Background
Trauma is a major and costly public health problem around the world, and it is the fourth leading cause of death among young adults in China [
1,
2]. Infectious disease, sepsis, and multiple organ dysfunction syndrome (MODS) remain important complications for patients who survive major trauma [
3,
4]. Therefore, preventing sepsis and MODS is crucial in the treatment of patients who survive major trauma.
A single-nucleotide polymorphism (SNP) is a variation in a single nucleotide that occurs at a specific position in the genome where each variation is present to some appreciable degree within a population. Increasing evidence suggests that SNPs are critical for determining interindividual differences in both inflammatory responses and clinical outcomes in sepsis patients [
5,
6]. The previous results of our group and results reported by other groups have indicated that genetic variations within at least 38 genes from the proinflammatory signaling pathway, including pattern recognition receptors (
TLR1,
TLR2,
TLR4, and
TLR9), signal-transducing adaptor proteins (
cluster of differentiation 14 [CD14], LBP, MD2, HMG1), and inflammatory cytokines of the immune system (
IL1A,
IL1B,
IL1RN,
IL4,
IL6,
IL8,
IL10,
IL17F,
IL18, and
TNF-α), are critical determinants of the magnitude of the immune inflammatory response, which profoundly affects the proinflammatory response to trauma and predisposes trauma patients to susceptibility or resistance to sepsis and MODS [
7‐
14]. Therefore, identifying the polymorphisms and associated interindividual differences may assist with the risk stratification of trauma patients at the early stages of trauma and might contribute to developing new genetically based diagnostic and therapeutic interventions that alter host susceptibility to sepsis-related outcomes. However, these studies are not easily compared, owing to the relatively small sample sizes and different study populations [
13]. Furthermore, the outcomes of these studies are sometimes different, which makes it more difficult to pool or compare results between these studies. Although some SNPs clearly appear to be associated with the disease, no definitive conclusions currently can be drawn regarding the influence of specific sequence variations on outcomes in trauma patients [
13].
It has been demonstrated that an inappropriate immune inflammatory response contributes to the development of sepsis and MODS in major trauma patients [
15,
16]. The development and resolution of inflammation may involve both proinflammatory and anti-inflammatory mechanisms [
17,
18]. The resolution of inflammation is an active process that prevents damage to the host and is governed by specific proresolving mediators. Lipoxin A4 (LXA4) was a main endogenous stop signal of inflammation, and the responses were mediated by a specific G protein-coupled receptor called
formyl peptide receptor 2 (FPR2, which is also known as FPRL1 or ALXR). Human FPR2/ALX is highly expressed in myeloid cells and expressed at low levels in lymphocytes and dendritic, macrophage, and endothelial cells [
17,
18]. FPR2/ALX conveys proresolving properties by provoking detachment of neutrophils, facilitating neutrophil apoptosis, and promoting macrophage efferocytosis and release of anti-inflammatory cytokines [
19]. On the basis of these biological functions, activation of FPR2/ALX has been shown to be essential in models of experimental inflammation, such as sepsis [
20‐
23], cerebral inflammation [
24], acute lung injury [
25,
26], and stroke [
27]. Accumulating evidence shows that the FPR2/ALX expression level may have pathophysiological relevance. Additionally, the
Fpr2/3 gene (an orthologue to human
FPR2/ALX) deficiency in mice impairs bacterial clearance and aggravates the host response in polymicrobial sepsis [
28], which suggests that FPR2/ALX has a pivotal role in the development of sepsis. Simiele et al. have shown that a single-nucleotide mutation (A/G) located −220 bp upstream of the transcription start site reduces promoter activity by approximately 35% to 90% in vitro [
29]. In addition, the messenger RNA (mRNA) and protein levels of FPR2/ALX are reduced by approximately ten- and threefold, respectively, in neutrophils of individuals carrying the A/G mutation compared with cells from individuals expressing the wild-type allele [
29]. These findings suggest that genetic variants of
FPR2/ALX may play an important role in regulating gene expression and ultimately influence the anti-inflammatory processes in patients with sepsis. However, there has not been any research on the clinical relevance of all the genetic variants of the
FPR2/ALX gene with sepsis and MODS after major trauma.
