Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, remains a significant global health challenge. According to the Global Burden of Diseases report, sepsis affected approximately 49 million people worldwide in 2017, causing around 11 million deaths and accounting for nearly 20% of all global deaths (Rudd et al., 2020; Gavelli et al., 2021). The mortality rate of sepsis has gradually decreased with the timely use of antibiotics, fluid resuscitation, and supportive therapies for multiple organ dysfunction over the past two decades. However, there is still a significant mortality rate and considerable room for improvement. In addition to the high health-related burden, septic shock is one of the most expensive pathological conditions to treat. Its estimated annual healthcare cost is $24 billion (McBride et al., 2020). Current diagnostic and therapeutic approaches for sepsis have limitations. They cannot accurately predict outcomes or effectively target the underlying pathophysiological processes (Cecconi et al., 2018). Consequently, there is an urgent need for novel biomarkers and therapeutic targets to improve the diagnosis, prognosis, and treatment.

Necroptosis is a regulated form of necrotic cell death. It has emerged as a critical player in the pathogenesis of various inflammatory diseases, including sepsis (Linkermann and Green, 2014). Unlike apoptosis, necroptosis is characterized by cell membrane rupture and the release of damage-associated molecular patterns (DAMPs), which can exacerbate inflammation and tissue injury (Pasparakis and Vandenabeele, 2015). The key mediators of necroptosis, such as receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL), are upregulated in sepsis and contribute to organ damage and mortality (Newton et al., 2016). Inhibition of necroptosis in animal models of sepsis has been shown to reduce mortality and improve outcomes (Newton and Manning, 2016), suggesting that targeting necroptosis might be a promising therapeutic strategy for sepsis. However, the specific functions of necroptosis-related genes (NRGs) in the development and progression of sepsis remain unclear.

Immune system dysregulation plays a key role in the development of sepsis. During the initial phase of sepsis, the exaggerated inflammatory response triggers the recruitment of a large number of neutrophils, which play a crucial role in pathogenic bacterial clearance but also cause tissue damage (Ioannou et al., 2022). Damaged cells act as endogenous inflammatory inducers, thereby promoting the inflammatory response. Furthermore, dendritic cells (DCs) can activate toll-like receptors (TLRs) and produce excessive pro-inflammatory factors that amplify the immune response. These hyperactive immune responses ultimately trigger a cytokine storm, severely impairing normal immune function (Xiao et al., 2018). With the development of sepsis, the over-activation stage gradually transforms into the immune paralysis stage (also called immunosuppression) (Zhang et al., 2019; Forceville et al., 2021). Therefore, improving the immune microenvironment is crucial for treating septic patients. Immunotherapy holds broad potential for clinical application in sepsis. However, in sepsis, the relationship between immune cell infiltration characterization and necroptosis remains unknown.

Necroptosis plays a pivotal role in inflammation and cell death. Understanding how NRGs are expressed and function in sepsis could offer valuable insights into the disease’s pathophysiology. In this study, we investigated the differential expression of NRGs in sepsis and their impact on immune characteristics. We utilize gene expression profiles from peripheral blood cell samples of sepsis patients and healthy controls. Additionally, single-cell RNA sequencing data were used to identify differentially expressed genes (DEGs) and perform functional enrichment analysis as well as evaluate immune cell infiltration patterns.

By utilizing gene expression data from the GEO database, we performed differential gene expression analysis, immune cell infiltration analysis, and also analyzed single-cell RNA sequencing data. Using these bioinformatics methods, we identified 157 necroptosis-related DEGs, clarified their functions, and examined their links to immune cell infiltration in sepsis. The study has been published online in preprint form (Wang, 2024). Our findings provide new insights into the molecular mechanisms of sepsis and highlight potential biomarkers and therapeutic targets for sepsis, a devastating condition.

