INTRODUCTION
The innate immune system constitutes the first line of host defense during infection and therefore plays a crucial role in the early recognition and subsequent triggering of a proinflammatory response to invading pathogens (242). The adaptive immune system, on the other hand, is responsible for elimination of pathogens in the late phase of infection and in the generation of immunological memory. Whereas the adaptive immune response is characterized by specificity developed by clonal gene rearrangements from a broad repertoire of antigen-specific receptors on lymphocytes, the innate immune response is mediated primarily by phagocytic cells and antigenpresenting cells (APCs), such as granulocytes, macrophages, and dendritic cells (DCs), and has been regarded as relatively nonspecific (151).
The innate immune response relies on recognition of evolutionarily conserved structures on pathogens, termed pathogen-associated molecular patterns (PAMPs), through a limited number of germ line-encoded pattern recognition receptors (PRRs), of which the family of Toll-like receptors (TLRs) has been studied most extensively (7, 242).
PAMPs are characterized by being invariant among entire classes of pathogens, essential for the survival of the pathogen, and distinguishable from “self” (153). However, in certain cases, PRRs also recognize host factors as “danger” signals, when they are present in aberrant locations or abnormal molecular complexes as a consequence of infection, inflammation, or other types of cellular stress (32, 236) (Fig. 1).
FIG. 1. Principles in innate immune recognition by PRRs. During microbial infection or breakdown of tolerance, pathogen-specific molecules, aberrant localization of foreign or self molecules, or abnormal molecular complexes are recognized by PRRs. This event triggers PRR-mediated signaling and induction of an innate immune response, which ultimately results in resolution of infection but also may cause inflammatory diseases or autoimmunity.
Upon PAMP recognition, PRRs present at the cell surface or intracellularly signal to the host the presence of infection and trigger proinflammatory and antimicrobial responses by activating a multitude of intracellular signaling pathways, including adaptor molecules, kinases, and transcription factors (6).
PRR-induced signal transduction pathways ultimately result in the activation of gene expression and synthesis of a broad range of molecules, including cytokines, chemokines, cell adhesion molecules, and immunoreceptors (7), which together orchestrate the early host response to infection and at the same time represent an important link to the adaptive immune response.
The relatively recent understanding of the nature of pathogen recognition and signaling mechanisms in innate immune defenses has significantly changed previous ideas about this system. Janeway was the first to propose the existence of a class of innate immune receptors recognizing conserved microbial structures or “patterns,” even prior to the molecular identification of such a system (153).
However, the immunostimulatory activity of nucleic acids had long been recognized. Already in 1963, two separate groups reported the observation that DNA and RNA derived from pathogens or host cells were capable of inducing interferon (IFN) production in fibroblasts (156, 309), but cellular receptors for nucleic acids, as well as for other microbial components, have remained unknown until a few years ago (12, 76, 119, 121, 243, 357, 393).
Accordingly, the current view on pathogen recognition has been shaped only during the last two decades, initiated by Janeway’s hypothesis and further stimulated by the identification of TLRs in 1997 (154, 243). Given the fact that sensing and defeating microbial infection is essential for mammalian species, PRRs and the signal transduction pathways they activate belong to an old and evolutionarily conserved system (211, 242).
Pathogens of quite different biochemical composition and with entirely different life cycles, including viruses, bacteria, fungi, and protozoa, are recognized by slightly different yet surprisingly similar and overlapping mechanisms by host PRRs (7), and the unraveling of these common principles has contributed significantly to the understanding of these systems. Thus, recent studies have demonstrated that the innate immune system has a greater specificity than was previously thought and that it appears to be highly developed in its ability to discriminate between self and foreign. The innate immune system is therefore no longer regarded as a primitive, nonspecific system involved only in destroying and presenting antigen to cells of the adaptive immune system.
