Clinical relevance of elevated soluble ST2, HSP27 and 20S proteasome in patients with COVID-19

Clinical relevance of elevated soluble ST2, HSP27 and 20S proteasome in patients with COVID-19 Ralph Wendt1 *, Marie-Therese Lingitz2,3*, Maria Laggner2, Michael Mildner2,4 Denise Traxler2,5, Alexandra Graf6, Pavla Krotka6, Bernhard Moser2,7,, Konrad Hoetzenecker7, Sven Kalbitz1, Christoph Lübbert1,8, Joachim Beige1,9, Hendrik Jan Ankersmit2,7 * contributed equally 1 Department of Infectious Diseases, Tropical Medicine, Nephrology and Rheumatology, St. Georg Hospital, Delitzscher Str. 141, D-04129 Leipzig, Germany 2 Laboratory for Cardiac and Thoracic Diagnosis, Department of Surgery, Regeneration and Applied Immunology, Medical University of Vienna, Research Laboratories Vienna General Hospital, Waehringer Guertel 18-20, 1090 Vienna, Austria 3 Department of Anaesthesia, Intensive Care Medicine and Pain Medicine, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria 4 Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria 5 Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria 6 Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of Vienna, Spitalg. 23, 1090 Vienna, Austria 7 Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Waehringer Guertel 1820, 1090 Vienna, Austria 8 Division of Infectious Diseases and Tropical Medicine, Department of Medicine II, Leipzig University Medical Center, Liebigstr. 20, D-04103 Leipzig, Germany 9 Martin-Luther-University Halle-Wittenberg, Halle/Saale, Germany Corresponding author: HJ Ankersmit Department of Thoracic Surgery, Medical University of Vienna Waehringer Guertel 18-2


Introduction
The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic is one of the largest challenges the world faces today. It remains difficult to estimate the frequencies of severe disease courses and fatalities linked to Coronavirus Disease 2019 , due to the number of undiagnosed and asymptomatic infections, which varies with differences in susceptibility and vulnerability among different populations. The reported hospitalization rates for patients with diagnosed SARS-CoV-2 infections varies between 7 and 25% [1], [2]. The Robert Koch Institute reported 23% mortality among patients hospitalized with COVID-19 in Germany [2]. Among patients that required intensive care in Germany, mortality rates were 30% and 36% for patients that received ventilation support [2]. Clinical deterioration or the development of severe or critical disease typically occurs later in the disease course, at least 7 to 10 days after the infection. Although several immunopathological mechanisms that lead to severe COVID-19 have been discovered, the exact immunopathogenesis remains incompletely understood. Several studies have indicated that pulmonary inflammation in COVID-19 was associated with high systemic plasma levels of inflammatory cytokines, which resulted in the so-called "cytokine storm" [3]- [6]. Inflammatory cytokines are released by infected cells, alveolar macrophages, recruited T lymphocytes, monocytes, and neutrophils. They increase the permeability of lung epithelium and reduce its barrier function. As a result, pulmonary edema fills the alveolar spaces, followed by the formation of hyaline membranes, similar to the early-phase features of acute respiratory distress syndrome (ARDS) [7], [8]. Indeed, cytokine overproduction is a characteristic feature of ARDS; consequently, several studies have suggested that a cytokine storm may be the main pathogenic factor in COVID-19-related ARDS. [9], [10]. Additionally, the overproduction of inflammatory cytokines is involved in the subsequent development of microthrombosis, disseminated intravascular coagulation, and multiorgan-failure. This imbalance between pro-and anti-coagulants was commonly found in patients that died of COVID-19, along with elevated d-dimer concentrations [11]- [13].
arises from a shift from Th1 to Th2-biased cell proliferation, Brunner et al. [22] showed an increase in the levels of soluble suppressor of tumorigenicity (sST2), a member of the interleukin-33 (IL-33) family. Indeed, sST2 is predominantly expressed in type-2 T helper (Th2) cells, but not Th1 cells, which suggests that sST2 plays a role in cell proliferation and the promotion of the Th2 immune response. Additionally, Mildner et al [23] showed that sST2 was mainly produced in alveolar epithelial cells in the lung and cardiac myocytes. They also detected a strong augmentation of sST2 serum levels within 24 h post-LPS infusion, which implied a strong inflammatory response, accompanied by the secretion of inflammatory cytokines, such as IL-6 and TNF-α. Moreover, heatshock-protein 27 (HSP27), a member of the small heat shock protein family, was identified as an early marker of pulmonary damage and disease severity in patients with chronic obstructive pulmonary disease (COPD) [24], [25].
Considering these observations, we hypothesized that concentrations of the above-mentioned proteins might also be elevated in patients with severe COVID-19, and they might act as prognostic markers of survival. In the present study, we investigated the survival of 245 patients with COVID-19 and determined serum levels of sST2, TNF1 receptor (TNFR1), cCK18, HSP27, and 20S proteasome in specimens collected at hospital admission. We then explored which inflammatory markers were associated with a poorer prognosis in overall survival, 30-, 60and 90-day survival and in-hospital mortality. In addition, we investigated which parameters could indicate higher risk for patients requiring ventilatory assistance or oxygen therapy.