Sepsis is a life-threatening condition characterized by a dysregulated host response to infection that leads to organ dysfunction and high mortality rates (Singer et al., 2016). Despite a 52.8% decline in mortality from 1990 to 2017, sepsis still impacted 48.9 million people in 2017 alone. It resulted in 11.0 million lives annually; this accounts for 19.7% of global deaths (Rudd et al., 2020). This burden is magnified in pediatric populations, where sepsis drives approximately 33% of fatalities in the Pediatric Intensive Care Unit (PICU) in the United States, with an approximate 25% mortality rate (Weiss et al., 2015). Furthermore, refractory septic shock is associated with a 34% mortality rate (Weiss et al., 2017). Importantly, microbial invasion initiates pathological cascades that involve anti-inflammatory cytokines become central drivers of immune failure.

The disease process develops through interactions between pathogens and the host, which disrupt inflammatory balance. Following microbial invasion, anti-inflammatory mediators such as IL-10 paradoxically amplify immunosuppression. Elevated IL-10 levels correlate with the severity of septic shock and dysfunctional pro-inflammatory responses in critically ill patients (Ng et al., 2003). IL-10 acts as a potent suppressor of cytokine synthesis in T cells and macrophages. However, it also exacerbates immunosuppressive effects, contributing to immune dysfunction. Within this regulatory axis, neutrophils are the primary infiltrating cells that combat pathogens like Klebsiella pneumoniae (KPn) and normally restore tissue homeostasis by clearing apoptotic cells via efferocytosis. However, KPn infection subverts this process (Jondle et al., 2018) through the concerted activation of neutrophil necroptosis machinery—a regulated form of inflammatory cell death—along with concurrent suppression of apoptosis-associated phosphatidylserine (PS) externalization. Additionally, KPn infection enhances flippase activity, which anchors PS to the plasma membrane inner leaflet. These combined effects culminate in impaired macrophage efferocytosis of infected neutrophils. This coordinated dysregulation of cell death pathways further interacts with IL-27-mediated metabolic reprogramming deficits, together exacerbating the pathological cascade. Neonatal models show that IL-27 deficiency improves bacterial clearance, reduces inflammation, and enhances survival during E. coli infection (Povroznik et al., 2025). The KPn pneumonia model further corroborates that targeting the necroptosis pathway (via RIPK1/RIPK3 inhibitors) not only restores efferocytic capacity but also significantly improves disease outcomes (Jondle et al., 2018). Collectively, these findings show that abnormal reprogramming of immune cell death pathways works together with imbalances in the cytokine network. This synergy drives the progression of organ failure.

These pathological processes converge on a critical bottleneck. Although sepsis is recognized as a dysregulated host response, the specific mechanisms in different cell types that cause immune dysfunction remain unclear. Necroptosis, in particular, represents a key process that amplifies tissue damage while simultaneously reprogramming immune cell functionality in a lineage-dependent manner. Necroptosis emerges as a critical bottleneck that exacerbates tissue damage and reprograms immune cell functionality in a lineage-dependent manner. This phenomenon is vividly exemplified by the death modality switching in neutrophils during Klebsiella pneumoniae (KPn) infection. In this context, apoptosis, which should routinely occur, is replaced by necroptosis, compromising efferocytosis and driving persistent inflammation. To address this, our study uses multi-modal transcriptomics to investigate how necroptosis-associated genes regulate immune subpopulation dysregulation, spatial immunometabolism, and clinical heterogeneity in sepsis.

Necroptosis is a form of programmed cell death distinct from apoptosis. It has been implicated in various inflammatory diseases and is thought to play a critical role in the pathogenesis of sepsis (Linkermann and Green, 2014). Necroptosis should be considered as an early surge of cell death that could lead to acute or sustained inflammation. Consequently, targeting the molecular mediators of necroptosis represents a promising therapeutic direction for sepsis (Qu et al., 2022). Previous bioinformatics analyses have demonstrated a correlation between changes in immune cell infiltration and sepsis (Xu et al., 2020). However, despite the known roles of necroptosis and immune infiltration in sepsis, few bioinformatics studies have specifically explored the relationship between necroptosis and sepsis.