Moreover, it now appears that innate and adaptive immune responses are much more intimately connected than initially believed, and several important findings support the idea that the innate immune system, besides being essential for early pathogen recognition, is also involved in the activation and shaping of adaptive immunity (151).
In this review, mechanisms of pathogen recognition and proinflammatory signal transduction in innate immune defenses are presented. To illustrate the complexity, yet similarities in overall principles, of these signaling pathways, viral, bacterial, fungal, and protozoan recognition by PRRs will be covered. Moreover, interference with pathogen-induced inflammatory responses by endogenous mechanisms and by pathogens is described. Finally, medical implications, including understanding of the role of PRRs in primary immunodeficiencies and in the pathogenesis of infectious diseases and autoimmunity, as well as the possibilities of therapeutic intervention with pathogen recognition and innate immune signaling are discussed.
INNATE IMMUNE DEFENSES AND PRRs
The innate immune system is based principally on physical and chemical barriers to infection, as well as on different cell types recognizing invading pathogens and activating antimicrobial immune responses (31, 242). Physical and chemical defense mechanisms are represented by epidermis, ciliated respiratory epithelium, vascular endothelium, and mucosal surfaces with antimicrobial secretions (31).
Likewise, the cellular components of innate immunity include antigen-presenting DCs, phagocytic macrophages and granulocytes, cytotoxic natural killer (NK) cells, and T lymphocytes (31). The important ability of the innate immune system to recognize and limit microbes early during infection is based primarily on employment of complement activation, phagocytosis, autophagy, and immune activation by different families of PRRs (Fig. 2).
FIG. 2. Cellular PRRs. TLRs are membrane-bound receptors localized at the cellular or endosomal membranes, recognizing PAMPs via the LRR domain and transducing signals to the intracellular environment through the TIR domain. RLRs with a C-terminal helicase domain bind RNA and become activated to transduce CARD-dependent signaling. The dsRNA-activated kinase PKR is an intracellular PRR that senses RNA through binding to two N-terminal dsRNA-binding domains. DAI and AIM2 are intracellular DNA sensors. NLRs are a class of intracellular proteins characterized by a central NOD domain and a C-terminal LRR domain, the latter of which serves as a pattern recognition domain. Signals are transduced through N-terminal domains, including CARD and pyrin (PYD) domains.
Whereas the focus in this review is on pathogen recognition and PRR-mediated proinflammatory signal transduction pathways, other components of innate immune defenses have been reviewed elsewhere, for instance, by Basset et al. (31). The different families of PRRs are shown in Fig. 2, and general principles of pathogen recognition by PRRs are presented below.
TLRs
The family of TLRs is the major and most extensively studied class of PRRs. TLRs derived their name and were originally discovered based on homology to the Drosophila melanogaster Toll protein (243), which plays a role in dorso-ventral patterning during embryogenesis as well as in the antifungal response in Drosophila (211). Structurally, TLRs are integral glycoproteins characterized by an extracellular or luminal ligand-binding domain containing leucine-rich repeat (LRR) motifs and a cytoplasmic signaling Toll/interleukin-1 (IL-1) receptor homology (TIR) domain (280). Ligand binding to TLRs through PAMP-TLR interaction induces receptor oligomerization, which subsequently triggers intracellular signal transduction. To date, 10 TLRs have been identified in humans, and they each recognize distinct PAMPs derived from various microbial pathogens, including viruses, bacteria, fungi, and protozoa (7) (Table 1).
