ABSTRACT
Multiple studies have identified an association between neutrophils and COVID-19 disease severity; however, the mechanistic basis of this association remains incompletely understood. Here we collected 781 longitudinal blood samples from 306 hospitalized COVID-19+ patients, 78 COVID-19- acute respiratory distress syndrome patients, and 8 healthy controls, and performed bulk RNA-sequencing of enriched neutrophils, plasma proteomics, cfDNA measurements and high throughput antibody profiling assays to investigate the relationship between neutrophil states and disease severity or death. We identified dynamic switches between six distinct neutrophil subtypes using non-negative matrix factorization (NMF) clustering. At days 3 and 7 post-hospitalization, patients with severe disease had an enrichment of a granulocytic myeloid derived suppressor cell-like state gene expression signature, while non-severe patients with resolved disease were enriched for a progenitor-like immature neutrophil state signature. Severe disease was associated with gene sets related to neutrophil degranulation, neutrophil extracellular trap (NET) signatures, distinct metabolic signatures, and enhanced neutrophil activation and generation of reactive oxygen species (ROS). We found that the majority of patients had a transient interferon-stimulated gene signature upon presentation to the emergency department (ED) defined here as Day 0, regardless of disease severity, which persisted only in patients who subsequently died. Humoral responses were identified as potential drivers of neutrophil effector functions, as enhanced antibody-dependent neutrophil phagocytosis and reduced NETosis was associated with elevated SARS-CoV-2-specific IgG1-to-IgA1 ratios in plasma of severe patients who survived. In vitro experiments confirmed that while patient-derived IgG antibodies mostly drove neutrophil phagocytosis and ROS production in healthy donor neutrophils, patient-derived IgA antibodies induced a predominant NETosis response. Overall, our study demonstrates neutrophil dysregulation in severe COVID-19 and a potential role for IgA-dominant responses in driving neutrophil effector functions in severe disease and mortality.
ABSTRACT
A recent estimate suggests that one in five deaths globally are associated with sepsis1. To date, no targeted treatment is available for this syndrome, likely due to substantial patient heterogeneity2,3 and our lack of insight into sepsis immunopathology4. These issues are highlighted by the current COVID-19 pandemic, wherein many clinical manifestations of severe SARS-CoV-2 infection parallel bacterial sepsis5-8. We previously reported an expanded CD14+ monocyte state, MS1, in patients with bacterial sepsis or non-infectious critical illness, and validated its expansion in sepsis across thousands of patients using public transcriptomic data9. Despite its marked expansion in the circulation of bacterial sepsis patients, its relevance to viral sepsis and association with disease outcomes have not been examined. In addition, the ontogeny and function of this monocyte state remain poorly characterized. Using public transcriptomic data, we show that the expression of the MS1 program is associated with sepsis mortality and is up-regulated in monocytes from patients with severe COVID-19. We found that blood plasma from bacterial sepsis or COVID-19 patients with severe disease induces emergency myelopoiesis and expression of the MS1 program, which are dependent on the cytokines IL-6 and IL-10. Finally, we demonstrate that MS1 cells are broadly immunosuppressive, similar to monocytic myeloid-derived suppressor cells (MDSCs), and have decreased responsiveness to stimulation. Our findings highlight the utility of regulatory myeloid cells in sepsis prognosis, and the role of systemic cytokines in inducing emergency myelopoiesis during severe bacterial and SARS-CoV-2 infections.
ABSTRACT
BACKGROUND: The aim of the present study was to determine whether certain components of nonmyeloablative regimens for hematopoietic cell transplantation might compromise the growth of hematopoietic progenitors. METHODS: Porcine peripheral blood progenitor cells (PBPC) were cytokine-mobilized, collected by leukapheresis, and cryopreserved using 5% dimethyl sulfoxide and 6% hydroxyethyl starch. The influence of cryopreservation on PBPC was tested in vitro by enumeration of colony-forming units (CFUs) in methylcellulose and cobblestone area-forming cell (CAFC) subsets in stromal-associated long-term cultures on fresh and frozen PBPC. The effects of mycophenolate mofetil (MMF) on porcine PBPC and baboon and human bone marrow (BM) were tested in vitro by adding varying doses of MMF to the CFU assays. One baboon was treated with increasing doses of MMF (100-500 mg/kg per day continuously intravenous), and sequential BM aspirations were tested for CFU content. RESULTS: Fresh cytokine-mobilized PBPC had similar frequencies of progenitor cells when compared with porcine BM. Freezing-thawing of PBPC had no effect on porcine CFUs but reduced the recovery of CAFCs by more than 90%. In vitro, MMF completely inhibited the development of porcine and human CFUs at a concentration of 1 microg/mL and of baboon CFUs at levels between 10 and 100 microg/mL. Plasma-free mycophenolic acid levels of 10 to 30 microg/mL were associated with decreased CFUs in the BM. CONCLUSIONS: Cryopreservation and MMF potentially prevent engraftment of porcine PBPC by reducing the content or development of progenitor cells. These results indicate that the use of fresh PBPC might improve the induction of mixed hematopoietic chimerism and raise the possibility that use of high doses of MMF in the poststem cell transplant may compromise engraftment.
