruitment of TRIF to induce transcription of inflammatory cytokines and type I interferons through the IKK complex and TBK1/IKKi, respectively, via the activation of NF-κB and IRF3/IRF7. RIG-I-like receptor (RLR) signaling. Recognition of PAMPs by cytosolic sensors, such as RIG-I and Mda5, activates signaling through the mitochondria-localized adaptor protein IPS-1 leading to the activation of NF-κB and IRF3/IRF7 through the IKK complex and TBK1/IKKi, respectively, which results in the production of inflammatory cytokines and type I interferons. LGP2, another member of the RLR family, potentiates the RIG-I- and MDA5-mediated signaling pathways. Cytosolic DNA sensor-dependent signaling. PAMPs in the cytoplasm of cells are sensed by cytosolic DNA sensors, or DAI, which activate NF-κB and IRF3/IRF7 via the IKK complex and TBK1/IKKi, respectively, and the ER-localized protein, STING. Recognition of DNA by AIM2 also induces the maturation of proIL-1β to IL-1β through an inflammasome complex consisting of ASC and caspase-1. Nod-like receptor (NLR) signaling. Recognition of PAMPs by NOD1 and NOD2 initiates the recruitment of RICK, which activates NF-κB via the IKK complex. Another member of the NLR family constitutes the inflammasome. The inflammasome is a multi-protein complex required for the maturation or activation of pro-IL-1 family cytokine to its bioactive IL-1 family cytokine. Activation of the inflammasome requires two steps. First, NF-κB-dependent up-regulation of the pro-forms of the cytokine and, second, conversion of the inactive form of the cytokine to a bioactive form by the inflammasome. International Reviews of Immunology Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. Pathogen Recognition by Innate Immunity activation. For TRIF-dependent signaling, TLR4 is trafficked to the endosome via dynamin-dependent endocytosis and forms a complex with TRAM and TRIF. This complex initiates the TRIF-dependent signaling required for IRF3 activation, which induces type I interferon and this signaling pathway activates second-phase of NFκB and MAPK activation for the induction of inflammatory cytokines [26]. In cDCs, UNC93B1 (a protein localized to the endoplasmic reticulum (ER)) plays a critical role in the transportation of endosome-localized TLRs, such as TLR3, TLR7, and TLR9. Mice with a mutation in this protein show complete abrogation of all cytokine production after stimulation with their respective PAMPs [27–29]. MyD88-DEPENDENT SIGNALING PATHWAYS Stimulation of macrophages and DCs with TLR PAMPs initiates MyD88-dependent signaling via recruitment of IRAK-family signaling proteins (Fig. 1) [2, 3, 6, 20]. First, IRAK4 is activated and recruited by MyD88. Then, IRAK1 and IRAK2 are sequentially activated and recruited to form an active signaling complex which interacts with TRAF6 (an E3 ligase required for Lys63 (K63)-linked poly-ubiquitination). Recently, the crystal structure of the MyD88-IRAK4-IRAK2 complex was reported and shows a left-handed helical oligomeric signaling complex, which consists of six, four, and four molecules of MyD88, IRAK4, and the IRAK2 death domain, respectively [30]. MyD88 then recruits IRAK4 and this complex recruits the IRAK4 substrates, IRAK2 or IRAK1. Formation of this signaling complex facilitates the phosphorylation of all these kinases, resulting in the activation of downstream signaling molecules. TRAF6, along with E2 ubiquitin-conjugating enzymes, such as Ubc13 and Uev1A, ubiquitinates both itself and IRAK1 to activate TAK1 [31]. Activated TAK1 then activates NF-κB and MAP kinases to initiate the transcription and translation of various proinflammatory cytokines, chemokines, interferons, and other TLR-inducible genes. TLR4-mediated signaling also recruits TRAF3 to the MyD88 multiprotein complex, where it undergoes K48-linked ubiquitination and degradation. This results in TAK1 activation and subsequent induction of inflammatory cytokines [32]. Of the TLR inducible-genes, several (IkBz, IkB-NS, ATF3, C/EBP, and the zincfinger proteins Zc3h12a and tristetraprolin) have been characterized [33–38]. IκBζ and C/EBPd positively regulate the expression of IL-6 and IL-12p40. In contrast, IκBNS and ATF3 negatively regulate NF-κB-driven proinflammatory cytokines, such as IL-6, IL-12p40, and TNFα. The zinc finger protein, Zc3h12a, consists