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Delirium is a neurologic syndrome characterized by inattention and cognitive impairment frequently encountered in the medically ill. Peripheral inflammation is a key trigger of delirium, but the patient-specific immune responses associated with delirium development and resolution are unknown. This retrospective cohort study of prospectively collected biospecimens examines RNA sequencing from peripheral blood mononuclear cells of adults hospitalized for COVID-19 to better understand patient-specific factors associated with delirium (n = 64). Longitudinal transcriptomic analyses highlight persistent immune dysregulation in delirium, marked by increasing expression trajectories of genes linked to innate immune pathways, including complement activation, cytokine production, and monocyte/macrophage recruitment. Genes involved adaptive immunity showed a declining trajectory over time in patients with delirium. Although corticosteroid treatment suppressed some aspects of immune hyperactivation, aberrant responses contributing to delirium were exacerbated. Delirium resolution was characterized by normalization of key transcripts such as CCL2 and innate immune markers. Novel associations with delirium were found in genes related to stress granule assembly and DUSP2 and KLF10 , which mediate T-cell responses. These findings provide insights into the peripheral immune responses accompanying delirium and their modulation by corticosteroids. Future trials targeting aberrant inflammatory responses may mitigate the severe outcomes associated with delirium due to COVID19.

Following SARS-CoV-2 infection, ~10-35% of COVID-19 patients experience long COVID (LC), in which often debilitating symptoms persist for at least three months. Elucidating the biologic underpinnings of LC could identify therapeutic opportunities. We utilized machine learning methods on biologic analytes and patient reported outcome surveys provided over 12 months after hospital discharge from >500 hospitalized COVID-19 patients in the IMPACC cohort to identify a multi-omics "recovery factor". IMPACC participants who experienced LC had lower recovery factor scores compared to participants without LC. Biologic characterization revealed increased levels of plasma proteins associated with inflammation, elevated transcriptional signatures of heme metabolism, and decreased androgenic steroids in LC patients. The recovery factor was also associated with altered circulating immune cell frequencies. Notably, recovery factor scores were predictive of LC occurrence in patients as early as hospital admission, irrespective of acute disease severity. Thus, the recovery factor identifies patients at risk of LC early after SARS-CoV-2 infection and reveals LC biomarkers and potential treatment targets., Competing Interests: The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines influenza virus vaccines and influenza virus therapeutics which lists Florian Krammer as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, Castlevax, to develop SARS-CoV-2 vaccines. Florian Krammer is co-founder and scientific advisory board member of Castlevax. Florian Krammer has consulted for Merck, Curevac, Seqirus, GSK and Pfizer and is currently consulting for 3rd Rock Ventures, Sanofi, Gritstone and Avimex. The Krammer laboratory is also collaborating with Dynavax on influenza vaccine development and with VIR on influenza virus therapeutics development. Viviana Simon is a co-inventor on a patent filed relating to SARS-CoV-2 serological assays (the “Serology Assays”). Ofer Levy is a named inventor on patents held by Boston Children’s Hospital relating to vaccine adjuvants and human in vitro platforms that model vaccine action. His laboratory has received research support from GlaxoSmithKline (GSK) and Pfizer, and he is a co-founder of and advisor to Ovax, Inc that develops opioid vaccines. Charles Cairns serves as a consultant to bioMerieux and is funded by a grant from Bill & Melinda Gates Foundation. James A Overton is a consultant at Knocean Inc. Jessica Lasky-Su serves as a scientific advisor of Precion Inc. Scott R. Hutton, Greg Michelloti and Kari Wong are employees of Metabolon Inc. Vicki Seyfer- Margolis is a current employee of MyOwnMed. Nadine Rouphael reports grants or contracts with Merck, Sanofi, Pfizer, Vaccine Company and Immorna, and has participated on data safety monitoring boards for Moderna, Sanofi, Seqirus, Pfizer, EMMES, ICON, BARDA, and CyanVan, Imunon Micron. N.R. has also received support for meetings/travel from Sanofi and Moderna and honoraria from Virology Education and Krog consulting. Chris Cotsapas is a current employee of Vesalius Therapeutics. Adeeb Rahman is a current employee of Immunai Inc. Steven Kleinstein is a consultant related to ImmPort data repository for Peraton. Nathan Grabaugh is a consultant for Tempus Labs and the National Basketball Association. Akiko Iwasaki is a consultant for 4BIO, Blue Willow Biologics, Revelar Biotherapeutics, RIGImmune, Xanadu Bio, Paratus Sciences. Monika Kraft receives research funds paid to her institution from NIH, ALA; Sanofi, Astra-Zeneca for work in asthma, serves as a consultant for Astra-Zeneca, Sanofi, Chiesi, GSK for severe asthma; is a co-founder and CMO for RaeSedo, Inc, a company created to develop peptidomimetics for treatment of inflammatory lung disease. Esther Melamed received research funding from Babson Diagnostics and honorarium from Multiple Sclerosis Association of America and has served on the advisory boards of Genentech, Horizon, Teva, and Viela Bio. Carolyn Calfee receives research funding from NIH, FDA, DOD, Roche-Genentech and Quantum Leap Healthcare Collaborative as well as consulting services for Janssen, Vasomune, Gen1e Life Sciences, NGMBio, and Cellenkos. Wade Schulz was an investigator for a research agreement, through Yale University, from the Shenzhen Center for Health Information for work to advance intelligent disease prevention and health promotion; collaborates with the National Center for Cardiovascular Diseases in Beijing; is a technical consultant to Hugo Health, a personal health information platform; cofounder of Refactor Health, an AI-augmented data management platform for health care; and has received grants from Merck and Regeneron Pharmaceutical for research related to COVID-19. Grace A McComsey received research grants from Rehdhill, Cognivue, Pfizer, and Genentech, and served as a research consultant for Gilead, Merck, Viiv/GSK, and Janssen. Linda N. Geng received research funding paid to her institution from Pfizer, Inc. Catherine Hough receives support through her institution from the NIH and CDC. David Hafler has received research funding from BristolMyers Squibb, Novartis, Sanofi, and Genentech. He has been a consultant for Bayer Pharmaceuticals, Repertoire Inc, Bristol Myers Squibb, Compass Therapeutics, EMD Serono, Genentech, Novartis Pharmaceuticals, and Sanofi Genzyme.