This study focuses on the expression of necroptosis-related genes in sepsis and their immunological characteristics, aiming to provide new insights into the disease’s pathogenesis and potential therapeutic targets. By integrating multiple bioinformatics approaches, this study identifies differentially expressed necroptosis-related genes and their associated pathways. These findings highlight the roles of these genes in the pathogenesis and immune modulation of sepsis. Functional enrichment analyses, including GO and KEGG, reveal key biological pathways involved in sepsis. Moreover, GSVA and GSEA provide a comprehensive view of pathway alterations between sepsis patients and healthy controls. To further explore these findings at a finer resolution, the study’s single-cell transcriptomic analysis elucidates the heterogeneity of immune cell populations and their necroptosis gene expression profiles, offering valuable insights into the immune landscape of sepsis. Our findings suggest that these genes may contribute to the dysregulated immune response observed in sepsis, potentially serving as new targets for therapeutic intervention. By integrating multi-omics data and advanced bioinformatics techniques, this study sheds light on molecular alterations in sepsis, paving the way for improved diagnostic and therapeutic strategies (Tang et al., 2019).

Single-cell data analysis revealed that six necroptotic genes were differentially expressed in immune cells. These genes are CTSS, TXN, MYH9, FPR1, FMR1, and MPRIP. Among these, CTSS was highly expressed in various types of immune cells. CTSS, also known as Cathepsin S, is a cysteine protease that degrades proteins within lysosomes. It is predominantly expressed in immune cells, such as professional antigen-presenting cells (APCs), B cells, dendritic cells (DCs) and macrophages (Riese and Chapman, 2000). With predominant expression in both DCs and B cells, which are professional APCs, CTSS plays an important role in the immune response. Specifically, CTSS activity fundamentally drives MHC class II maturation, antigen processing and presentation (Smyth et al., 2022). In the context of sepsis, CTSS has been implicated in modulating immune responses and inflammation. The increased expression of CTSS might contribute to the excessive inflammation and tissue damage in sepsis. It promotes the degradation of extracellular matrix components and facilitating the infiltration of immune cells into tissues, —both of which are hallmarks of sepsis (Turk et al., 2012). CTSS involvement in inflammation is also evident in necroptosis, a form of programmed cell death that plays an intrinsic role in inflammatory processes (Dhuriya and Sharma, 2018). CTSS has been shown to cleave RIP1 kinase, which limiting necroptosis within macrophages. Therefore, inhibiting CTSS along with caspase-8 may offer a promising treatment strategy for various inflammatory conditions (McComb et al., 2014). This is consistent with our findings that CTSS is one of the key DEGs in sepsis; however, this is the first time it has been found to play a role in the necroptotic process of sepsis.

Thioredoxin (TXN) is a small redox protein. It plays a pivotal role in maintaining cellular redox homeostasis by reducing oxidized proteins. And it is involved in various cellular processes, such as DNA synthesis, repair, and apoptosis (Holmgren, 1985). In sepsis, TXN exerts protective effects by mitigating oxidative stress and modulating inflammatory responses (Nakamura et al., 1997). Previous studies have suggested that TXN is a key regulator in maintaining endoplasmic reticulum (ER) homeostasis in sepsis. Additionally, it has been identified as a diagnostic biomarker strongly associated with immune cell infiltration in sepsis (Zhou et al., 2023b). Building on these findings, our results indicate that TXN is highly expressed in CD4⁺ T cells, CD8⁺ T cells, monocytes, neutrophils, and natural killer (NK) cells. The upregulation of TXN during sepsis may represent a compensatory mechanism to counteract excessive oxidative stress and inflammation.

