TLR-signaling networks: an integration of adaptor molecules, kinases, and cross-talk

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.

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.

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.

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.

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).

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.

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.