The post-acute sequelae of SARS-CoV-2 (PASC), also known as long COVID, remain a significant health issue that is incompletely understood. Predicting which acutely infected individuals will go on to develop long COVID is challenging due to the lack of established biomarkers, clear disease mechanisms, or well-defined sub-phenotypes. Machine learning (ML) models offer the potential to address this by leveraging clinical data to enhance diagnostic precision. We utilized clinical data, including antibody titers and viral load measurements collected at the time of hospital admission, to predict the likelihood of acute COVID-19 progressing to long COVID. Our machine learning models achieved median AUROC values ranging from 0.64 to 0.66 and AUPRC values between 0.51 and 0.54, demonstrating their predictive capabilities. Feature importance analysis revealed that low antibody titers and high viral loads at hospital admission were the strongest predictors of long COVID outcomes. Comorbidities, including chronic respiratory, cardiac, and neurologic diseases, as well as female sex, were also identified as significant risk factors for long COVID. Our findings suggest that ML models have the potential to identify patients at risk for developing long COVID based on baseline clinical characteristics. These models can help guide early interventions, improving patient outcomes and mitigating the long-term public health impacts of SARS-CoV-2.

Secondary bacterial pneumonia (2°BP) is associated with significant morbidity following respiratory viral infection, yet remains incompletely understood. In a prospective cohort of 112 critically ill adults intubated for COVID-19, we comparatively assess longitudinal airway microbiome dynamics and the pulmonary transcriptome of patients who developed 2°BP versus controls who did not. We find that 2°BP is significantly associated with both mortality and corticosteroid treatment. The pulmonary microbiome in 2°BP is characterized by increased bacterial RNA mass and dominance of culture-confirmed pathogens, detectable days prior to 2°BP clinical diagnosis, and frequently also present in nasal swabs. Assessment of the pulmonary transcriptome reveals suppressed TNFα signaling in patients with 2°BP, and sensitivity analyses suggest this finding is mediated by corticosteroid treatment. Further, we find that increased bacterial RNA mass correlates with reduced expression of innate and adaptive immunity genes in both 2°BP patients and controls. Taken together, our findings provide fresh insights into the microbial dynamics and host immune features of COVID-19-associated 2°BP, and suggest that suppressed immune signaling, potentially mediated by corticosteroid treatment, permits expansion of opportunistic bacterial pathogens., (© 2024. The Author(s).)

In the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, epithelial populations in the distal lung expressing Angiotensin-converting enzyme 2 (ACE2) are infrequent, and therefore, the model of viral expansion and immune cell engagement remains incompletely understood. Using human lungs to investigate early host-viral pathogenesis, we found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations. Human alveolar macrophages (AMs) reliably expressed ACE2 allowing both spike-ACE2-dependent viral entry and infection. In contrast to Influenza A virus, SARS-CoV-2 infection of AMs was productive, amplifying viral titers. While AMs generated new viruses, the interferon responses to SARS-CoV-2 were muted, hiding the viral dissemination from specific antiviral immune responses. The reliable and veiled viral depot in myeloid cells in the very early phases of SARS-CoV-2 infection of human lungs enables viral expansion in the distal lung and potentially licenses subsequent immune pathologies.

Dexamethasone is the standard of care for critically ill patients with COVID-19, but the mechanisms by which it decreases mortality and its immunological effects in this setting are not understood. Here we perform bulk and single-cell RNA sequencing of samples from the lower respiratory tract and blood, and assess plasma cytokine profiling to study the effects of dexamethasone on both systemic and pulmonary immune cell compartments. In blood samples, dexamethasone is associated with decreased expression of genes associated with T cell activation, including TNFSFR4 and IL21R. We also identify decreased expression of several immune pathways, including major histocompatibility complex-II signaling, selectin P ligand signaling, and T cell recruitment by intercellular adhesion molecule and integrin activation, suggesting these are potential mechanisms of the therapeutic benefit of steroids in COVID-19. We identify additional compartment- and cell- specific differences in the effect of dexamethasone that are reproducible in publicly available datasets, including steroid-resistant interferon pathway expression in the respiratory tract, which may be additional therapeutic targets. In summary, we demonstrate compartment-specific effects of dexamethasone in critically ill COVID-19 patients, providing mechanistic insights with potential therapeutic relevance. Our results highlight the importance of studying compartmentalized inflammation in critically ill patients., (© 2024. The Author(s).)

