TLR-signaling Networks

Abstract

Toll-like receptors play a critical role in innate immunity by detecting invading pathogens. The ability of TLRs to engage different intracellular signaling molecules and cross-talk with other regulatory pathways is an important factor in shaping the type, magnitude, and duration of the inflammatory response. The present review will cover the fundamental signaling pathways utilized by TLRs and how these pathways regulate the innate immune response to pathogens. Abbreviations: TLR, Toll-like receptor; PRR, pattern recognition receptor; PAMP, pathogen-associated molecular pattern; LPS, lipopolysaccharide; APC, antigen-presenting cell; IL, interleukin; TIR, Toll/IL-1R homology; MyD88, myeloid differentiation factor 88; IFN, interferon; TRIF, TIR-domain-containing adapter-inducing interferon-β; IRAK, IL-1R-associated kinase; TAK1, TGF-β-activated kinase; TAB1, TAK1-binding protein; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B-cells; MAPK, mitogen-activated protein kinase; NLR, NOD-like receptors; LRR, leucine-rich repeats; DC, dendritic cell; PI3K, phosphoinositide 3-kinases; GSK3, glycogen synthase kinase-3; mTOR, mammalian target of rapamycin; DAF, decay-accelerating factor; IKK, IκB kinase; IRF, interferon regulatory factors; TBK1, TANK-binding kinase 1; CARD, caspase activation and recruitment domain; PYD, pyrin N-terminal homology domain; ATF, activating transcription factor; and PTEN, phosphatase and tensin homolog.

Introduction

The ability of the host’s immune system to initially recognize and respond to microbial components is largely mediated by the innate immune system via the expression of a family of type I transmembrane receptors, Toll-like receptors (TLRs) (Medzhitov et al., 1997, 1998; Yang et al., 1998). Activation of TLRs by microbial products leads to the engagement of a diverse number of intracellular signaling pathways that dictate the host inflammatory response (Kawai and Akira, 2007; Kumar et al., 2009a,b). Although the production of inflammatory cytokines upon TLR activation plays an important role in mediating the initial host defense against invading pathogens (Dinarello, 2000), an inability to regulate the nature, magnitude, or duration of the host’s inflammatory response can often mediate detrimental effects, as observed in chronic inflammatory diseases. In this review, we will cover TLR signaling pathways, TLR cross-talk, and how TLR signaling can be amplified or suppressed.

Toll-Like Receptors (TLRs)

TLRs are among the most well-studied and well-characterized pattern recognition receptors (PRRs), due to their ability to detect a variety of pathogen-associated molecular patterns (PAMPs), such as lipids, proteins, lipoproteins, and nucleic acids (Medzhitov et al., 1997, 1998; Yang et al., 1998). To date, 10 human TLRs have been identified, and each TLR has a specific set of ligands that it can detect (Fig. 1) (Kawai and Akira, 2007; Kumar et al., 2009a,b). Specifically, TLR4 recognizes the lipopolysaccharide (LPS) of Gram-negative bacteria. TLR2 can heterodimerize with TLR1 or TLR6, and recognize peptidoglycan, lipopeptide, and lipoproteins (Takeuchi et al., 1999, 2001, 2002). TLR3 recognizes double-stranded RNA (dsRNA). TLR5 has been shown to recognize bacterial flagellin. TLR7 and TLR8 can recognize imidazoquinolines and single-stranded RNA. TLR9 recognizes bacterial and viral CpG DNA motifs and the malaria pigment hemozoin. Although the ligand for TLR10 is currently unknown, it has been demonstrated that TLR10 can heterodimerize with TLR1 or TLR2 (Hasan et al., 2005). Upon recognition, TLRs can induce inflammatory cytokines, co-stimulatory molecules, type I interferons (IFNs), and chemokines. The ability of TLRs to facilitate the activation of and induction of immunomodulatory cytokines from antigen-presenting cells (APCs) such as dendritic cells or macrophages is critical in shaping the adaptive immune response. Distinct differences have been reported between the ability of TLR2 and TLR4 to favor the induction of Th2- or Th1-type immune responses, respectively, due to the capacity of TLR4 to promote IL-12 p70 production from APCs (Re and Strominger, 2001). Whereas all TLRs can mediate the production of inflammatory cytokines, activation of TLR3, TLR4, TLR7, TLR8, and TLR9 can result in the production of type I IFNs that are important for antiviral immune responses.

Figure 1.

