Outline

1. Introduction of Toll-Like Receptors (TLRs) 
   1. TLRs structure, location and ligands
2. The Role of TLR-4 in Signal Transduction 
   1. Cell Signaling through TLR-4
      1. The MyD88- Dependent Pathway
      1. The MyD88- Independent Pathway
   1. Negative Regulation of TLR4 in Signal Transduction            
3. TLR-4 Signaling in Immune System
   1. The Role of TLR-4 in Multiple Sclerosis
   1. The Role of TLR-4 in Tuberculosis Infections

Concluding Remarks and Future Perspectives

Abstract

Toll like receptors (TLRs) recognize foreign organism and activate innate immune system against to this organism. Pathogens have conserved PAMPs (pathogen-associated microbial patterns) that are detected by TLRs. There are ten types of TLRs are discovered in human and thirteen in mice. Different TLRs are stimulated by different ligands. Ligands could be DNA of bacteria, flagellin, or peptidoglycan. TLRs have extracellular domain that is leucine-rich region and cytoplasmic tail that include TIR domain. The cell could produce cytokines or interferons against to pathogens. The locations of TLRs change with respect to the type of the receptor, it could be on the surface or in the endosomal compartment. Here we focus on specifically TLR4 which is located on the surface of the cell and stimulated by LPS. The mechanism of TLR4, its regulation and the role of TLR4 in Multiple Sclerosis and Tuberculosis Infections are summarized. 

TLRs structure, location and ligands

Toll protein is firstly identified in Drosophila and named as Toll to show scientists’ excitement about discovering this protein. It comes from German scientists’ reaction: das ist ja toll, it means that is great. It was shown that this protein is important for early innate immune system and to specify dorsal-ventral axis during early developmental process in Drosophila. 

Toll like receptors (TLRs) are important to recognize pathogens and activate immune system. These receptors are part of pattern-recognition receptors (PRRs) (Krishnan et al.,2007). In order to carry out recognition of microorganisms, pathogen-associated microbial patterns (PAMPs) have an essential role. These patterns are well conserved throughout evolution and many different pathogens such as bacteria, fungi, and virus have these patterns. These patterns could be the part of cell wall, or could be a compound of pathogen such as protein, lipopeptide, lipopolysaccharide, peptidoglycan. Also, flagella and nucleic acids of pathogens could have these patterns (Lu et al.,2008) . These PAMPs are detected by TLRs and this interaction activates the immune system. After recognition foreign organism, the cell could produce cytokine, chemokine, or interferon (Krishnan et al.,2007).

TLRs are transmembrane proteins and they classified as type I. These receptors have extracellular and intracellular domain. The specific features of these domains are that extracellular domain has leucine rich repeats (LRR) and intracellular domain has Toll/IL receptor (TIR) domain (Lu et al.,2008) . Because of their function in immune response, these receptors are commonly found spleen, lung, mucosal epithelial cells and leukocytes (Krishnan et al.,2007). Up to now, ten types of TLRs was discovered in human and thirteen types in mice. The locations of TLRs depend on the types of receptors. As it is illustrated on Figure I, TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are found at the surface of the cell. On the other hand, TLR3, TLR7, TLR7, and TLR9 are found at the inside of the cell (Lu et al.,2008). 

Each TLR has different types of ligands. TLR2 is a specific receptor to sense the component of the cell wall of the Gram (+) bacteria. TLR3 senses viral ds-DNA. Lipopolysaccharide of Gram (-) bacteria is a ligand for TLR4. Flagella stimulate TLR5. TLR7 and TLR8 are part of viral infection response and detect antiviral molecules. TLR9 is important for recognition of CpG DNA without methylation. TLR11 detect the pathogenic type of E.coli and Toxoplasma gondii.