of a CCCH-type zinc-finger domain and an RNase domain and is involved in IL-6 mRNA and IL-12p40 mRNA degradation. Stimulation with TLR PAMPs induces large amounts of IL-6 and IL-12p40 in macrophages lacking Zc3h12a compared with wild-type cells. Furthermore, mice lacking Zc3h112a show elevated serum immunoglobulins and autoantibody production. Another protein, tristetraprolin, which regulates the degradation of TNFα mRNA by removing the poly(A) tail, plays a critical role in the development of autoimmune arthritis. In pDCs, MyD88 is required for the production of both proinflammatory cytokines and type I interferons. In pDCs stimulated with TLR7 and TLR9 PAMPs, MyD88 recruits various signaling proteins, such as IRAK4, TRAF6, TRAF3, IRAK1, and IKKa, which phosphorylate IRF7 to initiate the transcription of type I interferons. Other proteins, such as osteopontin (a protein induced after stimulation with TLR9), phosphoinositol 3 kinase (PI3K), mTOR (downstream of PI3K), and p70S6K, also play critical roles in IRF7 activation [2, 3, 6, 20]. pDCs and cDCs stimulated with CpG (a TLR9 PAMP) are transported to the endolysosome, where TLR9 is cleaved by cathepsins B, K, and L and an asparagine endopeptidase. This cleaved form triggers TLR9-mediated induction of proinflammatory cytokines and type I interferons. Stimulation with TLR7 PAMPs also requires C Informa Healthcare USA, Inc. Copyright H. Kumar et al. acidification of the endolysosomal compartment; however, it remains unclear whether TLR7 needs to be cleaved for PAMP recognition [39–43]. Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. TRIF-DEPENDENT SIGNALING PATHWAYS Macrophages and DCs stimulated with TLR3 and TLR4 PAMPs trigger TRIFdependent signaling, which leads to the production of proinflammatory cytokines and type I interferons via activation of NF-κB, MAP kinases and IRF3 (Fig. 1) [2, 3, 6, 20]. TRIF-dependent signaling is initiated through the recruitment of TRAF6 and RIP1 to the distinct domain of TRIF. The interaction of TRAF6 with TRIF (N-terminal TRAF-binding domain) activates TAK1 via mechanisms similar to those in the MyD88dependent pathway. However, TRIF interacts with RIP1 and undergoes K63-linked polyubiquitination. RIP1 also interacts with TRADD, and this multiprotein complex is required for NF-κB activation. TRIF recruits non-canonical IKKs, such as TBK1 and IKKi (IKK), via TRAF3 to phosphorylate IFR3. Phosphorylated IFR3 translocates to the nucleus and initiates the transcription of type I interferons. TRAF3 plays a critical role in the regulation of both MyD88-dependent and TRIF-dependent signaling because TRAF3 degradation via K48-linked ubiquitination activates MyD88-dependent signaling and suppresses TRIF-dependent signaling (and vice versa). Furthermore, NRDP1, a RING-containing E3 ligase, regulates both MyD88-dependent and TRIFdependent signaling via its interaction with TBK1. K63-linked ubiquitination activates TBK1 and suppresses the MyD88-mediated pathway. This suggests that cells have mechanisms for regulating both signaling pathways to avoid injurious effects due to overproduction of inflammatory cytokines during microbial infection. RIG-I-LIKE RECEPTORS The RLR family consists of three members, namely, RIG-I, MDA5, and LGP2. These sensors recognize the RNA from RNA viruses in the cytoplasm of infected cells and induce inflammatory cytokines and type I interferons. Inflammatory cytokines primarily initiate and co-ordinate various innate immune responses through recruitment of professional immune cells such as macrophages and dendritic cells [3, 6, 44]. Type I interferons consist of several structurally related IFN-α proteins and a single IFN-β protein, which can bind directly to infected cells in an autocrine or paracrine manner through a common receptor and initiate the transcription of several interferon-stimulated genes (ISGs). Type I interferons, together with ISGs, induce an antiviral state in all infected and healthy cells by altering various cellular processes. This inhibits viral replication, induces apoptosis in infected cells, increases the lytic capacity of natural killer cells, up-regulates the expression of MHC class I molecules and activates various components of the adaptive immune response. STRUCTURE OF RLRs RIG-I and MDA5 contain N-terminal