TLRs, TLR ligands, and localization of TLRs. TLR2 (TLR2/1 or TLR2/6), TLR4, and TLR5 are located on the outer membrane of cells, whereas TLR3, TLR4 (initially located on the outer membrane), TLR7, TLR8, and TLR9 are located on endosomes. TLR2 recognizes peptidoglycan, mycobacterial lipoarabinomannan, P. gingivalis LPS, Leptospira LPS, glycosylphosphatidyl inositol mucin from Trypanosoma, hemagglutinin from the measles virus, and phospholipomannan from Candida. TLR2 can heterodimerize with TLR1 or TLR6 and imparts specificity for triacyl (TLR2/1) or diacyl (TLR2/6) lipoproteins. TLR3 recognizes dsRNA, while TLR4 recognizes LPS. TLR5 recognizes bacterial flagellin. TLR7 and TLR8 can recognize imidazoquinolines and single-stranded RNA. TLR9 recognizes CpG DNA motifs from viruses and bacteria, the malaria pigment hemozoin, and dsDNA. TLR10 can heterodimerize with TLR1 or TLR2.

Structurally, all TLRs are type I integral membrane proteins consisting of an ectodomain comprised of leucine-rich repeats (LRRs) and a cytoplasmic domain containing a Toll/IL-1R homology (TIR) domain, which is required for signaling (Medzhitov et al., 1997, 1998; Medvedev et al., 2000; Kawai and Akira, 2007; Kumar et al., 2009a,b). Whereas TLR2 (TLR2/1 or TLR2/6), TLR4, and TLR5 are located on the outer membrane of cells, TLR3, TLR7, TLR8, and TLR9 are located on endosomes (Fig. 1). Activation of TLRs sets forth a diverse array of intracellular signaling pathways that dictate the magnitude, type, and duration of the inflammatory response. A fundamental basis of TLR signaling is dependent upon the recruitment and association of adaptor molecules that contain the structurally conserved Toll/interleukin-1 receptor (TIR) domain. In part, specificity of a given TLR can be imparted via the interaction of its TIR domain with myeloid differentiation factor 88 (MyD88), TIRAP, TRIF, or TRAM (Medzhitov et al., 1998; Kawai et al., 1999; Fitzgerald et al., 2001; Horng et al., 2001, 2002; Kaisho et al., 2001; Yamamoto et al., 2003a,b). In turn, these adaptor molecules provide the necessary framework to recruit and activate downstream kinases and transcription factors that regulate the host inflammatory response and type I IFN production.

MyD88 Signaling

All TLRs, with the exception of TLR3, utilize MyD88 (Fig. 2). The TLR4/MyD88 signaling pathway is predominantly used to induce the expression of pro-inflammatory cytokines (Fig. 2). Upon TLR4 activation, both MyD88 and TIRAP are recruited through TIR-TIR interactions to the TLR4 receptor, with PIP2 playing an important role in the recruitment process (Fitzgerald et al., 2001; Horng et al., 2001, 2002; Yamamoto et al., 2003a,b; Kagan and Medzhitov, 2006). MyD88 interacts with IL-1R-associated kinase (IRAK)-4 through its death domain. In turn, IRAK-4 activates other members of the IRAK family, like IRAK-1 (Cao et al., 1996; Muzio et al., 1997; Li et al., 2000; Li et al., 2002). This process results in the recruitment/activation of TRAF6, along with other E2 ubiquitin protein ligases, which activate a complex containing TGF-β-activated kinase 1(TAK1), TAK1-binding protein 1(TAB1), TAB2, and TAB3 (Chen, 2005). Activation of the TAK1/TAB complex triggers both the MAPK and NF-κB signaling pathways (Wang et al., 2001). IKK-β, along with IKK-α and NEMO (IKK-γ), make up the IKK complex that mediates the phosphorylation of IκB-α, a NF-κB inhibitory protein that masks the nuclear localization signal of NF-κB. Upon phosphorylation, IκB-α is ubiquitinated and degraded, freeing NF-κB to translocate into the nucleus and initiate the transcription of inflammatory cytokines (Ghosh et al., 1998). TAK1/TAB activity results in the activation of kinases critical for growth and cytokine production, including MAPKs ERK1/2, JNK1/2, and p38 (Wang et al., 2001). Activation of both NF-κB and MAPKs can occur in the presence or absence of MyD88, although the latter results in delayed kinetics. Other TLRs, including TLR2, TLR5, TLR7/8, and TLR9, utilize MyD88 for signaling, and these pathways are shown in detail in Fig. 2.

Figure 2.

