Targeting Doublecortin-Like Kinase 1 (DCLK1)-Regulated SARS-CoV-2 Pathogenesis in COVID-19.

Host factors play critical roles in SARS-CoV-2 infection-associated pathology and the severity of COVID-19. In this study, we systematically analyzed the roles of SARS-CoV-2-induced host factors, doublecortin-like kinase 1 (DCLK1), and S100A9 in viral pathogenesis. In autopsied subjects with COVID-19 and pre-existing chronic liver disease, we observed high levels of DCLK1 and S100A9 expression and immunosuppressive (DCLK1+S100A9+CD206+) M2-like macrophages and N2-like neutrophils in lungs and livers. DCLK1 and S100A9 expression were rarely observed in normal controls, COVID-19-negative subjects with chronic lung disease, or COVID-19 subjects without chronic liver disease. In hospitalized patients with COVID-19, we detected 2 to 3-fold increased levels of circulating DCLK1+S100A9+ mononuclear cells that correlated with disease severity. We validated the SARS-CoV-2-dependent generation of these double-positive immune cells in coculture. SARS-CoV-2-induced DCLK1 expression correlated with the activation of ß-catenin, a known regulator of the DCLK1 promoter. Gain and loss of function studies showed that DCLK1 kinase amplified live virus production and promoted cytokine, chemokine, and growth factor secretion by peripheral blood mononuclear cells. Inhibition of DCLK1 kinase blocked pro-inflammatory caspase-1/interleukin-1ß signaling in infected cells. Treatment of SARS-CoV-2-infected cells with inhibitors of DCLK1 kinase and S100A9 normalized cytokine/chemokine profiles and attenuated DCLK1 expression and ß-catenin activation. In conclusion, we report previously unidentified roles of DCLK1 in augmenting SARS-CoV-2 viremia, inflammatory cytokine expression, and dysregulation of immune cells involved in innate immunity. DCLK1 could be a potential therapeutic target for COVID-19, especially in patients with underlying comorbid diseases associated with DCLK1 expression. IMPORTANCE High mortality in COVID-19 is associated with underlying comorbidities such as chronic liver diseases. Successful treatment of severe/critical COVID-19 remains challenging. Herein, we report a targetable host factor, DCLK1, that amplifies SARS-CoV-2 production, cytokine secretion, and inflammatory pathways via activation of ß-catenin(p65)/DCLK1/S100A9/NF-κB signaling. Furthermore, we observed in the lung, liver, and blood an increased prevalence of immune cells coexpressing DCLK1 and S100A9, a myeloid-derived proinflammatory protein. These cells were associated with increased disease severity in COVID-19 patients. Finally, we used a novel small-molecule inhibitor of DCLK1 kinase (DCLK1-IN-1) and S100A9 inhibitor (tasquinimod) to decrease virus production in vitro and normalize hyperinflammatory responses known to contribute to disease severity in COVID-19.

Short Communication: S100A8 and S100A9, Biomarkers of SARS-CoV-2 Infection and Other Diseases, Suppress HIV Replication in Primary Macrophages.

S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils, form a heterodimer complex, and are secreted in plasma on pathogen infection or acute inflammatory diseases. Recently, both proteins were identified as novel biomarkers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibit HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads during SARS-CoV-2 co-infection.

Lipocalin-2, S100A8/A9, and cystatin C: Potential predictive biomarkers of cardiovascular complications in COVID-19.

