Alternative titles; symbols
HGNC Approved Gene Symbol: TLR4
Cytogenetic location: 9q33.1 Genomic coordinates (GRCh38) : 9:117,704,403-117,724,735 (from NCBI)
In Drosophila, the Toll transmembrane receptor plays a central role in the signaling pathways that control dorsal-ventral axis formation and the innate nonspecific immune response. TLR4, the human homolog of Drosophila Toll, is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat region and an intracellular domain homologous to that of human interleukin-1 receptor (IL1R; 147810). Signaling through TLR4 and Toll parallels the signaling pathway induced by IL1R in mammalian cells in that both signal through the NF-kappa-B (see 164011) pathway (summary by Medzhitov et al., 1997).
By searching an EST database for human Toll homologs, Medzhitov et al. (1997) identified a sequence encoding TLR4, or human Toll. The predicted 841-amino acid protein contains the characteristic Toll motifs: an extracellular leucine-rich repeat (LRR) domain and a cytoplasmic IL1R-like region. Northern blot analysis revealed that TLR4 was expressed as 4- and 5-kb transcripts in a tissue-specific pattern.
Independently, Rock et al. (1998) identified cDNA sequences from TLR4 and 4 other TLR genes. By Northern blot analysis, they determined the expression patterns of all 5 genes. TLR4 was expressed as a 7-kb mRNA in placenta only. Like TLR4, all the TLRs contain extracellular LRR domain arrays and a cytoplasmic signaling (Toll homology or TH) domain. Structural analysis predicted that the TH domain forms a parallel beta/alpha fold with an acidic active site. The predicted structure is topologically identical to the structures of response regulators in bacterial 2-component signaling pathways, leading Rock et al. (1998) to propose that a signaling route coupling morphogenesis and primitive immunity in insects, plants, and animals may have roots in bacterial 2-component pathways.
Rehli et al. (2000) determined that the amino acid sequence proposed by Rock et al. (1998) is preceded by an in-frame stop codon leading to a truncated, nonfunctional protein. Primer extension analysis detected a major start site residing 190 bp upstream of the start codon, with a minor start site 74 bp further upstream.
Rock et al. (1998) mapped the TLR4 gene to chromosome 9q32-q33 by fluorescence in situ hybridization.
Medzhitov et al. (1997) found that a constitutively active mutant TLR4 could activate NF-kappa-B and the expression of NF-kappa-B-controlled genes when expressed in human cells.
Noting that TLR4 expression is relatively high in the heart, Frantz et al. (1999) examined TLR4 mRNA and protein abundance in isolated cellular constituents of cardiac muscle and in normal and abnormal murine, rat, and human myocardium. Constitutive expression of this vertebrate Toll homolog was found in normal cardiac muscle, almost exclusively within cardiac myocytes. Dissociation of heart tissue, followed by isolation and primary culture of cardiac myocytes and coronary microvascular endothelial cells, was found to result in robust TLR4 expression in both cell types. In tissue sections from hearts of humans with cardiomyopathies and of rodents with experimental cardiac dysfunction, myocyte TLR4 expression became more focal and intense. Frantz et al. (1999) concluded that increased expression and signaling by TLR4, and perhaps other Toll homologs, may contribute to the activation of innate immunity in injured myocardium.
Using Northern blot analysis, Muzio et al. (2000) determined the differential expression pattern of the TLRs in leukocytes. TLR4, like TLR2 (603028) and TLR5 (603031), was expressed in myelomonocytic cells. Exposure to bacterial products or proinflammatory cytokines increased TLR4 expression, and IL10 (124092) blocked this effect.
Harju et al. (2001) found that expression of Tlr2 and Tlr4 increased in fetal mouse lung with age. Expression increased sharply after birth and into adulthood. In contrast, no developmental trends were detectable in liver. In placenta, expression of Tlr4 decreased during the second half of pregnancy, while Tlr2 expression was constant and significantly lower throughout gestation.
Lipopolysaccharide (LPS) binds to LBP (151990), an opsonin for membrane-anchored CD14 (158120). In a review, Aderem and Ulevitch (2000) underlined the importance of transmembrane Toll-like receptors, and of TLR4 in particular, in understanding how a transmembrane signal is linked to LPS-induced activation. The LPS-LBP-CD14 ternary complex activates TLR4, which signals by Toll homology domains through the adaptor protein MYD88 (602170). The N-terminal death domain of MYD88 undergoes homophilic interaction with the death domain of the serine kinase IRAK (300283); IRAK is then autophosphorylated and forms a complex with TRAF6 (602355), ultimately leading to I-kappa-B (see 164008) activation and NFKB translocation to the nucleus. A secreted protein, MD2 (LY96; 605243), binds to the extracellular domain of TLR4, where it facilitates LPS responsiveness, possibly by stabilizing TLR4 dimers. TLR4 appears to be the major, and possibly the exclusive, receptor for LPS isolated from most gram-negative organisms.
Noting the restriction of TLR4 expression to endothelial cells, B cells, and myeloid cells, and the importance of TLR4 in responses to LPS, Rehli et al. (2000) used promoter analysis to define the factors governing TLR4 expression in macrophages. By shotgun cloning and sequencing of a BAC, Rehli et al. (2000) obtained a 19-kb sequence containing the TLR4 gene and showed that the amino acid sequence proposed by Rock et al. (1998) is preceded by an in-frame stop codon leading to a truncated, nonfunctional protein. Only transcripts containing exons I, III, and IV yield the proper TLR4 protein. Primer extension analysis detected a major start site residing 190 bp upstream of the start codon, with a minor start site 74 bp further upstream. RT-PCR analysis indicated that TLR4 is mainly expressed by myeloid cells. Luciferase reporter assays localized the macrophage-specific gene expression regulatory region to sequences approximately 75 bp upstream of the major transcriptional start site. Sequence analysis revealed that the proximal promoter regions of mouse and human TLR4 are characterized by the absence of TATA, SP1 (189906), and CCAAT boxes, consensus initiator sequences, and GC-rich sequences, but instead contain several purine-rich elements with a 5-prime-GGAA-3-prime core on either strand, characteristic of many myeloid-specific genes. The proximal promoter regions also contain consensus binding sites for ETS (e.g., PU.1, or SPI1; 165170) transcription factors. Gel shift and supershift analyses determined that PU.1, interferon consensus sequence-binding protein-1 (ICSBP1; 601565), and, less strongly, PU.1-interacting protein (PIP), bind to adjacent elements within the -75 promoter and participate in the basal regulation of TLR4 expression in myeloid cells. Gamma-interferon (IFNG; 147570) does not enhance this activity, suggesting that TLR4 expression is constitutive rather than inducible.
Using RT-PCR and ELISA analysis, Kadowaki et al. (2001) defined the differential expression of TLR1 through TLR10 and the pathogen-associated molecular pattern recognition profiles and cytokine production patterns of monocytes and dendritic cell precursors. They concluded that neither monocytes nor dendritic cell precursors can respond to all microbial antigens and that they have limited functional plasticity.
Using agonists specific for TLR4 (LPS) and TLR2 (peptidoglycan of S. aureus), Re and Strominger (2001) showed differential cytokine expression patterns in dendritic cells (DCs) by RNase protection and ELISA analysis. Stimulation of TLR4 but not of TLR2 promoted expression of the Th1-inducing cytokine IL12 p70 heterodimer (see IL12B; 161561) and the IFNG inducible chemokine protein IP10 (CXCL10; 147310). TLR2 stimulation resulted in the release of the IL12 inhibitory p40 homodimer, which favors Th2 development, and the chemokines IL8 (146930) and IL23/p19 (IL23A; 605580). Re and Strominger (2001) suggested that the failure of TLR2-stimulated DCs to produce CXCL10 may result in defective recruitment of Th1 cells that preferentially express the CXCL10 receptor, CXCR3 (300574). They concluded that TLRs can translate the information regarding the nature of pathogens into differences in the cytokines and chemokines produced by DCs, which will in turn differentially polarize adaptive immune responses.
LPS interacts with LBP and CD14 to present LPS to TLR4, which activates inflammatory gene expression through NF-kappa-B and MAPK signaling. Bochkov et al. (2002) demonstrated that oxidized phospholipids inhibit LPS-induced but not TNF-alpha (191160)-induced or interleukin-1-beta (147720)-induced NF-kappa-B-mediated upregulation of inflammatory genes by blocking the interaction of LPS with LBP and CD14. Moreover, in LPS-injected mice, oxidized phospholipids inhibited inflammation and protected mice from lethal endotoxin shock. Thus, in severe gram-negative bacterial infection, endogenously formed oxidized phospholipids may function as a negative feedback to blunt innate immune responses. Furthermore, Bochkov et al. (2002) identified chemical structures capable of inhibiting the effects of endotoxins such as LPS that could be used for the development of new drugs for treatment of sepsis.