TLR adaptor molecules and signaling pathways. Signaling specificity of a given TLR can be imparted via the interaction of its TIR domain with myeloid differentiation factor 88 (MyD88), TIRAP, TRIF, or TRAM. TLR activation can induce pro-/anti-inflammatory cytokines, induction of co-stimulatory molecules, type I interferons (IFN-α/β), type II interferons (IFN-γ), and chemokines. All TLRs except TLR3 utilize MyD88 for the production of inflammatory cytokines or type I IFNs (TLR7, TLR8, and TLR9). TLR2 and TLR4 recruit MAL/TIRAP and MyD88 to their TIR domain for activation of NF-κB and MAPKs that regulate pro- and anti-inflammatory cytokine production. TLR5 signals by MyD88 for the activation of NF-κB and MAPKs. TLR4 can signal independently of MyD88 via the recruitment of TRAM and TRIF that activate IRF3 and delayed activation of MAPKs and NF-κB for the production of type I IFNs. TLR7, TRL8, and TLR9 signal by MyD88 for both the activation of IRF7 and NF-κB that regulates the production of type I IFNs and inflammatory cytokines, respectively. Upon ligand recognition by the TLR complex, TIRAP (utilized by TLR2 or TLR4) along with MyD88 is recruited to the TIR domain of TLR2 or TLR4. Sequentially, MyD88 recruits and activates IRAK4, which in turn can activate other IRAK family members, such as IRAK1. The downstream activation of TRAF6 by IRAK4/1 leads to the activation of a complex consisting of TAK1 and the TAB proteins. The TAK1/TAB complex activates both the MAPK and NF-κB pathways. Activation of the IKK complex (IKK-α, IKK-β, and NEMO) leads to IκB-α degradation, which exposes the NF-κB nuclear localization sequence and allows for NF-κB to translocate to the nucleus and initiate transcription. MyD88-independent signaling involves the recruitment of TRIF (for TLR3) to the TIR domain of TLR3 upon ligand recognition. To recruit TRIF, TLR4 requires an additional adaptor protein, TRAM. TRIF-mediated activation of TRAF3 leads to the activation of the non-canonical IKKs, TBK1 and IKKi. The subsequent activation of IRF3 leads to type I IFN production along with IL-10. TRIF recruitment and activation of TRAF6 and RIP1 lead to NF-κB activity through TAK1.

MyD88-Independent Signaling

In contrast to type II IFNs (IFN-γ) that require MyD88-signaling for their synthesis, the major outcome of MyD88 independent signaling for TLR4 is the production of type I IFNs (Fig. 2). TLR4 requires TRAM for the activation of TRIF that associates with TRAF3 and TRAF6 through a binding domain present on its N-terminus (Wang et al., 2001; Yamamoto et al., 2003a,b). The activation of TRAF6 and RIP1 by TRIF leads to NF-κB activation. TRAF3 mediates IRF3 activation by activating TBK1 and IKKi, both non-canonical IKKs. The phosphorylation of IRF3 by TBK1/IKKi leads to the nuclear translocation of IRF3 and the induction of IFN-β (Doyle et al., 2002). Activation of TRAF3 has been shown to be critically involved in regulating the production of type I interferons (IFNs) and the anti-inflammatory cytokine interleukin-10 (IL-10) (Cao et al., 1996; Hacker et al., 2006). However, recent work suggests that TRAF3 differentially controls MyD88- and TRIF-signaling, imparting specificity on the repertoire of cytokines produced (Tseng et al., 2010). In MyD88-dependent signaling, the ubiquitination and degradation of TRAF3 were demonstrated to be essential for the activation of MAPKs and inflammatory cytokine production. Interestingly, whereas the degradation of TRAF3 was needed for the production of pro-inflammatory cytokines, it was not required for type I IFN production (Tseng et al., 2010).

NOD/TLR Receptor Cross-Talk

NOD-like receptors (NLRs) are a cytoplasmic PRR family consisting of 3 domains: a leucine-rich repeats (LRR) domain utilized for ligand sensing, a NATCH domain required for self-oligomerization and activation, and an effector domain at the N-terminus that mediates interactions with other signaling proteins (Kanneganti et al., 2007) (Fig. 3). The NLR family possesses different effector domains that alter protein interactions. For example, the NOD subfamily contains a CARD effector domain, while the NALP possesses a PYD domain. NOD1 and 2, members of the NOD subfamily of NLR, recognize bacterial molecules involved in the metabolism of peptidoglycan such as IE-DAP and MDP and induce the transcription of pro-inflammatory cytokines via the activation of NF-κB (Fig. 3) (Chamaillard et al., 2003; Girardin et al., 2003). In addition, TLR activation induces the synthesis of IL-1β mRNA by activating NF-κB (Fig. 3). Pore-forming toxins, ATP-mediated activation of the pannexin-1 pore, or type III or type IV secretion systems allow for bacteria or bacterial products to gain entry into the cytosol (Fig. 3). Flagellin from Salmonella and Legionella can induce the Ipaf-dependent activation of caspase-1 that is required for processing the pro-form of IL-1β into the biologically active mature form of IL-1β. Anthrax toxin and MDP activate caspase-1 in a NALP1-dependent manner (Fig. 3). Nalp3 induces the activation of caspase-1 in response to whole bacterium, bacterial RNA, and endogenous components like uric acid or ATP (Fig. 3).

Figure 3.

