Exploring the biomarkers and potential therapeutic drugs for sepsis via integrated bioinformatic analysis

Sepsis is a fatal organ dysfunction produced by a dysregulated response to infection [1]. Sepsis is responsible for an increasing number of deaths worldwide each year [2]. According to the World Health Organization, sepsis causes around 6 million deaths worldwide each year and the majority of which can be prevented [3]. The World Health Organization adopted a resolution in 2017 to enhance sepsis prevention, detection, and treatment, recognizing it as a global health priority [4]. According to a newly published comprehensive review, among all patients treated for sepsis in hospital, HA (hospital acquired) sepsis accounted for 23.6% of cases, with a 95% confidence interval of 17% to 31.8%, and a range spanning from 16% to 36.4%. Within the intensive care unit (ICU), 24.4% of sepsis cases involving organ dysfunction occurred during the patient’s stay in the ICU, and this estimate had a 95% confidence interval of 16.7% to 34.2%, along with a range from 10.3% to 42.5%. Additionally, nearly half of all cases of sepsis, specifically 48.7%, originated within the hospital, with a 95% confidence interval of 38.3% to 59.3%, and a range extending from 18.7% to 69.4% [5]. The complicated host immune response during and after sepsis includes an early, excessively inflammatory reaction of the host in response to infection resulting in tissue damage, organ failure, and impaired endothelial function [6]. However, decades of attempts to reduce the harmful effects of this excessive inflammation through anti-inflammatory treatment methods have failed, which prompting medical professionals and academics to reevaluate the biology of sepsis [7]. Treatment for sepsis remains largely supportive, with simple measures; we still don't have a single treatment that can consistently save the lives of patients with sepsis [8‐10]. Therefore, it is better to further elucidate the mechanism of sepsis to find more efficient drugs for effective and precise treatment reducing unnecessary costs, mortality and complications.

The use of biomarkers is extremely important for identifying, diagnosing, therapy, following up, stratification and predicting outcomes of diseases like sepsis. Biomarkers can provide important information because they can indicate the severity of sepsis, guide the clinicians to rapid diagnosis and treatment beyond the standard therapy and provide ongoing information on disease activity [11]. A wide range of sepsis biomarkers (cytokines, cell membrane receptors, metabolites, chemokines, cell proteins, complement component system etc.) has been described. However, their effectiveness in many instances is limited by insufficient specificity or sensitivity [12]. In addition, no single biomarker has been found to have sufficient diagnostic power to be used as a standard diagnostic tool. Consequently, there is a need to search for effective biomarkers in sepsis to improve early diagnosis, monitor therapeutic efficacy and improve prognosis.

Early diagnosis is crucial for prompt treatment, enhancing sepsis outcomes [13]. Delaying sepsis treatment increases the chance of mortality [14]. Sepsis is a highly heterogeneous syndrome with complex pathophysiology, excessive inflammation and immunosuppression [9]. The occurrence and progression of sepsis are significantly influenced by the immunological response of immune cells, such as T cells, NK cells, macrophages, and others [9]. In sepsis, severe lymphopenia and apoptosis of lymphocytes may be a significant cause of death [15]. Therefore, the differences in molecular expression patterns linked to immunological and inflammatory pathways require deep consideration and rigorous research.

Infection prevention is the only way to prevent sepsis, and vaccines are an important tool in reducing the risk of infections. Vaccines work by imitating the viral infection, causing the body to produce t-lymphocytes and antibodies that can recognize and destroy the invading organism [16, 17]. Vaccination can be an effective way to prevent infections that can lead to sepsis. Many infections that can lead to sepsis are becoming resistant to antibiotics, so preventing them by vaccination is becoming increasingly important [17, 18].

Despite advancements in the last ten years in describing sepsis-induced immunological dysfunctions, many unanswered concerns still exist, and several issues require additional studies [1]. Since high-throughput technologies generate large amounts of data, there is a need for effective bioinformatics tools that enable us to comprehend how molecules interact and control the various biological processes of health and disease [19‐21]. The molecular understanding of sepsis has opened a new chapter because of transcriptome-based research. The transcriptome is the collection of all RNA molecules transcribed by the genome of a specific cell at a specific physiological or pathological condition [22, 23]. Each of these specific molecules presents a different functional spectrum in the cell and responds differently to environmental stimuli [24, 25]. By analyzing the gene expression, researchers can explore the molecular basis of sepsis in multiple ways and provide information about sepsis progression, as well as the discovery of new and previously unknown biomarkers.

Consequently, it is very important and urgent to explore the genetic changes that occur during the pathogenesis of sepsis. Finding a biomarker and panel of biomarkers could be a new avenue to provide new approaches to treat sepsis. Based on the above-discussed facts, the current research aims to explore the genetic changes associated with disease development and understand the pathophysiology of sepsis to find novel biomarkers and candidate drugs that may be useful for sepsis therapy. We hope these biomarkers will reveal important insights and impact on sepsis treatment.

