Novel mechanisms of thrombo-inflammation during infection: spotlight on neutrophil extracellular trap-mediated platelet activation.

A state-of-the-art lecture titled "novel mechanisms of thrombo-inflammation during infection" was presented at the ISTH Congress in 2022. Platelet, neutrophil, and endothelial cell activation coordinate the development, progression, and resolution of thrombo-inflammatory events during infection. Activated platelets and neutrophil extracellular traps (NETs) are frequently observed in patients with sepsis and COVID-19, and high levels of NET-derived damage-associated molecular patterns (DAMPs) correlate with thrombotic complications. NET-associated DAMPs induce direct and indirect platelet activation, which in return potentiates neutrophil activation and NET formation. These coordinated interactions involve multiple receptors and signaling pathways contributing to vascular and organ damage exacerbating disease severity. This state-of-the-art review describes the main mechanisms by which platelets support NETosis and the key mechanisms by which NET-derived DAMPs trigger platelet activation and the formation of procoagulant platelets leading to thrombosis. We report how these DAMPs act through multiple receptors and signaling pathways differentially regulating cell activation and disease outcome, focusing on histones and S100A8/A9 and their contribution to the pathogenesis of sepsis and COVID-19. We further discuss the complexity of platelet activation during NETosis and the potential benefit of targeting selective or multiple NET-associated DAMPs to limit thrombo-inflammation during infection. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.

Role of lymphoid lineage cells aberrantly expressing alarmins S100A8/A9 in determining the severity of COVID-19.

BACKGROUND: Alarmins S100A8 and S100A9 are recognized as hallmarks of severe COVID-19 and are primarily produced in myeloid cells, such as monocytes and neutrophils. As single-cell RNA-sequencing (scRNA-seq) data from patients with COVID-19 revealed the expression of S100A8/A9 in lymphoid cells in patients with severe COVID-19. OBJECTIVE: We investigated the characteristics of lymphoid cells expressing S100A8/A9 in COVID-19 patients. METHODS: Publicly available scRNA-seq data from patients with mild (N = 12) or severe (N = 7) COVID-19 were reanalyzed. The data were further divided into the following two groups based on the time of sample collection (from infection-onset): within 6 days (early phase) and after 6 days (late phase). Differential expression and gene set enrichment analyses were performed between S100A8/A9High and S100A8/A9Low lymphoid cells. Finally, cell-cell interaction analysis was performed to investigate the role of lymphoid cells expressing high levels of S100A8/A9 in COVID-19. RESULTS: S100A8/A9 overexpression was observed in lymphoid cells, including B cells, T cells, and NK cells, in patients with severe COVID-19 (compared to patients with mild COVID-19). Cells exhibiting strong interferon/cytokine responses were found to be associated with the severity of COVID-19. Furthermore, differences in S100A8/A9-TLR4/RAGE interactions were confirmed between patients with severe and mild disease. CONCLUSIONS: Lymphoid cells overexpressing S100A8/A9 contribute to the dysregulation of the innate immune response in patients with severe COVID-19, specifically during the early phase of infection. This study fosters a better understanding of the hyper-induction of pro-inflammatory cytokine expression and the generation of a cytokine storm in response to COVID-19 infection.

Dynamics of circulating calprotectin accurately predict the outcome of moderate COVID-19 patients.

BACKGROUND: Severe COVID-19 is associated with a high circulating level of calprotectin, the S100A8/S100A9 alarmin heterodimer. Baseline calprotectin amount measured in peripheral blood at diagnosis correlates with disease severity. The optimal use of this biomarker along COVID-19 course remains to be delineated. METHODS: We focused on patients with a WHO-defined moderate COVID-19 requiring hospitalization in a medical ward. We collected plasma and serum from three independent cohorts (N = 626 patients) and measured calprotectin amount at admission. We performed longitudinal measures of calprotectin in 457 of these patients (1461 samples) and used a joint latent class mixture model in which classes were defined by age, body mass index and comorbidities to identify calprotectin trajectories predicting the risk of transfer into an intensive care unit or death. FINDINGS: After adjustment for age, sex, body mass index and comorbidities, the predictive value of baseline calprotectin in patients with moderate COVID19 could be refined by serial monitoring of the biomarker. We discriminated three calprotectin trajectories associated with low, moderate, and high risk of poor outcome, and we designed an algorithm available as online software (https://calpla.gustaveroussy.fr:8443/) to monitor the probability of a poor outcome in individual patients with moderate COVID-19. INTERPRETATION: These results emphasize the clinical interest of serial monitoring of calprotectin amount in the peripheral blood to anticipate the risk of poor outcomes in patients with moderate COVID-19 hospitalized in a standard care unit. FUNDING: The study received support (research grants) from ThermoFisher immunodiagnostics (France) and Gustave Roussy Foundation.