NOD ligands and signaling pathways. The NLR family possesses different effector domains that alter protein interactions. The NOD and IPAF subfamily contains a CARD effector domain while the NALP subfamily possesses a PYD domain. NOD1 and NOD2 recognize bacterial molecules involved in the metabolism of peptidoglycan such as IE-DAP and MDP. Several NALPs have been shown to form inflammasomes upon activation leading to the processing of IL-1β. Pore-forming toxins, ATP-mediated activation of the pannexin-1 pore, or type III or type IV secretion systems allow for bacteria or bacterial products to gain entry into the cytosol. Flagellin from Salmonella and Legionella can induce the Ipaf-dependent activation of caspase-1. Anthrax toxin and MDP activate caspase-1 in a NALP1-dependent manner. Nalp3 induces the activation of caspase-1 in response to whole bacterium, bacterial RNA, and endogenous components like uric acid or ATP. Formation of the inflammasome complex leads to the recruitment of ASC. Since ASC contains both a CARD and a PYD domain, it mediates docking of other CARD-containing proteins into the inflammasome, such as caspase-1. Caspase 1 recruitment and activation lead to the processing of the proform of IL-1β. TLR stimulation induces NF-κB activation that leads to the production of pro-IL-1β.

The NALP subfamily is the largest among the NOD family, with over 14 members, each possessing a PYD domain (Tschopp et al., 2003). Several NALPs have been shown to form inflammasomes upon activation, leading to the processing of key inflammatory cytokines such as IL-1β (Agostini et al., 2004; Mariathasan et al., 2004, 2006) (Fig. 3). Formation of the inflammasome complex leads to the recruitment of ASC. Since ASC contains both a CARD and a PYD domain, it mediates docking of other CARD-containing proteins into the inflammasome, such as caspase-1. Caspase recruitment and activation lead to the processing of the proform of IL-1β.

TLRs and PI3K

The family of PI3K enzymes is largely responsible for the phosphorylation of phosphatidylinositol lipids in response to cellular stimuli. PI3K is a heterodimeric enzyme that consists of a regulatory (p85) and a catalytic (p110) subunit (Cantley, 2002). PI3K is often referred to as a dual-specific kinase, because it can phosphorylate both lipids and proteins. Activation of PI3K results in the generation of phosphatidylinositol 3, 4, 5 triphosphate (PIP3) from phosphatidylinositol 4, 5 bisphosphate (PIP2) (Fig. 4). The generation of PIP3 allows for the recruitment of signaling proteins, i.e., Akt, that possess pleckstrin homology domains. The phosphatase and tensin homolog (PTEN) negatively regulates the production of PIP3 by converting PIP3 to PIP2 (Fig. 4). Direct evidence for the involvement of PI3K in TLR-signaling was initially shown by Arbibe et al. (2000), who demonstrated that site-directed mutagenesis of specific tyrosine residues within the cytosolic domain of TLR2 resulted in a loss in the ability of p85 to associate with TLR2 and abrogated the ability of the TLR2 to induce NF-κB transcriptional activity. These studies were invaluable in defining that TLR-stimulated innate immune cells activated the PI3K pathway and gave insight into one of the several mechanisms by which TLRs can recruit and activate PI3K (Arbibe et al., 2000; Sarkar et al., 2004; Rhee et al., 2006; Santos-Sierra et al., 2009). Although these studies clearly identified that TLR-signaling resulted in the activation of the PI3K pathway, the functional relevance of this pathway was not immediately clear. Subsequent studies by Guha et al. (Guha and Mackman, 2002) demonstrated that the inhibition of PI3K resulted in the elevated production of several pro-inflammatory cytokines, and this effect was associated with increased NF-κB p65 activity. However, it was not until the generation of PI3K KO mice that the impact of the PI3K pathway on the host inflammatory response was appreciated. Studies by Fukao et al. (2002a,b) reported that mice deficient in the regulatory subunit of class I_A PI3K (p85-α) exhibited enhanced T-helper 1 (Th1)-like immune responses to an intestinal nematode and were unable to clear the infection. Also, p85-α KO mice on the BALB/c background were shown to be resistant to Leishmania major infection, unlike wild-type control mice, and exhibited augmented Th1-associated immunity. An assessment of the innate immune response in PI3K KO mice identified that the alterations in Th1 and Th2 immunity were likely the result of elevated IL-12 production by dendritic cells (Fukao et al., 2002a,b). It was subsequently demonstrated that the activation of the PI3K pathway by a TLR2-agonist exhibited a differential effect on the production of the prototypical anti-inflammatory cytokine, IL-10, and the pro-inflammatory cytokine IL-12 (Martin et al., 2003). Specifically, these studies showed that the inhibition of PI3K activity drastically reduced the production of IL-10, whereas IL-12 levels were enhanced (Martin et al., 2003). With both pharmacological and genetic approaches, it was shown that inhibition of PI3K resulted in the loss of several downstream targets within the PI3K pathway that were activated in LPS-stimulated cells (Guha and Mackman, 2002; Martin et al., 2003, 2005). LPS was shown to induce the phosphorylation of Akt on both threonine 308 and serine 473 in which the blockade of PI3K attenuated this site-specific phosphorylation (Guha and Mackman, 2002; Martin et al., 2003, 2005; Schabbauer et al., 2004). The direct inhibition of Akt in monocytes resulted in a pro-inflammatory phenotype similar to that observed with PI3K inhibition (Martin et al., 2005). The identification of which isoform of Akt was involved in regulating the downstream signaling properties of PI3K in innate immune cells has recently been elucidated by the findings of Androulidaki et al. using mice deficient in Akt1 or Akt2 (Androulidaki et al., 2009). Akt1 KO mice exhibited a hyper-inflammatory response to LPS and did not develop tolerance to endotoxin. Interestingly, while mice deficient in Akt1 produced elevated levels of inflammatory mediators in response to LPS, mice deficient in Akt2 did not exhibit the same phenotype.