Severe coronavirus (SARS-COV-2) infection often leads to systemic inflammation accompanied by cardiovascular complications including venous thromboembolism (VTE). However, it is largely undefined if inflammatory markers such as lipocalin-2 (LNC2), calprotectin (S100A8/A9), and cystatin C (CST3), previously linked with VTE, play roles in cardiovascular complications and advancement of COVID-19 severity. To investigate the same, hospitalized moderate and severe (presented pneumonia and required intensive care) COVID-19 patients were recruited. The levels of plasma LNC2, S100A8/A9, CST3, myoglobin, and cardiac Troponin I (cTnI) were assessed through enzyme-linked immunosorbent assay (ELISA). The investigation revealed a significantly upregulated level of plasma LNC2 at the moderate stage of SARS-CoV-2 infection. In contrast, the levels of S100A8/A9 and CST3 in moderate patients were comparable to healthy controls; however, a profound induction was observed only in severe COVID-19 patients. The tissue injury marker myoglobin was unchanged in moderate patients; however, a significantly elevated level was observed in the critically ill COVID-19 patients. In contrast, cTnI level was unchanged both in moderate and severe patients. Analysis revealed a positive correlation between the levels of S100A8/A9 and CST3 with myoglobin in COVID-19. In silico analysis predicted interactions of S100A8/A9 with toll-like receptor 4 (TLR-4), MyD88 LY96, and LCN2 with several other inflammatory mediators including MMP2, MMP9, TIMP1, and interleukins (IL-6, IL-17A, and IL-10). In summary, early induction of LCN2 likely plays a role in advancing the COVID-19 severity. A positive correlation of S100A8/A9 and CST3 with myoglobin suggests that these proteins may serve as predictive biomarkers for thromboembolism and tissue injury in COVID-19.

Hematopoietic progenitor cell liabilities and alarmins S100A8/A9-related inflammaging associate with frailty and predict poor cardiovascular outcomes in older adults.

Frailty affects the physical, cognitive, and social domains exposing older adults to an increased risk of cardiovascular disease and death. The mechanisms linking frailty and cardiovascular outcomes are mostly unknown. Here, we studied the association of abundance (flow cytometry) and gene expression profile (RNAseq) of stem/progenitor cells (HSPCs) and molecular markers of inflammaging (ELISA) with the cardiorespiratory phenotype and prospective adverse events of individuals classified according to levels of frailty. Two cohorts of older adults were enrolled in the study. In a cohort of pre-frail 35 individuals (average age: 75 years), a physical frailty score above the median identified subjects with initial alterations in cardiorespiratory function. RNA sequencing revealed S100A8/A9 upregulation in HSPCs from the bone marrow (>10-fold) and peripheral blood (>200-fold) of individuals with greater physical frailty. Moreover higher frailty was associated with increased alarmins S100A8/A9 and inflammatory cytokines in peripheral blood. We then studied a cohort of 104 more frail individuals (average age: 81 years) with multidomain health deficits. Reduced levels of circulating HSPCs and increased S100A8/A9 concentrations were independently associated with the frailty index. Remarkably, low HSPCs and high S100A8/A9 simultaneously predicted major adverse cardiovascular events at 1-year follow-up after adjustment for age and frailty index. In conclusion, inflammaging characterized by alarmin and pro-inflammatory cytokines in pre-frail individuals is mirrored by the pauperization of HSPCs in frail older people with comorbidities. S100A8/A9 is upregulated within HSPCs, identifying a phenotype that associates with poor cardiovascular outcomes.

The prognostic value of S100A calcium binding protein family members in predicting severe forms of COVID-19.