tandem CARDs that are essential for downstream signaling. LGP2 lacks the CARD domain, suggesting a negative regulatory role for LGP2 in RIG-I- and MDA5-mediated signaling [3, 6, 44, 45]. However, genetic studies show that LGP2 acts as a positive regulator of RIG-I- and MDA5-mediated signaling [46]. RIG-I contains a repressor domain (RD), which is required for the regulation of RIG-I-dependent downstream signaling. All three members contain an intermediate DExD/H-box RNA helicase domain, which is required for ligand recognition or binding. This domain also contains ATPase activity and mice lacking ATPase activity (due to a point mutation) in the LGP2 molecule do not produce type I interferons upon International Reviews of Immunology Pathogen Recognition by Innate Immunity Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. TABLE II RLR Ligands and Cellular Location RLRs Cellular localization RIG-I Cytoplasm MDA5 Cytoplasm RIG-I and MDA5 Cytoplasm Ligand and RNA viruses recognized by sensors Short double-stranded RNA (up to 1 kb) with triphosphate or monophosphate at 5 end; short length of poly IC, Newcastle disease virus (negative-sense, single strand); Sendai virus (negative-sense, single strand); vesicular stomatitis virus (negative-sense, single strand); respiratory syncytial virus (negative-sense, single strand); influenza A virus (negative-sense, single strand); Ebola virus (negative-sense, single strand); Japanese encephalitis virus (positive-sense, single strand); hepatitis C virus (positive-sense, single strand) Long length of poly IC, encephalomyocarditis virus (positive-sense, single strand); mengovirus (positive-sense, single strand); Theiler’s virus (positive-sense, single strand); poliovirus (positive-sense, single strand) Reovirus (double strand); dengue virus (positive-sense, single strand); West Nile virus (positive-sense, single strand) viral infection, suggesting that the ATPase activity of RLR members may be essential for antiviral responses [46]. PAMPs OR VIRUSES RECOGNIZED BY RLR RIG-I mainly recognizes members of paramyxoviridae family of viruses such as Newcastle Disease Virus (NDV) and Vesicular stomatitis virus (VSV). RIG-I-deficient cells show reduced production of inflammatory cytokines and type I interferons [47]. In addition, RIG-I recognizes members of the flaviviridae family, such as Japanese Encephalitis Virus (JEV) and Hepatitis C Virus (HCV) (Table II). MDA5 recognizes members of the picornaviridae family, such as the Polio Virus and encephalomyocarditis virus (EMCV) (Table II) [48, 49]. RIG-I also recognizes enzymatically synthesized RNA in vitro. It was thought that single-stranded RNAs bearing a 5 triphosphate were the only essential component for this recognition [50, 51]; however, recent studies suggest that, during the enzymatic synthesis of RNA, a secondary structure for double stranded RNA is generated, which acts as a RIG-I ligand [52, 53]. Chemically synthesized single-stranded RNA bearing a 5 triphosphate cannot induce inflammatory cytokines and type I interferons, whereas chemically synthesized double-stranded RNA can, irrespective of whether there is a triphosphate or monophosphate group at the 5 end [54, 55]. MDA5 also recognizes the synthetic double stranded RNA analog, poly IC, when this molecule is introduced into the cell [48]. Interestingly, enzymatically shortened poly IC is preferentially recognized by RIG-I, rather than by MDA5, suggesting that RIG-I and MDA5 recognize different lengths of double stranded RNA. Extrapolation of these results may provide a possible clue for the differential recognition of viruses. Furthermore, some viruses, such as dengue virus and West Nile Virus, require recognition by both RIG-I and MDA5 to generate a robust innate immune response [55]. The third member of the RLR family, LGP2, was considered as a negative regulator of RIG-I- and MDA5-mediated signaling [56–58]. However, in vivo studies using LGP2deficient mice and mice harboring an inactive ATPase in the DExD/H-box RNA helicase domain show that LGP2 acts as a positive regulator of RIG-I- and MDA5-mediated signaling after infection by RIG-I- and MDA5-specific RNA viruses. This suggests that C Informa Healthcare USA, Inc. Copyright H. Kumar et al. LGP2 might facilitate the accessibility of viral RNA by RIG-I or MDA5 to induce robust responses [46]. Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. RLR SIGNALING Viral infection is initiated either through direct introduction of viral nucleic acids (PAMPs) or through the receptor mediated endocytosis and subsequent liberation of viral nucleic acids (PAMPs) into the cytoplasm of the host cells [3, 6, 44, 45]. In the cytoplasm, RIG-I and MDA5, along with LGP2, sense the viral RNA, which probably leads to conformational changes within these sensors exposing the CARD domains of RIG-I and MDA5, which then interact with the CARD-containing adaptor protein, IPS-1 (also known as MAVS, Cardif, and VISA) [59–64]. IPS-1 localizes to the mitochondria, a process important for downstream signaling [60]. Recently, it has been shown that IPS-1 is also localized on peroxisomes, and that peroxisomal IPS-1 and mitochondrial IPS-1 are required for robust antiviral responses. Peroxisomal IPS-1 induces early responses through the induction of interferon-stimulating genes (ISGs) via the transcription factor, IRF1, whereas mitochondrial IPS-1 induces delayed responses through induction of ISGs and type I interferons via IRF3 [65]. Upon recruitment of RIG-I and MDA5, IPS-1 activates the IKK-related kinase, TBK1/IKKi, which activates IRF3/IRF7 and the subsequent transcription of type I interferons via TRAF3. IPS-1 also activates NF-κB through recruitment of TRADD, FADD, caspase-8, and caspase-10 [59, 66]. Recently, an IPS-1-interacting protein, EYA4, was found to enhance interferon induction upon NDV and VSV infection [67]. EYA4 has phosphatase activity for phosphotyrosine and phosphothreonine and this activity is required for antiviral responses (Fig. 1). IPS-1 is negatively regulated by an NLR family protein known as NLRX1, which is localized in mitochondria. NLRX1 interacts with IPS-1 at the outer membrane and inhibits IPS-1-mediated antiviral responses [68]. Furthermore, IPS-1 is also negatively regulated by the autophagy-related proteins, Atg5 and Atg12. MEF and DCs lacking autophagosome components, such as Atg5 and Atg12, show enhanced production of type I interferons after RNA virus infection because malfunctioning mitochondria and the IPS-1 associated with these mitochondria lead to the production of reactive oxygen species (ROS) [69]. Enhanced ROS production increases the production of type I interferons. The initial recognition by RIG-I is positively regulated by TRIM25 and RNF135 (a E3 ubiquitin ligase) via K63-linked ubiquitination [70, 71]. However, K48-linked ubiquitination of RIG-I by RNF125 leads to the down regulation of RIG-I-mediated signaling [72]. Recently, the mechanism underlying the activation of RIG-I was reported. The CARDs within RIG-I require K63-linked ubiquitination in the presence of RNA and ATP. Furthermore, free K63-linked ubiquitin chains activate RIG-I-dependent downstream signaling, suggesting that free ubiquitin chains are the endogenous ligands of RIG-I [73]. TLR INDEPENDENT DNA RECOGNITION Introduction of DNA, plasmid DNA (used for DNA vaccines), or DNA from dying cells into immune or non-immune cells, or infection with DNA viruses or pathogenic bacteria such as Listeria monocytogenes and Legionella pneumophila, induces the production of TLR9-independent type I interferons [3, 6, 74–76]. This indicates that cells possess an additional sensor(s) for DNA. Studies show that induction of type I interferons in response to DNA requires TBK1/IKKi. DAI (also known as DLM1 and ZBP1) is a cytoplasmic DNA sensor. In vitro studies, show that DAI is essential for type International Reviews of Immunology Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. Pathogen Recognition by Innate Immunity I interferon production via TBK1/IKKi [77]; however, genetic studies show that DAI is not essential for type I interferon production after the introduction of DNA, suggesting that cells possess an unidentified DNA sensor [78]. Recently, the mechanism for poly dA:dT-induced type I interferon production was elucidated by two independent groups [79, 80]. The results show that poly dA:dT introduced into cells is first transcribed into RNA by polymerase III, and this RNA has a double stranded conformation. This dsRNA is then sensed by RIG-I, which activates the RIG-I-IPS-1 signaling axis to induce type I interferons [79, 80]. However, introduction