TLRs can be divided into subfamilies primarily recognizing related PAMPs; TLR1, TLR2, TLR4, and TLR6 recognize lipids, whereas TLR3, TLR7, TLR8, and TLR9 recognize nucleic acids (7). Moreover, it appears that TLRs can recognize PAMPs either through direct interaction or via an intermediate PAMP-binding molecule. Thus, TLR1/2, TLR3, and TLR9 directly bind to triacetylated lipopeptides, double-stranded RNA (dsRNA), and CpG DNA, respectively (159, 202, 216), whereas TLR4 recognizes lipopolysaccharide (LPS) through the accessory molecule MD2 (187). Intriguingly, some TLRs are endowed with the capacity to recognize structurally and biochemically unrelated ligands, as exemplified by the ability of TLR4 to recognize such divergent structures as LPS, the fusion protein of respiratory syncytial virus (RSV), and cellular heat shock proteins (HSPs) (7). The molecular basis of this phenomenon may be the ability of different regions of the extracellular portion of TLRs to bind their cognate ligands or the involvement of different PAMP-binding molecules, such as MD2 (187, 216). Further distinction between different PAMPs is accomplished through the formation of heterodimers between TLR2 and either TLR1 or TLR6 (287). Another way of grouping TLRs is based on their cellular distribution. Certain TLRs (TLR1, -2, -4, -5, -6, and -10) are expressed at the cell surface and mainly recognize bacterial products unique to bacteria and not produced by the host, whereas others (TLR3, -7, -8, and -9) are located almost exclusively in intracellular compartments, including endosomes and lysosomes, and are specialized in recognition of nucleic acids, with self versus nonself discrimination provided by the exclusive localization of the ligands rather than solely based on a unique molecular structure different from that of the host (151). The most important cell types expressing TLRs are APCs, including macrophages, DCs, and B lymphocytes (151). In different experimental systems, however, TLRs have been identified in most cell types, expressed either constitutively or in an inducible manner in the course of infection (151, 252, 269). Although patterns of TLR expression in different cell types and anatomical tissue locations, as well as mechanisms regulating TLR gene expression in response to inflammatory mediators, are of great relevance and potential profound bio logical significance, many of these aspects remain poorly characterized. As a general rule, gram-negative bacteria are recognized by TLR4 via the lipid A portion of LPS (296), whereas lipoteichoic acid, lipoproteins, and peptidoglycan of gram-positive bacteria are detected by TLR2 (328, 395). However, most gram-positive and -negative bacteria can activate additional TLRs via alternative PAMPs present in the cell membrane, cell wall, or intracellularly (262), as illustrated in Fig. 3. For instance, flagellin, the major constituent of the motility apparatus of flagellated bacteria, is recognized by TLR5 (117, 118). With respect to TLR-mediated recognition of nucleic acids in intracellular compartments, TLR3 recognizes dsRNA produced during viral replication (12), whereas TLR7 and TLR8 are activated by single-stranded RNA (ssRNA) (76, 119). Finally, TLR9 is responsible for the detection of unmethylated CpG DNA present in the genomes of both viruses and bacteria (121) as opposed to methylated DNA present in mammalian cells, which generally does not activate the immune system. A fundamental property of this system is that a given pathogen can activate several different TLRs via different PAMPs, and likewise, several structurally unrelated pathogens can activate any given TLR. The net result of TLR engagement of a relevant PAMP is the triggering of downstream signaling pathways, ultimately resulting in the generation of an antimicrobial proinflammatory response. More details on PRRs and their ligands are given in Table 1.
Cytosolic PRRs
Since TLRs are expressed at either the cell surface or the luminal aspect of endo-lysosomal membranes, they do not seem capable of recognizing intracellular cytosolic pathogens and their derivatives, such as viral ssRNA, dsRNA, and DNA, as well as components of internalized or intracellular bacteria.
Additionally, data from animal studies indicated the existence of other classes of PRRs. More specifically, evidence suggested that receptors other than TLR3 and TLR9 were able to induce type I IFN (IFN- α and IFN- β) production in response to RNA, DNA, or viral infections (81, 127, 149, 344).
Subsequent studies revealed that TLR-independent recognition of pathogens is accomplished by a large group of cytosolic PRRs, which can be broadly divided into retinoid acid-inducible gene I (RIG-I)- like receptors (RLRs) (393) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (169).
