This study demonstrates the existence of proteomic patterns associated with sepsis in comparison to patients with NISIRS. Thirty proteins have been detected with varying degrees of association with sepsis. Among these, those with the strongest association are PPBP (+ 0.09), CRL1 (+ 0.08), C5 (+ 0.07), VWF (+ 0.05), and SERPINA4 (+ 0.05), while proteins with weaker associations include RBP4 (0), PRX2 (0), and SERPINA6 (0). Furthermore, the different proteins observed in this study participate in various physiological metabolic pathways, highlighting sepsis as a highly complex process involving diverse biological processes.
Early diagnosis and prognosis of sepsis are crucial in medical research. Mass spectrometry (MS) is a highly powerful and sensitive analytical technique that enables the identification and measurement of molecules based on their mass. Using MS, it is possible to obtain information about multiple identified molecules simultaneously through a targeted approach or even analyze hundreds or thousands of compounds using a broader approach. In the initial research stages, non-targeted MS approaches are often employed to compare samples from different populations. Once a compound that exhibits differential levels between these populations is identified, a targeted approach can be used in a later stage to thoroughly analyze the suspected biomarkers [22].
Proteolysis
For decades, it has been known that sepsis induces proteolysis [23]. Bauzá-Martínez et al. observed that the plasma of patients with septic shock showed an approximately three-fold increase in the total peptide count compared to healthy individuals, indicating elevated proteolysis above physiological levels in septic shock [24].
SERPINA4 or Kallistatin is a glycoprotein with protective activity in vascular injury due to its anti-inflammatory and antioxidant effects [25]. In our study we have observed that this protein has an important role in sepsis with a shap value of 0.05. Chao et al. had already observed low levels of Kallistatin in 10 sepsis patients [26]. Lin et al. also observed reduced levels of Kallistatin in 54 patients with Community-Acquired Pneumonia, establishing a direct correlation between the levels of this glycoprotein and the severity of sepsis [27].
GSN or Gelsolin is a protein that inhibits cell apoptosis and can regulate macrophage function in inflammatory processes [28] and that in our study has an intermediate role in sepsis (+ 0.04). Halis et al. observed a significant decrease in plasma gelsolin levels in septic patients. After resolution of the septic process, these levels normalized, concluding that plasma gelsolin could be a useful marker for severe sepsis. [29]. It has even been observed, in a murine study, that the administration of recombinant plasma gelsolin can modulate the inflammatory response while increasing the host's antibacterial activity [30].
FN1 or Fibronectin, also in our study with an intermediate role in sepsis (+ 0.03), is a high molecular weight glycoprotein involved in many processes, including cell adhesion, proliferation, embryonic development, and matrix remodeling [31], with its primary functions related to the host's response to infection [32]. Ruiz Martín et al. observed a decrease in plasma FN1 levels in septic patients compared to a control group [33]. The same conclusion was reached by Lemańska-Perek et al. in 71 septic patients compared to a control group. Furthermore, lower FN1 levels were associated with higher mortality, giving FN1 a prognostic role [34].
Alpha-1 Antitrypsin or SERPINA3, in our study with a shap value of 0.03, is a glycoprotein whose main function is to inhibit serine proteases such as chymotrypsins, cathepsin G, and mast cell chymases by binding to them in a stable complex, preventing their proteolytic activity [35]. Čaval et al. observed changes in the glycosylation of this protein at various stages in septic patients. These changes are even observed in the early stages of sepsis, leading to its dysfunctionality and, consequently, proteolytic activation [36]. Sehgal et al observed that in cirrhotic patients with sepsis, the SERPINA3 protein was downregulated, leading to increased activation and autophagy of neutrophils [37].