Study population
From August 2020 to February 2021, serum samples were collected from patients infected with SARS-CoV-2 that were treated in the Department of Infectious Diseases and Tropical Medicine at St. Georg Hospital , in Leipzig. The treating unit included a standard hospital ward (33 beds) and an intermediate-care unit with 7 beds.
At the time of admission, routine laboratory parameters were determined, including complete blood counts and C-reactive protein (CRP). In addition, laboratory parameters and clinical information, such as comorbidities and the course of disease, were derived from the medical history of each patient. Among the 270 serum samples, 25 were excluded, due to duplicate samples and patients with missing values. Thus, 245 patient samples were included in this study.
None of the patients had been vaccinated for SARS-CoV-2 prior to study inclusion. Patients that required extracorporeal membrane oxygenation procedures were transferred to another hospital, which was considered a discharge in our analyses.
All patients provided written informed consent prior to blood collection and participation.

Serum samples
Venous blood was drawn within 48 h of hospital admission. Whole blood was centrifuged at 4075g for 4 minutes at room temperature. After centrifugation, aliquoted samples were stored at -80 °C, until tests were performed. significant predictors from the univariable analyses. Due to the exploratory character of the study, no correction for multiplicity was performed and p-values <0.05 were considered statistically significant.

Study population characteristics
After excluding duplicate samples and patients with missing data, we included serum samples of 245 patients with SARS-CoV-2 infections. Demographic, clinicopathological, and laboratory details are shown in Table 1.
The study population displayed a female to male ratio of 46.1:53.9 with a median age of 76 years (IQR: 64-83).
The majority of the study population comprised patients with pneumonic infiltrates, detected by thorax CT, and most (n=137, 55.9%) required oxygen supplementation at hospital admission, which corresponded to a WHO outcome classification of 4, based on the Ordinal Scale for Clinical Improvement [29]. hospitalization. Twenty-six patients with shorter follow-up than 60 days and 39 patients that already died in the hospital were excluded for this analysis.
Soluble ST2 serum content is significantly associated with worse overall survival To assess a potential prognostic impact of investigated inflammatory cytokines on Overall Survival, a univariable Cox-Regression model was performed. In our univariable analysis, age (measured in years) and sST2 (measured in ng/ml) are associated with worse overall survival in our study cohort (HR=1.364, p=0.001).
Elevated sST2 levels remained significantly associated with higher mortality adjusting for age in the multivariable analysis (HR=1.353, p=0.002). All other investigated inflammatory markers showed no significant association with worse overall survival. Detailed results are listed in Table 2.
Elevated sST2 acts as an potential prognostic factor for 30-, 60-and 90-days mortality, elevated 20S proteasome levels were associated with worse 30-days mortality To investigate the association of assessed inflammatory markers with 30-, 60-and 90-day mortality, uni-and multivariable logistic regression models were computed. Since their follow up time was shorter than 30 days, 24 patients were excluded from the analysis. Within 30 days, 44 patients died. Increased levels of serum sST2 and 20S proteasome were associated with higher 30-days mortality in a univariable analysis (sST2: OR=1.536, p<0.001; 20S proteasome: OR=1.037, p=0.004). None of the other assessed biomarkers were significantly associated with worse prognosis in 30-days mortality. In multivariable analysis with inclusion of sST2, 20S proteasome and age, only sST2 remained significantly associated with the outcome (OR=1.457, p=0.004) ( Table   3) ROC Curves and AUC values are depicted in Figure S1 and Table S1.
For 60-day mortality outcome, 25 patients were excluded since their follow up time was shorter than 60 days.
Only sST2 was significantly associated with increased 60-day mortality risk (univariable: OR=1.335, p=0.007; multivariable: 1.298, p=0.016), whereas the other markers showed no significant effect on the 60-day outcome (Table 4). ROC curves and AUC are shown in Figure S2 and Table S2.
For the analysis of 90-day mortality, 40 patients were excluded due to shorter follow-up time. In our study cohort, 52 patients died during the first 90 days. In the univariable and multivariable logistic regression model, increased values of sST2 were significantly associated with higher 90-day mortality risk in both, the univariable (OR=1.335, p=0.007) and multivariable (OR=1.304, p=0.014) analyses. (Table 5) ROC Curves and AUC are presented in Figure S3 and Table S3.  (Table 6) ROC Curves and AUC values are depicted in Figure S4 and Table S4.
Serum content of sST2, HSP27 and 20S proteasome might predict risk of intubation and requirement of oxygen therapy We further evaluated the impact of inflammatory markers on endpoints such as the requirement of invasive ventilation or oxygen therapy. In our study cohort, 43 patients required invasive ventilation during hospitalization and oxygen therapy at hospital admission was needed in 179 cases. HSP27, sST2 and 20S proteasome serum levels were elevated in patients requiring invasive ventilation or oxygen therapy.
Increased values of these cytokines were also significantly associated with the need for oxygen therapy in the multivariable analysis (OR=1.134, 1.175 and 1.147, p=0.037, 0.046 and 0.004, respectively) ( Table 8).
Correlations between sST2 with 20S proteasome, HSP27, and TNFR1 To identify potential correlations between the investigated biomarkers, we performed the Spearman rank correlation analyses. We found that ST2 was positively correlated with 20S proteasome, HSP27, and TNFR1 and that HSP27 was positively correlated with 20S proteasome (Table 9).  [32]. We also identified sST2 as an potential prognostic marker of overall mortality, 30-,60-and 90-day mortality, and in-hospital mortality. Serum sST2, 20S proteasome and HSP27 were also associated with the need of oxygen therapy or invasive ventilation.
To our knowledge, this study was the first to examine HSP27 and 20S proteasome concentrations in SARS-CoV-2 positive patients. Moreover, we showed that elevated serum sST2 and 20S proteasome levels at hospital admission were significantly associated with increased risk of in-hospital mortality in uni-and multivariable logistic regression.