RLRs and other cytosolic nucleotide sensors. RIG-I and melanoma differentiation-associated gene 5 (MDA5) are IFNinducible RNA helicases that play a pivotal role in sensing of cytoplasmic RNA (392, 393). These RNA helicases contain an N-terminal caspase recruitment domain (CARD) and a central helicase domain with ATPase activity required for RNA-activated signaling (393).
Binding of dsRNA or 5'-triphosphate RNA to the C-terminal domains of RLRs (65, 356) triggers signaling via CARD-CARD interactions between the helicase and the adaptor protein IFN- β promoter stimulator 1 (IPS-1) (179, 246, 329, 383), ultimately resulting in an antiviral response mediated by type I IFN production (172, 393).
The importance of CARDs is evidenced by the third helicase, LGP2, which is devoid of such domains and hence does not induce signaling but rather prevents RIG-I signaling (310), whereas the specific role of LGP2 in MDA5-activated signaling remains unresolved, with indications of a positive function (374).
Although RIG-I and MDA5 function by similar mechanisms, studies have suggested differential roles of these two helicases, with RIG-I being essential for the response to paramyxoviruses and influenza virus, whereas MDA5 seems to be critical for the response to picornavirus and norovirus (174, 238). At the biochemical level, these differences may be due to length-dependent binding of dsRNA by these two RLRs (173).
Specifically, RIG-I and MDA5 recognize short and long dsRNAs, respectively (173), and in addition, RIG-I detects 5'-triphosphate RNA (174, 295).
Furthermore, one report has demonstrated that in addition to viral RNA, RLRs can recognize self-derived small RNAs generated by RNase L, thus amplifying the IFN response (221). Viral RNA can also be recognized by the IFN-inducible dsRNA-activated protein kinase (PKR), which represents a major mediator of the antiviral and antiproliferative activities of IFN (270, 312, 389). Binding of dsRNA, 5'-triphosphate RNA, or poly(I-C) induces conformational changes in PKR, resulting in autophosphorylation, dimerization, and subsequent substrate phosphorylation (50, 270, 306). The best-characterized PKR substrate is the eukaryotic initiation factor eIF2 α , the phosphorylation of which leads to inhibition of protein synthesis (312). Moreover, PKR has been assigned a role in proinflammatory signal transduction as an upstream kinase involved in mediating dsRNA-dependent nuclear factor (NF)- κ B activation (396).
However, although PKR was originally considered the principal molecule responsible for cellular recognition of dsRNA, this perception was questioned by data from PKR-deficient mice, which did not show considerable impairment in their response to viral infection (2, 131, 389). The subsequent identification of RLRs has resolved some of this paradox.
The prevailing view is that the major contribution to dsRNA-activated responses is mediated by RLRs, with recent data suggesting that PKR may be able to amplify RLR signaling (237, 399), thus illustrating cross talk between these different cellular dsRNA-sensing systems involved in antiviral defense.
Cytoplasmic localization of DNA is recognized by the innate immune system independently of TLRs, RLRs, and NLRs (338, 345) and seems to be involved in mounting a response to both bacteria and DNA viruses (55, 208, 274, 301, 344).
Recently, the identification of the first cytosolic DNA sensor, DAI (DNA-dependent activator of IFN-regulatory factors), was reported (357). DNAs from various sources were demonstrated to bind to DAI, thereby inducing DNA-mediated induction of type I IFN and the products of other genes involved in innate immunity (357). However, DAI is probably not the only cytosolic DNA receptor triggering the IFN response, since inhibition of DAI by small interfering RNA had little or no effect on the IFN response to different types of DNA (357, 379).
This is supported by a report demonstrating induction of type I IFN by group B streptococcus via intracellular recognition of its DNA independently of DAI (55). Although cytosolic DNA has been ascribed particularly important roles in activation of the IFN response (55, 208, 274, 301, 344), members of this class of PAMPs also activate other parts of the innate immune response (268). Recently a cytosolic DNA receptor stimulating proinflammatory signaling and maturation of pro-IL-1 β has been reported and named AIM2 (absent in melanoma 2) (136). Considering the large and heterogeneous group of proteins belonging to the family of PRRs, it will not be surprising if even more cytoplasmic DNA receptors are identified.