of poly dA:dT into RIG-I-deficient cells results only in a marginal reduction in type I interferon production suggesting that cells requires other DNA sensors for poly dA:dT recognition in addition to RIG-I, which senses the secondary metabolic products derived from the introduced DNA. Furthermore, another ER-localized protein, STING (also known as TMEM173, ERIS, or MITA), was identified by functional screening for IFN-β promoter activators [81–83]. STING interacts with TBK1 and induces type I interferon production after stimulation by DNA and RNA. However, STING does not interact directly with DNA, suggesting that STING acts downstream of an unidentified DNA sensor. STING-deficient mice are highly susceptible to the DNA virus, HSV, and the RNA virus, VSV, suggesting a role for STING in innate immune defense against various viruses [84]. Recently, ATG9a was implicated in the regulation of STING-mediated TBK1 activation (Fig. 1). Cells lacking ATG9a show increased activation of TBK1 and production of type I interferons by promoting the assembly of STING with TBK1 [85]. NLRs NOD-like receptors (NLRs) are a family of molecules that sense a wide range of ligands within the cytoplasm of cells. This family comprises 23 members in humans and approximately 34 in mice. Among NLRs member functions of several NLR members are well characterized [3, 6, 86–88]. These sensors comprise three domains: the C-terminal domain consists of several (LRRs) and is thought to be involved in the recognition of microbial PAMPs, or endogeneous host molecules; the N-terminal domain consists of a death effector domain (DED), a Pyrin domain (PYD), a CARD, baculovirus inhibitor repeats (BIRs) and an acidic domain (which is required for homotypic interactions with downstream signaling proteins); and an intermediate domain consisting of nucleotide-binding and oligomerization (NACHT) domains, which are required for ligand-induced, ATP-dependent oligomerization of the sensors and formation of active receptor complexes for activation of downstream signaling. Upon recognition of PAMPs, these sensors either activate NF-κB or MAP kinases to induce the production of inflammatory cytokines, or activate a multiprotein-complex, the “inflammasome,” which either initiates the proteolytic cleavage (or maturation) of various caspases resulting in the maturation and production of inflammatory cytokines, such as IL-1β and IL-18, or initiates cell death. NOD1 AND NOD2 NOD1 and NOD2 (also known as CARD4 and CARD15, respectively) comprise Cterminal LRRs, a central oligomerization domain and an N-terminal domain containing either one (NOD1) or two (NOD2) CARDs. These proteins are mainly expressed in the cytosol of various cells. However, expression on the plasma membrane has also been reported [89, 90]. NOD1 and NOD2 recognize peptidoglycans, a major component of bacterial cell walls. NOD1 and NOD2 recognize iE-DAP and MDP, respectively [91]; however, evidence for the direct recognition of these PAMPs is lacking. NOD1 and NOD2 also recognize various pathogenic microbial pathogens (Table III) [6]. NOD2 C Informa Healthcare USA, Inc. Copyright H. Kumar et al. TABLE III NLR Ligands and Cellular Location NLRs Cellular localization Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. NOD1 (CARD4) Cytoplasm NOD2 (CARD15) Cytoplasm NLRP1 (NALP1) Cytoplasm NLRP3 (NALP3, Cytoplasm CIAS1 and Cryopyrin) NLRC4 (IPAF, CLAN) Cytoplasm Ligand or pathogen γ -D-glutamyl-meso-diaminopimelic acid (iE-DAP) (a dipeptide) from Bacillus subtilis, Listeria monocytogenes, entropathogen (Escherichia coli), Shigella Flexneri, Pseudomonas aeruginosa, Chlamydia pneumoniae, Campylobacter jejuni and Helicobacter pylori Muramyl dipeptide (MDP) from Streptococcus pneumonia, Mycobacterium tuberculosis, Listeria monocytogenes, Salmonella typhimurium, Shigella Flexneri and Staphylococcus aureus Muramyl dipeptide (MDP) is recognized by human NLRP1, lethal toxin from Bacillus anthracis is recognized by mouse NLRP1b Crystals (uric acid, calcium pyrophosphade dehydrate), extracellular ATP, fibrillar amyloid-β peptide, hyaluronan, pollutants (silica and asbestos), bacterial and viral RNA, poly IC, antiviral compound (R837 and R848), toxins (nigericin and maitotoxin), UV light, skin irritant (picryl chloride and 2,4-dinitrofluorobenzene), vaccine