SARS-CoV-2 is a novel coronavirus that causes acute respiratory distress syndrome (ARDS), death and long-term sequelae. Innate immune cells are critical for host defense but are also the primary drivers of ARDS. The relationships between innate cellular responses in ARDS resulting from COVID-19 compared to other causes of ARDS, such as bacterial sepsis is unclear. Moreover, the beneficial effects of dexamethasone therapy during severe COVID-19 remain speculative, but understanding the mechanistic effects could improve evidence-based therapeutic interventions. To interrogate these relationships, we developed an scRNA-Seq and plasma proteomics atlas (biernaskielab.ca/COVID_neutrophil). We discovered that compared to bacterial ARDS, COVID-19 was associated with distinct neutrophil polarization characterized by either interferon (IFN) or prostaglandin (PG) active states. Neutrophils from bacterial ARDS had higher expression of antibacterial molecules such as PLAC8 and CD83. Dexamethasone therapy in COVID patients rapidly altered the IFNactive state, downregulated interferon responsive genes, and activated IL1R2+ve neutrophils. Dexamethasone also induced the emergence of immature neutrophils expressing immunosuppressive molecules ARG1 and ANXA1, which were not present in healthy controls. Moreover, dexamethasone remodeled global cellular interactions by changing neutrophils from information receivers into information providers. Importantly, male patients had higher proportions of IFNactive neutrophils, a greater degree of steroid-induced immature neutrophil expansion, and increased mortality benefit compared to females in the dexamethasone era. Indeed, the highest proportion of IFNactive neutrophils was associated with mortality. These results define neutrophil states unique to COVID-19 when contextualized to other life-threatening infections, thereby enhancing the relevance of our findings at the bedside. Furthermore, the molecular benefits of dexamethasone therapy are also defined, and the identified pathways and plasma proteins can now be targeted to develop improved therapeutics. Competing Interest Statement The authors have declared no competing interest. Footnotes * 1. Validation, using immunofluorescence staining for immature and polarization state proteins, to confirm neutrophil states previously identified in silico. 2. New evidence that the novel neutrophil polarization signatures revealed in our COVID-19 group accurately predict outcomes when applied to a validation cohort of 103 bulk RNA-Seq samples (where 17 cases were fatal). Here we demonstrate that our neutrophil polarization states are a superior predictor of 28-day mortality compared to clinical severity scales such as sequential organ failure assessment (SOFA) across all classification thresholds. Moreover, these signatures are specifically suppressed in the dexamethasone group supporting our evidence of the beneficial mechanisms underpinning this therapy. 3. Evidence at the level of the lung tissue, supporting our studies using blood, of an expansion of IFNactive neutrophils in severe COVID-19 relative to mild/moderate disease (1.5 FC) and in COVID-19 patients relative to those with bacterial pneumonia (4.7 FC). Here, we used inferred neutrophil composition in bronchoalveolar microenvironments by projecting two high-quality BALF sc-RNA-Seq datasets onto our peripheral blood reference. 4. Serum proteomics screen for SARS-CoV-2 viral proteins from COVID-19 patients captured in our study. A proteomic screen for SARS-CoV-2 specific viral proteins in COVID-19 patient serum revealed robust detection of one or more viral proteins in COVID-19. 5. Serum shotgun proteomics using mass spectrometry verified several interferon response elements identified in our scRNAseq analyses. It also revealed dexamethasone-induced alterations in neutrophil antimicrobial proteins and serine proteases, further strengthening our claim that neutrophil-related inflammatory processes are a critical driver of dexamethasone action. In addition, we identify dexamethasone-induced suppression of 10 host plasma proteins that have previously been identified as biomarkers of COVID-19 severity and mortality. Full host proteome (comprising 633 proteins detected) is made queryable through our Online Atlas. 6. New evidence of mortality differences between males and females with life threatening COVID-19 at an epidemiological level. Using a retrospective province-wide (Alberta, Canada) ICU data repository, we compared the mortalities of pre-dexamethasone ICU patients (51 M, 21 F) versus post-dexamethasone (1013 M, 568 F) treated ICU patients. This revealed a statistically significant survival benefit in male COVID-19 patients treated with dexamethasone. We stratified all COVID-19 ICU patients by sex and separated them by the dates when dexamethasone became standard of care and further observed that males had reduced mortalities since dexamethasone became standard of care, but females did not derive any benefit in terms of mortality. * http://www.biernaskielab.ca/COVID_neutrophil...

An Immune Cell Atlas Reveals Dynamic COVID-19 Specific Neutrophil Programming Amenable to Dexamethasone Therapy

10.1101/2021.04.18.440366