Using microarray technology to compare gene expression profiles of mouse B lymphocytes stimulated with CD40LG (300386) or LPS, Doyle et al. (2002) identified IRF3 (603734) as a factor specifically induced by stimulation of TLR3 (603029) or TLR4, but not by TLR2, TLR9 (605474), or CD40 (109535). The primary response genes induced by this activation were coregulated by the NFKB pathway, common for both TLRs and TNFRs, and the IRF3 pathway. Additional secondary response genes were activated by autocrine and paracrine secretion of IFNB (147640). Selective TLR3/TLR4-IRF3 pathway activation potently inhibited viral replication. Doyle et al. (2002) concluded that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.
Hsu et al. (2004) observed that apoptosis induced by live pathogenic gram-positive and gram-negative bacteria required both TLR4 and PKR (176871), possibly representing a major mechanism for pathogenic bacteria that use specific virulence factors to avoid detection and destruction by the innate immune system. Hsu et al. (2004) proposed that TLR4 activates PKR and triggers apoptosis through TRIF (607601) and TRAM (608321) adaptor proteins and that inhibition of PKR may augment macrophage-mediated antibacterial responses.
Using confocal microscopy, Blander and Medzhitov (2006) showed that different phagosomes in the same DC contained either apoptotic host cells, which did not induce surface major histocompatibility class (MHC) II expression or DC maturation, or bacteria or particles expressing the TLR4 ligand LPS, which did induce MHC II expression and DC maturation. Only mature DCs were able to stimulate T cells to secrete IL2 (147680). Blander and Medzhitov (2006) concluded that there is TLR-dependent and phagosome-autonomous selection of antigens for presentation by DCs to T-cell receptors, allowing for discrimination of self and nonself antigens at the subcellular level.
Seimon et al. (2006) found that Sra (MSR1; 153622) ligands failed to trigger apoptosis in endoplasmic reticulum (ER)-stressed macrophages from Tlr4 -/- mice. Sra ligands could stimulate Sra -/- ER-stressed macrophages through Tlr4 to activate Nfkb and Jnk (see 601158). Cytoplasmic calcium was required for Tlr4-induced Jnk activation and apoptosis in ER-stressed macrophages. Apoptosis also required Sra-dependent suppression of Irf3-Ifnb signaling, which is involved in cell survival. Seimon et al. (2006) concluded that combinatorial signaling between TLR4 and SRA results in a functional outcome, macrophage apoptosis, that does not occur with either receptor alone.
Brinkmann et al. (2004) reported that activated neutrophils release granule proteins and chromatin that form extracellular fibers that bind bacteria. These 'neutrophil extracellular traps' (NETs) degrade virulence factors and kill the organisms. Using flow cytometric analysis, Clark et al. (2007) demonstrated that LPS bound human platelets expressing TLR4. Fluorescence microscopy showed that platelets activated by LPS bound neutrophils, whereas other platelet activators, such as thrombin (F2; 176930), had no effect. Using a mouse model of endotoxemia, Clark et al. (2007) showed that platelets and neutrophils adhered to liver sinusoids in a neutrophil-dependent manner. However, in postsinusoidal venules, only neutrophil adherence could be detected. LPS-induced platelet-neutrophil interactions caused marked neutrophil degranulation. Confocal microscopy revealed that septic plasma- or LPS-stimulated platelets caused NET formation under flow conditions. The NETs promoted trapping of bacteria, and the trapping could be reduced by DNase treatment. Clark et al. (2007) proposed that platelet TLR4 is a threshold switch for bacterial trapping in severe sepsis.
Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Apetoh et al. (2007) demonstrated that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. Apetoh et al. (2007) described in both mice and humans a previously unrecognized pathway for the activation of tumor antigen-specific T-cell immunity that involves secretion of the high mobility group box-1 (HMGB1; 163905) alarmin protein by dying tumor cells and the action of HMGB1 on TLR4 expressed by dendritic cells. During chemotherapy or radiotherapy, dendritic cells require signaling through TLR4 and its adaptor MYD88 (602170) for efficient processing and crosspresentation of antigen from dying tumor cells. Patients with breast cancer who carried a TLR4 loss-of-function allele, D299G (603030.0001), relapsed more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. Apetoh et al. (2007) concluded that their results delineated a clinically relevant immunoadjuvant pathway triggered by tumor cell death.
Vogl et al. (2007) demonstrated that mice lacking Mrp8-Mrp14 (123885, 123886) complexes are protected from endotoxin-induced lethal shock and Escherichia coli-induced abdominal sepsis. Both proteins are released during activation of phagocytes, and Mrp8-Mrp14 complexes amplify the endotoxin-triggered inflammatory responses of phagocytes. Mrp8 is the active component that induces intracellular translocation of MYD88 and activation of interleukin-1 receptor-associated kinase-1 (IRAK1; 300283) and nuclear factor-kappa-B (NFKB; see 164011), resulting in elevated expression of tumor necrosis factor-alpha (TNF-alpha; 191160). Using phagocytes expressing a nonfunctional TLR4, HEK293 cells transfected with TLR4, CD14 (158120), and MD2 (605243), and by surface plasmon resonance studies in vitro, Vogl et al. (2007) demonstrated that MRP8 specifically interacts with the TLR4-MD2 complex, thus representing an endogenous ligand of TLR4. Vogl et al. (2007) concluded that MRP8-MRP14 complexes are novel inflammatory components that amplify phagocyte activation during sepsis upstream of TNF-alpha-dependent effects.
Using RT-PCR and confocal microscopy, Weindl et al. (2007) detected comparable constitutive expression of all TLRs in Candida albicans-infected reconstituted epithelium (RHE) and polymorphonuclear cells (PMNs). However, expression of TLR4 was markedly upregulated in RHE when PMNs were added to the RHE culture. Electron microscopy and immunogold labeling revealed that TLR4 recognized C. albicans directly and was expressed intracellularly. TLR4 upregulation protected the epithelial cell layer from fungal infection, and TLR4 expression and protection could be reversed by anti-TLR, TLR4 small interfering RNA (siRNA), or TNF neutralization. Weindl et al. (2007) proposed that PMN-mediated, TLR4-dependent protection of RHE occurs in 3 separate phases: initial priming of RHE by the pathogen and secretion of chemokines and cytokines that recruit and stimulate PMNs, followed by TNF initiation of PMN-mediated TLR4 upregulation in RHE and, finally, direct protection of the oral mucosa from fungal invasion and cell injury by TLR4.
By stimulating mouse peritoneal macrophages with purified bacterial FimH, Mossman et al. (2008) showed potent induction of innate immune responses that were Myd88-dependent and occurred only in cells expressing Tlr4. Mossman et al. (2008) proposed that TLR4 is a functional receptor for FimH, the adhesin portion of bacterial type-1 fimbria.
Trompette et al. (2009) noted that the main allergen of the house dust mite, Der p 2, has structural homology with MD2. Using human and mouse cells, they found that the mite allergen also exhibited functional homology with MD2, in that it facilitated signaling through direct interactions with the TLR4 complex and reconstituted LPS-driven TLR4 signaling in the absence of MD2. Airway sensitization and challenge with the mite allergen led to experimental allergic asthma, as shown by eosinophil numbers in bronchoalveolar lavage fluid, IgE production, and histopathologic analysis, in wildtype and Md2-deficient mice, but not in Tlr4-deficient mice. Trompette et al. (2009) proposed that Der p 2 and other allergens belonging to the MD2-like lipid-binding family have intrinsic adjuvant activity together with their accompanying lipid cargo and may thus account for the phenomenon of allergenicity.
Delgado et al. (2009) investigated the formalin-inactivated respiratory syncytial virus (FIRSV) vaccine that failed to generate protective neutralizing antibodies and instead induced enhanced respiratory disease (ERD) in seronegative vaccinated children subsequently exposed to RSV in the 1960s. They found that wildtype RSV induced protective immunity in mice, whereas FIRSV or RSV inactivated by ultraviolet light (UVRSV) instead induced ERD. Protective immunity was associated with antibody affinity maturation and increased antibody avidity. Multiple injections of UVRSV, but not FIRSV, could induce appropriate protective antibody responses. Adding Tlr4, Tlr3, and Tlr7 (300365) agonists (LPS, poly(I:C), and polyU, respectively), but not alum adjuvant, simultaneously with UVRSV significantly increased antibody affinity and neutralizing capacity. Delgado et al. (2009) concluded that safe and effective RSV vaccines for infants will require neutralizing antibodies with similar avidity for protective antigens to that elicited by live virus inoculation.
