Sepsis (sepsis) is a dysfunctional host response to infectiont, which is characterized by the abnormal secretion of peripheral inflammatory factors such as IL-1β, IL-6, and TNF-α (1–3). This condition is increasingly relevant in critically ill patients due to a rising incidence, which may reflect factors like an aging population and the prevalence of comorbidities (2, 4). These peripheral inflammatory factors act as powerful pro-inflammatory mediators, which not only drive local inflammatory responses, but also spread through the whole body, leading to the outbreak of cytokine storms, which ultimately lead to multi-organ dysfunction and failure (5) ( Figure 1B ). These peripheral inflammatory factors act as powerful pro-inflammatory mediators that not only drive localized inflammatory responses, but also spread systemically, leading to an outbreak of cytokine storms, which in turn triggers systemic inflammatory response syndrome (SIRS) and ultimately leads to multi-organ dysfunction and failure (5, 6).The persistent elevation of peripheral inflammatory factors is an important pathological feature in the development of sepsis, reflecting the state of immune imbalance (2, 6–8). In sub-Saharan Africa, the incidence of sepsis was reported to be 1,772 cases/100,000 people with a hospital mortality rate of 23.7% during 2013-2016 (9). The pathologic progression of this disease is triggered by pathogen-associated molecular patterns (PAMP) and damage-associated molecular patterns (DAMP), which induce an inflammatory response through the activation of host pattern-recognition receptors (PRRs), forming a pro-inflammatory cascade (10, 11).

Ubiquitination, a key post-translational modification of proteins, regulates the function and localization of target proteins through the synergistic action of E1, E2, and E3 enzymes, while deubiquitinating enzymes (DUBs) reverse this regulatory process by excising the ubiquitin chain (12, 13). Ubiquitination and deubiquitination dynamically modulate necroptosis by regulating inflammation-related proteins involved in apoptosis and pyroptosis. This regulation influences the release of peripheral inflammatory mediators, ultimately shaping the inflammatory response in sepsis patients. Both PAMPs and DAMPs activate pro-inflammatory signaling pathways via PRRs, while ubiquitination finely tunes the intensity and duration of this inflammatory response by modulating key proteins (e.g., NF-κB) within these signaling pathways. DUBs such as CYLD and A20 control the inflammatory response by removing the K63 of the RIPK1 chain ubiquitin and inhibit the overactivation of downstream pro-inflammatory signaling to prevent uncontrolled inflammation, revealing that the dynamic balance between ubiquitination and deubiquitination is critical for the regulation of inflammation in sepsis (14). This process holds significant clinical promise as a potential therapeutic target.

The aim of this review is to delve into the multilevel regulatory mechanisms of ubiquitination and deubiquitination in sepsis, focusing on the process by which PAMP and DAMP trigger inflammatory responses through activation of PRRs, and how they regulate the activity and function of key inflammatory proteins (e.g., RIPK1 and NF-κB) via the ubiquitination pathway. We will elaborate how these regulatory mechanisms affect cell death and immune cell activation (15–17).

Ubiquitin is a small protein consisting of 76 amino acids and is present in all tissues of eukaryotes (18). With only three amino acid differences between mammals, yeast and plants, it shows significant evolutionary conservation. It acts as a modifier by covalently attaching to cellular proteins via an enzymatic cascade, which involves three classes of enzymes called E1 (activation), E2 (binding), and E3 (ligation) (19). The process of ubiquitin labeling of proteins is called ubiquitination, and the process involves three major steps: first, E1 activating enzymes activate the ubiquitin molecule; then, E2 binding enzymes receive and transfer the ubiquitin; and finally, E3 ligase covalently attaches ubiquitin to the lysine residues of the target protein. Ubiquitination is one of the most versatile cellular regulatory mechanisms known to be used to control cell death and immune-inflammatory responses ( Figure 1A ). In sepsis, E3 ligases such as TRAF6 amplify the inflammatory response by activating the NF-κB and MAPK signaling pathways through K63 chain ubiquitination. RING-type and HECT-type E3 ligases play different roles in the recognition and modification of different inflammation-associated proteins (20, 21). Dysregulation of ubiquitination often results in a dysfunctional inflammatory response, exacerbating inflammation in sepsis. Out of control, exacerbating the cytokine storm in sepsis.