Figure 4.

The role of the PI3K pathway in TLR-signaling. TLR stimulation activates the PI3K/Akt signaling pathway. Upon TLR activation, PI3K is recruited and converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3), which allows for the recruitment of signaling molecules that possess a plekstrin homology domain, i.e., Akt. Activation of Akt by PDK1 (Thr308) and mTORC2 (Ser473) results in its full activation. Activated Akt can phosphorylate GSK3-α (Ser21) or GSK3-β (Ser9), and this site-specific phosphorylation results in the inactivation of GSK3. Inactivation of GSK3 promotes increased nuclear levels of CREB (Ser133) that displace NF-κB p65 (Ser273) from the co-activator of transcription CBP. The enhanced transcriptional activity of CREB and reduced transcriptional activity of NF-κB p65 result in increased IL-10 production while concurrently suppressing the levels of pro-inflammatory cytokines. Alternately, activated Akt can inhibit GSK3 activity indirectly through mTORC1. Active Akt inhibits the TCS2/1 complex. Once the TSC2/1 complex is inhibited, mTORC1 is activated, which, in turn, phosphorylates and activates both S6K isoforms, p70 and p85 S6K. S6K has been shown to affect the phosphorylation of GSK3, but it is unknown if the phosphorylation of GSK3 is due to a direct or indirect effect of S6K activity. Inhibition of mTORC1 with rapamycin attenuates phosphorylation of GSK3 and STAT3, augments NF-κB activation, and increases pro-inflammatory cytokine production (TNF and IL-12).

Both PI3K and Akt were identified as kinases involved in the ability of TLRs to mediate the phosphorylation and inactivation of GSK3-β (serine 9) (Guha and Mackman, 2002; Martin et al., 2003). siRNA-mediated knockdown of GSK3-β or pharmacological inhibition of GSK3 with a panel of different GSK3-selective inhibitors suppressed the production of IL-1β, IL-6, TNF, and IL-12, whereas the production of IL-10 was increased in TLR2-, TLR4-, TLR5- and TLR9-stimulated monocytes (Martin et al., 2005). The importance of the GSK3-β isoform in controlling the inflammatory response was confirmed in mouse embryonic fibroblasts that were deficient in GSK3-β (Fig. 4). Due to the capacity of GSK3 to differentially control the production of IL-10 and IL-12, Ohtani et al. set forth to determine the in vivo relevance of GSK3 inhibition by assessing the ability of GSK3 to alter Th1- and Th2-responses in a Listeria major infection model (Ohtani et al., 2008). For these studies, Ohtani et al. used PI3K (p85α) KO mice on the BALB/c background, which, unlike their wild-type BALB/c littermate controls, are able to mount an effective Th1 response. In contrast to PI3K KO mice, inhibition of GSK3 in PI3K KO mice resulted in enhanced footpad swelling, and these mice were unable to control the infection to L. major.

Studies by Hu et al. have identified that GSK3 is involved in the ability of IFN-γ to suppress TLR2-induced IL-10 production (Hu et al., 2006). These studies reported that IFN-γ treatment of macrophages augmented the degradation of the inhibitory protein molecule IκB-α and increased NF-κB binding in TLR2-stimulated cells. In contrast, IFN-γ treatment suppressed the phosphorylated levels of GSK3-α/GSK3-β, increased the kinase activity of GSK3, and attenuated the IL-10-dependent phosphorylation of STAT3 in TLR2-stimulated macrophages. Inhibition of GSK3 abolished the ability of IFN-γ to suppress the levels of IL-10 produced by TLR2-stimulated macrophages. With siRNA targeting GSK3-β, or chimeric mice reconstituted with GSK3-β-deficient fetal liver cells, it was shown that the capability of IFN-γ to increase GSK3-β but not GSK3-α activity was responsible for the reduced IL-10 levels produced by TLR2-stimulated cells (Fig. 4). These findings identified GSK3-β as an instrumental regulator of the inflammatory potential of IFN-γ-signaling and helped explain how IFN-γ dampened the production of the anti-inflammatory cytokine IL-10.