Their function could change upon dimerization with another type of TLR. If TLR2 dimerize with TLR6, it detects diacylated lipoproteins. If TLR2 dimerize with TLR1, it detects triacylated lipoproteins. The dimerization of TLR1 with TLR4 causes decreasing LPS recognition of TLR4. (Lu et al.,2008)

Cell Signaling through TLR-4

TLR4 needs to specific PAMPs for activation. Not only LPS but also F protein that is found in respiratory syncytial virus and protein that is recognized on the envelope of the mouse mammary tumor virus could stimulate the TLR4 (Krishnan et al.,2007). Also, TLR4 could be affected by molecules that are found endogenous part of the cell such as fibronectin, HSP, β-defensin2 (Krishnan et al.,2007) . Though LPS is not the only ligand for TLR4, it is one of the most important one. It is found on outer membrane of Gram (-) bacteria. Three subparts determine LPS structure which are lipid A, core oligosaccharide, and O side chain. Among these subparts, lipid A is crucial because it participates in recognition of PAMPs by TLR4. The recognition of LPS by TLR4 is the process that involves a couple of proteins. As illustrated on Figure II, this process starts with binding LPS binding protein (LBP) to LPS. This protein carries the LPS to CD14 protein which is close to MD2/TLR4 complex (Lu et al.,2008). CD14 protein helps to recognition of LPS by MD2/TLR4 complex. After recognition process complete, TLR4 has to activate many other intracellular compounds. The TIR domain of the TLR4 has the main role to transmit the extracellular signal through inside the cell. The role of TIR domain is that pulling adaptor proteins to receptor that is bound to plasma membrane by using protein-protein interaction. The adaptor proteins also should have this TIR domain to carry out interaction. MyD88 (myeloid differentiation primary response gene 88), TRIF (TIR domain-containing adaptor inducing IFN-β), TRAM (TRIF-related adaptor molecule), TIRAP (TIR domain containing adaptor protein, Mal), and SARM (sterile α and heat-armadillo motifs) are the five of the adaptor protein that involve TIR domain. TRAM is involved in MyD88-independent pathway with TRIF. TRAM is adaptor protein between TLR4 and TRIF. TRAM is also associated with membrane with the help of myristoylation reaction (Lu et al.,2008). SARM is adaptor protein that is thought as an inhibitor; however, the researches still continue to clarify its role. TIRAP is associated with plasma membrane with the help of its phosphatidylinositol 4,5-biphosphate domain (PIP2). TIRAP is an adaptor protein between TLR4 and MyD88. One of the most important one is MyD88 that is used by all TLRs although there is one exception: TLR3. There are two different pathways that are MyD88-dependent pathway and MyD88-independent pathway to convey the signal throughout inside the cell. The response of cells could produce either proinflammatory cytokines as a result of MyD88-dependent pathway or Type I interferons as a result of MyD88-independent pathway. 

MyD88-dependent Pathway

Recognition of PAMPs causes another interaction between adaptor protein and TIR domain of TLRs. First intracellular interaction occurs between MyD88 and TIRAP that is interacted with TLR4. In this pathway, MyD88 is an adaptor protein that has not only TIR domain but also death domain. Interaction between TIR domain and MyD88 effect IRAK4 (interleukin 1 receptor associated kinase 4) that is also has death domain. IRAK4 additionally has kinase activity and IRAK1 is phosphorylated by IRAK4 (Lim et al.,2013). This phosphorylation causes activation of IRAK1. After activation of IRAK1, IRAK1 and IRAK4 dissociate from MyD88-TLR complex and bind to TRAF6 (TNF receptor associated factor 6) which is a part of a complex include UBC13 (ubiquitin conjugating enzyme 13) and UEV1A (ubiquitin conjugating enzyme E2 variant 1 isoform A) (Lu et al.,2008). This binding leads to ubiquitination of TRAF6. Upon this ubiquitination, TAB2, TAB3, TAK1, and TRAF6 become a complex and this complex is needed to activate TAK1 (transforming growth factor β activated kinase 1) (Kawai et al.,2006). TAK1 is effective on IKK complex which has IKKα, IKKβ and IKKγ subunits (Uematsu et al.,2007). This complex phosphorylates IKB inhibitor. This inhibition allows translocation of NFkB transcription factor to nucleus. This pathway results in producing proinflammatory cytokine. TAK1 also activates MAPK (mitogen-activated protein kinase). The activation of this pathway has an effect on AP-1 that is transcription factor affecting expression of proinflammatory cytokines. NFkB and MAPK are not only involved in MyD88-dependent pathway, they could also be used in MyD88independent pathway. IRF5 (interferon regulatory factor) is another regulatory factor that use this pathway to produce proinflammatory cytokines (Lu et al.,2008). However, its mechanism is being still investigated.

