Sepsis is the major cause of morbidity and death in the intensive care unit (ICU) (1). Sepsis-1 defined sepsis as a host’s systemic inflammatory response syndrome (SIRS) to infection, and the clinical criterion was suspected infection plus SIRS (2). There are many causes of sepsis, such as bacterial infections, influenza, pneumonia, and food poisoning (3). ICUs are rare recourses worldwide. The definition was then revised and validated with new clinical criteria. The third international consensus on the definition of sepsis and septic shock defined sepsis as a syndrome of physiological, pathological, and biochemical abnormalities of the body induced by infection (4). Sepsis-3 criteria defined sepsis as “Life-threatening organ dysfunction caused by a dysregulated host response to infection” (5). Inflammation resulting from an overreaction to an infectious agent can induce fatal diseases such as hypotensive septic shock or septic shock, which have a high mortality rate (6). Currently, there is a large amount of transcriptomic data on sepsis in public databases, however, the molecular mechanisms of sepsis remain unclear. The heterogeneity of sepsis conditions and diverse sources of infection may contribute to this result (7). Conventional single-gene-based approaches are ineffective in distinguishing sepsis from traditional markers such as procalcitonin (PCT) and interleukin-8 (IL-8) (8). Recombinant human activated protein C (rhAPC) is currently the only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of severe sepsis. Therefore, it is necessary to develop molecular diagnostic markers based on a higher level. We propose to use the KEGG pathway as a marker in the diagnosis, classification, treatment, and prognosis of sepsis and provide potential therapeutic drugs accordingly. This method provides a novel idea from disease mechanism research to clinical translation. Here, we identify KEGG pathways associated with sepsis, survival, immune cells, and infection types, and discover potential disease-related drugs to provide important information for follow-up research.

Sepsis is a heterogeneous disease, as diverse immune cell types are involved and many microorganisms can cause the disease. Current analysis of sepsis is mainly focused on single-source data. We integrated bulk transcriptome, single-cell transcriptome, and methylation data to find potential disease biomarkers and mechanisms. The integration of single-cell data is necessary as researchers cannot discriminate which cell produces the signal in bulk transcriptome data. Knowing the cell source of gene expression is necessary for the studies on mechanisms and treatment (18). Due to disease heterogeneity, the result of a single gene-based analysis sometimes lacks reproducibility (18). Therefore, we propose transcriptome analysis at the pathway level to identify differential pathways associated with sepsis. We analyzed pathways associated with survival, the relationship between pathways, immune cells, and the source of infection. We found T cell dysfunction in sepsis and used the T cell exhaustion-related pathway to mine related therapeutic drugs. This article provides useful ideas for the study of sepsis mechanisms, diagnosis, classification, and drug treatment.

Gene set-based analysis has been reported to yield more consistent results (19). This paper systematically analyzed the expression of the KEGG pathway in sepsis ( Table S1 ). The identified differentially expressed pathways are supported by the literature. For example, cardiac dysfunction and hypotension are hallmarks of septic shock, which correspond to myocardial contractions and vascular smooth muscle contractions identified here (20). VEGF signaling is down-regulated in a rat model of sepsis, and this is consistent with our findings (21). In severe sepsis, protein breakdown occurs in skeletal muscle, leading to malnutrition in the body, as well as respiratory muscle dysfunction and pulmonary complications. This process is mainly involved in the ubiquitin-proteasome protein degradation pathway (22). This is consistent with the proteasome up-regulation identified here. Apoptosis, exhaustion, and function decline of T cells can lead to immunosuppression. Immune dysfunction is an important reason for the high mortality of sepsis. The acquired immune system dominated by T cells plays a key role in the late stage of sepsis (23). This is consistent with the down-regulation of the TCR signaling pathway found in this paper. LTP is the main molecular mechanism underlying learning and memory, and cognitive function and synaptic plasticity are decreased in septic mice (24). This is consistent with the LTP down-regulation found in this paper. Cytosolic free DNA as a danger signal is recognized by the DNA sensor cyclic GMP-AMP synthase (cGAS) and activates downstream signals to promote the production of type I interferons and other cytokines, thereby generating a corresponding immune response (25). We found that this pathway is up-regulated, which may be related to the inflammatory response. Furthermore, cluster analysis revealed intrinsic heterogeneity of the KEGG pathway in septic patients, suggesting the complexity of the septic disease. This may be caused by a variety of factors, such as the patient’s own genetic and physiopathological characteristics, differences in the period of infection, and different types of bacterial or viral infections. Therefore, it is difficult to carry out clinical diagnosis and treatment through a single molecule, but various factors should be considered comprehensively, and different treatment strategies should be formulated for patients with different characteristics.