Post-acute sequelae of SARS-CoV-2 (PASC) is a significant public health concern. We describe Patient Reported Outcomes (PROs) on 590 participants prospectively assessed from hospital admission for COVID-19 through one year after discharge. Modeling identified 4 PRO clusters based on reported deficits (minimal, physical, mental/cognitive, and multidomain), supporting heterogenous clinical presentations in PASC, with sub-phenotypes associated with female sex and distinctive comorbidities. During the acute phase of disease, a higher respiratory SARS-CoV-2 viral burden and lower Receptor Binding Domain and Spike antibody titers were associated with both the physical predominant and the multidomain deficit clusters. A lower frequency of circulating B lymphocytes by mass cytometry (CyTOF) was observed in the multidomain deficit cluster. Circulating fibroblast growth factor 21 (FGF21) was significantly elevated in the mental/cognitive predominant and the multidomain clusters. Future efforts to link PASC to acute anti-viral host responses may help to better target treatment and prevention of PASC., (© 2024. The Author(s).)

GCLiPP is a global RNA interactome capture method that detects RNA-binding protein (RBP) occupancy transcriptome-wide. GCLiPP maps RBP-occupied sites at a higher resolution than phase separation-based techniques. GCLiPP sequence tags correspond with known RBP binding sites and are enriched for sites detected by RBP-specific crosslinking immunoprecipitation (CLIP) for abundant cytosolic RBPs. Comparison of human Jurkat T cells and mouse primary T cells uncovers shared peaks of GCLiPP signal across homologous regions of human and mouse 3' UTRs, including a conserved mRNA-destabilizing cis-regulatory element. GCLiPP signal overlapping with immune-related SNPs uncovers stabilizing cis-regulatory regions in CD5, STAT6, and IKZF1., (© 2023. The Author(s).)

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGE hi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS., Competing Interests: J.A.H., H.S., J.V.H., C.O., L.O., X.G., N.W., A.Q., D.C., A.C, D.F.C., T.R., J.M.M., F.C., A.T., M.B., L.T., A.R., S.B.K., R.N.B., and C.M.R. were employees of Genentech, Inc. at the time of this study and own equity in Roche. The COMET study was supported in part by Genentech funding. C.J.Y. is a scientific advisory board member for and holds equity in Related Sciences and ImmunAI, a consultant for and holds equity in Maze Therapeutics, and a consultant for TRex Bio. C.J.Y. has received research support from Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, and Genentech. C.S.C. has received research funding from Roche-Genentech for an unrelated project as well as from NIH, DOD, and Quantum Leap Healthcare Collaborative. C.S.C. is a consultant for Vasomune, Quark, and GEn1E Lifesciences. C.H. is a consultant for Spring Discovery but does not have any financial interest in the company nor is the work related to what is covered in this manuscript. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and, until 31 July 2020, was a scientific advisory board member of Thermo Fisher Scientific, Syros Pharmaceuticals, Asimov, and Neogene Therapeutics. A.R. is a named inventor on multiple patents related to single-cell and spatial genomics filed by or issued to the Broad Institute., (© 2023 The Authors.)

Dexamethasone is the standard of care for critically ill patients with COVID-19, but the mechanisms by which it decreases mortality and its immunological effects in this setting are not understood. We performed bulk and single-cell RNA sequencing of the lower respiratory tract and blood, and plasma cytokine profiling to study the effect of dexamethasone on systemic and pulmonary immune cells. We find decreased signatures of antigen presentation, T cell recruitment, and viral injury in patients treated with dexamethasone. We identify compartment- and cell- specific differences in the effect of dexamethasone in patients with severe COVID-19 that are reproducible in publicly available datasets. Our results highlight the importance of studying compartmentalized inflammation in critically ill patients.

Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy., Competing Interests: Conflict of interests The authors have no competing interests. A patent “INHIBITORS OF SARS-COV-2 INFECTION AND OTHER CORONA VIRUS INFECTIONS” SF-2021-201-2-PCT-0 has been filed., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The coronavirus SARS-CoV-2 causes the severe disease COVID-19. SARS-CoV-2 infection is initiated by interaction of the viral spike protein and host receptor angiotensin-converting enzyme 2 (ACE2). We report an improved bright and reversible fluorogenic reporter, named SURF (split UnaG-based reversible and fluorogenic protein-protein interaction reporter), that we apply to monitor real-time interactions between spike and ACE2 in living cells. SURF has a large dynamic range with a dark-to-bright fluorescence signal that requires no exogenous cofactors. Utilizing this reporter, we carried out a high-throughput screening of small-molecule libraries. We identified three natural compounds that block replication of SARS-CoV-2 in both Vero cells and human primary nasal and bronchial epithelial cells. Cell biological and biochemical experiments validated all three compounds and showed that they block the early stages of viral infection. Two of the inhibitors, bruceine A and gamabufotalin, were also found to block replication of the Delta and Omicron variants of SARS-CoV-2. Both bruceine A and gamabufotalin exhibited potent antiviral activity in K18-hACE2 and wild-type C57BL6/J mice, as evidenced by reduced viral titres in the lung and brain, and protection from alveolar and peribronchial inflammation in the lung, thereby limiting disease progression. We propose that our fluorescent assay can be applied to identify antiviral compounds with potential as therapeutic treatment for COVID-19 and other respiratory diseases., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements. These sequences were functionally validated and optimized via massively parallel reporter assays (MPRAs) for IL-13-inducible activity. The top secretory cell-selective sequence from the MPRA, a novel, distal enhancer of the sterile alpha motif pointed domain containing E-26 transformation-specific transcription factor ( SPDEF ) gene, was utilized to drive CRISPRi and knock down SPDEF or mucin 5AC ( MUC5AC ), both involved in pathologic mucus production in asthma. Our work provides a catalog of cell type-specific genes and regulatory elements involved in IL-13 bronchial epithelial response and showcases their use for therapeutic purposes., Competing Interests: K.D.K. (Genentech), L.R.B. (AstraZeneca), and J.S. (Genentech) have additional affiliations as employees. N.A. is the cofounder and a member of the scientific advisory board of Regel Therapeutics and receives funding from BioMarin Pharmaceutical Inc., (© 2022 The Author(s).)

Rationale: Autopsy and biomarker studies suggest that endotheliopathy contributes to coronavirus disease (COVID-19)-associated acute respiratory distress syndrome. However, the effects of COVID-19 on the lung endothelium are not well defined. We hypothesized that the lung endotheliopathy of COVID-19 is caused by circulating host factors and direct endothelial infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Objectives: We aimed to determine the effects of SARS-CoV-2 or sera from patients with COVID-19 on the permeability and inflammatory activation of lung microvascular endothelial cells. Methods: Human lung microvascular endothelial cells were treated with live SARS-CoV-2; inactivated viral particles; or sera from patients with COVID-19, patients without COVID-19, and healthy volunteers. Permeability was determined by measuring transendothelial resistance to electrical current flow, where decreased resistance signifies increased permeability. Inflammatory mediators were quantified in culture supernatants. Endothelial biomarkers were quantified in patient sera. Measurements and Main Results: Viral PCR confirmed that SARS-CoV-2 enters and replicates in endothelial cells. Live SARS-CoV-2, but not dead virus or spike protein, induces endothelial permeability and secretion of plasminogen activator inhibitor 1 and vascular endothelial growth factor. There was substantial variability in the effects of SARS-CoV-2 on endothelial cells from different donors. Sera from patients with COVID-19 induced endothelial permeability, which correlated with disease severity. Serum levels of endothelial activation and injury biomarkers were increased in patients with COVID-19 and correlated with severity of illness. Conclusions: SARS-CoV-2 infects and dysregulates endothelial cell functions. Circulating factors in patients with COVID-19 also induce endothelial cell dysfunction. Our data point to roles for both systemic factors acting on lung endothelial cells and viral infection of endothelial cells in COVID-19-associated endotheliopathy.

Background: Studies quantifying SARS-CoV-2 have focused on upper respiratory tract or plasma viral RNA with inconsistent association with clinical outcomes. The association between plasma viral antigen levels and clinical outcomes has not been previously studied. Our aim was to investigate the relationship between plasma SARS-CoV-2 nucleocapsid antigen (N-antigen) concentration and both markers of host response and clinical outcomes., Methods: SARS-CoV-2 N-antigen concentrations were measured in the first study plasma sample (D0), collected within 72 h of hospital admission, from 256 subjects admitted between March 2020 and August 2021 in a prospective observational cohort of hospitalized patients with COVID-19. The rank correlations between plasma N-antigen and plasma biomarkers of tissue damage, coagulation, and inflammation were assessed. Multiple ordinal regression was used to test the association between enrollment N-antigen plasma concentration and the primary outcome of clinical deterioration at one week as measured by a modified World Health Organization (WHO) ordinal scale. Multiple logistic regression was used to test the association between enrollment plasma N-antigen concentration and the secondary outcomes of ICU admission, mechanical ventilation at 28 days, and death at 28 days. The prognostic discrimination of an externally derived "high antigen" cutoff of N-antigen ≥ 1000 pg/mL was also tested., Results: N-antigen on D0 was detectable in 84% of study participants. Plasma N-antigen levels significantly correlated with RAGE (r = 0.61), IL-10 (r = 0.59), and IP-10 (r = 0.59, adjusted p = 0.01 for all correlations). For the primary outcome of clinical status at one week, each 500 pg/mL increase in plasma N-antigen level was associated with an adjusted OR of 1.05 (95% CI 1.03-1.08) for worse WHO ordinal status. D0 plasma N-antigen ≥ 1000 pg/mL was 77% sensitive and 59% specific (AUROC 0.68) with a positive predictive value of 23% and a negative predictive value of 93% for a worse WHO ordinal scale at day 7 compared to baseline. D0 N-antigen concentration was independently associated with ICU admission and 28-day mechanical ventilation, but not with death at 28 days., Conclusions: Plasma N-antigen levels are readily measured and provide important insight into the pathogenesis and prognosis of COVID-19. The measurement of N-antigen levels early in-hospital course may improve risk stratification, especially for identifying patients who are unlikely to progress to severe disease., (© 2022. The Author(s).)