sST2
ST2 is a member of the IL-1 receptor family. It is expressed in two main isoforms: a transmembrane cellular (ST2L) and a soluble (sST2) form. ST2 is the receptor for IL-33, which is secreted by living cells in response to cell damage [27], [33]. Alveolar epithelial cells in the lung and cardiac myocytes were previously identified as main sources of sST2 [23]. Because sST2 is inducible by inflammatory stimuli, it is elevated in a large number of diseases, including sepsis, trauma, burns, and heart failure [27], [34]- [36]. Another study assessed serum sST2 levels in patients with chronic heart failure [27] and showed that the median sST2 concentration was 3.631.ng/ml. In contrast, serum sST2 levels were greater than 400 ng/ml in patients on day 1 after a left ventricular assist device (LVAD) implantation, and subsequently, levels normalized by the end of the first postoperative week [37]. In comparison, the median sST2 value in our study cohort was 7.853 ng/ml (IQR: 6.584-8.935). Therefore, the median serum sST2 levels in patients with COVID-19 were twice as high as the levels found in patients with chronic heart failure. This finding suggested that patients with COVID-19 infections had undergone a remarkable production of inflammatory cytokines by the time they were admitted to the hospital. Moreover, we found sST2 as a potential prognostic factor for in-hospital mortality, 30-, 60-and 90day mortality. This finding supported the hypothesis that patients with severe COVID-19 exhibited strong inflammatory activation [5], [6], [8], and it confirmed previous findings that identified sST2 as a marker that clearly distinguished between patients with increased risk and patients with low risk of mortality [5], [6], [8], [38].