NLRs and the inflammasome. NLRs belong to a family of innate immune receptors which have gained increasing interest over the past few years and are now considered key sensors of intracellular microbes and danger signals and therefore believed to play an important role in infection and immunity.
NLRs are defined by a centrally located NOD that induces oligomerization, a C-terminal LRR that mediates ligand sensing (in analogy with TLRs), and an N-terminal CARD that is responsible for the initiation of signaling (169). The two bestcharacterized members of the NLR family are NOD1 and NOD2, which sense bacterial molecules derived from the synthesis and degradation of peptidoglycan (169).
Whereas NOD1 recognizes diaminopimelic acid produced primarily by gram-negative bacteria (53, 101), NOD2 is activated by muramyl dipeptide (MDP), a component of both gram-positive and -negative bacteria (102). It is currently unresolved whether NOD1 and NOD2 serve as direct receptors of PAMPs or instead detect modifications of host factors as a consequence of the presence of microbial molecules in the cytosol (169).
Irrespective of the specific mechanism, activation of NOD proteins induces oligomerization and recruitment of downstream signaling molecules and transcriptional upregulation of inflammatory genes (169). Whereas NOD1 and NOD2 stimulation results primarily in activation of proinflammatory gene expression, other NLR proteins are involved in activation of caspases (169).
During infection, microbes induce TLR-dependent cytosolic accumulation of inactive IL-1 β precursor and activation of caspase-1, the latter of which catalyzes the cleavage of the IL-1 precursor pro-IL-1 β (226, 231). A protein complex responsible for this catalytic activity has been identified by Martinon et al. and was termed the inflammasome (231). This inflammasome is composed of the adaptor ASC (apoptosis-associated speck-like protein containing a CARD), pro-caspase-1, and an NLR family member, such as Ipaf (Ice protease-activating factor), NALP (NAcht LRR protein) 1, or NALP3/Cryopyrin (226, 231).
Oligomerization of these proteins through CARDCARD interactions results in activation of caspase-1, which subsequently cleaves the accumulated IL-1 precursor, eventually resulting in secretion of biologically active IL-1 (5, 169). Several families of inflammasomes have been identified, each recognizing different danger signals or PAMPs through their respective NLR (169) (Fig. 3).
FIG. 3. Recognition of PAMPs from different classes of microbial pathogens. Viruses, bacteria, fungi, and protozoa display several different PAMPs, some of which are shared between different classes of pathogens. Major PAMPs are nucleic acids, including DNA, dsRNA, ssRNA, and 5'-triphosphate RNA, as well as surface glycoproteins (GP), lipoproteins (LP), and membrane components (peptidoglycans [PG], lipoteichoic acid [LTA], LPS, and GPI anchors). These PAMPs are recognized by different families of PRRs.
For instance, NALP3 has been ascribed a role in recognition of ATP (226), uric acid crystals (232), viral RNA (168), and bacterial DNA (268), whereas both NALP3 and NALP1 have been demonstrated to mediate caspase-1 activation in response to bacterial MDP (85, 230). Intriguingly, NALP1 engages in a protein complex with NOD2 to mediate caspase-1 activation in response to MDP recognition (140), thus implying that NOD2 plays a dual role in both pro-IL-1 synthesis and caspase-1-dependent IL-1 β maturation. Recent data suggest that the composition of the inflammasome may be even more complex than first anticipated, since the cytosolic DNA receptor AIM2 was found to associate with ASC and form a caspase-1-activating inflammasome (136).