Background: Better understanding of the association between characteristics of patients hospitalized with coronavirus disease 2019 (COVID-19) and outcome is needed to further improve upon patient management., Methods: Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective, observational study of 1164 patients from 20 hospitals across the United States. Disease severity was assessed using a 7-point ordinal scale based on degree of respiratory illness. Patients were prospectively surveyed for 1 year after discharge for post-acute sequalae of COVID-19 (PASC) through quarterly surveys. Demographics, comorbidities, radiographic findings, clinical laboratory values, SARS-CoV-2 PCR and serology were captured over a 28-day period. Multivariable logistic regression was performed., Findings: The median age was 59 years (interquartile range [IQR] 20); 711 (61%) were men; overall mortality was 14%, and 228 (20%) required invasive mechanical ventilation. Unsupervised clustering of ordinal score over time revealed distinct disease course trajectories. Risk factors associated with prolonged hospitalization or death by day 28 included age ≥ 65 years (odds ratio [OR], 2.01; 95% CI 1.28-3.17), Hispanic ethnicity (OR, 1.71; 95% CI 1.13-2.57), elevated baseline creatinine (OR 2.80; 95% CI 1.63- 4.80) or troponin (OR 1.89; 95% 1.03-3.47), baseline lymphopenia (OR 2.19; 95% CI 1.61-2.97), presence of infiltrate by chest imaging (OR 3.16; 95% CI 1.96-5.10), and high SARS-CoV2 viral load (OR 1.53; 95% CI 1.17-2.00). Fatal cases had the lowest ratio of SARS-CoV-2 antibody to viral load levels compared to other trajectories over time (p=0.001). 589 survivors (51%) completed at least one survey at follow-up with 305 (52%) having at least one symptom consistent with PASC, most commonly dyspnea (56% among symptomatic patients). Female sex was the only associated risk factor for PASC., Interpretation: Integration of PCR cycle threshold, and antibody values with demographics, comorbidities, and laboratory/radiographic findings identified risk factors for 28-day outcome severity, though only female sex was associated with PASC. Longitudinal clinical phenotyping offers important insights, and provides a framework for immunophenotyping for acute and long COVID-19., Funding: NIH., Competing Interests: Declaration of interests J.S reports funds paid to institution by the National Institute of Health - National Institute of Allergy and Infectious Diseases for the completion of this manuscript. C.S.C reports funds from NHLBI, has grants/contracts from Bayer, Roche-Genentech, Quantum Leap Healthcare Collaborative and received consulting fees from Vasomune, Gen1e Life Sciences, Cellenkos, Janssen. C.B.C reports funds paid to institution from the National Institute of Health - National Institute of Allergy and Infectious Diseases for the completion of this manuscript. C.B.C also reports a grant paid to institution by the Bill & Melinda Gates Foundation for Covid-19 work. C.B.C is a consultant for bioMerieux on clinical biomarkers. He reports his participation on a data safety monitoring board or advisory board for the Convalescent plasma Covid-19 study for the National Heart, Lung and Blood Institute (NHLBI). CBC is also President, Board of Directors for the National Foundation of Emergency Medicine (NFEM), a non-profit supporting emergency medicine research and researchers. M.K reports funds paid to institution for grants or contracts from the National Institute of Health. L.B reports grant awarded to institution from the National Institute of Health - National Institute of Allergy and Infectious Diseases. A.C.S reports funds paid to institution by the National Institute of Health for the completion of this manuscript (NIH U19 AI089992 – NIH K24 AG042489). F.KR has received funds from the National Institute of Allergy and Infectious Diseases Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051, the National Institute of Health - Centers of Excellence for influenza Research and Response, (CEIRR), 75N93021C00014, the JPB Foundation and the Open Philanthropy Project (research grant 2020-215611, 5384), the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024, Task Order No. 75N91020F00003 and Research funding from Pfizer for development of animal models for SARS-CoV-2. F.KR receives royalties from Avimex, and receives consulting fees from Pfizer, Seqirus, Avimex and Third Rock Ventures. F.KR has received several payments or honoraria for academic lectures over the past two years. F. KR is listed as a co-inventor on patents filed for applications relating to SARS-CoV-2 serological assays (the “Serology Assays”) and NDV-based SARS-CoV-2 vaccines. H.vB reports receipt of the following grants paid to institution: National Institute of Health Centers of Excellence for influenza Research and Response, (CEIRR - 75N93021C00014), and National Institute of Health (Dengue Human Immunology Project Consortium - Mount Sinai IMPACC COVID-19 Cores - U19 AI118610 S1). D. E reports funds paid to institution for grants or contracts from the National Institute of Health. D.E is a statistician on the Data Safety Monitoring Board for the COLSTAT Trial. D.E is on the board of Directors for the Society for Clinical Trials. V.S is listed as a co-inventor on a patent filed relating to SARS-CoV-2 serological assays (the “Serology Assays”). R.M reports a grant from the National Institute of Health for the completion of this manuscript (AI 089992). R. M served as a Counselor for the Society of Leukocyte Biology from 2018 to 2021. S.K is a personal consultant related to ImmPort data repository for Peraton. O.L reports funds paid to institution by the National Institute of Health - National Institute of Allergy and Infectious Diseases for the completion of this manuscript (1-U19-AI118608-01A1). O.L received payments in the past 36 months from Midsized Bank Coalition of America (MBCA) and Moody's Analytics for presentations regarding the coronavirus pandemic. D.J.E reports funds paid to institution for grants or contracts from the National Institute of Health. K.C.N reports having received grants paid to institution from the following institutes in the past 36 months: National Institute of Allergy and Infectious Diseases (NIAID), National Heart, Lung, and Blood Institute (NHLBI), National Institute of Environmental Health Sciences (NIEHS), and Food Allergy Research & Education (FARE). K.C.N receives consulting fees from Excellergy, Eli Lilly, Red Tree Ventures, and Phylaxis. K.C.N has one Licensee: Alladapt and Before Brands (Application number: US15/048,609) for Mixed allergen composition and methods for using the same. K.C.N has two patents issued: Granulocyte-based methods for detecting and monitoring immune system disorders (Application number: US12/686,121), and Methods and Assays for Detecting and Quantifying Pure Subpopulations of White Blood Cells in Immune System Disorders (Application number: US12/610,94). K.C.N is the director of the World Allergy Organization Center of Excellence for Stanford and a co-founder of Seed Health, IgGenix, ClostraBio, and ImmuneID. K.C.N is also a co-founder of Before Brands, Alladapt, and Latitude and an advisor for Cour Pharma. K.C.N is a member of the national scientific committee for the Immune Tolerance Network (ITN) and the National Institutes of Health (NIH) clinical research centers. C.L.H reports a grant awarded to institution by the National Institute of Health and the American Lung Association. C.L.H also reports having received consulting fees from the National Institute of Health. C.L.H has received payment for medical grand rounds at institution and support for attending meetings and/or travel from Critical care trialists, critical care reviews and multiple universities. C.L.H is on the data safety monitoring board of QuantumHealth for iSPY COVID and is on the board of directors for the American Thoracic Society. W.M reports a grant from the National Institute of Health – National Institute of Allergy and Infectious Diseases for the completion of this manuscript (NIH NIAID R01AI14583). J.P.M reports funds paid to institution for grants or contracts from the National Institute of Health for the completion of this manuscript (NIH Grant # 3U19AI062629-17S2). M.A.A reports a grant from the National Institute of Health - National Institute of Allergy and Infectious Diseases for the completion of this manuscript (5U54AI142766-03). L.E reports grant awarded to institution from the National Institute of Health (NIH R01AI104870-S1). E.M reports funds paid to institution for grants or contracts from the National Institute of Health for the completion of this manuscript (NIH R01 AI104870S1). E.M also reports having received the following grants paid to institution in the past 36 months: the Babson Diagnostics Grant, the Austin Public Health Grant, and K08 26-1616-11. E.M received payments for lectures for the MS Association of America. E. M received payments for the participation in the advisory boards of Genentech, Horizon, Teva and Viela Bio. G.M receives consulting fees from Gilead. B.PE reports a grant from the National Institute of Health - National Institute of Allergy and Infectious Diseases for the completion of this manuscript. E.F.R reports funds paid to institution by the National Institute of Health - National Institute of Allergy and Infectious Diseases for the completion of this manuscript (NIAID U19AI12891303). M.C.A reports funds received from the National Institute of Health for the completion of this manuscript (NIH – R01AI32774) and funding by the National Institute of Allergy and Infectious Diseases for travel to present data related to this study. P.M.B is a federal employee serving as project scientist for this project but has no role in funding decisions or oversight for relevant grants. N.R reports funds paid to institution by the National Institute of Health - National Institute of Allergy and Infectious Diseases for the completion of this manuscript. N.R reports contracts with Lilly and Sanofi for conduct of Covid-19 clinical trials. N.R receives consulting fees from ICON EMMES for consulting on safety for Covid-19 clinical trials. N.R is an associate editor for Clinical infectious diseases. The remaining authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