BACKGROUND: Excessive inflammation has been implicated in the immunopathogenesis of coronavirus disease 2019 (COVID-19). In the current study, the involvement of S100 calcium binding protein S100A4, S100A9, and S100A10 in the inflammatory settings of COVID-19 patients were evaluated. METHODS: Peripheral blood samples were obtained from 65 COVID-19 subjects and 50 healthy controls. From the blood samples, RNA was extracted and cDNA was synthesized, and then the mRNA expression levels of S100A4, S100A9, and S100A10 were measured by Real-time PCR. RESULTS: The mRNA expression of S100A4 (fold change [FC] = 1.45, P = 0.0011), S100A9 (FC = 1.47, P = 0.0013), and S100A10 (FC = 1.35, P = 0.0053) was significantly upregulated in COVID-19 patients than controls. The mRNA expression of S100A4 (FC = 1.43, P = 0.0071), (FC = 1.66, P = 0.0001), and S100A10 (FC = 1.63, P = 0.0003) was significantly upregulated in the severe COVID-19 subjects than mild-to-moderate subjects. There was a significant positive correlation between mRNA expression of S100A4 (ρ = 0.49, P = 0.030), S100A9 (ρ = 0.55, P = 0.009), and S100A10 (ρ = 0.39, P = 0.040) and D-dimer in the COVID-19 patients. The AUC for S100A4, S100A9, and S100A10 mRNAs were 0.79 (95% CI 0.66-0.92, P = 0.004), 0.80 (95% CI 0.67-0.93, P = 0.002), and 0.71 (95% CI 0.56-0.85, P = 0.010), respectively. CONCLUSIONS: S100A4, S100A9, and S100A10 play a role in the inflammatory conditions in COVID-19 patients and have potential in prognosis of severe form of COVID-19. Targeting these modules, hopefully, might confer a therapeutic tool in preventing sever symptoms in the COVID-19 patients.

Targeting S100A9 Reduces Neutrophil Recruitment, Inflammation and Lung Damage in Abdominal Sepsis.

S100A9, a pro-inflammatory alarmin, is up-regulated in inflamed tissues. However, the role of S100A9 in regulating neutrophil activation, inflammation and lung damage in sepsis is not known. Herein, we hypothesized that blocking S100A9 function may attenuate neutrophil recruitment in septic lung injury. Male C57BL/6 mice were pretreated with the S100A9 inhibitor ABR-238901 (10 mg/kg), prior to cercal ligation and puncture (CLP). Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested for analysis of neutrophil infiltration as well as edema and CXC chemokine production. Blood was collected for analysis of membrane-activated complex-1 (Mac-1) expression on neutrophils as well as CXC chemokines and IL-6 in plasma. Induction of CLP markedly increased plasma levels of S100A9. ABR-238901 decreased CLP-induced neutrophil infiltration and edema formation in the lung. In addition, inhibition of S100A9 decreased the CLP-induced up-regulation of Mac-1 on neutrophils. Administration of ABR-238901 also inhibited the CLP-induced increase of CXCL-1, CXCL-2 and IL-6 in plasma and lungs. Our results suggest that S100A9 promotes neutrophil activation and pulmonary accumulation in sepsis. Targeting S100A9 function decreased formation of CXC chemokines in circulation and lungs and attenuated sepsis-induced lung damage. These novel findings suggest that S100A9 plays an important pro-inflammatory role in sepsis and could be a useful target to protect against the excessive inflammation and lung damage associated with the disease.

S100A8/A9 in COVID-19 pathogenesis: Impact on clinical outcomes.

Coronavirus disease 2019 (COVID-19) has a broad range of clinical manifestations, highlighting the need for specific diagnostic tools to predict disease severity and improve patient prognosis. Recently, calprotectin (S100A8/A9) has been proposed as a potential biomarker for COVID-19, as elevated serum S100A8/A9 levels are associated with critical COVID-19 cases and can distinguish between mild and severe disease states. S100A8/A9 is an alarmin that mediates host proinflammatory responses during infection and it has been postulated that S100A8/A9 modulates the cytokine storm; the hallmark of fatal COVID-19 cases. However, it has yet to be determined if S100A8/A9 is a bona-fide biomarker for COVID-19. S100A8/A9 is widely implicated in a variety of inflammatory conditions, such as cystic fibrosis (CF) and chronic obstructive pulmonary disorder (COPD), as well as pulmonary infectious diseases, including tuberculosis and influenza. Therefore, understanding how S100A8/A9 levels correlate with immune responses during inflammatory diseases is necessary to evaluate its candidacy as a potential COVID-19 biomarker. This review will outline the protective and detrimental roles of S100A8/A9 during infection, summarize the recent findings detailing the contributions of S100A8/A9 to COVID-19 pathogenesis, and highlight its potential as diagnostic biomarker and a therapeutic target for pulmonary infectious diseases, including COVID-19.