adjuvant (alum), fungi (Candida albican and Saccharomyces cerevisiae), β-glucan, bacteria (Listeria monocytogenes and Staphylococcus aureus), Viruses (Sendai virus, adenovirus and influenza virus) Shigella flexneri, Salmonella typhimurium, Pseudomonas aeruginosa, Legionella pneumophila, Flagellin delivered into the macrophages (In addition to NLRC4, NAIP5 recognize flagellin. Furthermore, TLR5 also recognizes flagellin on surface of cells to induce proinflammatory cytokine through NFκB and MAP kinases) is also important for defense against pathogenic protozoal parasites, such as Toxoplasma gondii [92]. PAMP recognition initiates oligomerization of these sensors, which subsequently recruit a CARD-containing adaptor protein known as RIP2 (RICK) via CARD-CARD interactions, and activate NF-κB and MAP kinases to induce the transcription of inflammatory cytokines (Fig. 1) [89, 90]. In humans, NOD1 and NOD2 variants have been reported; NOD1 variants are associated with elevated levels of IgE, and a propensity toward asthma and atopic eczema [93, 94]. NOD2 variants are associated with chronic inflammation of the intestine, and these patients are susceptible to Crohn’s disease [95, 96]. NLRC5 Another member of the NLR family, NLRC5 (also known as NOD27), has been recently characterized. NLRC5 mRNA and protein expression are induced by stimulation with IFN-γ , LPS, poly IC and viral infection of myeloid and lymphoid cells lineages. This protein comprises a CARD and an LRR domain and is localized in the cytoplasm and/or nucleus [97–99]. In vitro studies show that NLRC5 regulates antiviral innate and adaptive immune responses through regulation of inflammatory cytokines International Reviews of Immunology Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. Pathogen Recognition by Innate Immunity and/or type I interferons and transcriptional regulation of MHC class I molecules in lymphoid and epithelial cell lines, respectively [97–101]. NLRC5 regulates antiviral innate responses through its association with IKK-α and IKK-β and inhibits their activation, thereby serving as a negative regulator of NF-κB activation. Moreover, NLRC5 interacts with RIG-I and MDA5 and suppresses both NF-κB and IRF3 activation [97]. In contrast, another study showed that NLRC5 overexpression induces IFN-β promoter activation and enhances RIG-I- and MDA5-mediated antiviral responses [97–99]. In addition, overexpression of NLRC5 induces caspase-1-dependent maturation of IL-1 family cytokines [102]. However, in vivo studies of NLRC5-deficient mice suggest that NLRC5 is not necessary for NF-κB- or IRF3-mediated production of inflammatory cytokines and type I interferons by various cell types including macrophages and DCs. Moreover, both NLRC5-deficient and wild-type mice intra-peritoneally challenged with poly IC show comparable levels of inflammatory cytokine and type I interferon production [102]. Collectively, in vivo studies suggest that NLRC5 is not essential for inflammatory cytokine and type I interferon production. However, NLRC5 may form a protein complex, the inflammasome, with caspase-1 and this inflammasome may sense some unknown pathogen or host ligand and induce the maturation of IL-1 family cytokines. One possible explanation for the differences between in vitro and in vivo studies may be species differences. INFLAMMASOMES Stimulation of immune cells, such as macrophages and dendritic cells, with microbial PAMPs initiates the assembly of a protein complex known as the inflammasome, which is composed of NLR members (e.g., NLRP3, NLRC4, NLRP1), non-NLR proteins, AIM2 and ASC [6, 88, 103, 104]. This protein complex associates with an inactive form of caspase-1 (procaspase-1) and promotes its proteolytic activation to yield caspase1, which, in turn, promotes proteolysis of the zymogan form of IL-1 family cytokines (Fig. 1). THE NLRP3 INFLAMMASOME The NLRP3 inflammasome is the most widely studied inflammasome and, to date, numerous PAMPs from all classes of pathogens (viruses, bacteria, and fungi) and host DAMPs (DAMPs [danger-associated molecular patterns] comprise molecules, such as heat shock proteins and BCL2, which are derived from necrotic or traumatized host cells and sensed by PRRs that induce inflammatory cytokines) have been reported (Table III), which activate the