Increased peripheral blood neutrophil activation phenotypes and NETosis in critically ill COVID-19 patients (preprint)

BackgroundIncreased inflammation is a hallmark of COVID-19, with pulmonary and systemic inflammation identified in multiple cohorts of patients. Definitive cellular and molecular pathways driving severe forms of this disease remain uncertain. Neutrophils, the most numerous leukocytes in blood circulation, can contribute to immunopathology in infections, inflammatory diseases and acute respiratory distress syndrome (ARDS), a primary cause of morbidity and mortality in COVID-19. Changes in multiple neutrophil functions and circulating cytokine levels over time during COVID-19 may help define disease severity and guide care and decision making. MethodsBlood was obtained serially from critically ill COVID-19 patients for 11 days. Neutrophil oxidative burst, neutrophil extracellular trap formation (NETosis), phagocytosis and cytokine levels were assessed ex vivo. Lung tissue was obtained immediately post-mortem for immunostaining. ResultsElevations in neutrophil-associated cytokines IL-8 and IL-6, and general inflammatory cytokines IP-10, GM-CSF, IL-1b, IL-10 and TNF, were identified in COVID-19 plasma both at the first measurement and at multiple timepoints across hospitalization (p < 0.0001). Neutrophils had exaggerated oxidative burst (p < 0.0001), NETosis (p < 0.0001) and phagocytosis (p < 0.0001) relative to controls. Increased NETosis correlated with both leukocytosis and neutrophilia. Neutrophils and NETs were identified within airways and alveoli in the lung parenchyma of 40% of SARS-CoV-2 infected lungs. While elevations in IL-8 and ANC correlated to COVID-19 disease severity, plasma IL-8 levels alone correlated with death. ConclusionsCirculating neutrophils in COVID-19 exhibit an activated phenotype with increased oxidative burst, NETosis and phagocytosis. Readily accessible and dynamic, plasma IL-8 and circulating neutrophil function may be potential COVID-19 disease biomarkers.

Natural SARS-CoV-2 infection in kept ferrets, Spain (preprint)

We found SARS-CoV-2 RNA in 6 of 71 ferrets (8.4%) and isolated the virus from one rectal swab. Natural SARS-CoV-2 infection does occur in kept ferrets, at least under circumstances of high viral circulation in the human population. However, small ferret collections are probably unable to maintain prolonged virus circulation.

Increased IL-8, Neutrophil Activation Phenotypes and NETosis in Critically Ill COVID-19 Patients (preprint)

Background: Increased inflammation is a hallmark of COVID-19, with pulmonary and systemic inflammation identified in multiple cohorts of patients. Definitive cellular and molecular pathways driving severe forms of this disease remain uncertain. Neutrophils, the most numerous leukocytes in blood circulation, can contribute to immunopathology in infections, inflammatory diseases and acute respiratory distress syndrome (ARDS), a primary cause of morbidity and mortality in COVID-19. Neutrophilia, elevated neutrophil:lymphocyte ratios, and elevated neutrophil-associated cytokines are present in COVID-19, but changes in neutrophil functions have not been characterized. Here we analyzed the functional state of circulating neutrophils in COVID-19.Methods: Blood was obtained from critically ill COVID-19 patients over two weeks and healthy controls across multiple timepoints. Plasma cytokine profiles were assessed by bead array. Neutrophils were isolated and tested ex vivo for oxidative burst, neutrophil extracellular trap formation (NETosis) and phagocytosis. Lung tissue was obtained immediately post-mortem from COVID-19 patients for immunostaining.Results: Elevations in neutrophil-associated cytokines IL-8 and IL-6 were identified in COVID-19 plasma both at the first measurement and across their hospitalization (p < 0.0001). Elevations in cytokines IP-10, GM-CSF, IL-1b, IL-10 and TNF were also present at the first measurement and across hospital stays. Functionally, circulating neutrophils from COVID-19 patients had exaggerated oxidative burst (p < 0.0001), NETosis (p < 0.0001) and phagocytosis (p < 0.0001) relative to controls. Increased NETosis was found to be correlated with both leukocytosis and neutrophilia in COVID-19 patients. Neutrophils and NETs were identified within airways and alveoli in lung parenchyma. While elevations in IL-8 and ANC correlated to COVID-19 disease severity, plasma IL-8 levels alone correlated with death.Conclusions: Circulating neutrophils in COVID-19 exhibit an activated phenotype with increased oxidative burst, NETosis and phagocytosis. Readily accessible and dynamic, plasma IL-8 and circulating neutrophil function can be explored as potential COVID-19 disease biomarkers.Funding Statement: This work was supported by the Department of Veterans Affairs (salary support and VA Merit Award, PI Crotty Alexander) and NIH NHLBI (PI Crotty Alexander).Declaration of Interests: The authors report no conflicts of interest.Ethics Approval Statement: The research protocol was approved by the UCSD, VASDHS and Rady Children’s Hospital institutional review boards (IRBs) and all participants or designated family member gave written informed consent.

AI-guided discovery of the invariant host response to viral pandemics (preprint)

We sought to define the host immune response, a.k.a, the cytokine storm that has been implicated in fatal COVID-19 using an AI-based approach. Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a seed gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. Surprisingly, this 166-gene signature was conserved in all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViPand severe-ViPsignatures, respectively. The ViPsignatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determines severity/fatality. Precise therapeutic goals were formulated and subsequently validated in high-dose SARS-CoV-2-challenged hamsters using neutralizing antibodies that abrogate SARS-CoV-2/ACE2 engagement. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine tracked with disease severity. Thus, the ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.