Comparative transcriptome analyses reveal genes associated with SARS-CoV-2 infection of human lung epithelial cells.

During 2020, understanding the molecular mechanism of SARS-CoV-2 infection (the cause of COVID-19) became a scientific priority due to the devastating effects of the COVID-19. Many researchers have studied the effect of this viral infection on lung epithelial transcriptomes and deposited data in public repositories. Comprehensive analysis of such data could pave the way for development of efficient vaccines and effective drugs. In the current study, we obtained high-throughput gene expression data associated with human lung epithelial cells infected with respiratory viruses such as SARS-CoV-2, SARS, H1N1, avian influenza, rhinovirus and Dhori, then performed comparative transcriptome analysis to identify SARS-CoV-2 exclusive genes. The analysis yielded seven SARS-CoV-2 specific genes including CSF2 [GM-CSF] (colony-stimulating factor 2) and calcium-binding proteins (such as S100A8 and S100A9), which are known to be involved in respiratory diseases. The analyses showed that genes involved in inflammation are commonly altered by infection of SARS-CoV-2 and influenza viruses. Furthermore, results of protein-protein interaction analyses were consistent with a functional role of CSF2 and S100A9 in COVID-19 disease. In conclusion, our analysis revealed cellular genes associated with SARS-CoV-2 infection of the human lung epithelium; these are potential therapeutic targets.

The associations of serum S100A9 with the severity and prognosis in patients with community-acquired pneumonia: a prospective cohort study.

BACKGROUND: Previous studies found that S100A9 may involve in the pathophysiology of community-acquired pneumonia (CAP). However, the role of S100A9 was unclear in the CAP. The goal was to explore the correlations of serum S100A9 with the severity and prognosis of CAP patients based on a prospective cohort study. METHODS: A total of 220 CAP patients and 110 control subjects were recruited. Demographic and clinical data were collected. Serum S100A9 and inflammatory cytokines were measured. RESULTS: Serum S100A9 was elevated in CAP patients on admission. Serum S100A9 was gradually elevated parallelly with CAP severity scores. Additionally, inflammatory cytokines were increased and blood routine parameters were changed in CAP patients compared with control subjects. Correlation analysis found that serum S100A9 was positively associated with CAP severity scores, blood routine parameters (WBC, NLR and MON) and inflammatory cytokines. Further, logistic regression analysis demonstrated that there were positive associations between serum S100A9 and CAP severity scores. Besides, the prognosis of CAP was tracked. Serum higher S100A9 on the early stage elevated the death of risk and hospital stay among CAP patients. CONCLUSION: Serum S100A9 is positively correlated with the severity of CAP. On admission, serum higher S100A9 elevates the risk of death and hospital stay in CAP patients, suggesting that S100A9 may exert a certain role in the pathophysiology of CAP and regard as a serum diagnostic and managing biomarker for CAP.

Neutrophil calprotectin identifies severe pulmonary disease in COVID-19.

Severe cases of coronavirus disease 2019 (COVID-19) are regularly complicated by respiratory failure. Although it has been suggested that elevated levels of blood neutrophils associate with worsening oxygenation in COVID-19, it is unknown whether neutrophils are drivers of the thrombo-inflammatory storm or simple bystanders. To better understand the potential role of neutrophils in COVID-19, we measured levels of the neutrophil activation marker S100A8/A9 (calprotectin) in hospitalized patients and determined its relationship to severity of illness and respiratory status. Patients with COVID-19 (n = 172) had markedly elevated levels of calprotectin in their blood. Calprotectin tracked with other acute phase reactants including C-reactive protein, ferritin, lactate dehydrogenase, and absolute neutrophil count, but was superior in identifying patients requiring mechanical ventilation. In longitudinal samples, calprotectin rose as oxygenation worsened. When tested on day 1 or 2 of hospitalization (n = 94 patients), calprotectin levels were significantly higher in patients who progressed to severe COVID-19 requiring mechanical ventilation (8039 ± 7031 ng/ml, n = 32) as compared to those who remained free of intubation (3365 ± 3146, P < 0.0001). In summary, serum calprotectin levels track closely with current and future COVID-19 severity, implicating neutrophils as potential perpetuators of inflammation and respiratory compromise in COVID-19.

