The common promoter polymorphism rs11666254 downregulates FPR2/ALX expression and increases risk of sepsis in patients with severe trauma

Han Zhang, Yao Lu, Guixiang Sun, Fang Teng, Nian Luo, Jianxin Jiang, Aiqing Wen, Han Zhang, Yao Lu, Guixiang Sun, Fang Teng, Nian Luo, Jianxin Jiang, Aiqing Wen

Abstract

Background: Formyl peptide receptor 2-lipoxin receptor (FPR2/ALX) modulates the anti-inflammatory response and therefore may be a target for treating sepsis. The purpose of this study was to investigate the association between genetic variants of the FPR2/ALX gene and sepsis after severe trauma as well as to further analyze the functions of sepsis-related genetic polymorphisms.

Methods: Three tag single-nucleotide polymorphisms (tag SNPs) that captured all common alleles across the FPR2/ALX genomic region were genotyped using pyrosequencing in an initial sample consisting of 275 patients with severe trauma. The rs11666254 polymorphism, which had statistical significance, was genotyped in an additional 371 patients, and logistic regression analysis was performed to determine associations between the FPR2/ALX gene polymorphism and sepsis susceptibility after severe trauma. The messenger RNA (mRNA) and protein levels of FPR2/ALX in the lipopolysaccharide-stimulated white blood cells of trauma patients were determined by performing quantitative polymerase chain reactions and Western blot analysis. Tumor necrosis factor (TNF)-α production was measured by enzyme-linked immunosorbent assay. The effects of the promoter polymorphism rs11666254 on the transcription activity of FPR2/ALX were analyzed using a luciferase reporter assay.

Results: Among the three tag SNPs, only the rs11666254 polymorphism was found to be significantly associated with sepsis in trauma patients, and this association persisted after a pooled analysis of all 646 trauma patients, which showed that patients who carried the A allele of rs11666254 had a significantly higher risk of developing sepsis than individuals who carried the G allele. This SNP was also significantly associated with lower FPR2/ALX mRNA and protein expression as well as higher TNF-α production from the peripheral blood leukocyte response to bacterial lipoprotein stimulation. In addition, the rs11666254 polymorphism could significantly decrease the promoter activity of the FPR2/ALX gene.

Conclusions: The rs11666254 polymorphism in the FPR2/ALX gene is a functional SNP that increases sepsis susceptibility in patients after traumatic injury.

Keywords: FPR2/ALX; Promoter polymorphism; Sepsis; Severe trauma.

Conflict of interest statement

Ethics approval and consent to participate

The protocol for this study was approved by the ethical and protocol review committee of the Third Military Medical University. Informed consent was obtained from the participants or their next of kin.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
The single-nucleotide polymorphism rs11666254 inhibits lipopolysaccharide (LPS)-induced FPR2/ALX messenger RNA (mRNA) and protein expression. Data are presented as the mean and SD. The whole-blood samples collected from 98 trauma patients were mixed 1:1 (vol/vol) with RPMI 1640 culture medium and incubated with 100 ng/ml of Escherichia coli LPS (O26:B6) at 37 °C for 4 h. (a) FPR2/ALX mRNA and (b) protein expression in the peripheral leukocytes were assayed using quantitative polymerase chain reactions (presented as relative abundance) and flow cytometry (presented as mean fluorescence intensity [MFI]), respectively. One-way analysis of variance was used to assess statistical significance. a For mRNA expression, P = 0.002 for dominant association (GG vs GA + AA) and P = 0.025 for recessive effect (GG + GA vs AA). P = 0.012 for GG vs GA; P = 0.002 for GG vs AA; P = 0.151 for GA vs AA. b For protein expression (MFI), P = 0.007 for dominant association (GG vs GA + AA), and P = 0.035 for recessive effect (GG + GA vs AA). P = 0.029 for GG vs GA, P = 0.004 for GG vs AA, P = 0.163 for GA vs AA. There was no significant difference between genotypes before LPS stimulation
Fig. 2
Fig. 2
rs11666254 polymorphism and lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production. The whole-blood samples collected from 98 trauma patients were treated as shown. TNF-α production was determined using a sandwich enzyme-linked immunosorbent assay. One-way analysis of variance was used to assess statistical significance. P = 0.001 for dominant association, GG vs GA + AA, P = 0.003 for recessive association. P = 0.029 for GG vs GA; P = 0.006 for GG vs AA; P = 0.187 for GA vs AA. There was no significant difference between genotypes before LPS stimulation
Fig. 3
Fig. 3
rs11666254 polymorphism and the transcription activity of the FPR2/ALX promoter. Relative luciferase activity (RLA) was used in human embryonic kidney cells transfected with rs11666254G or rs11666254A constructs as described in the Methods section. Luciferase activity was normalized for transfection efficiency using the control plasmid pRL-cytomegalovirus. These results are expressed as fold increases in RLA of the FPR2/ALX promoter construct vector compared with pGL3-basic (mean ± SD). *P = 0.03 vs the wild-type construct. LPS Lipopolysaccharide

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