Innate immune response
In our study, C1RL or Component C1r subcomponent-like shows a significant association with sepsis (+ 0.08). The function of this protein is not clear, but it appears to be involved, among other, in the proteolytic cleavage of a proform of haptoglobin [38]. Haptoglobin is a glycoprotein whose concentration increases during inflammation and also has several immunomodulatory functions, including the regulation of T cell-mediated immune responses [39]. Although this protein has been associated with the development of several types of cancers [40, 41], in this study we describe for first time the association of C1RL with sepsis. LBP or Lipopolysaccharide Binding Protein is an acute-phase protein that binds to lipopolysaccharides (LPS), a component of the outer wall of gram-negative bacteria, and triggers immune response activation [42]. Our study demonstrates an intermediate role in sepsis (+ 0.04). This protein has been studied in multiple studies as a sepsis biomarker [43–45], although in a meta-analysis, LBP showed low sensitivity and specificity in sepsis detection, so it is not recommended in clinical practice as a standalone biomarker [46].
FCN3 or Ficolin-3, in our study with a low association in sepsis (+ 0.02), is a protein produced in various organs of the body that binds to a wide spectrum of microorganisms, suggesting its involvement in host defense against a broad range of microbial infections [47]. Schlapbach et al. observed that low FCN3 concentration was associated with a higher risk of neutropenic fever, particularly with bacteremia, in children undergoing cancer chemotherapy. Therefore, low levels of FCN3 represent a new risk factor for chemotherapy-related infections [48]. Snipsøyr et al. studied patients with infectious endocarditis and observed a downregulation of this protein [49].
CD14 (low association in sepsis with a shap value of + 0.01) is an antigen present on the membrane of monocytes, macrophages, and dendritic cells, whose function is to bind to the lipopolysaccharides of Gram-negative bacteria [50]. Chen et al. conducted a murine study where, in mice induced with sepsis after cecal puncture, an elevation in CD14 expression was observed [51]. Furthermore, Edge et al. demonstrated that combined inhibition of C5 and CD14 efficiently attenuates organ inflammation induced by E. coli sepsis in pigs [52].
Complement activation
Another group of proteins that play a significant role in sepsis is the complement system, a key component of the innate immune system against pathogens [53]. In our study we have observed that there are various complement-associated proteins that have varying degrees of association with sepsis. Just as CRL1 (+ 0.08), already mentioned in the previous section as C5 (+ 0.05) play an important role in sepsis with respect to patients with NISIRS, others such as C6 (+ 0.03), C3 (+ 0.02) and FCN3 (0.02), its association with sepsis is minor. Complement activation (via the classical or alternative pathway) triggers proteases that cleave C3 and C5 into C3a and C5a, leading to the formation of the terminal complement complex. This complex binds to infected cells and induces cell lysis [54]. More than two decades ago, Stöve et al. observed that C3a levels and the C3a/C3 ratios, during the first 24 hours following the clinical onset of sepsis, were significantly higher in sepsis patients compared to those with SIRS. C3 levels for septic or SIRS patients were significantly lower than those of healthy donors. Interestingly, the C3a levels of septic patients decreased with appropriate treatment. Furthermore, they found that complement activation was much lower in SIRS patients than in septic patients [55]. De Nooijer et al. also observed that in septic patients, there is a decrease in complement components C3 and C5 and an increase in C3a, C3c, C5a [56]. In addition, no differences were observed in complement factor concentrations between patients with different immunological endotypes: hyperinflammation or immunoparalysis. Consequently, no correlation was observed with other inflammatory parameters, disease severity, or mortality.