Circulating Calprotectin as a Biomarker of COVID-19 Severity.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although demographic and clinical parameters such as sex, age, comorbidities, genetic background and various biomarkers have been identified as risk factors, there is an unmet need to predict the risk and onset of severe inflammatory disease leading to poor clinical outcomes. In addition, very few mechanistic biomarkers are available to inform targeted treatment of severe (auto)-inflammatory conditions associated with COVID-19. Calprotectin, also known as S100A8/S100A9, MRP8/14 (Myeloid-Related Protein) or L1, is a heterodimer involved in neutrophil-related inflammatory processes. In COVID-19 patients, calprotectin levels were reported to be associated with poor clinical outcomes such as significantly reduced survival time, especially in patients with severe pulmonary disease. AREAS COVERED: Pubmed was searched using the following keywords: Calprotectin + COVID19, S100A8/A9 + COVID19, S100A8 + COVID-19, S100A9 + COVID-19, MRP8/14 + COVID19; L1 + COVID-19 between May 2020 and 8 March 2021. The results summarized in this review provide supporting evidence and propose future directions that define calprotectin as an important biomarker in COVID-19. EXPERT OPINION: Calprotectin represents a promising serological biomarker for the risk assessment of COVID-19 patients.

Epigenetic Landscapes of Single-Cell Chromatin Accessibility and Transcriptomic Immune Profiles of T Cells in COVID-19 Patients.

T cells play a critical role in coronavirus diseases. How they do so in COVID-19 may be revealed by analyzing the epigenetic chromatin accessibility of cis- and trans-regulatory elements and creating transcriptomic immune profiles. We performed single-cell assay for transposase-accessible chromatin (scATAC) and single-cell RNA (scRNA) sequencing (seq) on the peripheral blood mononuclear cells (PBMCs) of severely ill/critical patients (SCPs) infected with COVID-19, moderate patients (MPs), and healthy volunteer controls (HCs). About 76,570 and 107,862 single cells were used, respectively, for analyzing the characteristics of chromatin accessibility and transcriptomic immune profiles by the application of scATAC-seq (nine cases) and scRNA-seq (15 cases). The scATAC-seq detected 28,535 different peaks in the three groups; among these peaks, 41.6 and 10.7% were located in the promoter and enhancer regions, respectively. Compared to HCs, among the peak-located genes in the total T cells and its subsets, CD4+ T and CD8+ T cells, from SCPs and MPs were enriched with inflammatory pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway and tumor necrosis factor (TNF) signaling pathway. The motifs of TBX21 were less accessible in the CD4+ T cells of SCPs compared with those in MPs. Furthermore, the scRNA-seq showed that the proportion of T cells, especially the CD4+ T cells, was decreased in SCPs and MPs compared with those in HCs. Transcriptomic results revealed that histone-related genes, and inflammatory genes, such as NFKBIA, S100A9, and PIK3R1, were highly expressed in the total T cells, CD4+ T and CD8+ T cells, both in the cases of SCPs and MPs. In the CD4+ T cells, decreased T helper-1 (Th1) cells were observed in SCPs and MPs. In the CD8+T cells, activation markers, such as CD69 and HLA class II genes (HLA-DRA, HLA-DRB1, and HLA-DRB5), were significantly upregulated in SCPs. An integrated analysis of the data from scATAC-seq and scRNA-seq showed some consistency between the approaches. Cumulatively, we have generated a landscape of chromatin epigenetic status and transcriptomic immune profiles of T cells in patients with COVID-19. This has provided a deeper dissection of the characteristics of the T cells involved at a higher resolution than from previously obtained data merely by the scRNA-seq analysis. Our data led us to suggest that the T-cell inflammatory states accompanied with defective functions in the CD4+ T cells of SCPs may be the key factors for determining the pathogenesis of and recovery from COVID-19.