This paper analyzed the relationship between differential KEGG pathways and survival, and verified the importance of differential pathways ( Figure 3 ). Compared with healthy subjects, antigen processing and presentation are down-regulated in sepsis patients, but their expression is positively correlated with patient survival, suggesting that this pathway plays a protective role in sepsis. Antigen processing and presentation is an important component of acquired immunity and is involved in the recognition of pathogenic antigens (26). Sepsis is an important factor in the death of patients with CML, and the CML pathway is down-regulated in sepsis (27). Cytokine-cytokine receptor interaction pathway plays a dual role in sepsis. Moderate inflammation can fight off foreign pathogens, and excessive inflammatory response can lead to tissue damage, organ failure, and even death (28). Leishmania infection was considered SIRS, and its clinical manifestations and biochemical analysis are similar to those of sepsis (29). The PPAR signaling pathway acts on multiple links of the inflammatory signal transcription pathway, inhibits the inflammatory response, and thus has a certain protective effect on sepsis (30). The clinical features of primary immunodeficiency resemble severe sepsis, with severe cases associated with fever, shock, and multiple organ failure (31). Progesterone improves sepsis syndrome by reducing inflammatory cytokines, IL-6 and TNF-α and by restoring the antioxidant defense system. Therefore, progesterone may help control inflammation in sepsis, and this pathway is up-regulated in sepsis (32). Vibrio vulnificus sepsis has a high mortality rate (33). Therefore, related pathways can serve as potential therapeutic targets and prognostic signals.

This paper analyzes the relationship between the KEGG pathway and immune cell type ( Figure S1 ). For example, CD4⁺ T cell abundance has the highest correlation with the TCR signaling pathway, which is positively correlated with 28-day survival. Neutrophils, a type of polymorphonuclear leukocytes, are the most abundant circulating leukocyte population in the human immune system, accounting for 50% to 70% of all circulating leukocytes in healthy adults. Neutropenia patients are more prone to microbial infection (34). We found that the number of neutrophils, which is highly positively correlated with the TLR signaling pathway, was positively correlated with survival. Neutrophil cell abundance was up-regulated in sepsis, suggesting its protective roles ( Figure S2 ). We found macrophages were associated with survival, but with opposite directions in total and M0 or M1 macrophages, indicating that anti-inflammatory M2 macrophages may play a protective role (35). It has been reported that macrophage polarization can induce cytokine storm and immune paralysis, and macrophage activation can lead to early death in sepsis (36; (37). Therefore, these cells are potential cellular markers or targets for subtyping or risk assessment, such as the high abundance of certain immune cells with a lower risk of death ( Figure 4 ).

We integrated multi-omics data to mine the potential molecular mechanisms of sepsis. We focused on the well-known TCR signaling pathway, which is down-regulated in sepsis ( Figure 5A ). Cell level analysis identified a down-regulated abundance of T cells ( Figure S2 ). DNA methylation analysis revealed the differentially methylated genes were enriched with GTPase activity ( Figure S3 ), which controls T cell activation (38). A recent study showed that bacteria can exploit eukaryotic Rho GTPase signaling cascades to promote invasion and proliferation within their host (39). Among our identified top differentially methylated genes, TPST1 can be induced by lipopolysaccharides (LPS) in macrophages (40). KCNJ15 was found to be involved in bacteria clearance in infection (41). The two genes were mainly expressed on monocyte/macrophage (data not shown), and we found they were hypomethylated and up-regulated in sepsis.

At present, no research has proposed a pathway that can distinguish different types of infection, and determining the type of infection is the prerequisite for formulating a reasonable clinical treatment plan. The multiple pathways identified in this paper have good performance in distinguishing bacterial, viral sepsis, and SIRS, and some of them are also prognostic for patient survival, such as primary immunodeficiency, antigen processing and presentation, etc ( Figure 7 ). The latest study found that plasma tyrosine biosynthesis was significantly decreased in rats with acute lung injury in sepsis (42). While we found that this pathway is up-regulated in viral sepsis, it can be inferred that plasma tyrosine levels may serve as a promising molecular marker for distinguishing bacterial from viral sepsis. Viral sepsis can cause down-regulation of cardiac muscle contraction, while viral infections have long been associated with heart damage. These results suggest that clinicians should formulate differential diagnoses and treatment strategies for different subtypes/causes of sepsis to effectively improve the treatment effect (43).

