Study On The Genetic Association And Mechanism Of TLR4 Gene Polymorphism And Susceptibility To Sepsis

Background and Objective: Sepsis, a life-threatening condition to human life and health, is the leading cause of death in critically ill patients. However, the pathogenesis of sepsis is unknown yet. It is believed to be the result of a variety of factors, such as genetic background, pathogenic microorganism and environmental factors, among which genetic background draws more and more attention due to its role played in the body response to pathogenic microorganism and environmental factors.As a pattern recognition receptor identifying the composition of the Gram-negative bacteria, TLR4 plays a crucial role in the occurrence and development of sepsis by combining with lipopolysaccharide(LPS) and triggering the signal transduction in the inflammatory cells, so as to mediate the nonspecific inflammatory reaction to gram-negative bacteria.This study aimed to explore the association between TLR4 polymorphisms and the susceptibility and severity to sepsis in Chinese Han from Jiangsu Province, China and then try to verify the functional significance of positive SNP by biological methods.Materials and methods: A case-control study was conducted to analyze the tag SNPs in TLR4 gene by using Sequenom Mass ARRY detection methods. The associations of TLR4 genotypes with the susceptibility to sepsis and its severity(including severe sepsis, septic shock, multiple organ dysfunction syndrome and death, etc.) were evaluated by non-conditional logistic regression, adjusted by age, sex, smoking, drinking, APACHEⅡscore and primary disease status. For the significantly correlated SNPs, flow cytometry was performed to observe the expression patterns of CD14, TLR4 and IL-6R on the surface of human monocytes before and after LPS stimulation, so as to explore the different responses of the three different genotypes of TLR4 rs11536889 to LPS stimulation.Results: In this study, 15 tag SNPs in TLR4 gene were detected in the blood samples from 280 patients with sepsis and 224 control individuals. All SNPs were successfully genotyped, among which the frequency distribution of rs10983744 did not conform to the Hardy- Weinberg equilibrium. By genetic association analysis, we found rs11536889 SNP in the 3’-untranslated region of TLR4 gene and gender interaction was closely related to the severity of sepsis. As stratified by gender, male patients with CC genotype in male were more likely to suffer septic shock(in recessive mode: GG + GC vs. CC, OR = 9.36, 95% CI = 1.26- 69.83) and subsequent multiple organ dysfunction syndrome(in recessive mode: GG + GC vs. CC, OR = 19.10, 95% CI = 2.31-157.66) than that in female. Regarding the above results, we carried out a functional experiment on human peripheral monocytes. After the in vitro LPS stimulation on the whole blood with different concentrations, the expression of CD14 on the surface of monocyte increased and reached the peak at 1h and then decreased, but the expression of IL- 6R decreased to the lowest and then gradually rose. The mean expression of TLR4 on the surface of monocyte showed no significant change after LPS stimulation, but the high standard deviation indicated great diversity among the individuals. As stratified by genotypes, LPS stimulation for 1h at the dose of 1μg could trigger different TLR4 expression on the monocytes with TLR4 CC, CG and GG genotypes, but showing no statistically significant difference(p>0.05). In LPS stimulation group, the difference values of TLR4 expression(?TLR4LPS = TLR4 LPS 1h- TLR4 LPS 0h) between before and after LPS stimulation among the three genotypes showed significant differences. It decreased less significantly in monocyte with CC genotype than that in monocyte with GC or GG genotype(reduction of mean fluorescence intensity(MFI): 19.2±10.7 vs. 45.9±22.2 or 50.3±20.1, P < 0.05), while the reduction of MFI in the control group(?TLR4baseline = TLR4 baseline 1h- TLR4 baseline 0h) revealed no significant difference among the three genotypes(12.8±9.1 vs. 8.6±4.9 vs. 9.9±6.0, P > 0.05).Conclusion: Our results from case-control trial indicated that rs11536889 loci in TLR4 gene may be associated with the severity of sepsis in Chinese male patients.The in vitro experiment illustrated that LPS stimulation can reduce the expression of TLR4 on the surface of monocyte with different genotypes at difference extent. The reduction in monocyte with CC genotype was less than that in monocyte with GC or GG genotype, suggesting that individuals with CC genotype has poorer feedback control ability to TLR4 expression level on the surface of monocyte, which is easy to cause relative increase of TLR4 protein level in monocyte and thereby increasing the inflammatory reactions and exacerbating the severity of sepsis.