MyD88-independent Pathway

In this pathway, TRIF and TRAM have main role. Both have TIR domain and TRIF is used as adaptor protein instead of MyD88. TRIF has an effect on transcription factors which are IRF3, NFkB, and MAPK (Lu et al.,2008). To carry out NFkB activation, TRIF interacts with RIP1 with the help of Rip homotypic interaction motif (RHIM) that is found on both molecules (Lu et al.,2008). TRAF3 should be activated by TRIF for IRF3 activation. TRAF3 is important to activate TANK (TRAF family member associated NFkB activator), TBK1 (TANK binding kinase 1), and IKKi (Kawai et al.,2006). This complex helps to translocate of IRF3. This pathway results in producing Type I interferons by the translocation of IRF3 and NFkB.

Negative Regulators of TLR-4 Signalling

 Adapters are the main important factors in TLR signalling. The checkpoint systems in immune responses were created at diverse levels during TLR signalling which lead limitation in the duration and/or amount of the immune responses. Adapter molecules play important inhibitory roles during this checkpoint occurs. ST2 (suppression of tumorigenicity 2) is member of IL1R subgroup of TIR proteins separates Myd88 and MAL by causing inhibition in IL1R and TLR4 signalling. These events cannot inhibit TLR3 signalling (Brint et al., 2004). Myd88s is the splice variant of Myd88 prevents NFKb activation by displacing Myd88. The interaction of Myd88 does not occur with IRAK4 since Myd88 has lack interaction region for IRAK4 recruitment.  Therefore, IRAK4 cannot interact with IRAK1 by causing in the inhibition of IRAK1 phosphorylation. Finally, NKFb activation fails. 

 Viruses as the target of adapters negatively regulates signal transduction through TLR signalling. IL-1 and TLR4 signalling are blocked by vaccina virus A46R. Actually, A46R is the blocker for multiple TIR adapters where TIR separates MAL, Myd88, TRIF and TRAM (Stack et al., 2005) Another crucial protein NS3/4A in the negative regulation of TLR4 signalling is released by Hepatisis C virus result in proteolysis of TRIF (Li et al., 2005) Therefore, the evolution of viruses is one hallmark in antiviral immunity through TLR. 

 Another important inhibition in TLR4 signalling occurs through endothelial cell zinc finger proteins A20. The inhibition occurs by the activated TRAF6 interference with NFKb.A1 and A20 proteins negatively regulates LPS induced TLR4 signalling by means of PI3K signalling crosstalk. In the inhibition of PI3K signalling, IRAKM prevents TRAF6 and IRAK association (Kobayashi et al., 2002). Inhibitory adapters such as DOK1 and DOK2 inhibits LPS induced ERK activation. It is important to note that, designing selective inhibitors of adapters might be used therapeutically where they suppress TRAF6 by negatively regulating LPS signalling in TLRs.