Sepsis is a clinical syndrome defined as "life-threatening organ dysfunction caused by a dysregulated host immune response to infection" [41]. The host response to sepsis is characterized by both pro-inflammatory responses and anti-inflammatory immune suppressive responses [42]. Sepsis clearly alters the innate and adaptive immune responses for sustained periods of time after clinical recovery, with immune suppression, chronic inflammation, and immune paralysis being common [43]. Deficits in the adaptive immune response may play a major role in sepsis patient mortality. The adaptive immune response involves a number of cell types including T cells, B cells, and dendritic cells, all with immunoregulatory roles aimed at limiting damage and returning immune homeostasis after infection [41, 44]. Our ability to discriminate adaptive and maladaptive immune responses in sepsis is limited. The dysregulated host immune response activated during sepsis may persist for up to 1 year [44]. Transcriptomic research turns out to be an effective tool for elucidating the molecular processes that regulate sepsis. This study aimed to identify significant genes and molecular dysregulation pathways associated with sepsis by applying bioinformatics analysis to sequencing data related to sepsis. Previous studies have limitations of the negligible amount of individuals in each study due to the high cost of these techniques, as well as the differences between different analysis and their platforms which are challenging to interpret and compare the results between different research groups. Three publicly accessible datasets were used in the analysis for this study. A total of 537 shared DEGs with similar expression patterns were discovered by identifying the overlapping DEGs that were present in all three datasets. One of these DEGs, MCEMP1, for instance, is highly expressed in sepsis, its down-regulation inhibited the inflammation of septic mice [45]. In our study, it was shown that sepsis cases had a noticeably higher expression of CD177. The finding aligns with earlier research that connected neutrophil transmigration and CD177 to inflammatory diseases [46, 47]. It also protects the intestines from inflammation in IBD [46]. S100A12 is highly increased during inflammation, which induces monocyte activation [48‐50]. As mentioned in one literature review, ANXA3 was also up-regulated in sepsis, but its role is unclear [51]. It may enhance the prolonged survival of neutrophils and pathogen clearance in the early phase, but result in organ failure at a later stage [51]. GYG1 is an enzyme of glycogen synthesis, which was up-regulated in sepsis. Glycogen metabolism also regulated macrophage-mediated acute inflammatory responses [52]. Glycogen disorder is common in patients with severe sepsis [53].

This study demonstrated that different biological processes were significantly enriched in the DEGs, whether these genes were found in individual groups or were shared by several groups. T cell activation, leukocyte cell–cell adhesion, T cell differentiation, leukocyte differentiation, mononuclear cell differentiation, and pathways of hematopoietic cell lineage, as well as Th1, Th2 and Th17 cell differentiation were among these processes, but they were not limited to them. Furthermore, DEGs were also found to be enriched in pathways related to Staphylococcus aureus infection, among others [31‐33]. This strongly suggests that T cells are involved in the occurrence and development of sepsis. The pathophysiology of T cell modifications may involve both intrinsic processes that directly affect T cells as well as indirect mechanisms that affect antigen-presenting cell or immature neutrophil activities, according to previous studies [54‐57]. Th17 cells, a distinct subset of T helper (Th) cells recognized for their production of IL-17, have been strongly related to the onset and development of a number of inflammatory reactions and autoimmune diseases [58]. Additionally, the 28-day mortality among patients with severe sepsis and ICU-acquired infections have both been linked to a continually shifting Th2 / Th1 cell ratio [58]. Our findings strongly indicate that T cells, particularly Th1, Th2 and Th17, play a pivotal role in sepsis development.

In our study, the PPI network analysis and submodule analysis also suggested that these genes in most top submodules were also related to T cell activation, cell–cell adhesion as well as Th1, Th2 and Th17 cell differentiation. Moreover, we used the plug-in cytoHbba to disclose the essential hub genes in the PPI network. Five hub genes, CD3E, HLA-DRA, IL2RB, ITK and LAT were explored. CD3E forms the T-cell receptor-CD3 complex, which couples antigen recognition to intracellular signal transduction pathways and is down-regulated in sepsis [59]. Prior studies have highlighted the importance of HLA-DRA as a promising future biomarker for evaluating immunosuppression in sepsis [60]. IL2RB is a crucial mediator of Th1 and Th17 cellular immunity, which plays vital roles in the immune response against bacteria and fungi [61]. The expression level of IL2RB negatively correlates with mortality [61]. ITK is involved in regulating thermal homeostasis in mast cell responses in LPS-induced sepsis, and its lack leads to hypothermia exacerbation [62]. LAT is a crucial adaptor molecule in the TCR signaling pathway, and directly recruiting from cell surface LAT to microclusters is also critical for T-cell activation [63, 64]. The level of T cell activation may be influenced by the quantity of LAT on the cell surface [64, 65]. The T cell dysfunction may cause immunosuppression after acute sepsis [1]. Furthermore, KM analysis showed that sepsis with high expression values of these five hub genes had a better prognosis, suggesting that these five hub genes may serve as important therapeutic targets or biomarkers for sepsis. Our preliminary experiment found that CD3E, IL2R and HLA-DR were significantly reduced in sepsis when compared to healthy control.