C6 is the complement component that binds to complement C5b along with C7, C8, and C9, forming the membrane attack complex. This complex creates a pore in the membrane, leading to pathogen lysis [57]. Several studies demonstrate that patients with a deficiency in complement component C6 are associated with increased susceptibility to meningococcal infection. Petersen et al. observed that thirteen patients who had experienced at least one episode, typically two or more episodes, of bacteremia caused by Neisseria meningitidis or Neisseria gonorrhoeae had a deficiency in complement components C6, C7, or C8 [58]. Ellison et al. evaluated the complement system in 20 patients who presented with a first episode of meningococcal meningitis, meningococcemia, or meningococcal pericarditis and found that three of these six patients had a deficiency in a terminal pathway protein, C6 and C8 [59]. In contrast, a murine study involving mice in which the C6 protein had been eliminated observed defective innate responses in surface adhesion molecules, the generation of superoxide anion, the appearance of reactive oxygen species, and histone release after PMN activation, along with defective phagocytosis. When these mice were subjected to polymicrobial sepsis, they exhibited reduced organ dysfunction due to lower levels of proinflammatory cytokines and chemokines in the plasma and lower histone levels in the plasma [60].
Response to lipopolysaccharides
Lipopolysaccharides (LPS) are molecules present in the outer membrane of Gram-negative bacteria. LPS can be recognized by toll-like receptor 4, found in monocytes, macrophages, neutrophils, and dendritic cells, activating the immune system [61]. Of all the proteins found in our study, PPBP or Connective tissue-activating peptide III is the one with the strongest association with sepsis (+ 0.09). This is a peptide with demonstrated antimicrobial capabilities found inside platelets [62]. Tang et al, using proteomic techniques, were able to isolate 7 proteins, among which PPBP was included. In vitro, they were exposed to cultures with different bacteria (E. coli and Staphylococcus aureus) and fungi (Cryptococcus neoformans and Candida albicans) and observed that there was good antimicrobial activity against the first three germs with no activity against C. albicans [63]. Smith et al analyzed the expression of PPBP in patients with chronic cavitary pulmonary aspergillosis (CCPA) and allergic bronchopulmonary aspergillosis (ABPA), observing a significant increase in PPBP expression and protein levels (ABPA − 19.7 times and CCPA − 27.7 times) compared to control groups. They conclude that in patients with the described characteristics, PPBP levels could be used as a diagnostic biomarker in these types of patients [64].
Blood coagulation
VWF or Von Willebrand factor, with a shap value in our study of + 0.05, is a protein with prothrombotic and proinflammatory capabilities. Additionally, VWF serves as a binding site for ligands of bacteria that cause potentially life-threatening local and systemic infections, such as Staphylococcus aureus and Streptococcus pneumoniae [65]. In a study comparing forty patients with severe sepsis and septic shock with forty healthy controls, Kremer Hovinga et al observed that VWF antigen levels were significantly higher in septic patients compared to controls, with no correlation with disease severity, organ dysfunction, or outcome [66]. Singh et al reached the same conclusion in a recent study comparing septic and non-septic patients, although the difference from the previous study is that VWF antigen levels were higher in non-surviving patients compared to those who survived sepsis [67].
In our study, F12 or Coagulation factor XIIa heavy chain shows a weak association with sepsis compared to patients with NISIRS (+ 0.01). However, some studies suggest that this protein contributes to the host's pathological response to certain infectious organisms, leading to septic processes. Matsumoto et al observed, in a murine study, that the administration of a protease from Serratia marcescens resulted in activation of F12 and prekallikrein in guinea pigs, increasing vascular permeability due to inflammatory processes [68]. Kaminishi et al, in an in vitro study, exposed normal human plasma to proteases from Candida albicans, Pseudomonas aeruginosa and Serratia marcescens, observing that F12 activation occurred, leading to thrombin formation. This suggests that systemic blood circulation can be affected by the activation of the blood coagulation cascade by microbial infections, especially in septic patients, facilitating disseminated intravascular coagulation and multiorgan failure [69].