HSP27
HSP27 acts as an intracellular chaperone to maintain normal cell functions, but it is also active extracellularly.
HSPs are involved in apoptosis inhibition, cytoprotection, and immunomodulation [39]- [41]. Moreover, HSP27 may play a major role in platelet aggregation by modifying actin polymerization [10], [42]. Tian et al. [10] found an association between a high thrombus burden and elevated HSP27 levels in patients with ST elevation myocardial infarctions. In addition, recently, HSP27 was identified as prognostic marker in inflammatory diseases, such as COPD and trauma-related ARDS [24], [25], [43], [44]. Therefore, we hypothesized that, in patients with COVID-19, elevated HSP27 might be associated with lung injury. Furthermore, the increase in HSP27 might have been related to thromboinflammation, which is the coordinated activation of inflammatory and thrombotic responses. Indeed, this condition might explain some of the key aspects of morbidity and mortality observed in patients with COVID-19 [10], [45]. HSP27 serum levels were previously assessed in healthy volunteers, who exhibited a median concentration of 1.482 ng/ml [46]. In our study cohort, the median HSP27 level at hospital admission was 5.839 ng/ml (IQR: 4.070-8.316), which was about four-fold higher than the levels found in healthy controls. In our study cohort, HSP27 was significantly associated with increased risk for invasive ventilation or the need of oxygen therapy, which could indicate an extensive damage of the lung in these patients.

20S proteasome
Roth et al. [20] previously described elevated core 20S proteasome levels in critically ill patients, particularly those with sepsis. Core 20S proteasome is a marker of increased apoptotic turnover. Therefore, we investigated whether 20S proteasome levels could act as a prognostic marker for overall and in-hospital mortality.
Surprisingly, we found that increased 20S proteasome levels were not significantly associated with overall-, 30-,60-and 90-day mortality, but we found that 20S proteasome was a significant predictor for the risk of invasive ventilation or the need of oxygen therapy. Increased 20S serum proteasome was also significantly associated with higher in-hospital mortality risk. Elevated 20S proteasome was found in several other pathological conditions such as carcinoma [47], burn injuries [48] and autoimmune diseases [49]. These observations confirm our findings that patients in our study cohort with elevated 20S proteasome levels suffer from severe tissue damage and hyper-activated immune system, whereas these factors together lead to increased apoptosis and thus probably higher risk of invasive ventilation or higher in-hospital mortality [50].

Survival
Our population comprised patients primarily hospitalized in a non-intensive care unit (ICU) setting. Patients with severe diseases, up to WHO scale 5 (high-flow oxygen therapy), could be treated outside the ICU. Patients that required non-invasive or invasive ventilation had to be transferred to the ICU. Our cohort had a mortality of 19.91% after 30 days. However, the mortality of our study cohort increased to 23,64% after 90 days, respectively. Additionally, the fact that after discharge from the hospital, nearly 8% of patients died after initially surviving COVID-19, reflect the possibly longer-term effects of the SARS-CoV-2 infection.
The main limitation of this study was the lack of a matched reference group without a COVID-19 history, for survival estimations in this vulnerable group of older patients (median age 76 years). However, our survival results suggested that COVID-19 also had an impact on survival after successful treatment of the acute infection and clearance of the virological disease.

Conclusion
With this study we evidenced that high level of sST2 could serve as prognostic marker for disease severity and overall survival. We demonstrated that serum sST2 was an early prognostic marker for overall mortality, 30-, 60and 90 day mortality and in-hospital mortality, and the need for invasive ventilation or oxygen therapy.
Moreover, we showed that besides sST2, also 20S proteasome, and HSP27 were significantly associated with a subsequent requirement for invasive ventilation during hospitalization. In addition, we evidenced that the augmented release of these inflammatory cytokines was associated with the need for oxygen therapy at hospital admission. We also detected a significant increment in 20S proteasome and sST2 levels in patients that died during the hospital stay. These findings contributed to a broader understanding of the systemic inflammation response in patients with COVID-19 and might help identify high risk patients at admission.

Supplementary material
The following are available in the supplementary file: Figure S1 ROC Curves for the endpoint 30-day mortality of investigated parameters; Figure S2 ROC Curves for the endpoint 60-day mortality of investigated parameters, Figure S3 ROC Curves for the endpoint 90-day mortality of investigated parameters, Figure S4 ROC Curves for the endpoint in-hospital mortality of investigated parameters, Table S1 AUC of ROC curves for the endpoint 30day mortality of investigated parameters, Table S2 AUC of ROC curves for the endpoint 60-day mortality of investigated parameters, Table S3 AUC of ROC curves for the endpoint 90-day mortality of investigated parameters, Table S4 AUC of ROC curves for the endpoint in-hospital mortality of investigated parameters

Institutional Review Board Statement
The study was approved by the Ethics Committee of the German-Saxonian Board of Physicians, Dresden, Germany (EK-BR-88/20.1), and it was conducted in accordance with the current version of the Declaration of Helsinki.

Informed Consent Statement
All patients provided written informed consent prior to blood collection and participation.

Data Availability Statement:
All data and materials support our published claims and comply with field standards. All data are available upon request.