PATHOGEN RECOGNITION IN INNATE IMMUNITY
The repertoire of PRRs is very extensive, and similarly, the classes of pathogens recognized by PRRs are very diverse. A central feature of innate pathogen recognition is that microbes of quite different biochemical composition and with entirely different life cycles are recognized by relatively similar mechanisms by host PRRs (7). Moreover, an important property of this system is that no single class of pathogen is sensed by only one type of PRR. Rather, a number of different PRRs are engaged by a given pathogen via various PAMPs, hence securing a rapid and potent inflammatory response and also allowing for some specificity of the response.
Viruses
The outcome of virus-mediated PRR activation can range from an antiviral response that efficiently clears the infection to the establishment of a cellular environment that favors viral replication and spread (261). Viruses possess several structurally diverse PAMPs, including surface glycoproteins, DNA, and RNA species (261).
These immunostimulatory nucleotides may be present in the infecting virion or may be produced during viral replication, and the host is in possession of a broad range of viral nucleotide sensors. Whereas viral DNA is recognized by TLR9 and DAI, ssRNA is detected by TLR7 and TLR8, and finally, dsRNA and 5'-triphosphate RNA activate RLRs, TLR3, and PKR (261). Furthermore, several viral glycoproteins are recognized by TLR2 and TLR4 (7, 261).
For instance, the fusion protein from RSV activates TLR4 (199), whereas TLR2 is activated by different viruses or viral components, including measles virus hemagglutinin, cytomegalovirus, and herpes simplex virus (HSV) (35, 60, 198). Prominent examples of viral PAMPs and their recognition by PRRs are listed in Table 1, and a more detailed description of HSV recognition is presented below. HSV is an enveloped DNA virus with the two closely related subtypes HSV-1 and HSV-2. This important human pathogen can cause various diseases ranging from relatively mild illness, as in the case of gingivostomatitis, herpes labialis, and herpes genitalis, to severe and potentially fatal infections, including encephalitis, meningitis, and neonatal herpes infection (305). During an HSV infection, multiple mechanisms of pathogen recognition are operating, depending on the cell type and the stage of the viral replication cycle (301). First, HSV virions or virion surface glycoproteins interact with TLR2 on the cell surface (198). Although the molecular nature of the TLR2 agonist remains to be defined, TLR2 activation by HSV has been demonstrated to result in cytokine production (19) and to play a prominent role in HSV-1-associated immunopathology by contributing to lethal encephalitis in mice (198).
Another early response evoked by the incoming virus particle is a potent type I IFN response, which is induced by viral DNA and mediated by TLR9 (194, 220, 301). This response is independent of viral replication but is cell type specific and limited to plasmacytoid DCs (pDCs) (127, 301). It is interesting that recognition of viral DNA is dependent on the endosomal location of TLR9, suggesting that virion degradation in this compartment may be required for DNA to become accessible to TLR9 (30). The involvement of TLR-independent recognition systems in the early response to HSV infection has been strongly suggested by several individual reports (127, 223, 301) and further supported by in vivo data demonstrating that mice lacking TLR9 or myeloid differentiation primary-response gene 88 (MyD88) can still control HSV infection (194). Cell types other than pDCs may therefore have TLR-independent receptors that exert effective antiviral responses later during infection (127, 259, 301). Indeed, a recent study has demonstrated the involvement of RLRs in HSV-induced type I IFN production, which was abolished in fibroblasts unable to signal through this pathway (301). This is in agreement with the recent finding that dsRNA accumulates in the cytoplasm during infection of permissive cells with DNA viruses, including HSV, and thus represents a potential ligand for cytosolic dsRNA receptors (380). An alternative dsRNA-sensing molecule during HSV infection is PKR, which has been implicated in HSV-induced NF-B activation and IFN production in several studies, although mainly in non-pDCs. (223, 353). Finally, a role for DNAsensing proteins such as DAI has been suggested by data demonstrating that entry-dependent IFN production requires the presence of viral genomic DNA and proceeds through a mechanism independent of TLRs and viral replication (301). Collectively, HSV is detected by multiple cellular recognition systems, which operate in cell type- and time-dependent manners to trigger an antiviral response.