While studies have elucidated many pathophysiological elements of COVID-19, little is known about immunological changes during COVID-19 resolution. We analyzed immune cells and phosphorylated signaling states at single-cell resolution from longitudinal blood samples of patients hospitalized with COVID-19, pneumonia and/or sepsis, and healthy individuals by mass cytometry. COVID-19 patients showed distinct immune compositions and an early, coordinated, and elevated immune cell signaling profile associated with early hospital discharge. Intra-patient longitudinal analysis revealed changes in myeloid and T cell frequencies and a reduction in immune cell signaling across cell types that accompanied disease resolution and discharge. These changes, together with increases in regulatory T cells and reduced signaling in basophils, also accompanied recovery from respiratory failure and were associated with better outcomes at time of admission. Therefore, although patients have heterogeneous immunological baselines and highly variable disease courses, a core immunological trajectory exists that defines recovery from severe SARS-CoV-2 infection., Competing Interests: Declaration of interests M.H.S. is a board member and equity holder in Teiko.bio and has received research support from Roche/Genentech, Bristol Myers Squibb, Pfizer, and Valitor. C.S.C. has received funding from NHLBI, FDA, DOD, Genentech, and Quantum Leap Healthcare Collaborative and is on consulting/advisory boards for Vasomune, Gen1e Life Sciences, Janssen, and Cellenkos. C.M.H. has been consulting for Spring Discovery. P.G.W. has a contract from Genentech to study COVID-19., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic 1 , considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model 2,3 to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system., Competing Interests: Competing interests. The authors declare no competing interests.

