Clinical application of serum NLRP3 on the diagnosis and prognosis of sepsis patients complicated with acute respiratory distress syndrome

Introduction Acute respiratory distress syndrome (ARDS) is a common complication of sepsis, which significantly increases the mortality rate. This work explored the diagnostic value of serum NOD-like receptor family pyrin domain containing 3 (NLRP3) concentration in patients with sepsis for ARDS, and the predictive value of serum NLRP3 concentration at the time of diagnosis for death 28 days after treatment. Methods A total of 150 sepsis patients were included in this study, including age-matched two groups of patients, 75 patients with ARDS and 75 patients without ARDS. In addition, 60 age-matched healthy patients with physical examination were recruited in this study. Serum NLRP3 concentration was determined by enzyme-linked immunosorbent assay (ELISA). The diagnostic values of serum NLRP3 concentration for ARDS in sepsis patients were evaluated by receiver operating characteristics (ROC) analysis. Correlation of serum NLRP3 with APACHE II score and SOFA were performed by Spearman correlation analysis. Results Pulmonary infection, APACHE II score and serum NLRP3 concentration were risk factors for patients with sepsis complicated with ARDS. ROC curve results showed that the specificity of serum NLRP3 concentration was 74.67%, the sensitivity was 76.00%, and the area under the curve (AUC) was 0.82 (p<0.001). APACHE II score and SOFA were significantly positively correlated with serum NLRP3 concentration. Baseline serum NLRP3 levels had significant predictive value for 28-day mortality in sepsis patients complicated with ARDS. Conclusion Serum NLRP3 concentration has clinical value in the diagnosis of sepsis complicated with ARDS.


Introduction
Sepsis is an emergency condition caused by infection which might lead to acute respiratory distress syndrome (ARDS) (1).Sepsis patients who develop ARDS have a higher mortality rate compared to those without ARDS (2).It was reported that the mortality rate in sepsis patients with ARDS was 46%, compared to 25% in sepsis patients without ARDS (3).
ARDS is characterized by severe inflammation and damage to the lung tissue, and its pathogenesis is complex and multifactorial (4).The development of ARDS in sepsis patients is thought to be due to an exaggerated immune response that leads to inflammation and damage to the lung tissue (5).Early recognition and treatment of sepsis are critical in preventing the development of ARDS.The Surviving Sepsis Campaign guidelines recommend early recognition of sepsis (6).Treatment for sepsis patients with ARDS typically involves supportive care and lung-protective ventilation strategies (7).These strategies, such as low tidal volume ventilation, have been shown to improve outcomes in sepsis patients with ARDS (8).Other therapies that have been investigated for sepsis-associated ARDS include corticosteroids and extracorporeal membrane oxygenation (ECMO) (9,10).
NOD-like receptor family pyrin domain containing 3 (NLRP3) belongs to the NLR family of pattern recognition receptors (PRRs) (11).NLRP3 plays a critical role in the innate immune response, which senses danger signals and initiates an immune response (12).NLRP3 is involved in the formation of the NLRP3 inflammasome (13).Dysregulation of NLRP3 and the NLRP3 inflammasome has been found in the pathogenesis of various diseases, including autoimmune diseases and neurodegenerative diseases (14).Notably, previous studies have shown that dysregulation of NLRP3 is involved in the pathogenesis of sepsis.Previous studies have found that NLRP3 inflammasome participates in the pathogenesis of sepsis-induced kidney failure (15) and sepsisinduced liver injury (16).In addition, SIRT1/NF-kB/NLRP3 pathway has been shown to participate in acute lung injury-like diseases by regulating pyroptosis (17).TXNIP/NLRP3 and NF-kB have also been shown to regulate lipopolysaccharide-induced ARDS (18).Currently, there are no specific biomarkers available that can accurately and reliably diagnose ARDS in septic patients.While certain biomarkers, such as plasma cytokine levels and surfactant protein D, have been studied, their diagnostic utility is limited due to variability and lack of specificity (19).Therefore, the present study aimed to study the diagnostic value of serum NLRP3 concentration for ARDS in sepsis patients, and the predictive value of serum NLRP3 concentration at the time of diagnosis for death 28 days after treatment.