Recent studies have also identified that another downstream target of the PI3K pathway, mTORC1, is involved in differentially regulating the levels of pro- and anti-inflammatory cytokines produced by innate immune cells (Fig. 4) (Ohtani et al., 2008; Weichhart et al., 2008; Turnquist et al., 2010). Upon PI3K-Akt activation, Akt can phosphorylate and inactivate the tuberous sclerosis complex-(TSC1/TSC2) Rheb protein complex that impedes mTORC1 activity (Fig. 4). From studies with the mTORC1 inhibitor rapamycin, it has been reported that mTORC1 activity positively regulates IL-10 production while concurrently suppressing the levels of IL-12 and TNF produced by LPS-stimulated cells (Weichhart et al., 2008). Inhibition of mTORC1 in LPS-stimulated cells has been shown to attenuate the phosphorylation of several targets of MTOR, including p70S6K and 4-EBP1, as well as decrease the levels of phosphorylated STAT3 (Fig. 4) (Ohtani et al., 2008; Weichhart et al., 2008; Turnquist et al., 2010). In contrast, mTORC1 inhibition potently increased NF-κB activity, and this has been attributed to the ability of mTORC1 inhibition to increase the levels of IL-12 produced by LPS-stimulated cells (Weichhart et al., 2008). Interestingly, recent studies by Turnquist et al. have provided evidence that inhibiting mTORC1 with rapamycin resulted in the loss of GSK3-α/β (S21/S9), an event that is required to inactivate GSK3 (Frame et al., 2001; Turnquist et al., 2010). These studies also showed that the increased production of IL-12 by mTORC1-inhibited DC could be suppressed by use of the GSK3 inhibitor lithium (Turnquist et al., 2010). Thus, these studies suggest a possible convergence between mTORC1 and GSK3. Interestingly, studies by Gulati et al. demonstrated that siRNA-mediated knockdown in p70/p85S6K1, the downstream target of mTORC1, abrogated the phosphorylation of GSK3 (Fig. 4) (Gulati et al., 2008). Moreover, studies by Zhang et al. showed that p70/p85S6K1 regulated the phosphorylation of GSK3 when Akt activity was inhibited (Fig. 4) (Zhang et al., 2006). Clearly, future studies will be required to dissect the molecular mechanisms regulating the inflammatory properties of MTOR and how its ability to affect GSK3 activity is involved in this process.

TLRs and Complement

Complement and TLRs are rapidly activated by most pathogens upon encounter with the host, and common microbial molecules like bacterial lipopolysaccharide, CpG DNA, and yeast zymosan can act as both complement activators and TLR ligands. In this context, cross-talk between complement and TLRs is essential to appropriate coordination of the early innate response to infection (Hajishengallis and Lambris, 2010). Although Porphyromonas gingivalis inhibits the complement cascade regardless of the initiation mechanism (Popadiak et al., 2007), the bacterium employs its C5 convertase-like enzymes (HRgpA, and RgpB gingipains) to generate biologically active C5a (Wingrove et al., 1992; Popadiak et al., 2007). Upon C5aR binding, C5a stimulates Gαi-dependent intracellular Ca²⁺ signaling which synergistically enhances the cAMP response mediated by P. gingivalis-induced TLR2 activation (Fig. 5). Downstream events in this cross-talk pathway include the activation of the cAMP-dependent protein kinase A (PKA), which inactivates glycogen synthase kinase-3β (GSK3β) and impairs the inducible nitrogen synthase (iNOS)-dependent killing of P. gingivalis in macrophages (Fig. 5) (Wang et al., 2010). This exploited cross-talk pathway is also operational in vivo, and consequently C5aR-deficient mice or normal mice treated with a specific C5aR antagonist can clear P. gingivalis more effectively than untreated normal controls (Wang et al., 2010). Interestingly, unlike C5a, the C5b remnant is readily degraded to apparently prevent activation of the terminal complement pathway and formation of the membrane attack complex (Wingrove et al., 1992; Popadiak et al., 2007).

Figure 5.

TLR and complement cross-talk. Complement activation can suppress the production of IL-12 family member cytokines. Recently, it has been demonstrated that concomitant TLR2/1- and C5aR-signaling by P. gingivalis increased the intracellular levels of cAMP that resulted in the activation of PKA and the inactivation of GSK3-β, and suppressed iNOS-mediated killing. The fimbriae of P. gingivalis interact with TLR2 and CD14, inducing Rac1 and PI3K activity. The sequential activation of CD11b/CD18 enhances binding affinity to ICAM-1, which promotes monocyte-endothelial cell interactions.