Myosin Heavy Chain 9 (MYH9) is a non-muscle myosin that participates in several cellular processes such as cell motility, adhesion, and cytokinesis (Conti and Adelstein, 2008). Recent studies have suggested that MYH9 plays a role in immune function and inflammatory responses (Chung et al., 2019). In sepsis, MYH9 expression has been linked to altered immune cell dynamics and impaired immune responses. Our study identified significantly elevated expression of MYH9 in CD4⁺ T cells, CD8⁺ T cells, and HSC, as well as in monocytes, neutrophils and NK cells, indicating its potential involvement in the immune dysregulation observed in sepsis. The mechanisms by which MYH9 contributes to sepsis pathophysiology remain unclear. Furthermore, its role in cytoskeletal organization and cell signaling suggests it could be a critical mediator of immune cell function in the context of septic.

Formyl Peptide Receptor 1 (FPR1) is a member of the human G protein-coupled receptor (GPCR) family in humans and plays a key role in mediating the chemotaxis of immune cells, particularly neutrophils and macrophages, to sites of infection and inflammation (Le et al., 2002). FPR1 is crucial for host defense, regulating the migration and activation of immune cells in response to bacterial peptides (Ye and Boulay, 1997). Previous studies have demonstrated that FPR1 is vital in modulating the innate immune response through neutrophils (Lee et al., 2018), and elevated FPR1 expression contribute to excessive neutrophil infiltration (Chung et al., 2008). Recent reports indicate that FPR1 is also expressed in adaptive immune cells, in addition to innate immune cells involved in inflammation (Nagaya et al., 2017). Notably, FPR1 is expressed in the nuclei of naïve CD4 T cells and regulates their chemotactic migration (Lee et al., 2018). In the context of sepsis, dysregulated FPR1 expression leads to impaired immune cell recruitment and function; consequently, FPR1 has been suggested as a putative target for several diseases, including sepsis (Lee et al., 2017). Our findings show higher FPR1 expression in monocytes and neutrophils, which consistent with previous literature, but no significant difference was found in the expression levels in CD4⁺ T cells between SE and HC groups.

The Fragile X Mental Retardation 1 (FMR1) gene encodes the Fragile X Mental Retardation Protein (FMRP), which participates in synaptic function and neuronal development (Bagni and Greenough, 2005). Although primarily studied in neurological disorders, recent research indicates that FMR1 also play a role in immune regulation (Bhakar et al., 2012). In sepsis, FMR1 expression has been associated with altered immune cell function and inflammatory responses; moreover, the absence of FMRP induces TNF-mediated apoptosis and necroptosis in infections and liver diseases (Zhuang et al., 2020). Additionally, FMR1 has been shown to play a role in the stimulus-induced localization of mRNAs in dendritic cells (Dictenberg et al., 2008). Building on this, our findings of significantly higher FMR1 expression in CD8⁺ T cells suggest its contribution to the necroptosis in sepsis, warranting further investigation of the underlying mechanisms.

GO analysis revealed that these DEGs are prominently involved in regulation of apoptotic signaling pathways and the IκB kinase/NF-κB signaling pathway, both of which are critical for cell death, inflammation, and immune response modulation (Xu and Shi, 2007). Additionally, KEGG pathway enrichment analysis further highlighted the involvement of these DEGs in programmed cell death pathways, including necroptosis and apoptosis, as well as the NOD-like receptor signaling pathway, underscoring their pivotal roles in innate immunity (Zhou et al., 2024).

Necroptosis is a form of programmed cell death distinct from apoptosis and is inflammatory, potentially exacerbating the immune response in sepsis (Liu et al., 2021). The enrichment of DEGs in the necroptosis pathway suggests that this form of cell death contributes significantly to the pathogenesis of sepsis by promoting inflammation and tissue damage (Pasparakis and Vandenabeele, 2015). Similarly, the NOD-like receptor signaling pathway is essential for in recognizing pathogens and initiating immune responses in sepsis (Zhang and Ning, 2021). The significant enrichment of DEGs in the NOD-like receptor signaling suggests a potential mechanism through which sepsis may induce a robust immune response, leading to systemic inflammation and organ dysfunction (Foley et al., 2015).