Participants
A total of 150 sepsis patients were included in this study, including age-matched two groups of patients, 75 patients with ARDS and 75 patients without ARDS.In addition, 60 age-matched healthy patients with physical examination were recruited in this study.Our study is a retrospective study, and researchers who performed data collection (collecting clinical characteristics of patients, analyzing Serum NLRP3 concentration, following up clinical outcomes of patients, etc.) were blind to the grouping of patients in the two groups.The study was approved by the ethics committee of the Fourth Affiliated Hospital of China Medical University/China Medical University, and all the participants signed informed written consent.
Inclusion criteria: meet the diagnostic criteria for sepsis and ARDS; age ≥ 18 years; complete clinical data; informed consent of patients and their families.
Exclusion criteria: accompanied by other lung diseases, including bronchial asthma, active tuberculosis, bronchiectasis, pulmonary cystic fibrosis, lung abscess, and interstitial lung disease, etc.; combined with immune system diseases, mental illness, severe heart, brain, liver and kidney insufficiency, and blood system diseases; died within 24 hours of admission; suffering from malignant tumors.
Sepsis was diagnosed according to the "Guidelines for the Emergency Treatment of Sepsis and Septic Shock in China (2018)".

Serum concentration of NLRP3
Fasting venous blood (3 mL) were collected from patients on the morning of the second day after admission and from healthy subjects on the day of physical examination.The serum was separated by centrifugation at 1000 g/min for 10 min in anticoagulant tubes, and were stored at -80°C.They were stored and assessed as a batch.Serum samples were tested triplicate.The concentration of NLRP3 in the serum was determined by enzymelinked immunosorbent assay (ELISA).The kit was purchased from Wuhan Fine Biotech (EH4202), with a detection range of 0.781-50 ng/mL and a sensitivity of 0.469 ng/mL.

Patient information
General demographic information (age, gender) and clinical data of patients were collected, including basic medical history, whether septic shock occurred and multiple scores, etc.The Acute Physiology and Chronic Health Evaluation II (APACHE II) score is scored from three aspects: acute physiology, age, and chronic health.The acute physiology score was calculated based on the worse value of clinical indicators (body temperature, heart rate, respiration, oxygenation, mean arterial pressure, etc.) within 24 hours of admission.Chronic health is scored.The Sequential Organ Failure Assessment (SOFA) score is calculated.SOFA is a scoring system used to assess the extent of organ dysfunction in critically ill patients.It is widely employed in intensive care units (ICUs) to monitor and predict patient outcomes.The SOFA score provides a quantifiable measure of organ dysfunction by evaluating the function of six different organ systems: respiratory, cardiovascular, hepatic, coagulation, renal, and neurological.The scoring system assigns points based on the severity of dysfunction in each organ system.Higher scores indicate more severe dysfunction and are associated with poorer outcomes.The maximum score for each organ system is four, with a total possible score of 24.

Clinical treatment plan and prognosis judgment
On the basis of treating the primary disease, the treatment plan recommended by the "China Sepsis/Septic Shock Emergency Treatment Guidelines (2018)" formulated by the Chinese Medical Association Intensive Care Medicine Branch was adopted.Immediately after admission, the patient was given broadspectrum antibiotics for anti-infection, gastric mucosal protection to prevent stress ulcers, pulmonary hygiene, nutritional support, maintenance of electrolyte acid-base balance, mechanical ventilation, and possible treatment for the primary disease.At the same time, the patient was given subcutaneous 5000 IU lowmolecular-weight heparin sodium, 12 hours each time, for a total of 7 days.Follow-up was conducted for 28 days, and the survival of patients with sepsis complicated with ARDS was counted.

Statistical analysis
The data are shown as mean ± SD or n (percentage).The comparisons of data were done by Mann-Whitney test, Unpaired t test with Welch's correction or Fisher's exact test.The comparisons of data from multiple groups were done by Brown-Forsythe ANOVA test followed by Dunnett's T3 multiple comparisons test.Correlation of serum NLRP3 with APACHE II score and SOFA were performed by Spearman correlation analysis.ROC analysis (receiver operating characteristics) was used to determine the diagnostic values of serum NLRP3 concentration.*p < 0.05, **p < 0.01, ***p < 0.001.