Calprotectin and Imbalances between Acute-Phase Mediators Are Associated with Critical Illness in COVID-19.

The trajectory from moderate and severe COVID-19 into acute respiratory distress syndrome (ARDS) necessitating mechanical ventilation (MV) is a field of active research. We determined serum levels within 24 h of presentation of 20 different sets of mediators (calprotectin, pro- and anti-inflammatory cytokines, interferons) of patients with COVID-19 at different stages of severity (asymptomatic, moderate, severe and ARDS/MV). The primary endpoint was to define associations with critical illness, and the secondary endpoint was to identify the pathways associated with mortality. Results were validated in serial measurements of mediators among participants of the SAVE-MORE trial. Levels of the proinflammatory interleukin (IL)-8, IL-18, matrix metalloproteinase-9, platelet-derived growth factor (PDGF)-B and calprotectin (S100A8/A9) were significantly higher in patients with ARDS and MV. Levels of the anti-inflammatory IL-1ra and IL-33r were also increased; IL-38 was increased only in asymptomatic patients but significantly decreased in the more severe cases. Multivariate ordinal regression showed that pathways of IL-6, IL-33 and calprotectin were associated with significant probability for worse outcome. Calprotectin was serially increased from baseline among patients who progressed to ARDS and MV. Further research is needed to decipher the significance of these findings compared to other acute-phase reactants, such as C-reactive protein (CRP) or ferritin, for the prognosis and development of effective treatments.

Elevated Levels of Neutrophil Activated Proteins, Alpha-Defensins (DEFA1), Calprotectin (S100A8/A9) and Myeloperoxidase (MPO) Are Associated With Disease Severity in COVID-19 Patients.

Understanding of the basis for severity and fatal outcome of SARS-CoV-2 infection is of paramount importance for developing therapeutic options and identification of prognostic markers. So far, accumulation of neutrophils and increased levels of pro-inflammatory cytokines are associated with disease severity in COVID-19 patients. In this study, we aimed to compare circulatory levels of neutrophil secretory proteins, alpha-defensins (DEFA1), calprotectin (S100A8/A9), and myeloperoxidase (MPO) in COVID-19 patients with different clinical presentations. We studied 19 healthy subjects, 63 COVID-19 patients with mild (n=32) and severe (n=31) disease, 23 asymptomatic individuals identified through contact tracing programme and 23 recovering patients (1-4 months post-disease). At the time of disease presentation, serum levels of DEFA1 were significantly higher in patients with mild (mean230 ± 17, p<0.0001) and severe (mean452 ± 46, p<0.0001) disease respectively in comparison to healthy subjects (mean113 ± 11). S100A8/A9 proteins were significantly higher in COVID-19 patients (p<0.0001) irrespective of disease severity. The levels of DEFA1, S100A8/A9 and MPO reduced to normal in recovering patients and comparable to healthy subjects. Surprisingly, DEFA1 levels were higher in severe than mild patients in first week of onset of disease (p=0.004). Odds-ratio analysis showed that DEFA1 could act as potential biomarker in predicting disease severity (OR=11.34). In addition, levels of DEFA1 and S100A8/A9 were significantly higher in patients with fatal outcome (p=0.004 and p=0.03) respectively. The rise in DEFA1 levels was independent of secondary infections. In conclusion, our data suggest that induction of elevated levels of alpha-defensins and S100A8/A9 is associated with poor disease outcome in COVID-19 patients.

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.

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.

Induction of alarmin S100A8/A9 mediates activation of aberrant neutrophils in the pathogenesis of COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an unprecedented public health crisis. Evidence suggests that SARS-CoV-2 infection causes dysregulation of the immune system. However, the unique signature of early immune responses remains elusive. We characterized the transcriptome of rhesus macaques and mice infected with SARS-CoV-2. Alarmin S100A8 was robustly induced in SARS-CoV-2-infected animal models as well as in COVID-19 patients. Paquinimod, a specific inhibitor of S100A8/A9, could rescue the pneumonia with substantial reduction of viral loads in SARS-CoV-2-infected mice. Remarkably, Paquinimod treatment resulted in almost 100% survival in a lethal model of mouse coronavirus infection using the mouse hepatitis virus (MHV). A group of neutrophils that contributes to the uncontrolled pathological damage and onset of COVID-19 was dramatically induced by coronavirus infection. Paquinimod treatment could reduce these neutrophils and regain anti-viral responses, unveiling key roles of S100A8/A9 and aberrant neutrophils in the pathogenesis of COVID-19, highlighting new opportunities for therapeutic intervention.