In the absence of C5aR signaling, P. gingivalis uses a similar but less potent mechanism to undermine TLR2-dependent antimicrobial responses in macrophages (Wang et al., 2010). In this case, the cross-talk is between TLR2 and CXCR4 (Fig. 5) (Hajishengallis et al., 2008). Specifically, the concomitant activation of CXCR4 and TLR2 by P. gingivalis fimbriae induces cAMP-dependent PKA signaling, which in turn suppresses TLR2-dependent nitric oxide in response to the pathogen (Hajishengallis et al., 2008). The relevance of this in vitro evasion mechanism was confirmed in vivo. Indeed, mice treated with a specific CXCR4 antagonist display increased production of nitric oxide and enhanced ability to control P. gingivalis infection compared with untreated control mice (Hajishengallis et al., 2008).

Following activation by P. gingivalis, TLR2 induces distinct downstream signaling cascades (Hajishengallis et al., 2009). One of the cascades is MyD88-dependent, leads to induction of pro-inflammatory and antimicrobial responses, and represents the pathway that is undermined by the pathogen through C5aR and CXCR4 exploitation. The other cascade is a pro-adhesive pathway that culminates in the induction of the high-affinity conformation of CR3 (Hajishengallis et al., 2009). Specifically, P. gingivalis induces TLR2 inside-out signaling, which proceeds through Rac1, PI3K, and cytohesin-1 that transactivates CR3 (Fig. 5) (Harokopakis and Hajishengallis, 2005). This cross-talk is facilitated by the property of CR3 to cluster with TLR2 in lipid rafts of P. gingivalis-stimulated cells (Hajishengallis et al., 2006a). Once transactivated, however, CR3 becomes a target of subversive activity by P. gingivalis. P. gingivalis uses its fimbriae to bind CR3, which in turn mediates the uptake of this oral pathogen by macrophages (Hajishengallis et al., 2006b), but this phagocytic mechanism does not promote the killing of P. gingivalis (Wang et al., 2007). In contrast, when CR3 is blocked or genetically ablated, P. gingivalis phagocytosis via alternative receptors leads to dramatically enhanced intracellular killing (Wang et al., 2007). CR3 ligation by P. gingivalis also activates outside-in signaling and ERK1/2 activation, which in turn selectively inhibits mRNA expression of the IL-12 p35 and p40 subunits and production of IL-12 protein (Hajishengallis et al., 2007). In vivo, CR3-deficient mice elicit higher levels of IL-12 (and secondarily increased interferon-γ production) and display enhanced clearance of P. gingivalis infection compared with wild-type controls (Hajishengallis et al., 2007). Importantly, CR3 blockade with a specific antagonist suppresses P. gingivalis induction of periodontal bone loss in mice (Hajishengallis et al., 2007). In this evasion strategy, P. gingivalis has apparently co-opted a natural immunosuppressive mechanism. Specifically, CR3 is heavily committed with phagocytosis of iC3b-coated apoptotic cells, which are not normally recognized as dangerous (Mevorach et al., 1998; Kim et al., 2004). This does not warrant a vigorous host response, and, in fact, IL-12 induction production is inhibited following phagocytosis of apoptotic cells by macrophages (Kim et al., 2004). In general, CR3-mediated phagocytosis of iC3b-opsonized apoptotic cells induces a tolerogenic phenotype in phagocytes characterized by reduced expression of co-stimulatory molecules and pro-inflammatory cytokines and attenuated oxidative burst (Wright and Silverstein, 1983; Verbovetski et al., 2002; Vieira et al., 2002; Morelli et al., 2003; Kim et al., 2004; Lowell, 2006).

TLR Negative Regulation

Several mechanisms have been identified that negatively regulate TLR signaling, including the down-regulation of TLR expression, interference with ligand recognition, and inhibition of the intracellular TLR signaling pathway. Upon TLR stimulation, increased expression or activation of select proteins can diminish TLR responses (Fig. 6). TLR4 stimulation induces IRF4 expression that can interfere with MyD88/IRF5 interactions (Negishi et al., 2005). The increased competition for MyD88 binding leads to decreased IRF5 activation and reduced nuclear translocation. Early induction of the transcription factor ATF3 by LPS can suppress the expression of several TLR4 genes through a negative feedback loop (Gilchrist et al., 2006). Manipulation of chromatin structure is a likely mechanism for the suppression of IL-6 and IL-12 p40 production by ATF3. The inducible IκB protein IκBNS can also suppress late NF-κB gene expression. Interestingly, IκBNS -/- cells have elevated IL-6 and IL-12 p40 expression upon LPS stimulation, but other IRF3-mediated gene expression is unchanged (Kuwata et al., 2006).

Figure 6.