The Role TLR4 in Multiple Sclerosis

 Recent evidences suggest that the central nervous system (CNS) has the ability to mount inflammation following infections and tissue damages (Perry et al., 1994). Inflammation is initiated by the TLRs, inflammasomes and scavenger receptors. TLRs expressed in microglia and astrocytes and TLR mediated immune response is triggered by exogenous pathogen associated patterns and endogenous danger associated molecular patterns. Multiple sclerosis (MS) is a demyelinating immune mediated disease occurs with neurodegenerative pathology and chronic inflammation. The detrimental role of TLR4 in MS display in the onset and progression of the disease. During MS pathogenesis, axons, dendrites and neurons are lost due to demyelinated inflammatory responses and both genetic an environmental factors play role in the progression of the disease albeit ethology of the disease is unknown. Experimental autoimmune encephalomyelitis (EAE) is the murine model of the MS in mice. In EAE, TLR4 activation stimulates pathogenic functions of T cells. For example, Th1 cell response is increased by means of LPS induced TLR4 activation (Schanre et al., 2001). LPS on TLR4 result in damages and death in oligodendrocytes and neurons in heterogeneous glial population. LPS also cause oligodendrocyte lose and hypomyelination via production of IL1beta after intracerebral injection to pupils of rats (Lehnardt et al., 2002) However, another study demonstrates the suppressive role of LPS on EAE where early life exposure to LPS promotes tolerogenic dendritic cells and regulatory T cells. In more recent study, granulocyte macrophage colony stimulating factor (GM-CSF) production is induced by

LPS where secretion was observed in endothelial cells, monocytes, astrocytes and T cells.

Therefore, LPS-mediated inflammation is promoted by LPS in the CNS as a result of upregulation in CD14 and TLR in microglia cells (Parajuli et al., 2012) 

 The adjuvants in immunization process by TLR4 signalling plays role in EAE model. The pertussis toxin as an adjuvant substance in EAE model distributes the TLR4 signalling in terms of controlling its inducing effect of the disease (Racke et al., 2005). The pertussis toxin administration result in T cell infiltration into the CNS whereby observations show that the increase in P-selectin expression and interaction between endothelial cells and leukocytes. The pertussion toxin modulation of EAE shows its influence only in TLR4 through Myd88 dependent and Myd88 independent pathways (Racke et al., 2005). 

 MS patients show elevated level of TLR4 and TLR3 expression in their microglia cells and astrocytes. Early and late active lesion differs in terms of the expression of TLR4 and TLR3 in cell types. In early active lesions, TLR3 and TLR4 expression is observed on vesicles within microglial cells, however, the expression of TLR4 and TLR3 is seen on surface of astrocytes in late active lesions. This situation might be the proof of the similar TLR3 and TLR4 microglial signal occurs in early and late lesions. Jack et al, (2005) suggest that activation of TLR3 and TLR4 induces the release of chemoattractant named CXCL-10 for CD4+ T cells. 

 Since LPS induced TLR4 activation has potential outcome in MS, TLR4 inhibition might be the target for the treatment of MS. Therefore, targeting anti-LPS agents might be the promising strategy to reduce inflammatory responses occur in MS pathogenesis. Anti-LPS agents have a capability to bind LPS with their strong affinity. Hence, they sequester LPS by abrogating its toxicity (Peri and Pizza, 2012)

 One potential targeting strategy is to utilize from synthetic lipids as TLR4 antagonists. The synthetic lipid E5564 block the endotoxin binding to TLR4 by inhibiting TNF-alpha production in phase 1 clinical trials (Peri and Pizza, 2012). The other antagonizing agent ibudilast acts on TLR4 by producing pro-inflammatory cytokines and inducing antiinflammatory cytokines.  This anti-inflammatory effect of the compound acts by inhibiting glial cells and attenuating the inflammation (Ledeboer et al., 2007)

The Role of TLR4 in Tuberculosis Infections

 Myocobacterium tuberculosis (MTB)infection occurs when a bacillus type of bacterium infects mostly to lungs and cause approximately 1.5 million deaths worldwide. In vitro studies demonstrate that MTB can activate the cells by TLR4 or TLR2 signaling pathway in a CD14 dependent manner. One study shows the elevated levels of susceptibility to MTB occurs in TLR4 deficient mice.  In that study, TLR4 deficient mice might comprise additional LPS signal receptors rather than TLR4 receptors, therefore, compensation may occur for the lacking TLR4 according to genetic background of mice (Vogel et al, 1999) Still, there need to be an investigation the mode of action of TLRs in MTB infection.