The process of ubiquitination is reversible. Deubiquitination is the opposite process of ubiquitination, where mono- or polyubiquitin chains are removed from a modified protein to terminate the ubiquitination function of the protein. In simple terms, the degradation of ubiquitinated proteins is prevented and their stability is maintained (14). Currently DUBs number about 100 in humans and are classified into six structurally distinct DUB families including UCHs (ubiquitin carboxyl-terminal hydrolases) of human USPs (ubiquitin-specific proteases), OTUs (ovarian tumor proteases), MJDs (Machado- Josephin’s disease protein structural domain proteases), MINDYs (motifs with containing ubiquitin (MIU) and JAMMs (Zn-JAB1/MPN/MOV34 structural domain proteases) (22–25). The first five belong to the cysteine proteases (26). Among them, the USP sub family is the most diverse member of the DUB family (27, 28). For example, DUBs in the nucleus (e.g., USP10) regulate gene transcription and DNA repair, whereas DUBs located in the cytoplasm (e.g., CYLD and A20) regulate inflammation-associated signaling pathways, such as NF-κB and MAPK. In sepsis, cytoplasmic DUBs (CYLD and A20) ubiquitylize the K63 chain of key proteins, such as RIPK1 and NEMO, by removing the inhibit overactivation of NF-κB and MAPK signaling, thereby reducing cytokine release and inflammatory spread. This distribution and functional specificity suggests that DUBs finely regulate immune responses in different cellular regions, with potential clinical implications for the maintenance of immune homeostasis in sepsis patients.

DAMP (damage-associated molecular pattern) and PAMP (pathogen-associated molecular pattern) are key factors in triggering inflammatory responses and cell death. DAMP is released by damaged or dead cells, whereas PAMP originates from exogenous pathogens. Both initiate a strong innate immune response through activation of host pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerized structural domain-like receptors (NLRs), initiating a strong innate immune response. This activation not only leads to localized inflammation, but also induces SIRS, exacerbating the cytokine storm and ultimately leading to multi-organ damage. In sepsis, the persistence of DAMP and PAMP contributes to an uncontrolled inflammatory response, in which ubiquitination and deubiquitination mechanisms finely regulate inflammatory intensity and cell survival by modulating key signaling molecules (e.g., RIPK1, NF-κB).

Pattern recognition receptors (PRRs) are a central part of the innate immune system. PRRs, TLRs and NLRs, are the main sensing receptors in the initiation phase of sepsis ( Figure 1C ). Among them, TLRs are the most studied and relevant PRRs associated with cytokine storm and sepsis.

Toll-like receptor 4 (TLR4) is triggered by LPSand is the first mammalian paradigm for innate immune signaling (48). TLRs expressed at the plasma membrane (TLR1, 2, 4, 5, and 6) recognize a wide range of lipid- and protein-like ligands in the extracellular environment (49, 50). In particular, after recognizing LPS, TLR4 activates the NF-κB and MAPK signaling pathways, initiating a pro-inflammatory response that further triggers the sepsis-associated inflammatory cascade. It has been shown that TLR4 is particularly associated with sepsis, and TLR4-deficient mice exhibit reduced responsiveness in the face of LPS from Gram-negative bacteria. Further evidence for a central role of TLR4 in the development of sepsis (51).

In patients with sepsis, changes in the expression of TLR2 and TLR4 are more active on neutrophils than on monocytes. The expression of Toll-like receptor signaling genes in monocytes is reduced in more severe disease. In contrast, in the clinical phase of sepsis, neutrophil expression of these genes is upregulated (52). Like TLR4, TLR2 responds to the internalization of bacterial IL-12 (53).

The NLR is present in the cell and recognizes several types of ligands, including bacterial cell wall components, toxins, and host-derived molecules (e.g., ATP, uric acid, and damaged cell membranes), which in turn trigger the activation of different biological pathways. This ligand specificity stems from its NH2-terminal effector structural domain (54).

In the initial stage of sepsis, an intense inflammatory response is ignited, leading to a surge of large amounts of inflammatory factors (e.g., IL-1β, TNF-α), which in turn creates a cytokine storm. When local inflammation cannot be controlled, inflammation can spread throughout the body, leading to SIRS. As inflammation persists, immune function becomes progressively impaired and immunosuppression occurs, leading to viral reactivation, secondary infections, and ultimately increasing the risk of death in patients.

DAMPs released by necrotic apoptosis amplify the immune cell response to pathogens, while focal death exacerbates the cytokine storm in sepsis by releasing proinflammatory factors (e.g., IL-1β, IL-18). Consider the role of ubiquitination and deubiquitination at various stages of sepsis and control this process to prevent necrotic apoptosis and pyroptosis. Since ubiquitination and deubiquitination are independent yet interrelated processes, identification of specific deubiquitinating enzymes (DUB) corresponding to specific ubiquitinases may provide another effective strategy for regulating ubiquitination.

Multiple deubiquitinating enzymes are negative regulators of TLR signaling, but how they accomplish their job of inhibiting TLR overactivation and the associated cytokine storm has yet to be further explored. Sustained exposure to PAMPs (e.g., LPS released by bacteria) and DAMPs (e.g., molecules released by dying cells and damaged tissues of the host) leads to excessive and sustained activation of the TLR signaling pathway. With excessive activation of the TLR signaling pathway, cytokines such as TNF-α, IL-1β, and IL-6 are released in large quantities, ultimately triggering a cytokine storm. This cytokine storm drives the development of MODS by triggering a systemic inflammatory response, which in turn leads to organ damage and high mortality in sepsis patients. Therefore, in-depth studies on the regulatory mechanisms of TLR signaling are needed, especially on how to inhibit excessive cytokine storms by modulating the negative feedback or deubiquitination pathways of TLR signaling.