Negative regulation of TLR signaling. TLR signaling can be negatively controlled by multiple cellular mechanisms. Stimulation of TLR4 generates IRF4 that inhibits MyD88/IRF5 interactions. ATF3 can be generated during TLR4 signaling and can suppress the expression of IL-6 and IL-12 p40. The inducible IκB protein IκBNS inhibits NF-κB activity. The tyrosine kinases Dok1 and Dok2 are constitutively expressed, become activated upon TLR4 stimulation, and inhibit ERK1/2 signaling. β-arrestin interacts with TRAF6, preventing TRAF6 auto-ubiquitination, and this leads to diminished AP-1 and NF-κB activation. SOCS-1-mediated ubiquitination of TIRAP leads to its degradation and inhibits TLR2 or TLR4 signaling. Up-regulation of NF-κB p50 homodimers can inhibit NF-κB transcription, since NF-κB p50 lacks a transactivation domain. In endotoxin-tolerized cells, a defect is observed in the recruitment of MyD88 to TLR4 that results in suppressed MAPK and NF-κB activity.

Some proteins seem to inhibit only select TLRs. Dok1 and Dok2 are intracellular proteins that inhibit specific branches of TLR signaling (Fig. 6) (Shinohara et al., 2005). These tyrosine kinases are constitutively expressed and become activated shortly after TLR4 stimulation and inhibit ERK signaling, while p38, JNK1/2, and NF-κB are not affected. The inhibitory properties of the Doks seem to be exclusive to TLR4, despite the significant overlap in TLR signaling pathways.

β-arrestins are also potent inhibitors of TLR signaling (Fig. 6). Interactions between β-arrestins and TRAF6 prevent TRAF6 auto-ubiquitination and lead to diminished AP-1 and NF-κB activation (Wang et al., 2006). SOCS-1 is another potent inhibitor of TLR signaling. SOCS-1-mediated ubiquitination of TIRAP leads to its degradation and blunts both TLR2 and TLR4 signaling (Mansell et al., 2006).

An initial exposure to endotoxin, i.e., LPS, has been shown to induce a diminished inflammatory response upon subsequent exposure. This phenomenon is called ‘endotoxin tolerance’ and has been described in both human and murine models (Dobrovolskaia and Vogel, 2002; Cavaillon and Adib-Conquy, 2006; Biswas et al., 2007; Biswas and Tergaonkar, 2007; del Fresno et al., 2009). Tolerized macrophages produce reduced levels of pro-inflammatory cytokines and nitric oxide in response to LPS. Several mechanisms have been described to account for the diminished responses. Mice deficient in SOCS-1 or SHIP fail to develop endotoxin tolerance (Nakagawa et al., 2002; Sly et al., 2004; Rauh et al., 2005). However, these proteins were not correlated with the induction of endotoxin tolerance in humans (del Fresno et al., 2009). Recruitment of key upstream proteins in TLR signaling also is affected in endotoxin tolerance. Tolerized cells recruit less MyD88 to the TLR4 receptor, diminishing MyD88/ IRAK activation (Fig. 6) (Medvedev et al., 2002; Biswas et al., 2007; Piao et al., 2008). As a consequence, both NF-κB and MAPK activity is lower in endotoxin-tolerant cells.

Several other mechanisms have been implicated in the diminished activation of NF-κB in endotoxin tolerance. Up-regulation of p50 homodimers has been observed in tolerized cells (Fig. 6) (Adib-Conquy et al., 2000; Ziegler-Heitbrock, 2001). Since p50 homodimers have the ability to bind NF-κB sites, yet lack a transactivating domain, p50 homodimer binding to NF-κB sites can prevent binding of p65/p50 and attenuate NF-κB gene transcription. The in vivo relevance of p50 in endotoxin tolerance has also been noted in p50^-/- mice. Upon repeated treatment with LPS, p50^-/- mouse macrophages do not develop endotoxin tolerance.

A variety of cellular changes occurs in endotoxin-tolerized cells. Phagocytosis is increased in tolerized monocytes. The ability of tolerized cells to present antigen is also impeded (del Fresno et al., 2009). Down-regulation of the MHC Class II molecules has been noted in endotoxin-tolerant monocytes and results in impaired T-cell activation and proliferation (Wolk et al., 2000, 2003; Monneret et al., 2004). It is important to note, however, that endotoxin tolerance does not globally diminish the cytokine response. Production of anti-inflammatory cytokines such as IL-10, TGF-β, IL-1R agonist, and SOCS-1 is elevated in endotoxin-tolerized cells (Biswas and Lopez-Collazo, 2009).

Conclusion

The identification and characterization of the intracellular signaling pathways regulating TLR responses are just beginning to be elucidated. The ability of TLRs to engage different adaptor molecules, cross-talk with other regulatory pathways, and activate or suppress a plethora of kinases that are involved in a multitude of signaling cascades is an important factor in shaping the type, magnitude, and duration of the inflammatory response. The diverse number of signaling pathways affected by the innate immune system has clearly shifted the view of the innate immune response away from a somewhat one-dimensional response to a multifunctional immune response that displays an ability to finely tune the innate and adaptive system. Further examinations of these pathways are needed for a better understanding of how the host inflammatory response is regulated, as well as for identification of the cellular mechanisms by which pathogens exploit these signaling pathways to mediate disease.