Gram-Positive Bacteria
The cell walls of gram-positive bacteria consist mainly of peptidoglycan composed of linear sugar chains of alternating N-acetylglucosamine and N-acetylmuramic acid, cross-linked by peptide bridges to form a large macromolecular structure surrounding the cytoplasmic membrane (9). Other important components include the glycolipid lipoteichoic acid anchored in the cytoplasmic membrane as well as lipoproteins embedded in the bacterial cell wall (9). As previously described, TLR2 plays a major role in the detection of gram-positive bacteria via recognition of cell wall PAMPs, including lipoteichoic acid, lipoproteins, and peptidoglycan (328, 395), although the recognition of peptidoglycan by TLR2 remains controversial due to the possibility of endotoxin contamination (366).
The importance of TLR2 in host defense against gram-positive bacteria is evidenced by studies in TLR2-deficient mice, which display increased susceptibility to challenge with Streptococcus pneumoniae and Staphylococcus aureus compared to wild-type mice (80, 224, 359). In addition, bacterial CpG DNA represents an important PAMP of gram-positive bacteria and is recognized by TLR9 (121). In order to achieve a potent inflammatory response, gram-positive bacteria are also able to trigger cytosolic PRRs, including NOD2 and the NALP1 inflammasome, both activated by the peptidoglycan-derivative MDP (102) (85). Further evidence about the role of PRRs in protection from gram-positive bacterial infection is listed in Table 1 and presented below in a description of PRR-mediated recognition of S. pneumoniae. The spectrum of diseases caused by S. pneumoniae is diverse, with invasive pneumococcal diseases such as pneumonia, sepsis, and meningitis representing a significant burden of disease in both developing and developed countries (192).
S. pneumoniae is endowed with several PAMPs, and, perhaps due to the high incidence, mortality, and morbidity associated with pneumococcal diseases, recognition of this pathogen has been extensively studied (192). As is characteristic for gram-positive bacteria, TLR2 is activated by the cell wall components peptidoglycan and lipoteichoic acid (262, 327, 395). Moreover, some data suggest that the important virulence factor pneumolysin may stimulate TLR4, but controversy exists as to the importance of TLR4 in the immune response to pneumococci (40, 222, 262). Studies using preparations of live pneumococci have demonstrated activation of both TLR2 and TLR9 in vitro, whereas TLR4 did not contribute significantly to the production of proinflammatory cytokines (262). Such findings underscore the strength of using preparations of entire live organisms, which in many contexts seem to be physiologically more relevant (258, 262, 263), although at the expense of specificity in the molecular characterization of PAMP-TLR interactions. The importance of TLR9 in the generation of an inflammatory response to pneumococci is supported by detection of residual immune activation present in TLR2/TLR4 double-knockout mice (123). Further evidence of the involvement of TLR9 has been gained from studies with TLR9-deficient mice displaying increased susceptibility to pneumococcal respiratory tract infection, which was attributable to impaired pneumococcal uptake and killing by macrophages (10). Finally, NOD proteins have been demonstrated to recognize intracellular S. pneumoniae and thus to represent cytosolic innate immune receptors for this organism (281).
Gram-Negative Bacteria
The gram-negative bacterial cell wall contains a thin layer of peptidoglycan adjacent to the cytoplasmic membrane and an outer membrane consisting of LPS, phospholipids, and proteins. LPS, which is also termed endotoxin, is composed of an O-linked polysaccharide attached to the lipid A moiety via the core polysaccharide and for most bacteria is crucial for viability (11).