The TLR4 and Heat Shock Proteins

 The primary inflammatory mediators as a result of gram negative and gram positive bacteria reveal the immune response occurs through pattern recognition receptors. Khan et al. (2007), suggested that family of TLRs play role in those events. Endometriosis is the disease in pelvic environment stimulates inflammatory responses in the mode of different diseases. Heat shock proteins (HSPs) are the family of proteins which are produced by the response to stressful events in cells. Khan et al. (2008) looked for an answer to role of human HSPs and their association with the mechanism of TLRs in the immune cell mediated inflammation. Since human HSPs (HSP60, HSP70, HSP90) are expressed by human macrophages, endometrial cells, vascular endothelial cells and other target cell types, they investigated the recognition pattern of HSP60 and HSP70 in terms of TLR4 mediated cell signaling. Finally, they found HSP70 significantly stimulates the production of VEGF, HGF, IL-6 and TNFalpha from the endometrial macrophages in women with endometriosis. After following antiTLR4 antibody treatment, the endometrial cell growth significantly enhanced. These events suggest that HSP70 might be involved TLR4- mediated growth of endometrial cells and induction of pelvic inflammation. 

 The second challenging research by Bulut et al. (2005) demonstrated the role of MTB heat shock proteins in human endothelial cells. The purified Mycobacterim tuberculosis HSP65 and HSP70 induce NFkB activity in human endothelial cells. HSP65 signals are mediated with the interaction of  TLR4 during macrophage phagocytosis and Myd88, TIRAP,

TRIF and TRAM dependency is required in host-pathogen interactions and immune response. 

Concluding Remarks and Future Directions

 Although many of the knowledges have been investigated until now, there remains still some questions in terms of TLR signaling. Since we have a current knowledge on almost all mechanisms through cell signaling, the uncertainty occurs in terms of recognition of CpG oligonucleotides and bacterial DNA in TLR signaling against host DNA through the liberating events of the host DNA inside the body. Another consequence that we can gather from this review is that TLR2 and TLR4 is responsible signaling pathways in MTB infection. However, there is not so many knowledges in literature about the role of other TLRs. 

 In TLR4 signaling, both Myd88 dependent and independent pathways play important role. In EAE models for MS in mice, the LPS-induced TLR4 signaling elevates the neuroinflammation occurred. Therefore, one can utilize from TLR4 antagonists which are anti-LPS proteins, synthetic lipids and so on to reduce inflammation. In recent researches, literature also points out usage of chaperonins and humanized mAbs in terms of treating the MS. However, more clinical tests are needed to understand the role of those molecules during the treatment of MS. 

 Lastly, produced HSPs by both humans and bacteria in response to stress play important role in TLR4 signaling like the LPS. Recently, heat shock protein therapies emerged as very promising tools for the drug development (Soti et al., 2005). Therefore one can utilize those chaperones to prevent or reduce inflammation. Due to promising results in clinical phase trials, chaperone modulators might be the target as well as anti-LPS and TLR4 antagonists in terms delaying the prognosis of inflammatory diseases.

 As a result, we showed the role and mechanism of TLRs in the center of TLR4 in signaling pathways and associated diseases. 