Although mortality rates have improved, new drugs for sepsis are still required. Fourteen drugs targeting the above immune-related genes were obtained. Many of these compounds were previously approved and used as immunosuppressants or used to treat the diseases of immune cells in addition to cancer. Muromonab CD3, the treatment of acute solid organ transplant rejection, has proven an effective alternative and gives a substantial new perspective on immunosuppressive therapy [66]. Anti-CD20/CD3 T-cell dependent bispecific (TDB) antibody mosunetuzumab is entirely humanized full-length and assembled utilizing the knobs-into-holes technology [67, 68]. Mosunetuzumab and blinatumomab share a similar mode of action: B-cell lysis and T-cell activation result from mosunetuzumab's dual binding to CD20 on malignant B-cells and CD3 on T-cells [69]. Catumaxomab increases the activation of immune cells by combining the cancer cells, T cells, and auxiliary immune cells into proximity. It makes it easier for the immune system to kill cancer cells [70]. Teplizumab alters CD8⁺ T lymphocytes, which are believed to be the most critical effector cells that kill beta cells [71]. Interleukin-2 (Aldesleukin) has been licensed by the Food and Drug Administration (FDA) for the treatment of individuals with advanced forms of renal cell carcinoma (metastatic RCC) and melanoma [72]. Basiliximab or daclizumab in combination with triple treatment was an effective and safe immunosuppressive strategy, as indicated by a low incidence of acute rejections, excellent graft function, high survival rates, and an acceptable adverse event profile in adult patients one year after deceased donor renal transplantation [73]. A recombinant fusion protein called denileukin diftitox treats the cutaneous T cell lymphomas that express IL-2 receptors [57]. Human interleukin-2 (IL-2) is linked to diphtheria toxin fragments A and B [57]. Three out of 14 were BTK and ITK inhibitors such as Pazopanib, Fostamatinib and Zanubrutinib. Cytoplasmic tyrosine kinases BTK and ITK are essential for forming B and T cells, and loss-of-function mutations in either result in X-linked agammaglobulinemia and an increased risk of a severe, usually fatal Epstein-Barr virus infection, respectively [74]. Pazopanib, an approved medication for handling renal cell carcinoma and soft tissue sarcoma, is a VEGF receptor, platelet-derived growth factor receptor, fibroblast growth factor receptor, and stem cell receptor c-Kit inhibitor [75]. The abovementioned drugs are relevant to the immune balance in other diseases and may benefit sepsis patients. For example, we demonstrated in this study that catumaxomab and aldesleukin (agonists targeting CD3E and IL2R separately) effectively restore T cells' regulatory activity and suppress excessive inflammation, which is critical in reducing the occurrence of septic shock. At present, antibiotic treatment of sepsis is facing the problem of microbial resistance. Our research is based on drug targets for host immune regulation that do not develop antimicrobial resistance and have better application prospects than antibiotics. However, future studies based on the investigations of in vitro or animal models will be necessary to confirm these possibilities.

In conclusion, sepsis is a complicated clinical disease characterized by dysregulated immune responses that can linger long after the initial recovery. Immunological suppression, persistent inflammation, and immunological paralysis are frequently caused by this immune dysregulation, which may be a factor in patient death. The study used bioinformatics analysis and transcriptome research to investigate the complex biological mechanisms involved in sepsis. Analyzing publicly available data sets, the study identified 537 differentially expressed genes (DEGs) with similar patterns, revealing significant sepsis-related genes such as MCEMP1, CD177, S100A12, ANXA3, and GYG1. Furthermore, the study also showed a notable enrichment of biological pathways and processes involved in T cell activation, leukocyte adhesion, differentiation, and immunological responses, including Th1, Th2, and Th17 cell differentiation. This highlights the critical part T cells play in the onset and progression of sepsis, as well as their potential as biomarkers. The protein–protein interaction network analysis revealed hub genes, including CD3E, HLA-DRA, IL2RB, ITK, and LAT, which are all involved in T cell activation and immune regulation. Better outcomes for sepsis patients were associated with high expression of these hub genes. In addition, the study investigated potential drug candidates that target immune-related genes, some of which have demonstrated promise in immunosuppression and cancer treatment. These medications have the potential to treat sepsis, but more studies, especially those using in vitro and animal models, are required to validate their effectiveness.