Sepsis is found to be one of the fatal conditions with high morbidity and mortality in susceptible age groups. It is associated with inflammation [20], immunosuppression [21], immune dysfunction [22] and increased mortality [23]. The pathophysiology of the disease is not well established and demands further investigation. The current diagnostic methods in use fail to diagnose early sepsis, hence further thorough research is required to identify potent diagnostic markers. These markers may help predicting early sepsis for timely treatment of patients, which is critical in sepsis management. Hence, the basis of this meta-analysis study is to discover molecular markers associated with impaired immune function and immunosuppression through meta-analysis gene expression data, gene enrichment and network analysis.
We considered six GEO datasets from a public database to carry out a meta-analysis study. Analysis of datasets with GEO2R resulted in 233 common DEGs; 147 genes were upregulated and 87 were downregulated with p-value < 0.05 and log2 FC ≥ 1.5. The DEGs were subjected to network and functional enrichment analysis to find the hub genes, GO terms, and KEGG pathways. It was observed that several genes involved in adaptive and innate immune response were dysregulated, therefore interfering with normal immune function. In our study we found five MCODE clusters (Fig. 4) in which three clusters (Fig. 4a-c) were found to be significant. Cluster-1 genes are mainly involved in T cell biology. They regulate the pathways includes adaptive immune system, T cell differentiation and development. All genes in cluster-1 are downregulated, suggesting immunosuppression during sepsis. Cluster-2 and 3 genes participate in innate immune responses, antigen processing and presentation, neutrophil regulation and NET formation. Interestingly, all except three genes (CD74, HLA-DMA, and HLA-DMB) were upregulated. Targeting the cluster genes and corresponding pathways could lead to discovery of biomarkers associated with pathology of sepsis. Through network and functional enrichment analysis, 20 hub genes were identified that are involved in defense response against microbes, adaptive and innate immune response, regulation of T-cell and neutrophil differentiation and activation. Also these genes have significantly higher fold change and were found to be part of enriched GO terms (Fig. 5a) and KEGG pathways (Fig. 5b), which could be identified as key genes associated with sepsis condition.
Based on Venn diagram, four genes HK3, ITGA2B, MPO and TLR5 were found to be common in all six studies. These four genes were seen upregulated in all six studies. Hexokinase-3(HK3) and myeloperoxidase (MPO) are involved in neutrophil activation, regulation and degranulation. MPO is an enzyme present in the granules of polymorph nuclear (PMN) leucocytes associated with the formation of reactive oxygen species (ROS) and oxidation of biological material. Elevated neutrophil levels during sepsis increase MPO in the plasma; this could be used as a biomarker of neutrophil activity and inflammatory response during sepsis[24, 25]. HK-3 is found in HIF-1 signaling pathway that mediate glycolysis in neutrophils, also influencing increased PMNs chemotaxis and phagocytosis function [26]. ITGA2B is involved in regulation of hemopoiesis, hemostasis, coagulation, leukocyte migration, platelet activation and degranulation. Increased expression ITGA2B in circulating platelets during sepsis is associated with increased mortality, which could be used as potential marker to predict mortality [27]. TLR5 is associated with defense response to bacterium, positive regulation of IL-8 and cytokine production. It is a core member of the Toll -like receptor family. It induces the inflammatory cytokine production and immune response [28].
Immune cells like lymphocytes, granulocytes, monocytes and macrophages play a key role in host immune response and defense mechanism against pathogens. The differentiation and homeostasis of immune cells is tightly controlled, and any dysregulation will lead to abnormality in health conditions. The interaction and complex cross-talking between different immune cells is crucial in the development and pathophysiology of sepsis. As consistently observed in previous studies, the hypo- and hyper immune response in sepsis, our meta-analysis gene expression data revealed that the suppressed genes are involved adaptive immune response (CD3G, CD247,CD3E, CX3CR1, IL7R, CCR7 and CD40LG ) and massive activation of innate immune response genes (CD177, S100A12, ARG1, FCER1G, ELANE, S100A8, S100A9 and HP) during sepsis.
Almansa et al., have reported a similar pattern where expression of significant 55 genes involved in adaptive and innate immune response are associated with organ failure and mortality in sepsis [16]. In relation to this the genes associated with markedly enriched GO terms - innate immune response, defense response to bacteria and fungus, neutrophil activation and apoptosis were upregulated. Adaptive immune response, antigen processing and presentation, Immunoglobulin production, T-cell differentiation, selection, activation and regulation were found to be downregulated. Similar results were observed in KEGG pathway analysis, where innate immune response related pathways are upregulated and pathways related to adaptive immune response are downregulated.