Multi-omics analysis revealed the T cell dysfunction in sepsis. At the pathway level, we found the expression changes in pathways of TCR signaling and antigen processing and presentation. In single-cell analysis, we found that T cell exhaustion was present in sepsis. In methylation analysis, several T cell-related genes were altered, such as TNFSF8 and AKT3. In cell subtype level analysis, we found that gamma-delta T cells and Tregs were associated with bacterial and viral infections, and Tregs were associated with mixed infection. Indeed, publications have reported the associations between the gamma-delta T cells and sepsis severity (44), and the associations of Tregs and secondary infection (45). These results provide possible cellular targets for treatment.

Finally, we used the T cell-related KEGG pathway to mine potential drugs and provide important information for the clinical application of related drugs ( Table 1 ). For example, Gabapentin is an analog of gamma-aminobutyric acid (GABA), which has antiepileptic and analgesic effects, but it has the effect of lowering blood pressure and heart rate, so it may not be suitable for sepsis patients (33). Tesaglitazar, a dual PPAR agonist with an affinity for PPARɑ/γ, can increase insulin sensitivity and is used in the treatment of type 2 diabetes. We found that insulin signaling pathway was down-regulated in sepsis. Studies demonstrated that intensive insulin therapy could combat insulin resistance and decrease morbidity and mortality by modulating the proliferation, apoptosis, differentiation, and immune functions of certain immune cells, especially monocytes/macrophages, neutrophils, and T cells associated with sepsis (46). While PPARγ is a potential therapeutic target for sepsis, which can regulate cell growth and differentiation, and repress the TGFβ1, which is known to play immune suppression roles in cancer microenvironment (47; (48). Tesaglitazar can reduce the inflammatory response and has been used for glycemic control in burn patients (49). Interestingly, we found that LTBP1 was hypomethylated in sepsis, which encodes an important protein for the TGF-β1 activity regulation (47). We found a positive correlation between LTBP1 and TGF-β1. Elevated TGF-β1 could block naïve T cell differentiation towards a Th1 effector phenotype, and promotes their conversion towards the Treg which suppresses antigen presenting functions of dendritic cells (47). We found Treg is an unfavorable predictor for sepsis survival ( Figure 4 ). Interestingly, TGF-β was recently found to be a main driver of T cell exhaustion (50). We observed that exhausted T cells (TS3 subcluster) expressed high levels of TGF-β and CD14⁺ macrophage expressed high levels of TGF-β ( Figure 6E ), which may provide a clue for the treatment. A feature of the COVID-19 sepsis cytokine storm is an abnormally increased TGF-β activity (51). Various anti-TGF-β therapies should be explored in the future. While tesaglitazar treatment in the kidney results in a decrease in TGF-β1 mRNA (52). Therefore, tesaglitazar may not only alleviate inflammation but also abolish T cell suppression. Nimbolide is the main component of neem leaf extract, commonly used in cancer treatment. The latest study finds it protects against endotoxin-induced ARDS by suppressing the nitrosative-oxidative stress, inflammatory cytokines, and chemokines expression (53). ARDS is the most common fatal complication of sepsis, therefore, nimbolide can be a promising candidate for sepsis treatment. Melitten is the main component of bee venom, which can cause muscle contracture and increase blood pressure (54). A study found that melitten has toxicity and antibacterial effect on clinically extensively drug-resistant bacteria (55). However, safe dose levels of melittin could not exhibit antimicrobial activity, which hinders its application in clinical practice. Tanespimycin, a derivative of the antibiotic geldanamycin, is used in cancer therapy by inhibiting tumor Hsp90 expression. Interestingly, a study finds it prolongs survival, reduces inflammation, and reduces lung damage in mice with sepsis (56).

In conclusion, the work is the first to perform integrative analysis of multi-omics data and to globally characterize the expression changes of sepsis at the pathway level, providing molecular markers and targets for diagnosis, prognosis, and drug development.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

QL and MS: Methodology, Investigation, Writing – Review and editing. QZ: Validation, Writing – Review and editing. YL: Methodology, Formal analysis, Visualization, Writing – Original draft. JX: visualization, Writing – Review and editing. HF: Methodology, Supervision, Resources. All authors contributed to the article and approved the submitted version.