NLRP3 inflammasome [6, 103, 104]. In addition to PAMPs and DAMPs, environmental pollutants, such as silica and asbestos are also reported to activate the NLRP3 inflammasome. Although NLRP3 inflammasome biology has rapidly expanded, the mechanism by which biochemically diverse ligands are sensed by NLRP3 remains unknown. Furthermore, it is also unclear whether NLRP3 senses ligands directly or indirectly. The NLRP3 inflammasome is composed of NLRP3, ASC, and procaspase-1. Stimulation of cells with appropriate ligands induces oligomerization of NLRP3, which promotes the clustering of ASC with NLRP3 via a pyrin domain (PYD)-PYD interaction. The CARD of ASC and the CARD of procaspase-1 then interact to induce catalysis of procaspase-1 to yield caspase-1, consisting of a p10/p20 tetramer, which catalyzes the proteolysis of pro IL-1β (inactive) to IL-1β (active). Several models of NLRP3 activation have been proposed including ATP-induced efflux of potassium ions via P2X7 ion channels and pannexin-1, ROS induction, and lysosomal destabilization after phagocytosis of several crystalline and insoluble ligands (e.g., silica and amyloid-β), which leads to disruption of lysosomal membranes and the C Informa Healthcare USA, Inc. Copyright Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. H. Kumar et al. release of lysosomal proteins that activate the NLRP3 inflammasome [6, 88, 103, 104]. In addition, several studies in which mice were challenged with antigen and alum (a ligand for the NLRP3 inflammasome) suggest the involvement of the NLRP3 inflammasome in the regulation of adaptive immunity [105–107]; however, inconsistencies in the results were reported, which may be due to differences in the methods used and the immunization protocol. Therefore, it is difficult to establish the involvement of the NLRP3 inflammasome in adaptive immunity when alum is used as an adjuvant. Recently, several reports showed that the pathogenic fungus, Candida, and baker’s yeast (Saccharomyces) induced IL-1β via NLRP3, ASC and caspase-1, and further studies revealed that β-glucan, a major component of fungal cell walls, activates the NLRP3 inflammasome [108–115]. In addition, β-glucan-induced antibody responses are dependent upon the NLRP3 inflammasome. However, the IL-1β signaling pathway is not essential for antibody responses [111]. On other hand, when β-glucan is used as an adjuvant, T cell responses in both NLRP3-deficient and wild-type mice are comparable. Collectively these observations suggest that the NLRP3 inflammasome plays a pivotal role in antibody production by B cells, which is independent of IL-1β or TLR signaling. VIRUS-MEDIATED IL-1β PRODUCTION AND INNATE HOST DEFENSE The influenza A virus also induces IL-1β production through activation of inflammasomes composed of NLRP3, ASC, and caspase-1; however, the requirement for NLRP3 in inflammasome-induced IL-1β production remains controversial [116–120]. One group showed that the requirement for NLRP3 is cell-type specific, whereas other groups showed that all cells require NLRP3. However, in vivo studies reveal that ASCand caspase-1-deficient mice are more susceptible to the influenza A virus infection than wild-type mice or NLRP3-deficient mice after challenge with sub-lethal doses of the virus. Influenza virus-specific adaptive immunity has also been demonstrated; however, the results of these studies are dissimilar and the possible explanation for these differences may be the different titers of virus used [116–120]. More recently, the mechanism underlying influenza A virus-induced inflammasome activation was reported and showed that the M2 protein of the influenza virus, a proton-selective ion channel, is required for inflammasome activation and viral pathogenesis [121]. THE NLRC4 INFLAMMASOME The NLRC4 inflammasome is mainly composed of CARD-containing NLRC4 [122] (also known as IPAF) and procaspase-1, and is activated via direct interaction with the CARDs of caspase-1 and NLRC4 to induce pyroptosis through caspase-1 activation [123]. However, several studies suggest that ASC is also required for NLRC4 inflammasome activation and that ASC potentiates caspase-1 activation. These studies suggest that other PYD-containing NLR members may be involved in the formation of the NLRC4 inflammasome. The NLRC4 inflammasome is also