Clinical characteristics of sepsis participants complicated with ARDS or without ARDS
Table 1 is a comparison of the basic conditions of 75 sepsis patients with ARDS (SARDS) and 75 sepsis patients without ARDS (SNARDS) at the time of admission.Significant differences were observed between the two groups in pulmonary infection (p<0.001),mechanical ventilation (p=0.005),septic shock(p=0.022),APACHE II score (p<0.001),SOFA (p=0.002), and serum NLRP3 (p<0.001).In addition, the average age of 60 included healthy patients was 61.17 ± 9.62, including 33 males and 27 females, all of whom had no

Diagnostic values of serum NLRP3 concentration for ARDS in sepsis patients
We first compared the differences in serum NLRP3 concentrations in 75 sepsis patients with ARDS (SARDS), 75 sepsis patients without ARDS (SNARDS), and 60 age-matched healthy controls.It could be seen that NLRP3 was significantly upregulated in sepsis patients than that of controls, and the patients with ARDS were higher than those without ARDS (Figure 1A).ROC analysis showed the diagnostic values of serum NLRP3 concentration for ARDS in sepsis patients (Figure 1B).The ROC curve results showed that the cut off value was 4.29 ng/mL, the sensitivity was 76.00%, the specificity was 74.67%, and AUC was 0.82 (p<0.001).

Correlation of serum NLRP3 concentrations with risk factors for sepsis patients complicated with ARDS
We then analyzed the correlation of serum NLRP3 concentrations with sepsis severity and concurrent ARDS risk factors in all sepsis patients (n = 150).The results showed that APACHE II score (Figure 2A, r=0.47, p<0.001) and SOFA (Figure 2B, r=0.29, p<0.001) were significantly positively correlated with serum NLRP3 concentration.In addition, serum NLRP3 concentrations of sepsis patients with pulmonary infection (pneumonia with or without ARDS) were significantly elevated (Figure 2C, p<0.01).

The predictive value of serum NLRP3 concentration on the prognosis of sepsis
Subsequently, we followed up 75 patients with sepsis and ARDS for 28 days.During the follow-up, 36 cases died and 39 cases survived.We compared serum NLRP3 levels at baseline between the two groups, and analyzed the predictive value of baseline serum NLRP3 levels for 28-day mortality.It was found that patients with sepsis and ARDS had higher serum NLRP3 concentrations at baseline in patients who died at 28-day follow-up (Figure 3A, p<0.001), and the baseline serum NLRP3 levels had a significant predictive value for 28-day death (Figure 3B, p<0.001).The ROC curve results revealed that when the cut off value was 5.16 ng/mL, the sensitivity of serum NLRP3 concentration was 75.00%, the specificity was 69.23%, and the AUC was 0.75.In order to compare the changes of serum NLRP3 concentration in patients before and after treatment, we supplemented the concentration of NLRP3 in serum after treatment with retained blood samples of patients after 7 days of treatment.The results showed that after treatment in 75 sepsis patients with ARDS (SARDS) and 75 sepsis patients without ARDS (SNARDS), the concentration of NLRP3 in serum was significantly reduced (Figure S1).We also compared serum NLRP3 concentrations at admission between dead and alive subgroups, and the results are shown in Figure S2.It can be seen that the concentration of serum NLRP3 in the dead patients was higher on admission, but there was no significant difference.The area under the ROC analysis curve was 0.67, but it was not significant either (Figure S2).