Lipid metabolism
The role of lipoproteins in sepsis is increasingly recognized as a fundamental aspect of the host's early response to infection [70]. In our study, two lipoproteins are associated with sepsis, APOE and APOB, with the former showing the strongest association (+ 0.03). Fu et al conducted a study with 279 pediatric patients with various infections compared to 58 healthy controls. They observed that serum ApoE levels increased significantly in cases of bacterial infections, including sepsis, bacterial meningitis, and bacterial pneumonia, compared to healthy controls. No significantly elevated serum ApoE levels were observed in patients with aseptic meningitis or mycoplasma pneumonia [71]. Wang et al, also in a pediatric study, found that ApoE levels in cerebrospinal fluid (CSF) increased significantly in patients with bacterial meningitis (with an optimal cutoff value of CSF ApoE > 1.7 mg/L with a sensitivity of 85% and specificity of 100%), and serum ApoE was markedly elevated in patients with sepsis or bacterial meningitis (with an optimal serum cutoff value of > 42 mg/L with a sensitivity of 80% and specificity of 93%) compared to patients with other infections and uninfected children [72]. In a proteomics study on sepsis, Li et al identified several proteins, including APOE. They observed that the expression of this lipoprotein is increased in comparison to non-septic patients with an AUC of 0.619 (95% CI: 0.510–0.719) [73]. Tripp et al, in a study with rats, injected live colonies of Escherichia coli and found that ApoB production increased 2.6 times in the septic groups compared to uninfected rat controls (P = 0.037), while ApoE production decreased 2.9 times in the control group (P = 0.036) [74]. Conversely, Kumaraswamy observed that both severe sepsis and NISIRS led to a decrease in blood APOB levels, although in septic patients, it was a significantly greater decrease [75].
Other proteins
In our patients, we have observed other proteins with physiological functions different from the ones mentioned earlier. ORM1 or Alpha-1-acid glycoprotein 1 is the protein, in this section, that has a stronger association with sepsis (+ 0.03). It is a protein produced by the liver and peripheral tissues in response to systemic inflammation with immunomodulatory and substance transport capabilities related to inflammation [76]. Astrup et al, in a murine study, observed an increase of approximately 400–500 times in ORM1 in rats with cerebral infarction and cerebral abscesses compared to those with cerebral infarction alone [77]. In human studies, Lu et al observed that ORM1 expression was upregulated in sepsis patients compared to patients without sepsis upon discharge [78]. Similar results have been observed in a study with 25 sepsis patients compared to 25 healthy controls [79].
LUM or Lumican is a leucine-rich proteoglycan that binds to collagen and modifies the structure of collagen-rich connective tissues, in our study with an intermediate strength of association in sepsis of + 0.03. Subsequent studies have shown that it regulates the detection of bacterial lipopolysaccharides through the Toll-like receptor 4 signaling pathway and the innate immune response [80]. Maiti et al measured the abundance of LUM in the plasma of 11 sepsis patients and 17 healthy individuals and found a small but consistent increase in sepsis patients (26.41 ± 1.54 ng/ml versus 21.0 ± 1.19 ng/ml) [81].
In our study, LGR1 or Leucine rich alpha-2-glycoprotein 1 has a low association with sepsis (+ 0.02). Gong et al conducted a proteomic study on samples from 89 sepsis patients and 67 healthy controls. They observed that in septic patients, there were a series of genes upregulated upon discharge that are closely related and cooperate to respond to different types of insults, including those expressing LGR1 and ORM1 [82]. Similar results regarding LGR1 were found by Lai et al, comparing 233 sepsis patients and 70 healthy controls with AUCs exceeding 0.95 [83].
This study has several limitations. The first and main is that it only allows us to determine the degree of association that these proteins have with sepsis compared to patients with NISIRS, but we do not know the concentrations at which they are present in the patients. We cannot determine whether the expression is upregulated or downregulated compared to other studies.
Second, this is a single-center study. Our results only have internal validity due to the demographic characteristics of the patients. Therefore, it would be necessary to conduct multicenter studies to validate and generalize the findings found in this study.
Third, samples are collected upon sepsis code activation, and although they are taken early in the course of sepsis, we cannot rule out that all patients present the same stage of evolution at a pathophysiological level at the time of sample collection, which could affect the results.