Asthma is associated with chronic changes in the airway epithelium, a key target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many epithelial changes, including goblet cell metaplasia, are driven by the type 2 cytokine IL-13, but the effects of IL-13 on SARS-CoV-2 infection are unknown. We found that IL-13 stimulation of differentiated human bronchial epithelial cells (HBECs) cultured at air-liquid interface reduced viral RNA recovered from SARS-CoV-2-infected cells and decreased double-stranded RNA, a marker of viral replication, to below the limit of detection in our assay. An intact mucus gel reduced SARS-CoV-2 infection of unstimulated cells, but neither a mucus gel nor SPDEF, which is required for goblet cell metaplasia, were required for the antiviral effects of IL-13. Bulk RNA sequencing revealed that IL-13 regulated 41 of 332 (12%) mRNAs encoding SARS-CoV-2-associated proteins that were detected in HBECs (>1.5-fold change; false discovery rate < 0.05). Although both IL-13 and IFN-α each inhibit SARS-CoV-2 infection, their transcriptional effects differed markedly. Single-cell RNA sequencing revealed cell type-specific differences in SARS-CoV-2-associated gene expression and IL-13 responses. Many IL-13-induced gene expression changes were seen in airway epithelium from individuals with type 2 asthma and chronic obstructive pulmonary disease. IL-13 effects on airway epithelial cells may protect individuals with type 2 asthma from COVID-19 and could lead to identification of novel strategies for reducing SARS-CoV-2 infection.

Many studies have provided insights into the immune response to COVID-19; however, little is known about the immunological changes and immune signaling occurring during COVID-19 resolution. Individual heterogeneity and variable disease resolution timelines obscure unifying immune characteristics. Here, we collected and profiled >200 longitudinal peripheral blood samples from patients hospitalized with COVID-19, with other respiratory infections, and healthy individuals, using mass cytometry to measure immune cells and signaling states at single cell resolution. COVID-19 patients showed a unique immune composition and an early, coordinated and elevated immune cell signaling profile, which correlated with early hospital discharge. Intra-patient time course analysis tied to clinically relevant events of recovery revealed a conserved set of immunological processes that accompany, and are unique to, disease resolution and discharge. This immunological process, together with additional changes in CD4 regulatory T cells and basophils, accompanies recovery from respiratory failure and is associated with better clinical outcomes at the time of admission. Our work elucidates the biological timeline of immune recovery from COVID-19 and provides insights into the fundamental processes of COVID-19 resolution in hospitalized patients.

Cancers display significant heterogeneity with respect to tissue of origin, driver mutations, and other features of the surrounding tissue. It is likely that individual tumors engage common patterns of the immune system-here "archetypes"-creating prototypical non-destructive tumor immune microenvironments (TMEs) and modulating tumor-targeting. To discover the dominant immune system archetypes, the University of California, San Francisco (UCSF) Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub-compartments uncovered dominant patterns of immune composition across cancers. These archetypes were profound insofar as they also differentiated tumors based upon unique immune and tumor gene-expression patterns. They also partitioned well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune organization and provides a rational path forward to learn how to modulate these to improve therapy., Competing Interests: Declaration of interests M.F.K. is a founder and shareholder of PIONYR immunotherapeutic and FOUNDERY innovations. A.I.D. is a shareholder of Trex and Neuvogen, and he is also a member of the scientific advisory board of Neuvogen, Bristol-Myers and Squibb, Merck, Roche, Pfizer, Genentech, Incyte, Amgen, and Novartis. E.A.C. is a consultant at IHR Therapeutics, Valar, and Pear Diagnostics, reports receiving commercial research grants from Astra Zeneca, Ferro Therapeutics, Senti Biosciences, Merck KgA, and Bayer, and has stock ownership in Tatara Therapeutics, Clara Health, BloodQ, and Guardant Health., (Copyright © 2021 Elsevier Inc. All rights reserved.)

AU-rich elements (AREs) are 3' UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3' UTR sequences that conform to these motifs affect their function. Here, we use functional annotation of sequences from 3' UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3' UTR sequence fragments from 4653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3' UTRs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections including in patients with coronavirus disease 2019 (COVID-19). Using a combination of tracheal aspirate bulk and single-cell RNA sequencing we assessed lower respiratory tract immune responses and microbiome dynamics in 23 COVID-19 patients, 10 of whom developed VAP, and eight critically ill uninfected controls. At a median of three days (range: 2-4 days) before VAP onset we observed a transcriptional signature of bacterial infection. At a median of 15 days prior to VAP onset (range: 8-38 days), we observed a striking impairment in immune signaling in COVID-19 patients who developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients with VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. These findings suggest that COVID-19 patients who develop VAP have impaired antibacterial immune defense detectable weeks before secondary infection onset.