Deng et al. (2009) exposed human temporal artery-SCID mouse chimeras to the TLR4 ligand LPS or to the TLR5 ligand flagellin and found that the architecture of the resulting inflammation differed according to the ligand interacting with vascular DCs. Specifically, a transmural panarteritis developed after LPS exposure, whereas flagellin exposure promoted adventitial perivasculitis. The underlying mechanisms involved selective recruitment of T cells, with TLR4-mediated DC stimulation enhancing CCL20 (601960) production, which biased recruitment toward CCR6 (601835)-positive T cells. Adoptive transfer experiments showed that CCR6-positive T cells produced an arteritis pattern with media-invasive T cells damaging vascular smooth muscle cells, and this damage could be blocked by anti-CCR6. CCR6-positive T cells dominated the vasculitic infiltrates in patients with panarteritic giant cell arteritis. Deng et al. (2009) concluded that the original danger signal leads to vascular DC editing of the immune response to direct the inflammatory disease process.
Gray et al. (2010) showed that the short isoform of human MD2 (MD2s) interacted with LPS and TLR4 but failed to mediate LPS-induced NFKB activation and IL8 production. MD2s was upregulated by IFNG, IL6, and TLR4 stimulation and negatively regulated LPS-mediated TLR4 signaling. MD2s competitively inhibited binding of full-length MD2 to TLR4. Gray et al. (2010) proposed that MD2s may regulate TLR4 activation and have therapeutic potential to treat disease characterized by an excessive immune response to LPS.
Kasturi et al. (2011) demonstrated that immunization of mice with synthetic nanoparticles containing antigens plus ligands that signal through TLR4 and TLR7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with nanoparticles containing antigens plus a single TLR ligand. Consistent with this, there was enhanced persistence of germinal centers and of plasma cell responses, which persisted in the lymph nodes for more than 1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma cell response relative to that observed with single TLR ligands. Molecular profiling of activated B cells isolated 7 days after immunization indicated that there was early programming towards B-cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells, as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.
West et al. (2011) demonstrated that engagement of a subset of Toll-like receptors (TLR1, 601194; TLR2, 603028; and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mitochondrial reactive oxygen species (mROS) production. This response involves translocation of a TLR signaling adaptor, TRAF6 (602355), to mitochondria, where it engages the protein ECSIT (608388), which is implicated in mitochondrial respiratory chain assembly. Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT- and TRAF6-depleted macrophages have decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS levels by expressing catalase (115500) in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. West et al. (2011) concluded that their results revealed a novel pathway linking innate immune signaling to mitochondria, implicated mROS as an important component of antibacterial responses, and further established mitochondria as hubs for innate immune signaling.
Using flow cytometry and confocal microscopy in mouse cells, Zanoni et al. (2011) demonstrated that Cd14 chaperoned LPS to Tlr4, leading to Syk (600085)-dependent internalization of Tlr4 and signaling through Trif. Zanoni et al. (2011) concluded that pathogen recognition receptors induce both membrane transport and signal transduction.
Henao-Mejia et al. (2012) demonstrated that NLRP6 (609650) and NLRP3 (606416) inflammasomes and the effector protein IL18 (600953) negatively regulate nonalcoholic fatty liver disease/nonalcoholic steatohepatitis progression, as well as multiple aspects of metabolic syndrome via modulation of the gut microbiota. Different mouse models revealed that inflammasome deficiency-associated changes in the configuration of the gut microbiota are associated with exacerbated hepatic steatosis and inflammation through influx of TLR4 and TLR9 (605474) agonists into the portal circulation, leading to enhanced hepatic TNF-alpha (191160) expression, which drives NASH progression. Furthermore, cohousing of inflammasome-deficient mice with wildtype mice resulted in exacerbation of hepatic steatosis and obesity. Thus, Henao-Mejia et al. (2012) concluded that altered interactions between the gut microbiota and the host, produced by defective NLRP3 and NLRP6 inflammasome sensing, may govern the rate of progression of multiple metabolic syndrome-associated abnormalities, highlighting the central role of the microbiota in the pathogenesis of theretofore seemingly unrelated systemic autoinflammatory and metabolic disorders.
Shirey et al. (2013) reported that therapeutic administration of Eritoran (also known as E5564), a potent, well-tolerated, synthetic TLR4 antagonist, blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titers. CD14 (158120) and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2 (605243). Shirey et al. (2013) concluded that Eritoran blockade of TLR signaling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.
Millien et al. (2013) demonstrated that TLR4 is activated by airway proteinase activity to initiate both allergic airway disease and antifungal immunity. These outcomes were induced by proteinase cleavage of the clotting protein fibrinogen (see 134820), yielding fibrinogen cleavage products that acted as TLR4 ligands on the airway epithelial cells and macrophages. Thus, Millien et al. (2013) concluded that allergic airway inflammation represents an antifungal defensive strategy that is driven by fibrinogen cleavage and TLR4 activation.
Zhang et al. (2015) used in vivo aging analyses in mice to demonstrate that neutrophil proinflammatory activity correlates positively with their aging while in circulation. The authors found that aged neutrophils represent an overly active subset exhibiting enhanced alpha-M (120980)-beta-2 (600065) integrin activation and neutrophil extracellular trap formation under inflammatory conditions. Zhang et al. (2015) showed that neutrophil aging is driven by the microbiota via Toll-like receptor (TLR4 and TLR2, 603028)- and myeloid differentiation factor-88 (MYD88; 602170)-mediated signaling pathways. Depletion of the microbiota significantly reduced the number of circulating aged neutrophils and dramatically improved the pathogenesis and inflammation-related organ damage in models of sickle cell disease (603903) or endotoxin-induced septic shock. Zhang et al. (2015) concluded that their results identified a role for the microbiota in regulating a disease-promoting neutrophil subset.
Hu et al. (2015) found that WDFY1 (618080) played a crucial role in the TLR3/TLR4 signaling pathway. Overexpression and knockdown assays showed that WDFY1 promoted TLR3/TLR4 ligand-induced activation of IRF3 and NF-kappa-B, as well as production of IFN-beta and inflammatory cytokines in both human and mouse cell lines. In the TLR3 signaling pathway, TLR3 undergoes tyrosine phosphorylation and recruits TRIF, leading to activation of IRF3 and NF-kappa-B. WDFY1 acted at the level of TLR3, upstream of the kinase TBK1 (604834), and was associated with tyrosine-phosphorylated TLR3 in a ligand binding-dependent manner and mediated recruitment of TRIF to TLR3. WDFY1 associated with the cytosolic TIR domain of TLR3, and the second WD repeat domain of WDFY1 was required for its binding to TLR3. The FYVE domain of WDFY1 was essential for WDFY1 function in TLR3 signaling. The authors demonstrated that WDFY1 was also required for recruitment of TRIF to TLR4 and functioned in TLR4 signaling by the same mechanism as in TLR3 signaling.
Tang et al. (2017) identified TLR4 and the gut microbiome as critical stimulants of cerebral cavernous malformations (CCM; 116860) formation. Activation of TLR4 by gram-negative bacteria or lipopolysaccharide accelerated CCM formation, and genetic or pharmacologic blockade of TLR4 signaling prevented CCM formation in mice. Tang et al. (2017) found that TLR4 and CD14 expression parallels lesion burden in CCM. In CCM patients who carried a KRIT1 Q455X variant (604214.0004), polymorphisms that increase expression of the TLR4 gene (rs10759930, rs10759931) or its coreceptor CD14 (rs778587, rs778588) were significantly associated with increased CCM lesion number. These SNPs are in the 5-prime genomic region of each gene and constitute cis expression quantitative trait loci (QTLs) that positively regulate whole blood cell expression of TLR4 and CD14 in a dose-dependent manner corresponding with risk allele number. These results were corroborated using the GTEx Consortium data.
Using Western blot analysis in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) rat model, Chen et al. (2017) determined that LPS significantly induced the expression of Tlr4, MyD88, Trif (607601), and paralemmin-3 (PALM3; 621051). Formation of Tlr4/MyD88 and Tlr4/Trif complexes was significantly increased in lung tissues after LPS stimulation. Downregulation of Palm3 in lung tissue inhibited the interaction of Tlr4 with Trif or MyD88, but did not have an effect on their LPS-induced protein expression. Chen et al. (2017) concluded that the observed protective effect of downregulation of Palm3 against LPS-induced ALI and its mechanisms was partially associated with the modulation of inflammatory responses and inhibition of Tlr4/MyD88 and Tlr4/Trif complex formation.
Crystal Structure
Endotoxic lipopolysaccharide (LPS) with potent immunostimulatory activity is recognized by the receptor complex of MD2 and TLR4. Ohto et al. (2007) reported the crystal structure of human MD2 and its complex with the antiendotoxic tetra-acylated lipid A core of LPS at 2.0- and 2.2-angstrom resolutions, respectively. MD2 shows a deep hydrophobic cavity sandwiched by 2 beta sheets, in which 4 acyl chains of the ligand are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. Ohto et al. (2007) concluded that these structures suggested that MD2 plays a principal role in endotoxin recognition and provided a basis for antiseptic drug development.
