The common, co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms (SNPs), Asp299Gly and Thr399Ile, are associated with hyporesponsiveness to inhaled lipopolysaccharide (LPS) and increased susceptibility to Gram negative pathogens in humans. The purpose of this study was to identify the relative contributions of the Asp299Gly and the Thr399Ile variants in inhibiting the function of TLR4. 293/hMD2-CD14 cell line was transfected with lentiviral constructs containing human wild type (WT) TLR4-EGFP or TLR4-EGFP with Asp299Gly, Thr399Ile or Asp299Gly/Thr399Ile complementary DNA (cDNA). Multiple stable cell lines were established for each construct: three for WT TLR4, Asp299Gly, and Thr399Ile, and only two for Asp299Gly/Thr399Ile mutants and EGFP control. We did not observe a significant effect of polymorphisms on cell surface and intracellular TLR4 expression nor were there any significant differences in TLR4 and EGFP protein levels assessed by Western blotting and confocal microscopy among the multiple cell lines of each of the constructs. All cell lines had a dose-dependent responsiveness to LPS stimulation. However, compared to the WT TLR4, cells expressing TLR4 with Asp299Gly but not Thr399Ile polymorphism produced significantly less (P<0.05) IL-8 following LPS stimulation. Similarly, cells expressing TLR4 Asp299Gly but not Thr399Ile allele had significantly lower percentage of phosphorylated and total NF-κB P65 following LPS stimulation. While we could not do statistics on the Asp299Gly/Thr399Ile group, we observed a reduced responsiveness to LPS compared to WT TLR4. Taken together, we observed that the TLR4 Asp299Gly variant, but not the Thr399Ile variant, is responsible for impaired responsiveness of TLR4 to LPS and corresponding activation of NF-κB.
References
[1]
Vogel SN, Fenton M (2003) Toll-like receptor 4 signalling: new perspectives on a complex signal-transduction problem. Biochem Soc Trans 31: 664–668. doi: 10.1042/bst0310664
[2]
Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F (1999) Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 274: 10689–10692. doi: 10.1074/jbc.274.16.10689
[3]
Hirschfeld M, Weis JJ, Toshchakov V, Salkowski CA, Cody MJ, et al. (2001) Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect Immun 69: 1477–1482. doi: 10.1128/iai.69.3.1477-1482.2001
[4]
Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, et al. (2000) Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 1: 398–401.
[5]
Bulut Y, Faure E, Thomas L, Karahashi H, Michelsen KS, et al. (2002) Chlamydial heat shock protein 60 activates macrophages and endothelial cells through Toll-like receptor 4 and MD2 in a MyD88-dependent pathway. J Immunol 168: 1435–1440. doi: 10.4049/jimmunol.168.3.1435
[6]
Kuhns DB, Nelson EL, Alvord WG, Gallin JI (2001) Fibrinogen induces IL-8 synthesis in human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine or leukotriene B(4). J Immunol 167: 2869–2878. doi: 10.4049/jimmunol.167.5.2869
Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, et al. (2001) The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 276: 10229–10233. doi: 10.1074/jbc.m100099200
[9]
Jiang D, Liang J, Fan J, Yu S, Chen S, et al. (2005) Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nat Med 11: 1173–1179. doi: 10.1038/nm1315
[10]
Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, et al. (2002) Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 195: 99–111. doi: 10.1084/jem.20001858
[11]
Guillot L, Balloy V, McCormack FX, Golenbock DT, Chignard M, et al. (2002) Cutting edge: the immunostimulatory activity of the lung surfactant protein-A involves Toll-like receptor 4. J Immunol 168: 5989–5992. doi: 10.4049/jimmunol.168.12.5989
[12]
Tsan MF, Gao B (2004) Cytokine function of heat shock proteins. Am J Physiol Cell Physiol 286: C739–744. doi: 10.1152/ajpcell.00364.2003
[13]
Gay NJ, Gangloff M (2007) Structure and function of Toll receptors and their ligands. Annu Rev Biochem 76: 141–165. doi: 10.1146/annurev.biochem.76.060305.151318
[14]
Schumann RR, Leong SR, Flaggs GW, Gray PW, Wright SD, et al. (1990) Structure and function of lipopolysaccharide binding protein. Science 249: 1429–1431. doi: 10.1126/science.2402637
[15]
Tobias PS, Mathison JC, Ulevitch RJ (1988) A family of lipopolysaccharide binding proteins involved in responses to gram-negative sepsis. J Biol Chem 263: 13479–13481.
[16]
Tobias PS, Soldau K, Ulevitch RJ (1989) Identification of a lipid A binding site in the acute phase reactant lipopolysaccharide binding protein. J Biol Chem 264: 10867–10871.
[17]
Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249: 1431–1433. doi: 10.1126/science.1698311
[18]
Alexander C, Rietschel ET (2001) Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res 7: 167–202. doi: 10.1179/096805101101532675
[19]
Lorenz E, Frees KL, Schwartz DA (2001) Determination of the TLR4 genotype using allele-specific PCR. Biotechniques 31: 22–24.