In this study, to assess the comprehensive potential biological significance of all polymorphisms of the FPR2/ALX gene, a tag single-nucleotide polymorphism (tSNP) approach was used. The association of the tSNPs with the development of sepsis and MODS in patients with major trauma was evaluated.
Discussion
Trauma is one of the leading causes of death globally among young adults [
1,
2]. Patients surviving the initial period after trauma are at risk of developing, and possibly dying as a result of, sepsis and sepsis-associated multiple organ dysfunction and failure. The disturbance of the regulation of inflammatory self-restriction is an important pathogenetic mechanism of sepsis [
34,
35]. Proresolving mediators and their receptors (FPR2/ALX), which downregulate inflammation, are the brake signal molecules of inflammation. LXA4 was shown to inhibit polymorphonuclear neutrophil (PMN) migration, to induce chemotaxis in monocytes, and to promote the phagocytosis of apoptotic PMNs by macrophages [
35].
In this study, we identify, for the first time to our knowledge, an SNP (rs11666254) located in the promoter of FPR2/ALX that is associated with increased sepsis hypersensitivity in major trauma patients. Our results show that rs11666254 was closely related to the sepsis morbidity rate after major trauma in a test cohort, and we confirmed the results in a validation trauma cohort. We then demonstrated its functional significance with respect to gene expression and ex vivo biological responses.
Our study shows that patients carrying the A allele of rs11666254 have higher risk of developing sepsis than G allele carriers. Between sepsis and nonsepsis individuals, we found that sex, ISS, pathogen type, and rs11666254 polymorphism were the important variables affecting sepsis morbidity; therefore, we used multiple logistic regression analysis to test for an independent effect of the rs11666254 polymorphism on the associations. Thus, our interpretation of the observed associations is less likely to be influenced by unmeasured confounders. Compared with patients who carried the GG genotype, patients carrying the GA or AA genotype had a significantly higher risk of developing sepsis (OR 1.81 and 3.01, respectively). Of the other two SNPs (rs17695052 and rs17695064) that were evaluated, we did not observe a significant association. No association was observed between the rs11666254 polymorphism and multiple organ dysfunction in sepsis patients. One of the reasons might be polygenetic and multifactorial involvement in the development of multiple organ dysfunction after trauma.
Researchers in a few studies have explored the asso ciation of
FPR2/ALX polymorphisms with human disease. Gwinn et al. found that two SNPs (F110S and C126W), which were located in the open reading frame of
FPR2/ALX, were associated with juvenile periodontitis [
36]. Kim et al. observed that an intronic SNP (4209 T/G) was associated with the risk of asthma disease [
37]. These three SNPs (F110S and C126W, 4209 T/G) along with 220A/G variants (data in the study by Simiele et al. [
29]) were not analyzed in the present study. The reason is that in this study, the common SNPs in the
FPR2/ALX gene with MAF greater than or equal to 0.05 were selected for the analysis of tSNPs, whereas the frequencies of these SNPs in the Chinese population is below 5%. A total of 21 SNPs in the
FPR2/ALX gene from the HapMap database for the CHB population (Additional file
1) were enrolled. On the basis of analysis of SNP haplotypes in each block and tagging threshold of
r
2, rs11666254 is the tag SNP in block 1 and rs17695052 is the tag SNP in block 2. rs17695064 was still selected because it is located in the 3′-UTR of exon 2, which might regulate
FPR2/ALX gene transcription. Taken together, the three SNPs (rs11666254, rs17695052, and rs17695064) selected in this study for genotyping might capture most of the genetic variation of the entire
FPR2/ALX gene and might represent potential biological significance of the
FPR2/ALX genetic variations. Three SNPs in our study (rs11666254 [−1010G/A], rs7248161 [−1160G/G], and rs7256993 [398 T/T]) were not included in Kim et al.’s study [
37]. The reason may be the relatively low MAF of the three SNPs in their study population.
