Sepsis is a serious systemic inflammatory response caused by bacterial, viral and fungal infection, and is one of the major causes of death in critical care patients (Rello et al., 2017). Data from developed countries estimate more than 30 million cases of sepsis and about 20 million cases of severe sepsis globally every year, which may result in more than 5 million deaths. Sepsis detection mainly depends on clinical diagnosis, but early clinical characteristics are not specific, and the lack of timely and reliable early warning diagnosis indicators is an important reason behind the high mortality among sepsis patients (Napolitano, 2018).

During the pathophysiological process of sepsis, pathogens and their toxins invade the vascular circulation, activate the host’s cellular immune system, generate cytokines and cause systemic inflammatory response syndrome (Rubio et al., 2019). Sepsis can affect various organs and systems of the body, causing necrosis and dysfunction of tissues and cells, and even multiple organ dysfunction syndrome (Ismail Hassan et al., 2020). Vascular endothelial cell damage, platelet function and immune dysfunction are key factors affecting disease progression (Khakpour et al., 2015; Guo and Rondina, 2019). Thus, resolving knowledge of pathogen invasion in sepsis will benefit diagnosis and treatment to reduce mortality of patients.

Treatment of sepsis is mostly aimed at pathogen control, including the early use of broad-spectrum antibiotics (Zhou et al., 2019). Removing the pathogen toxin is also very important. Lipopolysaccharide (LPS) (Gram-negative bacteria), lipoteichoic acid (LTA) (Gram-positive bacteria) and mannan (PLM) (fungi) all bind to toll-like receptors (TLRs) of innate immune cells (Yang et al., 2018). The lipid part of LPS can bind to TLR4 on many cells, LTA can bind to TLR2 and TLR6, and PLM can bind to TLR2, TLR4 and TLR6, activating the nuclear transcription factor-B (NF-B) signal pathway and the subsequent pro-inflammatory/anti-inflammatory cytokine response (Salomao et al., 2008). Blocking the pathogen toxin is therefore a critical step for preventing disease progression in sepsis patients.

Conversely, studies have shown that the susceptibility and prognosis of sepsis are genetically related to the host (Chapman and Hill, 2012; Rautanen et al., 2015). Heterogeneous host genome variation plays an important role in sepsis patients, suggesting another critical factor for diagnosis and treatment of sepsis. Genomic variation can produce gene expression differences among individuals. The location of expression quantitative trait loci (eQTLs) has been analyzed to resolve the relationship between sepsis and the host immune response and various signaling pathways (Davenport et al., 2016). However, most reported single nucleotide polymorphisms (SNPs) are in non-coding regions and have no obvious biological significance. The development of genetics and epigenetics research has revealed that some SNPs can influence RNA modification to control RNA secondary structure or regulate RNA protein interaction, modifying the function of enhancers, silencers and exon splicing (Ramaswami et al., 2015; Mao et al., 2016; Wu and Hurst, 2016). These studies show that some SNPs as well as eQTLs affect the stability and function of RNA, which is the basic mechanism of host genome variation associated with the disease.

Methylation of the sixth N atom on the adenine base is part of an important regulatory mechanism affecting RNA stability and functionality (Yue et al., 2015). In eukaryotes, m⁶A is the most common post-transcriptional modification of RNA; it can affect the specific recognition of a protein complex and RNA and thus affect the intracellular transport, shear processing, degradation and translation of mRNA, finally regulating gene expression (Dominissini et al., 2012; Alarcon et al., 2015). Key regulatory factors of m⁶A RNA modification, such as METTL3, METTL 14 and FTO, can control the occurrence and development of many diseases by regulating the level of m⁶A RNA modification (Li et al., 2017; Lang et al., 2019; Wang et al., 2019; Yue et al., 2019).

m⁶A-SNPs can be considered an important functional genetic variation, providing new clues for understanding the molecular mechanism of genetic variation. A large number of m⁶A-SNPs related to bone mineral density, periodontitis, rheumatoid arthritis and coronary heart disease have been identified through integration analysis of the m⁶A-SNP list (Mo et al., 2018a; Mo et al., 2018b; Mo et al., 2018c; Lin et al., 2020). They integrated GWAS data and m6A methylation to scope its role in disease. However, there is no report on the role of m⁶A modification in the development of sepsis. Identification of m⁶A-eQTLs associated with sepsis will extend our knowledge of the genetic mechanism of m⁶A modification in sepsis. We therefore used the open eQTLs data set to explore the potential role of m⁶A-eQTLs in the pathogenesis of sepsis.

Disease-related genes show genetic variation in the population, which can affect the occurrence and development of the disease including cancer, sepsis and infectious. For a long time, research on the functional mechanism of these genetic variations has focused on changes in biological functional activity of the encoded proteins. However, a large number of SNPs found in genome-wide association analysis do not correspond to functional regions of proteins. Similarly, eQTLs are not located in promoter regions regulating gene expression. In recent years, a large number of m⁶A methylation modifications have been identified on mRNA of eukaryotes (Yue et al., 2015). These regulate a series of biological processes by affecting the splicing, translocation, degradation and translation of mRNA involved in the occurrence and development of many diseases. Some SNPs that do not change the coded amino acid can regulate mRNA function by affecting the level of m⁶A modification, thus changing protein abundance. m⁶A-SNPs can change m⁶A modification levels to affect mRNA degradation rate; for example, change in mRNA secondary structure caused by altered m⁶A modification can affect mRNA binding with translation machinery. Similarly, m⁶A-SNPs can also affect mRNA transport from nucleus to cytoplasm. When m⁶A-SNPs are located at splicing sites, they can affect production of mature mRNA with correct biological function. Thus, m⁶A-SNPs can cumulatively regulate the function of key regulatory genes in the process of disease occurrence and development through multiple effects.