References

Amanda L. Blasius and Bruce Beutler, (2010), Intracellular Toll-like Receptors, Immunity       (review article)

Khan KN, Kitajima M, Hiraki K, Fujishita A, Ishimaru T, Masuzaki H. Toll-like receptors in innate immunity: role of bacterial endotoxin and toll-like receptor 4 (TLR4) in endometrium, endometriosis and placenta. Inflamm Immun 2007;15:56–68. (review article)

Sõti, C., Nagy, E., Giricz, Z., Vígh, L., Csermely, P., & Ferdinandy, P. (2005). Heat shock proteins as emerging therapeutic targets. British journal of pharmacology, 146(6), 769-780 (review article)

Bulut, Y., Michelsen, K. S., Hayrapetian, L., Naiki, Y., Spallek, R., Singh, M., & Arditi, M. (2005). Mycobacterium tuberculosis heat shock proteins use diverse Toll-like receptor pathways to activate pro-inflammatory signals.Journal of Biological Chemistry, 280(22), 20961-20967.

Brint EK, Xu D, Liu H, Dunne A, McKenzie AN, O’Neill LA, Liew FY. ST2 is an inhibitör of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat Immunol 2004;5:373-9

Jayalakshmi Krishnan, Kumar Selvarajoo, Masa Tsuchiya, Gwang Lee and Sangdun Choi, (2007), Toll-like receptor signal transduction, Experimental and Molecular Medicine 

Khan, K. N., Kitajima, M., Imamura, T., Hiraki, K., Fujishita, A., Sekine, I., … & Masuzaki, H. (2008). Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70. Human reproduction, 23(10), 2210-2219.

Kian-Huat Lim, Louis M. Staudt, (2013), Toll-like Receptor Signaling, Cold Spring Harbor Perspectives in Biology (Research article)

Kobayashi K, Hernandez LD, Galan JE, Janeway CA Jr, Medzhitov R, Flavell RA. IRAK-M  is a negative regulator of Toll-like receptor signaling. Cell 2002;110:191-202

Lehnardt, S., Lachance, C., Patrizi, S., Lefebvre, S., Follett, P.L., Jensen, F.E., Rosenberg, P.A., Volpe, J.J., and Vartanian, T. (2002). The Toll-like receptor TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte injury in the CNS. J. Neurosci. 22, 2478–2486.

Li K, Foy E, Ferreon JC, Nakamura M, Ferreon AC, Ikeda M, Ray SC, Gale M Jr, Lemon  SM. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci USA 2005;102:2992-7

Parajuli, B., Sonobe, Y., Kawanokuchi, J., Doi, Y., Noda, M., Takeuchi, H., Mizuno, T., and  Suzumura, A. (2012). GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia. J. Neuroinflammation 9, 268.

Perry, V.H., Anthony, D.C., Bolton, S.J., and Brown H.C. (1997). The blood-brain barrier and  the inflammatory response. Mol. Med. Today 3, 335–341.

Racke, M.K., Hu, W., and Lovett-Racke, A.E. (2005). PTX cruiser: driving autoimmunity via  TLR4. Trends Immunol. 26, 289–291.

Satoshi Uematsu, Shizuo Akira, (2007), Toll-like Receptors and Type-I Interferons, Journal of  Biological Chemistry

Schnare, M., Barton, G.M., Holt, A.C., Takeda, K., Akira, S., and Medzhitov, R. (2001). Toll- like receptors control activation of adaptive immune responses. Nat. Immunol. 2, 947–950. Stack J, Haga IR, Schroder M, Bartlett NW, Maloney G, Reading PC, Fitzgerald KA, Smith  GL, Bowie AG. Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence. J Exp Med 2005;201:1007-18 Taro Kawai, Shizuo Akira, (2006), TLR Signaling, Elsevier

Vogel S, Johnson D, PereraPY, Medvedev A, Lariviere L, QureshiST, Malo D. 1999.  Functional characterization of the effect of the C3H/HeJ defect in mice that lack an Lps gene: in vivo evidence for a dominant negative mutation. J. Immunol. 162:5666–70

Yong-Chen Lu, Wen-Chen Yeh, Pamela S. Ohashi, (2008), LPS/TLR4 signal transduction pathway, Elsevier (Review article)