LPS, and in particular the lipid A portion, is a prominent feature of gram-negative bacteria, being one of the most potent PAMPs known and responsible for the inflammatory response observed during endotoxic shock (7, 367). Lipid A has a mono- or biphosphorylated disaccharide backbone acetylated with fatty acids, and the levels of both phosphorylation and acylation determine the immunostimulatory potency of lipid A and LPS (9). Different bacteria produce structurally different lipid A and LPS molecules with various phosphorylations, numbers of acyl chains, and fatty acid compositions, which can profoundly affect bacterial virulence and immunogenicity and thus constitute one of the determinants of whether a bacterial strain is pathogenic or nonpathogenic (24).
LPS liberated from gram-negative bacteria associates with the extracellular acute-phase protein LPS-binding protein and then binds to the coreceptor CD14 expressed at the cell surface. This event allows transfer of LPS to the accessory molecule MD2, which is associated with the extracellular domain of TLR4, and is followed by TLR4 oligomerization and signaling (7). Accordingly, C3H/HeJ mice with nonfunctional TLR4 display impaired LPS responses and are highly susceptible to infection with gram-negative bacteria, such as Neisseria meningitidis and Salmonella enterica serotype Typhimurium (296). Furthermore, many gram-negative bacteria are simultaneously recognized by several PRRs in addition to TLR4; for instance, peptidoglycan and bacterial membrane proteins also stimulate TLR2 (88, 234, 328). Flagellin, being part of some gram-negative bacteria, is a strong activator of TLR5 (118) as demonstrated in experimental models of infection with Salmonella species, Legionella pneumophila, and Escherichia coli (15, 115, 335), and this potent PAMP also activates the Ipaf inflammasome (249). Increasing evidence suggests that TLR9 activation by unmethylated CpG DNA derived from bacterial genomes also plays an important role during infection with gram-negative bacteria, frequently in cooperation with other PRRs (26, 262). Finally, peptidoglycan derivatives of gramnegative bacteria are recognized in the cytosol by NOD1 and NOD2 (53, 102). A complex pattern of TLR activation by a single microbe is illustrated by the gram-negative bacterium N. meningitidis, which is recognized by three different TLRs, namely, TLR2, -4, and -9 (262). N. meningitidis is responsible for conditions ranging from colonization of the nasopharynx to chronic meningococcemia or severe acute diseases, including fulminant meningococcal meningitis and sepsis (82, 307). During initial studies involving purified bacterial components from N. meningitidis, the activation of TLR2 by the outer membrane protein porin (234) as well as recognition of meningococcal lipooligosaccharide (LOS) by TLR4 was established (403). Different strains of N. meningitidis produce structurally different LOSs exhibiting varied biological activity (297), and this may be reflected in differences in the ability to induce TLR4-mediated inflammatory signaling and hence in bacterial pathogenicity (7, 263). Studies using preparations of live N. meningitidis have confirmed the involvement of TLR2 and TLR4 and additionally have established a role for TLR9 in the proinflammatory response induced by N. meningitidis (262). Moreover, it was demonstrated that only live as opposed to heat-inactivated meningococci are able to activate TLR9 (262). Meningococci have mechanisms to enter into cellular endocytic vacuoles (343), where TLR9 is also located, and this mechanism may be dependent on bacterial viability, thus possibly explaining why TLR9 activation by meningococcal DNA was observed only when cells were infected with live bacteria (262). An important role of TLR9 in vivo was recently described in a murine model of meningococcal sepsis, in which TLR9-deficient mice displayed reduced survival and elevated levels of bacteremia (336a). At the cellular level, reduced signaling to NF-B and diminished expression of cytokines was observed in pDCs but not macrophages and bone marrow-derived DCs (336a). Collectively, these studies analyzing immune recognition of N. meningitidis demonstrate the utilization of several TLRs to recognize a pathogen and initiate an inflammatory response. Theoretically, such a strategy may enhance the immune response by engaging TLRs on multiple cell types and in a sequential manner, thus inducing a synergistic response and possibly avoiding immune evasion by the pathogen (26, 262).