[20]
Ferwerda B, McCall MB, Alonso S, Giamarellos-Bourboulis EJ, Mouktaroudi M, et al. (2007) TLR4 polymorphisms, infectious diseases, and evolutionary pressure during migration of modern humans. Proc Natl Acad Sci U S A 104: 16645–16650. doi: 10.1073/pnas.0704828104
[21]
Lorenz E, Mira J, Frees K, Schwartz D (2002) Relevance of mutations in the TLR4 receptor in patients with Gram negative septic shock. Arch Int Med 162: 1028–1032. doi: 10.1001/archinte.162.9.1028
[22]
Arbour NC, Lorenz E, Schutte B, Zabner J, Kline J, et al. (2000) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 25: 187–191. doi: 10.1038/76048
[23]
Bhuvanendran S, Hussin HM, Meran LP, Anthony AA, Zhang L, et al. (2011) Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms and typhoid susceptibility in Asian Malay population in Malaysia. Microbes Infect 13: 844–851. doi: 10.1016/j.micinf.2011.04.007
[24]
Montes AH, Asensi V, Alvarez V, Valle E, Ocana MG, et al. (2006) The Toll-like receptor 4 (Asp299Gly) polymorphism is a risk factor for Gram-negative and haematogenous osteomyelitis. Clin Exp Immunol 143: 404–413. doi: 10.1111/j.1365-2249.2005.03002.x
[25]
Kiechl S, Lorenz E, Reindl M, Wiedermann CJ, Oberhollenzer F, et al. (2002) Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med 347: 185–192. doi: 10.1056/nejmoa012673
[26]
Tulic MK, Hurrelbrink RJ, Prele CM, Laing IA, Upham JW, et al. (2007) TLR4 polymorphisms mediate impaired responses to respiratory syncytial virus and lipopolysaccharide. J Immunol 179: 132–140. doi: 10.4049/jimmunol.179.1.132
[27]
Rallabhandi P, Bell J, Boukhvalova MS, Medvedev A, Lorenz E, et al. (2006) Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling. J Immunol 177: 322–332. doi: 10.4049/jimmunol.177.1.322
[28]
Ohto U, Yamakawa N, Akashi-Takamura S, Miyake K, Shimizu T (2012) Structural analyses of human Toll-like receptor 4 polymorphisms D299G and T399I. J Biol Chem.
[29]
Fageras Bottcher M, Hmani-Aifa M, Lindstrom A, Jenmalm MC, Mai XM, et al. (2004) A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children. J Allergy Clin Immunol 114: 561–567. doi: 10.1016/j.jaci.2004.04.050
[30]
Norata GD, Garlaschelli K, Ongari M, Raselli S, Grigore L, et al. (2005) Effect of the Toll-like receptor 4 (TLR-4) variants on intima-media thickness and monocyte-derived macrophage response to LPS. J Intern Med 258: 21–27. doi: 10.1111/j.1365-2796.2005.01509.x
[31]
Guo J, Loke J, Zheng F, Hong F, Yea S, et al. (2009) Functional linkage of cirrhosis-predictive single nucleotide polymorphisms of Toll-like receptor 4 to hepatic stellate cell responses. Hepatology 49: 960–968. doi: 10.1002/hep.22697
[32]
McGettrick AF, O'Neill LA (2010) Localisation and trafficking of Toll-like receptors: an important mode of regulation. Curr Opin Immunol 22: 20–27. doi: 10.1016/j.coi.2009.12.002
[33]
Lundberg A, Wikberg LA, Ilonen J, Vaarala O, Bottcher MF (2008) Lipopolysaccharide-induced immune responses in relation to the TLR4(Asp299Gly) gene polymorphism. Clin Vaccine Immunol 15: 1878–1883. doi: 10.1128/cvi.00241-08
[34]
Prohinar P, Rallabhandi P, Weiss JP, Gioannini TL (2010) Expression of functional D299G.T399I polymorphic variant of TLR4 depends more on coexpression of MD-2 than does wild-type TLR4. J Immunol 184: 4362–4367. doi: 10.4049/jimmunol.0903142
[35]
Balistreri CR, Caruso C, Listi F, Colonna-Romano G, Lio D, et al. (2011) LPS-mediated production of pro/anti-inflammatory cytokines and eicosanoids in whole blood samples: biological effects of +896A/G TLR4 polymorphism in a Sicilian population of healthy subjects. Mech Ageing Dev 132: 86–92. doi: 10.1016/j.mad.2010.12.005
[36]
Candore G, Aquino A, Balistreri CR, Bulati M, Di Carlo D, et al. (2006) Inflammation, longevity, and cardiovascular diseases: role of polymorphisms of TLR4. Ann N Y Acad Sci 1067: 282–287. doi: 10.1196/annals.1354.037
[37]
Senthilselvan A, Dosman JA, Chenard L, Burch LH, Predicala BZ, et al.. (2009) Toll-like receptor 4 variants reduce airway response in human subjects at high endotoxin levels in a swine facility. J Allergy Clin Immunol 123 : 1034–1040, 1040 e1031–1032.
[38]
Figueroa L, Xiong Y, Song C, Piao W, Vogel SN, et al. (2012) The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of MyD88 and TRIF. J Immunol 188: 4506–4515. doi: 10.4049/jimmunol.1200202
[39]
Rallabhandi P, Awomoyi A, Thomas KE, Phalipon A, Fujimoto Y, et al. (2008) Differential activation of human TLR4 by Escherichia coli and Shigella flexneri 2a lipopolysaccharide: combined effects of lipid A acylation state and TLR4 polymorphisms on signaling. J Immunol 180: 1139–1147. doi: 10.4049/jimmunol.180.2.1139
[40]
Liadaki K, Petinaki E, Skoulakis C, Tsirevelou P, Klapsa D, et al. (2011) Toll-like receptor 4 gene (TLR4), but not TLR2, polymorphisms modify the risk of tonsillar disease due to Streptococcus pyogenes and Haemophilus influenzae. Clin Vaccine Immunol 18: 217–222. doi: 10.1128/cvi.00460-10