FPR2/ALX is located on chromosome 19 [
38]. Alternative splicing gives rise to four mRNAs in which there are different truncations at the 5′-UTR and different exon cassettes [
29]. Along these lines, 15-epi-LXA4 biosynthesis and FPR2/ALX expression determine the magnitude and duration of the inflammatory reaction in humans [
39]. In a previous study, a rare single-nucleotide mutation (A/G) located −220 bp upstream of the transcription start site was found to reduce the promoter activity as well as the mRNA and protein levels of FPR2/ALX [
29].
How might the rs11666254 variant affect susceptibility to sepsis? To confirm the possible functional significance of the rs11666254 polymorphism, we further investigated the association of this polymorphism with
FPR2/ALX expression using ex vivo stimulation of whole blood with LPS in trauma patients. The results show that the A minor allele was significantly and negatively associated with FPR2 expression, indicating that FPR2/ALX production in subjects with A carriers was significantly lower than in G carriers. Neutrophils are the primary source of soluble annexin A1 in inflammatory resolution [
40]. Our finding that FPR2/ALX was highly expressed in PMNs (data not shown) supported the role of FPR2/ALX in signaling the critical step in resolution [
41]. These results further validated the biological function of rs11666254, and we can conclude that rs11666254 not only was a useful biomarker for sepsis susceptibility posttrauma but also was a functional SNP affecting FPR2/ALX expression.
TNF-α is one of the most well-defined proinflammatory cytokines. Various evidence has shown that high serum TNF-α levels were positively correlated with the severity and prognosis of inflammatory diseases [
42,
43]. Data derived from in vitro and animal experiment data have indicated that the
Fpr2/3 gene (an orthologue to human
FPR2/ALX) is crucial to enacting nonredundant functions including control of cell recruitment, phagocytosis, modulation of soluble mediator generation, and containment of bacteremia, which prevents spread to vital organs and opens new opportunities to manipulate the host response in sepsis. The anti-inflammatory effect occurred mainly through the regulation of TNF-α [
28]. The levels of FPR2/ALX expression might be an important determinant of LPS-induced TNF-α production [
44]. Lipoxin and aspirin-triggered lipoxin inhibit TNF-α secretion from activated T cells via FPR2. Given the clinical relevance of the rs11666254 polymorphism, and on the basis of our results, we further hypothesized that this SNP might be associated with TNF-α production in patients with major trauma. An association was also observed between the rs11666254 A allele and higher TNF-α production. These results correlated with the clinical relevance of rs11666254.
This SNP is located −1010 bp upstream of the transcription start site of the FPR2/ALX gene. To further determine that the association of the rs11666254 polymorphism with FPR2/ALX production is due to the direct effect of this polymorphism rather than the effect of other polymorphisms in linkage disequilibrium with other polymorphisms, we investigated the effect of the rs11666254 polymorphism on the FPR2/ALX promoter activity using a reporter gene assay system. Our results showed that the fold increase of relative luciferase activity is significantly lower when transfected with vectors containing the rs11666254 A allele. The results suggest that G-to-A variation could significantly reduce the transcriptional activity of the FPR2/ALX promoter. However, the exact mechanism needs to be studied further.
Despite a sophisticated design, our study also has limitations. One limitation was that rare variants in the FPR2/ALX gene were not investigated. We estimated that our study had 89.9% power to detect a moderate association (OR 1.5) between a common variant (e.g., rs11666254) and sepsis. However, it had only 58.6% and 36.1% power to detect a moderate association (OR 1.5) for a rare variant with MAFs of 0.1 and 0.05, respectively. This result shows that larger samples are required to assess the association between rare variants of FPR2/ALX and sepsis. Another limitation was that the in vivo association of rs11666254 with FPR2/ALX expression and TNF-α level could not be confirmed in this study because of difficulties in obtaining blood samples from some sepsis patients.
In summary, the association between the common variants of FPR2/ALX and sepsis was evaluated in severe trauma patients. It was demonstrated that a promoter polymorphism in FPR2/ALX was capable of decreasing the level of gene transcription activity, downregulating mRNA and protein expression, and increasing TNF-α production with LPS induction. These results suggest that rs11666254 might increase the incidence of sepsis in patients with severe trauma. This polymorphism may be an important biomarker that can be used in the early risk assessment of sepsis after major trauma. However, the clinical application of this polymorphism has yet to be studied.