The role of m⁶A modification on mRNA in the pathogenesis of sepsis has not previously been reported. Therefore, study of m⁶A-eQTLs might aid in resolving the underlying mechanism of the pathogenesis of sepsis. By analyzing results of published research on eQTLs associated with sepsis and comparing them with overlapping m⁶A-SNPs in the m⁶AVAR database, we identified a large number of m⁶A-eQTLs related to sepsis, including 15,720 cis-eQTLs and 381 trans-eQTLs. As we expected, these m⁶A-eQTLs were distributed across all chromosomes, suggesting that m⁶A methylation of mRNA plays an important role in sepsis as in other diseases.

Among genes corresponding to m⁶A-eQTLs, we found some suggested as key players in the pathogenesis of sepsis. HSPA1B, with 61 m⁶A-cis-eQTLs, also named HSP70, is involved in the inflammatory response, which is an important biological step in the development of sepsis ( Table S2 ). Research on different phases of sepsis found that HSPA1B can be considered a serum marker for the acute proinflammatory phase (Gasparotto et al., 2018). Study on Multiple organ dysfunction syndrome in sepsis found that increased HSPA1B has an anti-inflammatory effect (Wang et al., 2016). In rats, raloxifene prevents severe sepsis through induction of HSPA1B with an anti-inflammatory effect (Shen et al., 2017). Hesperidin can protect against lung injury induced by sepsis, also through induction of HSPA1B. Another protein, KNK43, also takes part in the HSPA1B pathway to coordinate the inflammatory response (Yuan et al., 2019). There is evidence that heat stress induces HSPA1B to prevent lethal sepsis (Matsathit et al., 2016). These results indicate that m⁶A-eQTLs exist in the key regulatory genes of sepsis and m⁶A methylation is closely involved in the process of sepsis, while genetic variation may cause a large number of individual differences through the mechanism of m⁶A methylation of mRNA.

We used GO enrichment analysis to characterize critical biological processes associated with an abundance of sepsis m⁶A-eQTLs. The platelet degranulation process was associated with 17 genes identified in this study. It is well accepted that platelet degranulation is a typical biomarker of sepsis. In the development of sepsis, platelet dysfunction plays a critical role in tissue injury (Assinger et al., 2019). Early onset neonatal sepsis can be predicted by platelet/lymphocyte ratio (Arcagok and Karabulut, 2019). A high level of platelet-derived growth factor B (PDGFB) is found in survivors of sepsis (Dou et al., 2019), and PDGFB can reduce the mortality of sepsis by blocking inflammatory responses (Dou et al., 2019). In sepsis-associated liver injury, AST/platelet ratio might be an early onset predictor (Dou et al., 2019). The underlying mechanism of platelet dysfunction in sepsis remains to be elucidated. Here, we revealed that genes corresponding to m⁶A-eQTLs are enriched in the platelet degranulation process, suggesting that m⁶A methylation is closely involved in the pathologies of sepsis. Published literature supports the relationship between the 17 platelet degranulation related genes corresponding to m⁶A-eQTLs and sepsis. ORM2 interacts with TLR2 signaling involved in the pathobiology of sepsis (Sumanth et al., 2019). CFD and VWF are important biomarkers of acute mortality for severe sepsis (Avriel et al., 2014; Gragnano et al., 2017). TIMP1 could be used as prognostic marker for the onset of sepsis (Nino et al., 2017). Sepsis is attenuated by inhibition of ABCC4 in rats (Xia et al., 2019). Reduction of CD63 increases the mortality rate of sepsis in mice through its function in the immune response (Yang et al., 2016; Alvarez-Estrada et al., 2019). The above evidence strongly supports our view that m⁶A methylation plays an extremely important role in the pathological basis of sepsis. We suggest that m⁶A-eQTLs may be a potential predictor of different clinical characteristics of the sepsis disease caused by genetic variation in individuals.

KEGG pathway enrichment analysis in this study revealed 12 genes corresponding to m⁶A-eQTLs associated with sepsis enriched in the S. aureus infection pathway. S. aureus is one of the major pathogenic bacteria in sepsis (Machado et al., 2019). Among the 12 genes corresponding to sepsis m⁶A-eQTLs in this pathway, platelet activating factor receptor (PTAFR) is induced in sepsis patients infected with Gram-negative bacteria (Dhainaut et al., 1994). Inhibition of PTAFR can block severe sepsis (Moreno et al., 2006). FPR2 is also induced in the early phase of sepsis and functions in cerebral inflammation (Gavins et al., 2012; Sordi et al., 2013). ITGB2 has been identified as an important factor in the inflammatory response to sepsis (Kragstrup et al., 2017). FCAR, a typical innate receptor for bacteria, is suggested to be an important protector against sepsis through mediation of bacterial phagocytosis (Chiamolera et al., 2001; de Tymowski et al., 2019). Such a high proportion of m⁶A-eQTLs related to S. aureus response genes indicates that m⁶A methylation plays an extremely important role in the occurrence and development of sepsis. The m⁶A-eQTLs we identified will be of value in the prognosis and diagnosis of sepsis.

In conclusion, we identified a large number of m⁶A-eQTLs potentially having critical functions in the pathogenesis and development of sepsis. These SNPs may contribute to the effect of genetic variation on the different outcomes of the disease. By mining the published literature, we found that these m⁶A-eQTLs are enriched in the platelet degranulation process and S. aureus infection pathway. Both are critical biological processes controlling the pathogenesis and development of sepsis, suggesting that mRNA m⁶A methylation plays a crucial role in sepsis.

All datasets generated for this study are included in the article/ Supplementary Material .

XS and NL designed the project and wrote the manuscript. XS performed bioinformatics analysis. YD, GT and YL helped analysis of data and revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.