Park et al. (2009) determined the crystal structure of the TLR4-MD2-LPS complex. LPS binding induced the formation of an m-shaped receptor multimer composed of 2 copies of the TLR4-MD2-LPS complex arranged symmetrically. LPS interacts with a large hydrophobic pocket in MD2 and directly bridges the 2 components of the multimer. Five of the 6 lipid chains of LPS are buried deep inside the pocket and the remaining chain is exposed to the surface of MD2, forming a hydrophobic interaction with the conserved phenylalanines of TLR4. The F126 loop of MD2 undergoes localized structural change and supports this core hydrophobic interface by making hydrophobic interactions with TLR4. Comparison with the structures of tetra-acylated antagonists bound to MD2 indicated that 2 other lipid chains in LPS displace the phosphorylated glucosamine backbone of approximately 5 angstroms towards the solvent area. This structural shift allows phosphate groups of LPS to contribute to receptor multimerization by forming ionic interactions with a cluster of positively charged residues in TLR4 and MD2.
Ohto et al. (2012) obtained the crystal structure of human TLR4 containing the asp299-to-gly (D299G; 603030.0001) and thr399-to-ile (T399I; 603030.0002) variants (see MOLECULAR GENETICS) in complex with its coreceptor MD2 and LPS at 2.4-angstrom resolution. The variant complex exhibited an agonistic 'm'-shaped 2:2:2 architecture similar to that of the wildtype complex. Substitution of more flexible and neutral residues by D299G introduced structural changes that may affect ligand binding, folding efficiency, cell surface expression, and protein stability, particularly during stimulation by weakly agonistic ligands. In contrast, the impact of T399I was minor, with almost no structural changes observed.
There is much variability between individuals in response to inhaled toxins. Arbour et al. (2000) investigated whether TLR4, which affects LPS responsiveness in mice, is related to variability in airway responsiveness to inhaled LPS in humans. They showed that common, cosegregating missense mutations (asp299 to gly, or D299G 603030.0001; thr399 to ile, or T399I 603030.0002) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans. Transfection of the THP-1 cell line demonstrated that the asp299-to-gly mutation, but not the thr399-to-ile mutation, interrupted TLR4-mediated LPS signaling. Moreover, the wildtype allele of TLR4 rescued the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Findings provided the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrated that gene-sequence changes can alter the ability of the host to respond to environmental stress.
The ability to mount a prominent inflammatory response to bacterial pathogens confers an advantage in innate immune defense but may signal an increased risk of atherosclerosis. The family of Toll receptors provide a critical link between immune stimulants produced by microorganisms and the initiation of host defense. For infections with gram-negative bacteria, LPS is the main source of inflammation, and TLR4 is crucial in mediating its effects. TLR4 is expressed in cardiomyocytes, macrophages, airway epithelia, and endothelial and smooth-muscle cells and in small amounts in most other tissues. Kiechl et al. (2002) assessed 2 TLR4 variants, D299G and T399I, in a random sample of the general population and analyzed relations among these polymorphisms, the level of systemic inflammation, the risk of severe infections, and the development of atherosclerosis. The extent and progression of carotid atherosclerosis was assessed by high-resolution duplex ultrasonography. Fifty-five of 810 persons studied had the D299G allele. These individuals had lower levels of certain proinflammatory cytokines, acute-phase reactants, and soluble adhesion molecules, such as interleukin-6 (147620) and fibrinogen (see 134820). Although these subjects were found to be more susceptible to severe bacterial infections, they had a lower risk of carotid atherosclerosis and a smaller intima-media thickness in the common carotid artery.
Lorenz et al. (2002) genotyped 91 patients with septic shock and 73 healthy blood donor controls for the presence of D299G and T399I. D299G was found only in patients with septic shock and not in controls. Patients with septic shock and both D299G and T399I had a higher prevalence of gram-negative infections.
As a central component of the human endotoxin sensor, TLR4 functions in the early detection and response to gram-negative infection. Therefore, Smirnova et al. (2003) examined a large collection of patients with meningococcal sepsis, comparing the frequency of rare TLR4 coding changes to those in an ethnically matched control population. The results were compared with sequences of TLR2. Using the method that obviates the confounding effect of linkage disequilibrium, they observed that rare heterozygous missense mutations of TLR4 contribute to the development of systemic meningococcal disease among white populations of the southern United Kingdom (P = 0.02; odds ratio 8.2). The results from all white populations were pooled, and overwhelming significant excess of such mutations were observed among individuals with disease, giving an odds ratio of 27.0. The common white TLR4 variant TLR4B, synonymous TLR4 substitutions, and variant TLR2 alleles were not significantly overrepresented among patients with systemic meningococcal infections. No single variant of TLR4 was significantly overrepresented in the meningococcal population. Collectively, however, rare TLR4 coding variants were markedly overrepresented. Thus, sensing via TLR4 probably contributes to the early containment of meningococcal infection, and sensing defects create increased risk of disease.
Balistreri et al. (2004) reported that the D299G allele of the TLR4 gene was overrepresented in 55 very old Sicilian men (mean age, 100 years) compared to controls (see 152430).
Mockenhaupt et al. (2006) noted that the Plasmodium falciparum glycosylphosphatidylinositol anchor induces signaling via TLR2 and TLR4, whereas the hemozoin activates the immune system through TLR9. In a case control study of 290 Ghanaian children with severe malaria, 290 infected but asymptomatic Ghanaian children, and 290 healthy matched controls, Mockenhaupt et al. (2006) found no common TLR2 variants and no associations between TLR9 promoter polymorphisms and malaria severity. However, they found that the D299G and T399I variants of TLR4 were significantly less frequent in healthy controls than in patients and conferred 1.5- and 2.6-fold increased risk of severe malaria, respectively.
Ferwerda et al. (2007) performed TLR4 polymorphism analysis on 2,491 individuals from 15 populations in Africa, Europe, Asia, and South America. Between 10 and 18% of Africans had a single copy of the D299G allele. Of these, 2% also had T399I, and T399I was always found in individuals with D299G. T399I and D299G were nearly absent in populations from Asia and South America, with only single individuals from Indonesia having T399I only or D299G only. In contrast, 6 to 14% of Indo-Europeans were double heterozygotes for T399I and D299G, and the frequency reached 18% for Basques. Analysis of European families confirmed inheritance of a heterozygous haplotype. The distribution of the polymorphisms suggested that they arose in Africa, with D299G arising first. Secretion of TNF, but not IL10, was enhanced in response to LPS in African D299G heterozygotes, but it was unchanged in European D299G/T399I double heterozygotes. Patients with gram-negative sepsis and healthy controls showed no difference in the presence of the D299G/T399I haplotype. Cameroonian children with malaria and the D299G allele had much higher parasitemia, but lower cerebral malaria, than those with the wildtype allele. Ferwerda et al. (2007) concluded that, in Africa, the beneficial effect of D299G in malaria appeared to override its negative effect in sepsis. They proposed that in the absence of malaria pressure in Europe, the D299G allele was eliminated due to the deleterious consequences of severe gram-negative bacterial infections. Ferwerda et al. (2007) attributed the variable prevalence of the D299G/T399I haplotype in European populations to genetic drift.
By analyzing SNPs in 4 TLR genes in a discovery study of 336 recipients of hematopoietic cell transplants and their unrelated donors, Bochud et al. (2008) found that 2 donor TLR4 haplotypes increased the risk of invasive aspergillosis (614079) in recipients. A validation study with 103 patients and 263 matched controls who received transplants from related and unrelated donors confirmed that the donor TLR4 S4 haplotype was associated with increased risk of invasive aspergillosis in unrelated recipients. TLR4 haplotype S4 is defined by 4 SNPs, including 2 coding SNPs, D299G and T399I, in strong linkage disequilibrium in exon 3 that influence TLR4 function. The discovery study indicated that donor or recipient cytomegalovirus (CMV) seropositivity, donor S4 positivity, or both, were associated with increased probability of invasive aspergillosis within 3 years and death unrelated to relapse compared with negative results for CMV and S4. Bochud et al. (2008) concluded that donor TLR4 S4 haplotype is associated with risk of invasive aspergillosis among unrelated recipients after allogeneic hematopoietic cell transplantation, and they proposed that the risk can be reduced for transplant recipients by identifying unrelated donors with an increased risk of severe infection. In a commentary, Pamer (2008) suggested that persons receiving transplants from donors with a high-affinity TLR4 variant may acquire an elevated innate immune tone, increasing general resistance to infection.