involved in Shigella flexneri-induced cell death, which is independent of ASC [124, 125]. Infection with Legionella pneumophila, Salmonella typhimurium, Pseudomonas aeruginosa, or Shigella flexneri [125, 128] suggests that a type III or type IV secretion system (this secretion system makes pores in host cells and introduces virulence factors into the cells to activate various effector responses, such as caspase-1 activation and cell death) is essential for the activation of the NLRC4 inflammasome. Another study showed that another NLR family member, NAIP5, is required for the recognition and replication of Legionella pneumophila [129]. Moreover, NAIP5 recognizes the C-terminal portion of flagellin. International Reviews of Immunology Pathogen Recognition by Innate Immunity Introduction of flagellin into the cytoplasm of cells is necessary for the activation of the NLRC4 inflammasome (which is independent of TLR5). Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. THE NLRP1 INFLAMMASOME The human NLRP1 inflammasome senses MDP and comprises NLRP1, ASC, and caspase-1 [130]. ASC is not a necessary component, but the presence of ASC potentiates caspase-1 activation [130]. Three paralogs of NLRP1 have been identified in the mouse genome, namely NLRP1a, NLRP1b, and NLRP1c. NLRP1b senses lethal toxins from Bacillus anthracis, which plays an important role in pathogenesis [131]. THE AIM2 INFLAMMASOME Introduction of DNA into cells, or infection with DNA viruses, induces the production of IL-1β via the AIM2 inflammasome. AIM2 is a HIN-200 family, PYD-containing protein and is a cytoplasmic DNA sensor that directly senses DNA and induces proteolytic activation of caspase-1. This results in the production and maturation of IL-1β via the PYD-PYD-mediated interaction between AIM2 and ASC [132–138]. Recently, the in vivo function of AIM2 was demonstrated by two independent groups that generated AIM2-deficient mice and showed that AIM2 is critical for caspase-1 activation, production of IL-1β and IL-18, and pyroptosis (induced cell death) after infection with Francisella tularensis, Vaccinia virus and MCMV. In addition, AIM2 plays a role after Listeria infection. AIM2-deficient mice also have a higher mortality compared with wild-type mice after infection with MCMV and Francisella [139–142]. CONCLUSION In this review, we have extensively discussed the ligands, PRRs express on/in the cell surface, cellular vesicles, or cytoplasm and the signaling pathways activated by TLRs, RLRs, NLRs, and DNA receptors. However, most studies were performed either with a pure ligand, or model laboratory pathogens, using mouse models. Therefore, these studies may not reflect the true picture of host–pathogen interactions in the context of human disease. To resolve these issues, further studies using human immune cells challenged with clinical isolates of pathogens will provide deep insight into the host–pathogen interactions involved in various infectious disease conditions. In addition, it is noted that pathogens consist of multiple ligands, which may activate multiple signaling pathways, and that this may lead to the crosstalk between various signaling pathways resulting in a wide range of innate immune responses. Therefore, we need to extend our present knowledge of innate immune immunobiology by stimulating immune cells with multiple ligands to understand the net outcome of innate immune responses and their influence on the development of adaptive immunity. To date, human studies suggest that TLRs, RLRs, NLRs, and DNA sensors are not sufficient to mount protective immune responses against a wide range of infectious agents, suggesting that unidentified PRRs play a key role in defense against an array of pathogens. Therefore, the identification and characterization of new PRRs is needed. Collectively, these studies will be important not only for the basic understanding of host–pathogen interactions, but also for the development of various therapeutic strategies and better adjuvants for existing and new vaccines. C Informa Healthcare USA, Inc. Copyright H. Kumar et al. ACKNOWLEDGMENTS Int Rev Immunol Downloaded from informahealthcare.com by Helmholtz Zentrum fuer Infektionsforschung GmbH -Bibliothek- on 10/15/13 For personal use only. 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