Discussion
Early diagnosis and accurate prognosis of sepsis complicated with ARDS are crucial to improving patient outcomes (1,20).In recent years, studies have focused on identifying reliable biomarkers for sepsis diagnosis and prognosis (21).However, the diagnosis and management of sepsis patients complicated with ARDS could be challenging (22).Currently, there is a need for biomarkers that could accurately diagnose and predict outcomes in these patients (23).
NLRP3 is a cytoplasmic protein involved in the function of the inflammasome, which participates in the innate immune response (24).In sepsis, the inflammasome is activated, which causes the release of multiple cytokines (25).Accumulating studies have suggested that serum levels of NLRP3 may have potential clinical applications in sepsis patient diagnosis (26).One study for diagnosing and predicting prognosis in sepsis, its clinical application is still in its infancy, and it remains uncertain whether it will eventually be adopted as a standard biomarker in clinical practice.
Our study demonstrated that the serum NLRP3 concentration might be a useful diagnostic and prognostic biomarker for sepsis patients complicated with ARDS.In this study, the best critical value of NLRP3 diagnosis of ARDS in sepsis patients is 4.29 ng/ml, the sensitivity is 76.00%, and the specificity is 74.67%, which suggests that the abnormal elevation of NLRP3 level has certain warning significance to ARDS in sepsis patients.Moreover, high serum NLRP3 levels at admission are associated with increased mortality, severity of sepsis, and risk factors for ARDS, which has important reference significance for clinical diagnosis and prognosis evaluation.Our data suggest that serum NLRP3 levels are significantly higher in sepsis patients complicated with ARDS and may be a potential biomarker for sepsis diagnosis and prognosis.Our findings provide promising insights into the potential utility of serum NLRP3 in the diagnosis and prognosis of sepsis complicated with ARDS.However, future studies should be done to investigate the mechanisms underlying the association between serum NLRP3 levels and sepsis complicated with ARDS.
In addition, it is important to note that this study have some limitations, including small sample sizes and retrospective designs.A sample size of 75 patients in each group might limit the statistical power and may not be representative of the larger population.Our results suggested that the concentration of serum NLRP3 in the dead patients was higher on admission, but there was no significant difference.This may be due to the bias caused by the low sample size.Also, a 28-day follow-up period might not be sufficient to determine long-term outcomes, especially for a severe condition like sepsis with ARDS.It is worth noting that more of the ARDS patients are noted to be on mechanical ventilation which could in itself cause an inflammatory response.While it can provide critical support, there are several factors that can contribute to the development of more serious complications in patients receiving mechanical ventilation.Mechanical ventilation itself can lead to lung injury, known as ventilator-associated lung injury (VALI).Factors such as high tidal volumes, high airway pressures, and excessive positive end-expiratory pressure (PEEP) can contribute to VALI.Mechanical ventilation can exert Excessive pressure on the lungs, leading to barotrauma and volutrauma.Patients on mechanical ventilation are at an increased risk of developing ventilatorassociated pneumonia (VAP).The presence of an artificial airway provides a pathway for bacteria to enter the lungs.Mechanical ventilation itself is also a factor affecting patient prognosis.Future studies were necessary for the determination of the clinical utility of serum NLRP3 in the diagnosis of sepsis patients with ARDS.

Conclusion
In conclusion, our data suggest that serum NLRP3 might be a useful diagnostic and prognostic biomarker for sepsis patients complicated with ARDS.High serum NLRP3 levels were associated with increased mortality rates, poor clinical outcomes, and lung injury severity in sepsis patients with ARDS.Therefore, targeting the NLRP3 inflammasome pathway might be a potential therapeutic strategy for sepsis patients with ARDS.
FIGURE 3 (A), Comparisons of serum NLRP3 between survival (n = 39) and death (n = 36) in sepsis patients complicated with ARDS after 28 days of follow-up.(B), ROC analysis of predictive values of serum NLRP3 for 28 days of death in sepsis patients complicated with ARDS.***p < 0.001.

TABLE 1
Clinical characteristics of sepsis patients complicated with acute respiratory distress syndrome (SARDS) or not (SNARDS).

TABLE 2
Multivariate logistic analysis for acute respiratory distress syndrome in sepsis patients.
The data are shown as mean ± SD or n (percentage).The comparisons of data were done by Mann-Whitney test, Unpaired t test with Welch's correction or Fisher's exact test.APACHE II, Acute Physiology and Chronic Health Evaluation II; SOFA, Sequential Organ Failure Assessment; NLRP3, NOD-like receptor thermal protein domain associated protein 3.