In response to the findings of Bochud et al. (2008), correspondents suggested several potential confounding factors to the study, including the immunomodulatory properties of the antifungal agent amphotericin B (Levitz et al., 2009), the potential of the cytomegalovirus association with aspergillosis to act as an intermediate variable (Cervera et al., 2009), and the fact that invasive aspergillosis still develops in Asian patients after undergoing allogeneic hematopoietic stem-cell transplantation, even though the D299G and T399I polymorphisms are absent in Asian populations (Asakura and Komatsu, 2009). Bochud et al. (2009) replied that the D299I polymorphism is also associated with cavitary aspergillosis in patients without exposure to amphotericin B, citing the findings of Carvalho et al. (2008). They pointed out that multivariate analysis in their study did not support an association with CMV. Bochud et al. (2009) acknowledged that inherited risks are likely to be multifactorial and to differ among specific ethnic groups, in part due to the complex cell wall structure of fungi that may interact with different receptors. They noted the influence of IL10 with susceptibility to invasive aspergillosis in Koreans (Seo et al., 2005).
Tulic et al. (2007) found that production of IL8, IL6, and other cytokines in response to RSV or LPS was reduced in bronchial epithelial cells transfected with TLR4 constructs containing G299 or I399 compared with cells expressing TLR4 with D299 or T399. Reduced cytokine expression was associated with normal levels of intracellular TLR4 with G299 or I399, but a failure to translocate the receptor to the cell surface. These findings were mirrored by blunted peripheral blood mononuclear cell responses to RSV in children expressing the same TLR4 variants. Tulic et al. (2007) concluded that compromised first-line defense against RSV at the airway-epithelial surface of children expressing TLR4 G299 or I399 may confer increased susceptibility to severe bronchiolitis after RSV infection.
RSV is a leading cause of infant mortality worldwide, and RSV fusion protein activates cells through TLR4. Awomoyi et al. (2007) evaluated the prevalence of the D299G and T399I SNPs in the TLR4 ectodomain in 105 DNA samples extracted from archived nasal lavage samples from high-risk infants/children with confirmed RSV disease who participated in vaccine trials. Both SNPs were highly associated with symptomatic RSV disease in this largely premature population, with 89.5% and 87.6% of patients being heterozygous for D299G and T399I compared with control frequencies of 10.5% and 6.5%, respectively. Awomoyi et al. (2007) concluded that heterozygosity for these 2 TLR4 SNPs is associated with symptomatic RSV disease in high-risk infants, and that the data support a dual role for TLR4 SNPs in prematurity and increased susceptibility to RSV.
By evaluating 4 TLR4 SNPs in 441 Ethiopian patients with leprosy (see 609888) and 197 healthy controls, Bochud et al. (2009) found that the minor alleles of the 896G-A (D299G; 603030.0001) and 1196C-T (T399I; 603030.0002) SNPs were associated with a significant protective effect against the disease. TLR4 SNPs were not significantly associated with disease type. Stimulation of untyped monocytes with Mycobacterium leprae partially inhibited their subsequent cytokine response to LPS. Bochud et al. (2009) proposed that TLR4 polymorphisms are associated with susceptibility to leprosy, possibly due to M. leprae-mediated modulation of TLR4 signaling.
Hawn et al. (2005) examined the 896G-A (D299G) and 1196C-T (T399I) TLR4 SNPs in 108 cases and 508 controls from a 1999 Legionnaire disease (LD; see 608556) outbreak in the Netherlands. Eighty-nine controls were matched to patients for age, sex, and geographic residence, whereas the remaining 421 controls were unmatched. Hawn et al. (2005) found that allele 896G was associated with resistance to LD, with a frequency of 2.5% in LD patients compared with frequencies of 6.5% in all controls (odds ratio = 0.36; P = 0.025) and 8.6% in matched controls (odds ratio = 0.27; P = 0.008). An analysis of genotype frequencies demonstrated similar protective associations for 896G. Allele 1196T cosegregated with 896G and therefore had identical associations. Hawn et al. (2005) concluded that, although these TLR4 SNPs had been reported to be associated with susceptibility to gram-negative infections, they may also be associated with resistance to gram-negative pathogens, such as Legionella pneumophila, the causative agent of LD.
Figueroa et al. (2012) complemented embryonic kidney cells with fluorescent wildtype TLR4 or TLR4 with the D299G or T399I polymorphisms and observed comparable total TLR4 expression, interactions with MD2, and LPS binding. FACS analysis showed that D299G had minimal impact on cell surface levels of TLR4. Cells expressing D299G TLR4, but not those expressing T399I TLR4, exhibited impaired LPS-induced phosphorylation of p38 (MAPK14; 600289) and TBK1 (604834), activation of NF-kappa-B and IRF3, and induction of IL8 and IFNB mRNA expression. Expression of D299G in Tlr4 -/- mouse macrophages failed to elicit LPS-induced expression of Tnfa and Ifnb mRNA. Coimmunoprecipitation analysis revealed diminished LPS-driven interaction of MYD88 and TRIF with D299G TLR4 compared with wildtype TLR4. Figueroa et al. (2012) concluded that the D299G polymorphism compromises recruitment of MYD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4-MD2 interaction, and LPS binding, suggesting that D299G interferes with TLR4 dimerization and intracellular docking platform assembly.
By analyzing the nucleotide sequences of 16 TLR-related genes in primates, Nakajima et al. (2008) identified the extracellular domain of TLR4 as a suggestive target of positive Darwinian selection in the course of primate evolution. They noted that the D299G mutation (603030.0001) in human TLR4 is associated with a blunted response to LPS and increased susceptibility to gram-negative bacteria. Nakajima et al. (2008) found that D299 is highly conserved in great apes and gibbons, but that D299 has been replaced by asparagine (confirmed by Nakajima (2009)) in Old World, but not New World, monkeys. They suggested that these species may have different responses to certain types of LPS.
Conservative estimates hold that in the United States alone, 20,000 persons die each year as a result of septic shock brought on by gram-negative infection. The lethal effect of a gram-negative infection is linked, in part, to the biologic effects of bacterial lipopolysaccharide (endotoxin), which is produced by all gram-negative organisms. LPS, a powerful activator of host mononuclear cells, prompts the synthesis and release of tumor necrosis factor (TNF) and other toxic cytokines that ultimately lead to shock in sepsis. Nonetheless, timely recognition of LPS by cells of the innate immune system permits effective clearance of a gram-negative infection before it becomes widely disseminated. Sultzer (1968), Michalek et al. (1980), and others found that mice of the C3H/HeJ strain had a defective response to bacterial endotoxin. Inquiry into the genetic basis of LPS resistance revealed a single locus (Lps), wherein homozygosity for a codominant allele, Lps(d), was responsible for the endotoxin-unresponsive state. A second mutation preventing responses to endotoxin was identified in mice of another strain. These mutations in the Lps gene selectively impeded LPS signal transduction, rendering the mice resistant to endotoxin yet highly susceptible to gram-negative infection. The codominant Lps(d) allele was shown by Poltorak et al. (1998) to correspond to a missense mutation in the third exon of the Tlr4 gene, predicted to replace proline with histidine at position 712 (P712H) of the polypeptide chain. Mice homozygous for the second Lps mutation were found to carry a null mutation of Tlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces the LPS signal across the plasma membrane. Destructive mutations of Tlr4 predispose to the development of gram-negative sepsis, leaving most aspects of immune function intact.
Kurt-Jones et al. (2000) determined that proinflammatory cytokine responses to respiratory syncytial virus (RSV) F protein were absent or diminished in mice with deletions of either Cd14 or Tlr4, respectively. Importantly, Tlr4 -/- mice had higher levels of infectious virus in their lungs and were either unable to clear the virus or cleared the virus several days later than wildtype mice. The authors concluded that TLR4 and CD14 appear to be important not only in recognizing bacterial structures such as LPS, but are important in innate immune responses to viruses as well.
Roger et al. (2001) showed that mouse macrophages transfected with antisense Mif mRNA and macrophages from Mif -/- mice are hyporesponsive to LPS stimulation, but not stimulation by gram-positive bacteria, as shown by reduced TNFA (191160) and IL6 (147620) production. Mif-deficient cells expressed reduced Tlr4, but not Tlr2 (603028), mRNA and protein. EMSA and promoter analysis indicated that deficient Mif expression impairs basal PU.1 (165170) transcription factor activity of the mouse Tlr4 gene, resulting in reduced Tlr4 protein expression and responsiveness to LPS and gram-negative bacteria. Roger et al. (2001) suggested that inhibition of MIF activity may benefit people with gram-negative septic shock.