The Inflammatory Factors Associated with Disease Severity to Predict COVID-19 Progression.

Coronavirus disease 2019 (COVID-19) is associated with immune dysregulation and cytokine storm. Exploring the immune-inflammatory characteristics of COVID-19 patients is essential to reveal pathogenesis and predict progression. In this study, COVID-19 patients showed decreased CD3+, CD4+, and CD8+ T cells but increased neutrophils in circulation, exhibiting upregulated neutrophil-to-lymphocyte and neutrophil-to-CD8+ T cell ratio. IL-6, TNF-α, IL-1ß, IL-18, IL-12/IL-23p40, IL-10, Tim-3, IL-8, neutrophil extracellular trap-related proteinase 3, and S100A8/A9 were elevated, whereas IFN-γ and C-type lectin domain family 9 member A (clec9A) were decreased in COVID-19 patients compared with healthy controls. When compared with influenza patients, the expressions of TNF-α, IL-18, IL-12/IL-23p40, IL-8, S100A8/A9 and Tim-3 were significantly increased in critical COVID-19 patients, and carcinoembryonic Ag, IL-8, and S100A8/A9 could serve as clinically available hematologic indexes for identifying COVID-19 from influenza. Moreover, IL-6, IL-8, IL-1ß, TNF-α, proteinase 3, and S100A8/A9 were increased in bronchoalveolar lavage fluid of severe/critical patients compared with moderate patients, despite decreased CD4+ T cells, CD8+ T cells, B cells, and NK cells. Interestingly, bronchoalveolar IL-6, carcinoembryonic Ag, IL-8, S100A8/A9, and proteinase 3 were found to be predictive of COVID-19 severity and may serve as potential biomarkers for predicting COVID-19 progression and potential targets in therapeutic intervention of COVID-19.

S100A9 blockade prevents lipopolysaccharide-induced lung injury via suppressing the NLRP3 pathway.

BACKGROUND: S100 calcium binding protein A9 (S100A9) is a pro-inflammatory alarmin associated with several inflammation-related diseases. However, the role of S100A9 in lung injury in sepsis has not been fully investigated. Therefore, the present study aimed to determine the role of S100A9 in a lipopolysaccharide (LPS)-induced lung injury murine model and its underlying molecular mechanisms. METHODS: LPS was utilized to induce sepsis and lung injury in C57BL/6 or NOD-like receptor family pyrin domain containing 3 (NLRP3)-/- mice. To investigate the effects of S100A9 blockade, mice were treated with a specific inhibitor of S100A9. Subsequently, lung injury and inflammation were evaluated by histology and enzyme­linked immunosorbent assay (ELISA), respectively. Furthermore, western blot analysis and RT-qPCR were carried out to investigate the molecular mechanisms underlying the effects of S100A9. RESULTS: S100A9 was upregulated in the lung tissues of LPS-treated mice. However, inhibition of S100A9 alleviated LPS-induced lung injury. Additionally, S100A9 blockade also attenuated the inflammatory responses and apoptosis in the lungs of LPS-challenged mice. Furthermore, the increased expression of NLRP3 was also suppressed by S100A9 blockade, while S100A9 blockade had no effect on NLRP3-/- mice. In vitro, S100A9 downregulation mitigated LPS-induced inflammation. Interestingly, these effects were blunted by NLRP3 overexpression. CONCLUSION: The results of the current study suggested that inhibition of S100A9 could protect against LPS-induced lung injury via inhibiting the NLRP3 pathway. Therefore, S100A9 blockade could be considered as a novel therapeutic strategy for lung injury in sepsis.

An immune-based biomarker signature is associated with mortality in COVID-19 patients.

Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.