It is observed that the genes involved in regulation of neutrophil activation are upregulated (CD177, ELANE, ANX1, S100A8 S100A9, CTSG and IL18RAP) resulting in increased inflammation. Regulation of T-cell activity and differentiation related genes (CD2, CD8A, CD3E, CD3G, LCK, CD247, CXCR3, CX3CR1, CD40LG, CCR7 and IL7R) were downregulated, which is a possible cause of suppressed immune response. This shows that genes involved in innate and adaptive immune response play a significant role in immune dysregulation and immune suppression during sepsis. Screening these key genes responsible for immune dysregulation during sepsis may lead to development of a panel of biomarkers for diagnosis of early sepsis.
T-cells are an essential part of the immune system that fight against the pathogen to protect the host. Several studies reported depletion of T-cells leading to reduced adaptive immunity and prolonged lymphopenia in sepsis patients. A study reported a decreased number of CD4 + and CD8 + T-cells in both adult and elderly sepsis patients. The CD28 expressing CD4 + T-cells were reduced, which is known to be immunocompetent, while immunosuppressed PD-1 + T-cells were increased [6]. From the analysis we found PD-L1 (CD274) was upregulated in five different studies, which could be correlated to reduced T-cell proliferation and increased apoptosis thus affecting their function. This could lead to increased secondary infections, multiple organ dysfunction and mortality in sepsis and septic shock [29] [30]. N. Patil et al., have reported that PD-L1 knockout mice showed improved function of T-cells and survival [31], thus indicating its key role in T-cell exhaustion and pathogenesis of sepsis.
IL7R is an important cytokine related gene found to be involved in hematopoietic cell lineage pathway that is downregulated in sepsis, which is an important gene for maintaining the homeostasis of immune cells. It plays a predominant role in the growth, differentiation and survival of T-cells. In addition to IL-7R, CD27 is also involved in protecting T cells from apoptosis, thus contributing for survival and expansion of T cell [32, 33]. Reduced expression of IL-7R and CD27 is observed in current aalysis, and this could be associated with impaired differentiation of T-cells. This is one of the cause for immunosuppression and enhanced susceptibility to infections during sepsis [34]. In addition to this, increased cell death during sepsis releases excess amounts of DAMPs, neutrophil extracellular traps (NETs) which trigger host immune response leading to increased inflammatory reaction. Hence DAMPs play a prominent role in sepsis progression [35]. S100 A8/A9/A12, types of DAMPs which are increased in sepsis condition and are associated with increased severity and death of sepsis patients [36]. The genes which are responsible for balanced functioning of T-cells (CD8A, IL7R, IL2 and CD28) and T-cell surface markers (CD3) are downregulated. Increased expression of PD-L1 (CD274) and apoptotic genes could result in immune dysfunction, reduced functioning of T-cells and increased cell death, which may lead to increased severity of sepsis and mortality.
Among all immune cells neutrophils play a crucial role in innate immunity, acting as the first line of defense in the clearance of pathogenic bacteria during infection. Neutrophils play a key role in controlling infection during normal conditions, but their antimicrobial activity is impaired and immune responses are dysregulated during sepsis [37]. They participate in chemotaxis, phagocytosis, oxidative burst and neutrophil extracellular traps. Neutrophils exert their function through the formation of NETs, which is a mechanism to capture and kill the pathogen extracellularly. In sepsis excessive NETosis is shown to be detrimental to the host. This NETosis induces histone dependent vascular damage leading to tissue damage and multiple organ dysfunction [38]. The neutrophil genes are found to be highly upregulated in meta-analysis data. The life span of circulating human neutrophil is estimated to be 5.4 days approx. But, in case of sepsis, the cell count is increased due to delayed apoptosis of neutrophils, thus increasing the lifespan and altering the phenotype and functional properties of neutrophils.
Several studies have reported the expression of PD-L1 (CD274) on neutrophils in various disease conditions stimulated by interferon-gamma (IFN-G) and granulocyte macrophage-colony stimulating factor (GM-CSF) [39, 40]. S. de Kleijn et al., reported the stimulation of neutrophils with IFN-G ex-vivo induces PD-L1 expression on neutrophils, which suppress the lymphocyte proliferation and induce the apoptosis of T-cells leading to immunosuppression [41]. Upregulation of ARG1 on neutrophils in sepsis worsen the immunosuppression by suppressing T-lymphocytes. It impairs the function of T-cells by downregulating T-cell receptor (TCR) associated CD3G and CD3E chains leading to immunosuppressive state [42].
Immune suppression is induced by depletion of immune cells, specifically T cells during sepsis. Overexpression of apoptotic genes and downregulation of anti-apoptotic genes have potent effect on T cell apoptosis. Increased T cell apoptosis and neutrophil survival aggravates the immune suppression and inflammatory reaction respectively, exploring apoptotic and inflammatory genes could be a potential targets to reverse immunosuppression and identify valuable biomarkers. In this study, through functional enrichment analysis, we identified 20 candidate genes known to play a potential role in sepsis condition. These genes need to be validated further to understand their expression at various stages of sepsis, which could be clinically useful in early diagnosis of sepsis.