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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.
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COVID-19 has caused over 1 million deaths globally, yet the cellular mechanisms underlying severe disease remain poorly understood. By analyzing several thousand plasma proteins in 306 COVID-19 patients and 78 symptomatic controls over serial timepoints using two complementary approaches, we uncover COVID-19 host immune and non-immune proteins not previously linked to this disease. Integration of plasma proteomics with nine published scRNAseq datasets shows that SARS-CoV-2 infection upregulates monocyte/macrophage, plasmablast, and T cell effector proteins. By comparing patients who died to severely ill patients who survived, we identify dynamic immunomodulatory and tissue-associated proteins associated with survival, providing insights into which host responses are beneficial and which are detrimental to survival. We identify intracellular death signatures from specific tissues and cell types, and by associating these with angiotensin converting enzyme 2 (ACE2) expression, we map tissue damage associated with severe disease and propose which damage results from direct viral infection rather than from indirect effects of illness. We find that disease severity in lung tissue is driven by myeloid cell phenotypes and cell-cell interactions with lung epithelial cells and T cells. Based on these results, we propose a model of immune and epithelial cell interactions that drive cell-type specific and tissue-specific damage in severe COVID-19.
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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.
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BackgroundTransmission of COVID-19 from people without symptoms poses considerable challenges to public health containment measures. The distribution of viral loads in individuals with and without symptoms remains uncertain. Comprehensive cross-sectional screening of all individuals in a given setting provides an unbiased way to assess viral loads independent of symptoms, which informs transmission risks. COVID-19 cases initially peaked in Massachusetts in mid-April 2020 before declining through June, and congregate living facilities were particularly affected during this early surge. We performed a retrospective analysis of data from a large public health-directed outbreak response initiative that involved comprehensive screening within nursing homes and assisted living facilities in Massachusetts to compare nasopharyngeal (NP) viral loads (as measured by RT-PCR cycle threshold (Ct) levels) in residents and staff to inform our ability to detect SARS-CoV-2 in individuals with or without symptoms in the population. MethodsBetween April 9 and June 9, 2020, we tested NP swabs from 32,480 unique individuals comprising staff and residents of the majority of nursing homes and assisted living facilities in Massachusetts. Under the direction of the MA Department of Public Health (MDPH), symptomatology at the time of sampling and demographic information was provided by each facility for each individual to facilitate reporting to health officials. NP swabs were collected, RNA extracted, and SARS-CoV-2 testing performed using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). ResultsThe nursing home and assisted living facilities resident cohort (N =16,966) was 65% female with a mean age of 82 years (SD 13 yrs). The staff cohort (N = 15,514) was 76% female with a median age of 45 (SD 15 yrs). A total 2654 residents (15.5%) and 624 staff (4.1%) tested positive for SARS-CoV-2. 12.7% of residents and 3.7% of staff without symptoms tested positive for SARS-CoV-2, compared to 53.1% of residents and 18.2% of staff with symptoms. Of the individuals who tested positive, 70.8% of residents and 92.4% of staff lacked symptoms at the time of testing. In aggregate, the distributions of Cts for viral probes used in the qRT-PCR assay were very similar, with a statistically but not meaningfully different mean ({Delta}Ct 0.71 cycles, p = 0.006) and a similar range (12-38 cycles), between populations with and without symptoms over the entire time period, across all sub-categories examined (age, race, ethnicity, sex, resident/staff). Importantly, the Ct mean values and range were indistinguishable between the populations by symptom class during the peak of the outbreak in Massachusetts, with a Ct gap appearing only later in the survey period, reaching >3 cycles (p [≤] 0.001) for facilities sampled during the last two weeks of the study. ConclusionsIn a large cohort of individuals screened for SARS-CoV-2 by qRT-PCR, we found strikingly similar distributions of viral load in patients with or without symptoms at the time of testing during the local peak of the epidemic; as the epidemic waned, individuals without symptoms at the time of testing had lower viral loads. The size of the study population, including both staff and residents spanning a wide range of ages, provides a comprehensive cross-sectional point prevalence measurement of viral burden in a study spanning 2 months. Because the distributions of viral loads in infected individuals irrespective of symptomatology are very similar, existing testing modalities that have been validated for detection of SARS-CoV-2 RNA in symptomatic patients should perform similarly in individuals without symptoms at the time of testing.