Using in situ hybridization, Wolfs et al. (2002) detected constitutive expression of Tlr2 and Tlr4 in mouse proximal and distal tubular renal epithelial cells. To gain insight into the regulation of TLR expression during inflammation, the authors used a mouse model of renal inflammation. During kidney inflammation, mRNA for both receptors was enhanced by TNFA and IFNG, with expression mainly localized in distal tubules, the thin limb of the loop of Henle, and collecting ducts. Western blot analysis showed enhanced renal Tlr4 expression. Wolfs et al. (2002) suggested that epithelial-derived TLR signaling has a role in the inflammatory response during ascending urinary tract infection.
Nagai et al. (2002) generated Md2 (605243)-deficient mice lacking the 37 amino acids encoded by exon 1. RT-PCR and flow cytometric analyses demonstrated expression of Tlr4 mRNA, but the Md2-Tlr4 complex on the cell surface was absent. Md2 -/- B cells, macrophages, and dendritic cells responded to CpG, anti-RP105 (602226), and proteoglycan, but not to LPS. The response to CpG indicated that Tlr9 was intact, and the response to proteoglycan indicated that Tlr2 was intact. In vivo, Md2-null mice, like Tlr4-null mice, failed to produce the acute-phase reactant, serum amyloid A. Md2-deficient mice all survived LPS challenge and did not produce inflammatory cytokines. However, the mutant mice showed enhanced susceptibility to Salmonella typhimurium, probably due to a defect in LPS sensing. Confocal microscopy demonstrated that mouse embryonic fibroblasts from Md2 -/- mice only expressed Tlr4 in the Golgi apparatus and not on the cell surface. Nagai et al. (2002) concluded that MD2 is essential for the correct intracellular distribution, cell surface expression, and LPS recognition of TLR4. They proposed that MD2 may be a target for neutralizing the toxic effects of endotoxin.
Supajatura et al. (2002) examined cytokine production by bone marrow-derived mast cells from mice deficient in either Tlr2 or Tlr4. Peptidoglycan (PGN) stimulated mast cells to produce Tnf, Il4 (147780), Il5 (147850), Il6, and Il13 (147683), but not Il1b, in a Tlr2-dependent manner. In contrast, LPS stimulated mast cells to produce Tnf, Il1b, Il6, and Il13, but not Il4 or Il5, in a Tlr4-dependent manner. Tlr2- but not Tlr4-dependent mast cell stimulation resulted in mast cell degranulation and calcium mobilization. Infection of Tlr4 -/- mice by cecal ligation and puncture revealed the necessity of Tlr4-mediated peritoneal mast cell activation and neutrophil recruitment for protection from gram-negative bacterial infection. Intradermal injection of PGN led to increased vasodilation and inflammation through Tlr2-mediated activation of skin mast cells, suggesting that Tlr2-dependent skin mast cell activation may exacerbate the inflammatory lesions of atopic dermatitis, in which gram-positive bacterial infection is common.
Streptococcus pneumoniae is one of the leading causes of invasive bacterial disease worldwide. Malley et al. (2003) tested the hypothesis that pneumolysin interacts with surface proteins of the TLR family other than TLR2. They presented evidence from several experiments indicating that the interaction of pneumolysin with TLR4 is critically involved in the innate immune response to pneumococcus. When compared with wildtype macrophages, macrophages from mice that carried a spontaneous mutation in Tlr4 (P712H) were hyporesponsive to both pneumolysin alone and the combination of pneumolysin with pneumococcal cell walls. Finally, these Tlr4-mutant mice were significantly more susceptible to lethal infection after intranasal colonization with pneumolysin-positive pneumococci than were control mice.
Andonegui et al. (2003) studied mice lacking Tlr4 in leukocytes or in endothelial cells. Following LPS administration, there was a dramatic recruitment of neutrophils to the lungs of leukocyte Tlr4 -/- mice, but not to the lungs of endothelium Tlr4 -/- mice. Intravital microscopy of peripheral microcirculation in the cremaster muscle revealed about 30-fold more leukocyte-endothelial cell interactions in LPS-treated endothelium Tlr4 -/- mice than in LPS-treated leukocyte Tlr4 -/- mice, a result consistent with less sequestration of leukocytes into the lungs of endothelium Tlr4 -/- mice. Andonegui et al. (2003) concluded that their data challenge the view that LPS directly activates neutrophils to trap in lungs.
To examine whether Toll-like receptor signaling regulates phagocytosis, Blander and Medzhitov (2004) compared macrophages from wildtype, Myd88 (602170)-null, and Tlr2-Tlr4 double-null mice. Myd-null and Tlr2-Tlr4 double-null macrophages were unresponsive to inactivated E. coli. Blander and Medzhitov (2004) found that activation of the Toll-like receptor signaling pathway by bacteria, but not apoptotic cells, regulated phagocytosis at multiple steps including internalization and phagosome maturation. Phagocytosis of bacteria was impaired in the absence of Toll-like receptor signaling. Two modes of phagosome maturation were observed, constitutive and inducible; their differential engagement depended on the ability of the cargo to trigger Toll-like receptor signaling.
Toll-like receptors and the downstream adaptor molecule Myd88 play an essential role in the innate immune responses. Michelsen et al. (2004) demonstrated that genetic deficiency of Tlr4 or Myd88 was associated with a significant reduction of aortic plaque areas in atherosclerosis-prone apolipoprotein E (APOE; 107741)-deficient mice, despite persistent hypercholesterolemia, implying an important role for the innate immune system in atherogenesis. ApoE-deficient mice that also lacked Tlr4 or Myd88 demonstrated reduced aortic atherosclerosis that was associated with reductions in circulating levels of proinflammatory cytokines IL12 (see 161560) or monocyte chemoattractant protein-1 (MCP1, or CCL2; 158105), plaque lipid content, number of macrophages, and cyclooxygenase-2 (COX2; 600262) immunoreactivity in their plaques.
Hollestelle et al. (2004) found that Tlr4 activation by LPS stimulated plaque formation and subsequent outward arterial remodeling in a femoral artery cuff model in atherosclerotic transgenic mice. Neointima formation and outward remodeling occurred in wildtype mice, but no outward arterial remodeling was observed independent of neointima formation in Tlr4-deficient mice. Carotid artery ligation in wildtype mice, but not Tlr4-deficient mice, resulted in outward remodeling without neointima formation in the contralateral artery, with increased Tlr4 expression and Eda (300451) and Hsp60 (118190) mRNA levels.
Oyama et al. (2004) found that Tlr4-deficient mice sustained significantly smaller infarctions compared with control mice following myocardial ischemia-reperfusion injury. Fewer neutrophils infiltrated the myocardium of Tlr4-deficient mice, and Tlr4-deficient myocardium had fewer lipid peroxides and less complement deposition compared with controls. Oyama et al. (2004) concluded that TLR4 serves a proinflammatory function in murine myocardial ischemia-reperfusion injury.
Li and Cherayil (2004) showed that peritoneal macrophages from C3H/HeJ mice, which carry a spontaneous point mutation in Tlr4 that leads to an inactivating substitution in the cytoplasmic domain, had impaired production of Tnf in response to the Tlr2 ligand peptidoglycan. The findings suggested that macrophages from wildtype mice are primed by chronically acting Tlr4 signals, probably resulting from exposure to environmental LPS, and that these signals are required for optimal Tnf production in response to Tlr2 stimulation.
To evaluate the role of TLRs in B-cell activation and antibody production, Pasare and Medzhitov (2005) transferred purified B cells from wildtype, Myd88-deficient, Tlr4-deficient, and Cd40-deficient mice into B cell-deficient mu-MT mice, which have a mutation in the Ighm gene (147020). They found that primary B-cell activation, including induction of IgM, IgG1, and IgG2 responses, but not IgE or, probably, IgA responses, required TLRs in addition to helper T cells. In contrast, Cd40 was required for isotype switching.
Tlr4 is expressed exclusively on microglia in the rodent central nervous system. Tanga et al. (2005) found that Tlr4-null mice and rats treated intrathecally with an antisense oligodeoxynucleotide to decrease Tlr4 expression had significantly attenuated behavioral hypersensitivity after nerve transection at L5 compared to controls. This was correlated with decreased expression of spinal microglial markers and proinflammatory cytokines. The findings suggested that TLR4 contributes to the initiation of neuroimmune activation after nerve injury and that neuropathic pain may result from an early CNS/microglial response to injury.