Our GSVA revealed notable differences between the HC and SE groups. Pathways such as oxidative phosphorylation, sphingolipid metabolism and glycosphingolipid biosynthesis (lacto and neolacto series) were downregulated in the SE group. Conversely, the Wnt signaling pathway was upregulated. Oxidative phosphorylation is essential for ATP production and the cellular energy metabolism. Consequently, the downregulation of oxidative phosphorylation may reflect mitochondrial dysfunction commonly observed in sepsis (Nedel et al., 2023). The analysis of the sphingolipid metabolism pathway revealed that plasma levels of plasmalogens, circulating antioxidant phospholipids, decreased in sepsis patients. These plasmalogens provide critical protection against oxidative insults by preserving cellular lipid integrity during stress (Chang et al., 2023). Similarly, the glycosphingolipid biosynthesis pathway (lacto and neolacto series) showed significant downregulation in sepsis. This finding therefore further supports the notion that sphingolipid metabolic reprogramming is a hallmark of this condition (Li et al., 2025).

The Wnt signaling pathway, known for regulating cell proliferation and differentiation, is upregulated and might indicate a compensatory mechanism or a shift in cellular dynamics in response to sepsis (Teo and Kahn, 2010). The Wnt signaling pathway is an evolutionarily conserved system that is essential for organ development and adult homeostasis. This pathway regulates proliferation, differentiation, apoptosis, motility, and polarization of cells (Schaale et al., 2011). Wnt signaling is classified into canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) pathways, with the canonical pathway capable of regulating anti-inflammatory responses (Schaale et al., 2011). Recent evidence identifies Wnt proteins and signaling components as key modulators of the immune system, involved in balancing between tolerance and immunity. DCs have been identified as direct targets of this regulation (Zhou et al., 2009; Manicassamy et al., 2010). The Wnt signaling pathway may coordinate the interaction between the body’s innate and adaptive immunity in response to infection (Brandenburg and Reiling, 2016). A recently published study showed that inhibition of Wnt/β-catenin signaling reduces inflammation and mitigates sepsis-induced organ injury in experimental models (Sharma et al., 2017). Based on these findings, targeting the Wnt/β-catenin pathway may provide a potential therapeutic approach for the treatment of sepsis.

These findings suggest metabolic dysregulation and altered signaling pathways in sepsis, which may contribute to the disease’s complex pathophysiology by disrupting cellular homeostasis and immune responses (Wasyluk and Zwolak, 2021). Additionally, GSEA further confirmed the enrichment of DEGs in pathways such as G alpha (s) signaling pathway and olfactory transduction. The G alpha (s) signaling pathway participates in various cellular processes, including hormone signaling and metabolic regulation. Disruption of this pathway could play a significant role in sepsis progression (Prossnitz and Barton, 2023). While olfactory transduction is primarily associated with the sense of smell, it has been implicated in immune responses and inflammation, suggesting a broader role in sepsis pathophysiology (Shepard and Pluznick, 2016).

Our ssGSEA revealed significant differences in immune cell infiltration between SE and HC groups, particularly in activated B cells and CD4⁺ T cells, indicating an imbalance that may drive the exaggerated inflammatory response and immune dysregulation in sepsis (Liu et al., 2022) and may be associated with the identified DEGs (Xue et al., 2023). Single-cell RNA sequencing allowed us to detail the cellular composition and revealed neutrophils as the predominant cell type in sepsis. This finding supports the established role of neutrophils in driving the inflammatory response during sepsis (Hong et al., 2022).