Hyaluronan, an extracellular matrix glycosaminoglycan with a repeating disaccharide structure, is produced after tissue injury, and impaired clearance results in unremitting inflammation. Jiang et al. (2005) noted that CD44 (107269) is essential for regulating turnover of hyaluronan, but it is not required for expression of chemokines by macrophages after lung injury. Using Tlr-deficient mouse macrophages, they found that hyaluronan fragments stimulated Mip2 (CXCL2; 139110), Mip1a (CCL3; 182283), and Kc (CXCL1; 155730) in a Tlr2- and Tlr4-dependent manner that also required Myd88. Mice deficient in Tlr2, Tlr4, or Myd88 showed impaired transepithelial migration of inflammatory cells, but decreased survival and enhanced epithelial cell apoptosis after lung injury. Lung epithelial cell overexpression of high molecular mass hyaluronan protected against acute lung injury and apoptosis, in part, through TLR-dependent basal activation of NFKB. Jiang et al. (2005) concluded that interaction of TLR2 and TLR4 with hyaluronan provides signals that initiate inflammatory responses, maintain epithelial cell integrity, and promote recovery from acute lung injury.
Zacharowski et al. (2006) found that the adrenal gland was significantly enlarged in Tlr4 -/- mice, with the enlargement occurring in the adrenal cortex rather than the medulla. Tlr4 -/- mice also showed significantly increased levels of adrenal corticosterone, but not adrenocorticotropic hormone (ACTH). Within the cortex of Tlr4 -/- mice, there was frequent direct contact of macrophages and endothelial cells with cortical cells, and there were altered structures within Tlr4 -/- adrenocortical cells. After LPS challenge, no elevation of plasma corticosterone, ACTH, or cytokines was detected in Tlr4 -/- mice, and Nfkb was not activated. Zacharowski et al. (2006) concluded that TLR4 has a key role in the hypothalamic-pituitary-adrenal axis. They proposed that TLR polymorphisms may contribute to impairment of the adrenal stress response in patients with sepsis.
In mouse adipocytes and macrophages, Shi et al. (2006) showed that nutritional fatty acids activated Tlr4 signaling and that induction of inflammatory signaling by fatty acids was blunted in Tlr4 -/- cells. Tlr4-null mice were substantially protected from the ability of systemic lipid infusion to suppress insulin signaling in muscle and to reduce insulin-mediated changes in systemic glucose metabolism. Tlr4-null female mice had increased obesity but were partially protected against high fat diet-induced insulin resistance. Shi et al. (2006) suggested that TLR4 is a molecular link between nutrition, lipids, and inflammation, and that the innate immune system participates in the regulation of energy balance and insulin resistance in response to changes in the nutritional environment.
Zhang et al. (2006) found that Tlr4 -/- mice exhibited pulmonary emphysema with age. The emphysema was independent of inflammation and was associated with decreased elastase inhibitory capacity in the face of normal levels of matrix metalloproteinases (e.g., MMP12; 601046), tissue inhibitors of metalloproteinase (e.g., TIMP1; 305370), and cathepsins (e.g., CTSS; 116845). Tlr4 -/- mice had decreased antioxidant activity, and the emphysema was reversed by antioxidants. Adoptive transfer experiments revealed that Tlr4 expression in lung structural cells was required for maintenance of normal lung architecture. Tlr4 -/- lung endothelial cells had increased NADPH oxidase activity due to induction of Nox3 (607105), resulting in increased oxidant generation and elastolytic activity. Treatment of Tlr4 -/- mice or endothelial cells with NADPH inhibitors or Nox3 siRNA reversed the observed phenotype. Zhang et al. (2006) concluded that TLR4 has a role in maintaining constitutive lung integrity by modulating oxidant generation.
Seki et al. (2007) found that wildtype mice developed overt hepatic fibrosis following bile duct ligation (BDL), whereas C3H/HeJ mice with the P712H missense mutation in Tlr4 did not. There was no difference in liver injury, as assessed by liver transaminase levels. Tlr4-mutant mice had reduced mRNA expression of the fibrogenesis markers Col1a1 (120150), Acta2 (102620), Tgfb (190180), and Timp1 and of the macrophage-recruiting chemokines Ccl2 and Ccl4 (182284). Antibiotic treatment to reduce intestinal microflora prior to BDL reduced hepatic fibrosis in wildtype mice, as demonstrated by decreased collagen accumulation, liver hydroxyproline levels, macrophage infiltration, and Tlr4 expression. Treatment of Kupffer cell-depleted mice with LPS caused increased expression of Nfkb in hepatic stellate cells (HSCs), but not other hepatocytes. LPS treatment of quiescent HSCs, the main precursors of liver myofibroblasts, activated Tlr4, upregulated chemokine secretion, induced Kupffer cell chemotaxis, and downregulated the Tgfb pseudoreceptor Bambi (604444). Tlr4 mediated Bambi downregulation through Myd88 and Nfkb and sensitized HSCs to Tgfb. Seki et al. (2007) concluded that intestinal microflora and functional TLR4, but not TLR2, are required for hepatic fibrogenesis.
Rolls et al. (2007) found that mice lacking Tlr2 had fewer Dcx (300121)-expressing adult hippocampal neural progenitor cells (NPCs), and Tlr2 appeared to be required for differentiation of NPCs into neurons via a Myd88- and Nfkb-dependent pathway. NPCs also expressed Tlr4, but inhibition of Tlr4 expression or deletion of Tlr4 increased sphere formation, clonal efficiency, and NPC differentiation via both Myd88-dependent and -independent pathways. Rolls et al. (2007) concluded that TLR2 and TLR4 are both involved in adult neurogenesis, with TLR2 primarily involved in cell fate determination and TLR4 primarily involved in neural stem cell self-renewal.
Using a middle cerebral artery ischemia-reperfusion model with line embolism in C3H/HeJ and CEH/OuJ mice, Cao et al. (2007) demonstrated that injury was less serious in mice of the C3H/HeJ strain than in mice of the C3H/OuJ strain, which carries the wildtype Tlr4 gene. Cao et al. (2007) proposed that TLR4 participates in the process of cerebral ischemia-reperfusion injury.
Using irradiated chimeric mice, Hammad et al. (2009) showed that Tlr4 expression on radioresistant lung structural cells, but not on DCs, was necessary and sufficient for DC activation in lung and priming of effector T-cell responses to house dust mite (HDM) allergen. Tlr4 triggering on structural cells caused production of the innate proallergic cytokines Tslp (607003), Gmcsf (CSF2; 138960), Il25 (606746), and Il33 (608678). Mice lacking Tlr4 on structural cells, but not on hematopoietic cells, did not develop HDM-driven airway inflammation, and a Tlr4 antagonist suppressed bronchial hyperreactivity and other features of asthma. Hammad et al. (2009) concluded that airway epithelial cells have an innate immune function that drives allergic inflammation through activation of mucosal DCs.
Using immunohistochemical analysis and a mouse footpad model to evaluate the architecture of the single draining lymph node from the site (the popliteal node), St John and Abraham (2009) observed disrupted B- and T-cell zones after infection by Salmonella typhimurium, but not by E. coli or Listeria monocytogenes. Disruption depended on the presence of the Salmonella msbB gene and intact Salmonella LPS (sLPS). Infection with wildtype Salmonella, but not a mutant lacking msbB, lowered expression of Ccl21 (602737) and Cxcl13 (605149), but not another T-cell zone chemokine, Ccl19 (602227), or another B-cell chemoattractant, Cxcl12 (600835). Reduced expression of Ccl21 and Cxcl13 induced by sLPS was Tlr4 dependent and involved Socs3 (604176) and Smad3 (603109). St John and Abraham (2009) concluded that sLPS is a virulence factor that exploits TLR4 signaling to disrupt host lymphoid tissue.
Maroso et al. (2010) showed that chemical induction of seizures in mice resulted in increased cytoplasmic expression of Hmgb1 (163905) in astrocytes in the hippocampus, as well as increased expression of Tlr4 in neurons within the pyramidal cell layers. The seizures most resembled temporal lobe epilepsy (TLE, see, e.g., ETL1; 600512) in humans. Brain tissue from patients with TLE showed increased HMGB1 and TLR4 expression compared to controls. The authors noted that HMGB1 can bind to and activate TLR4 (Apetoh et al., 2007). In vitro studies showed that neurons undergoing glutamate-induced cytotoxic cell death released Hmgb1. In mice, Hmgb1 was found to cause seizures in wildtype mice, but not in those with inactivation of the Tlr4 gene. Antagonists of Hmgb1 and Tlr4 retarded seizure precipitation and decreased acute and chronic seizure recurrence. Overall, the findings indicated that HMGB1-TLR4 signaling may contribute to the generation and perpetuation of seizures.
Andonegui et al. (2009) generated mice that expressed Tlr4 exclusively on endothelium (endothelium-Tlr4 mice). Wildtype and endothelium-Tlr4 mice exhibited comparable neutrophil recruitment in response to local LPS administration, but endothelium-Tlr4 mice showed reduced neutrophil infiltration in lungs in response to systemic LPS or intraperitoneal E. coli administration. Endothelium-Tlr4 mice instead mobilized neutrophils to primary sites of infection, cleared bacteria, and resisted a dose of E. coli that killed 50% of wildtype mice within 48 hours. Endothelium-Tlr4 mice failed to accumulate neutrophils in lungs after intratracheal LPS, a response that requires Tlr4 expression on bone marrow-derived immune cells. Andonegui et al. (2009) concluded that endothelial TLR4 functions as the primary intravascular sentinel system for detection of bacteria, whereas bone marrow-derived immune cells are critical for pathogen detection at barrier sites.