The initial inflammatory response to sepsis is primarily driven by innate immune cells in the immune system, including neutrophils, monocytes, and macrophages. These cells can release a variety of inflammatory cytokines (Muszynski et al., 2016). Neutrophils play a crucial role in sepsis-induced inflammation and immune imbalance, serving as the host’s first line of defense against infections (van der Poll et al., 2017). While neutrophils exhibit a powerful antimicrobial effect, they also have a double-edged role, serving as both vital guardians of host defenses and harmful facilitators of tissue damage in an uncontrolled inflammatory state (Liew and Kubes, 2019). The dual role of SAP has recently gained new validation; in Gram-negative bacterial pneumonic sepsis, the adaptor protein SAP enhances host defense by promoting neutrophil extracellular trap (NET) formation. SAP-deficient mice showed impaired NET generation, which led to compromised bacterial clearance and consequently significantly exacerbated lung damage and systemic infection (Tripathi et al., 2019). In septic patients, dysregulated neutrophil cell death can be detrimental, as it leads to immune-related organ failure, reducing host defenses and increasing susceptibility to hospital-acquired infections. Consequently, neutrophils shift from being potent antibacterial agents to harmful mediators that cause tissue damage and organ failure. Under septic conditions, spontaneous apoptosis of neutrophils is inhibited, resulting in increased longevity of these cells (Wang et al., 2021). The delayed apoptosis of neutrophils in sepsis is a significant causative factor in septic organ dysfunction. Research has demonstrated that anti-PD-L1 treatment can improve survival in septic mice (Wang et al., 2015), suggesting that promoting apoptosis in septic neutrophils may represent an effective therapeutic target for the treatment of sepsis. It is noteworthy that apoptosis is not the sole critical cell death pathway; neutrophil extracellular trap (NET) formation also serves as an essential antibacterial mechanism. Deficiency in the regulatory protein SAP impedes the release of DNA–antimicrobial protein complexes by neutrophils. Adoptive transfer experiments further reveal that SAP regulates NETosis indirectly, through mechanisms independent of neutrophils, unveiling novel therapeutic avenues for immunomodulation (Tripathi et al., 2019). In 2021, new developments emerged regarding disulfiram, an FDA-approved drug repurposed as a targeted inhibitor of neutrophil extracellular traps (NETs) and pyroptosis. This drug inhibits septic neutrophil gasdermin D (GSDMD) activation and reduces NET release, thereby improving sepsis-induced organ damage and survival in septic mice (Silva et al., 2021). This discovery, together with SAP research (Tripathi et al., 2019), clarifies that simultaneously targeting distinct cell death pathways—apoptosis, pyroptosis, and neutrophil extracellular trap (NET) formation—and their regulatory factors, such as PD-L1, GSDMD, and SAP, constitutes a synergistic therapeutic strategy for sepsis. Notably, as the first identified positive regulatory adaptor promoting NET formation, SAP facilitates extracellular entrapment mechanisms that enable innovative approaches to overcome immunoparalysis, a state of immune suppression commonly observed in sepsis. In summary, targeting neutrophil cell death may be a promising therapeutic strategy against sepsis.

Our study provides significant insights; however, it also has inherent limitations that should be considered. We identified differential expression patterns of NRGs in sepsis and their profound impact on immune dysregulation. However, these findings require validation in large-scale prospective cohorts using external datasets before clinical translation. We used ssGSEA, because of its sensitivity in quantifying various immune cell subsets and wide applicability. However, cross-validation with alternative algorithms (e.g., quanTIseq, xCell) was precluded by limitations in access to compatible raw data formats and computational resources. This highlights the need for future studies with larger sample sizes to validate immune infiltration patterns using integrated computational methods. Our transcriptomic analyses revealed correlations between NRGs and immune responses. However, establishing causal mechanisms requires further in vitro and in vivo experimental validation, especially to understand how NRGs regulate immune cell function and inflammatory cascades.

Overall, our findings provide a comprehensive understanding of the molecular mechanisms underlying sepsis, especially the critical role of necroptosis-related genes in immune responses, which underscore potential targets for therapeutic intervention. Further research is needed to explore these pathways in detail and to confirm their clinical relevance for sepsis management.