Kane et al. (2011) demonstrated that transmission of the retrovirus mouse mammary tumor virus (MMTV) required commensal intestinal microbiota and that MMTV was bound to LPS. Mice lacking Tlr4, Il6, Il10, or Cd14, but not those lacking Tlr2, eliminated MMTV in successive generations. Kane et al. (2011) concluded that LPS-induced TLR4 signaling drives a viral 'subversion' pathway via IL6-dependent IL10 production that promotes viral transmission to successive generations.
Apoe -/- mice are susceptible to development of atherosclerosis when fed a high-fat diet. Hayashi et al. (2012) found that mice lacking both Apoe and Tlr4 were susceptible to progressive narrowing of the innominate artery when orally infected with Porphyromonas gingivalis, whereas infected Apoe -/- mice and uninfected Apoe -/- and Apoe -/- Tlr4 -/- mice were unaffected. Lack of Tlr4 was associated with increased macrophage infiltration and expression of Tlr2 in aortic lesions in infected mice. Tlr4 deficiency also promoted a Th17 (see 603149)/regulatory T-cell imbalance in atherosclerotic lesions after infection, as well as altered IgG humoral immunity and Th1 responses. Hayashi et al. (2012) concluded that TLR4 has atheroprotective role in response to P. gingivalis infection.
To study the requirement for endothelial TLR4 in spontaneous cerebral cavernous malformation (CCM; 116860) formation, Tang et al. (2017) bred mice with floxed Tlr4 and Krit1 (604214) alleles using mice from the CCM-susceptible Krit1(ECKO) colony (endothelial-specific deletion of Krit1). Tang et al. (2017) observed that loss of a single endothelial Tlr4 allele resulted in an approximately 75% reduction in CCM lesion burden at postnatal day 10, whereas loss of both alleles resulted in virtually complete prevention of CCM lesion formation. Although less complete, global loss of Cd14, a soluble TLR4 coreceptor that binds lipopolysaccharide and facilitates TLR4 signaling, also prevented CCM formation in susceptible Krit1(ECKO) mice. Seven of 8 Krit1(ECKO) neonates who had been delivered and raised in a germ-free environment did not develop any CCM lesions. In contrast, all Krit1(ECKO) neonates who had been delivered in a germ-free environment but raised by conventional mothers exhibited robust CCM formation at postnatal day 10. Tang et al. (2017) found that CCM susceptibility associated with gram-negative bacteria, and that either Tlr4 blockade or altering the microbiome prevented CCM formation in susceptible mice. They concluded that while KRIT1 mutation predisposes to the formation of cerebral cavernous malformations, endothelial TLR4 and the microbiome drive their development.
Endotoxin Hyporesponsiveness
Arbour et al. (2000) showed that 2 common cosegregating missense mutations (asp299 to gly and thr399 to ile, 603030.0002) that affect the extracellular domain of the TLR4 receptor are associated with blunted response to inhaled lipopolysaccharide in humans. In transfection experiments, they found that the asp299-to-gly (D299G) mutation, but not the thr399-to-ile (T399I) mutation, interrupted TLR4-mediated LPS signaling. An A-to-G transition at nucleotide 896 of the TLR4 gene was responsible for the D299G amino acid change. Allele frequency of the 896G substitution was 6.6% in a population studied for airway responsiveness and 7.9% in a control population from Iowa. These findings indicated that the 2 populations were in Hardy-Weinberg equilibrium as to distribution of the TLR4 mutation. Findings demonstrated that the 896G substitution altered the ability of the host to respond to environmental stress; however, not all of the subjects who were hyporesponsive to LPS had mutations in TLR4, and not everyone with the TLR4 mutation was hyporesponsive to inhaled LPS.
In a study in Bruneck, Italy, Kiechl et al. (2002) found that out of 810 screened persons, 55 subjects had the D299G allele, with lower levels of certain proinflammatory cytokines, more susceptibility to severe bacterial infections, lower risk of carotid atherosclerosis, and a smaller intima-media thickness in the common carotid artery. The D299G allele was heterozygous in 53 and homozygous in 2 of the subjects. In 46 of these subjects, cosegregation with the T399I (603030.0002) polymorphism was observed, whereas 9 subjects had an isolated D299G polymorphism.
Colorectal Cancer, Susceptibility to
In a study of 89 Croatian patients with sporadic colorectal cancer (see 114500) and 88 sex- and age-matched Croatian controls, Boraska Jelavic et al. (2006) found that the gly299 allele of the TLR4 gene was more frequent in colorectal cancer patients than controls (p = 0.0269).
Macular Degeneration, Age-Related, 10, Susceptibility to
Zareparsi et al. (2005) examined the D299G and T399I (603030.0002) variants of TLR4 as contributors to susceptibility to age-related macular degeneration (ARMD10, 611488) in a sample of 667 unrelated Caucasian ARMD patients and 439 controls. Multiple logistic regression demonstrated an increased risk of ARMD in carriers of the G allele at TLR4 residue 299 (odds ratio = 2.65, P = 0.025), but lack of an independent effect by T399I variant. TLR4 D299G showed an additive effect on ARMD risk (odds ratio = 4.13, P = 0.002) with allelic variants of apolipoprotein E (APOE; 107741) and ATP-binding cassette transporter-1 (ABCA1; 600046), 2 genes involved in cholesterol efflux. The effect of TLR4, APOE, and ABCA1 variants on ARMD susceptibility was opposite to that of association with atherosclerosis risk.
Response to Anthracycline Therapy
Apetoh et al. (2007) analyzed the time to metastasis in a cohort of 280 patients with nonmetastatic breast cancer who were treated with anthracyclines after local surgery revealing lymph node involvement. The frequencies of heterozygous and homozygous germline D299G polymorphism (rs4986790) were 17.1% and 0.7%, respectively. Carriers of the polymorphism did not differ with respect to any classical prognostic factors from noncarriers. The frequency of metastasis by 5 years after surgery was statistically higher in the group carrying the D299G polymorphism (40% vs 26.5%). The Kaplan-Meier estimate of metastasis-free survival showed an overall significantly lower percentage of metastasis-free patients in the group of D299G carriers. Apetoh et al. (2007) found that the D299G SNP reduces the interaction between TLR4 and HMGB1 (163905) and abolishes the capacity of monocyte-derived dendritic cells to crosspresent dying melanoma cells to Mart1 (605513)-specific HLA-A2 (see 142800)-restricted cytotoxic T lymphocytes, a biologic property that depends on HMGB1 in wildtype monocyte-derived dendritic cells. Based on these and other findings, Apetoh et al. (2007) concluded that the D299G polymorphism in TLR4 may influence the immunologic component of anthracycline-based chemotherapy in human cancer. The authors stated that the D299G variant is found in 8 to 10% of Caucasians.
Myocardial Infarction
In Sicily, Balistreri et al. (2004) genotyped 105 young men with acute myocardial infarction, 127 male controls matched for age, and another control group of 55 very old men (mean age, 100 years) for the D299G polymorphism. They found significant differences among the 3 groups (p less than 0.001): the D299G allele was underrepresented in patients with acute myocardial infarction and overrepresented in very old men, with intermediate values in healthy young controls. After adjustment for risk factors, significant differences in allele frequency persisted between patients with acute myocardial infarction and very old men (p less than 0.002) and between young controls and very old controls (p less than 0.001).
Arbour et al. (2000) showed that 2 common cosegregating missense mutations (asp299 to gly, 603030.0001; and thr399 to ile) that affect the extracellular domain of the TLR4 receptor are associated with blunted response to inhaled lipopolysaccharide in humans. In transfection experiments, they found that the asp299-to-gly (D299G) mutation, but not the thr399-to-ile (T399I) mutation, interrupted TLR4-mediated LPS signaling. An A-to-G transition at nucleotide 896 of the TLR4 gene was responsible for the D299G amino acid change. Allele frequency of the 896G substitution was 6.6% in a population studied for airway responsiveness and 7.9% in a control population from Iowa. These findings indicated that the 2 populations were in Hardy-Weinberg equilibrium as to distribution of the TLR4 mutation. Findings demonstrated that the 896G substitution altered the ability of the host to respond to environmental stress; however, not all of the subjects who were hyporesponsive to LPS had mutations in TLR4, and not everyone with the TLR4 mutation was hyporesponsive to inhaled LPS.
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