Background: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there has been increasing urgency to identify pathophysiological characteristics leading to severe clinical course in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human leukocyte antigen alleles (HLA) have been suggested as potential genetic host factors that affect individual immune response to SARS-CoV-2. We sought to evaluate this hypothesis by conducting a multicenter study using HLA sequencing., Methods: We analyzed the association between COVID-19 severity and HLAs in 435 individuals from Germany ( n  = 135), Spain ( n  = 133), Switzerland ( n  = 20) and the United States ( n  = 147), who had been enrolled from March 2020 to August 2020. This study included patients older than 18 years, diagnosed with COVID-19 and representing the full spectrum of the disease. Finally, we tested our results by meta-analysing data from prior genome-wide association studies (GWAS)., Findings: We describe a potential association of HLA-C*04:01 with severe clinical course of COVID-19. Carriers of HLA-C*04:01 had twice the risk of intubation when infected with SARS-CoV-2 (risk ratio 1.5 [95% CI 1.1-2.1], odds ratio 3.5 [95% CI 1.9-6.6], adjusted p -value = 0.0074). These findings are based on data from four countries and corroborated by independent results from GWAS. Our findings are biologically plausible, as HLA-C*04:01 has fewer predicted bindings sites for relevant SARS-CoV-2 peptides compared to other HLA alleles., Interpretation: HLA-C*04:01 carrier state is associated with severe clinical course in SARS-CoV-2. Our findings suggest that HLA class I alleles have a relevant role in immune defense against SARS-CoV-2., Funding: Funded by Roche Sequencing Solutions, Inc., Competing Interests: Bettina Heidecker, MD reports support from Roche Sequencing Solutions, Inc; a project grant from the Swiss National Science Foundation; is an inventor on patents that use RNA for diagnosis of myocarditis. Juerg H. Beer, MD reports grants from the Swiss National Foundation of Science, the Swiss Heart Foundation, the Foundation Kardio, Baden; Grant support to the institution from Bayer not related to this study; and lecture fee from Daiichi Sankyo to the institution. Martin Witzenrath, MD reports grants from Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, Deutsche Gesellschaft für Pneumologie, European Respiratory Society, Marie Curie Foundation, Else Kröner Fresenius Stiftung, Capnetz Stiftung, International Max Planck Research School, Quark Pharma, Takeda Pharma, Noxxon, Pantherna, Silence Therapeutics, Vaxxilon, Actelion, Bayer Health Care, Biotest, and Boehringer Ingelheim; consulting fees from Noxxon, Pantherna, Silence Therapeutics, Vaxxilon, Aptarion, Glaxo Smith Kline, Sinoxa, and Biotest; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Astra Zeneca, Berlin Chemie, Chiesi, Novartis, Teva, Actelion, Boehringer Ingelheim, Glaxo Smith Kline, Biotest, and Bayer Health Care; patent EPO 12,181,535.1: IL-27 for modulation of immune response in acute lung injury issued 2012, patent WO/2010/094,491: Means for inhibiting the expression of Ang-2 issued 2010, and patent DE 102,020,116,249.9: Camostat/ Niclosamide cotreatment in SARS-CoV-2 infected human lung cells issued 2020/21. Alexander Rakitko, Valery Ilinsky, and Iaroslav Popov are employees of Genotek Ltd. Melina Müller declares support for the present manuscript from Roche Sequencing Solutions and Swiss National Science Foundation and Berlin Institutes of Health. Joseph Balnis and Ariel Jaitovich declare support from the National Institute of Health (NIH, K01-HL130704). Bernd Hinzmann, Mauricio A Salvo, Anja Blüher, and Sandra Siemann declare support from Roche Sequencing Solutions. Carolyn Calfee reports NIH payment to her institution; payment from Roche/Genentech Payment and Bayer to her institution for observational study in ARDS; payment from Quantum Leap Healthcare Collaborative to her institution for adaptive platform Phase 2 trial in COVID-19; and consulting fees for novel therapies for ARDS from Vasomune and Quark Pharmaceuticals Payment. David J Erle reports NIH Grants to UCSF. Prescott G Woodruff reports support from Roche Sequencing Solutions, Inc., Swiss National Science Foundation, and Berlin Institutes of Health; US National Institutes of Health grant to his institution (U19AI077439) Charles Langelier reports NIH payment to his institution. Federico García reports grants from ViiV, MSD, and Roche; payment from Abbvie, Gilead, ViiV, MSD, and Roche; support for attending meetings and/or travel from Abbvie and Gilead; participation on a Data Safety Monitoring Board or Advisory Board for Gilead, ViiV, and Thera. Joanna Zyla has been supported by the Silesian University of Technology grant for Support and Development of Research Potential. Terry C. Jones reports a grant from Wellcome Trust, UK, on unrelated research on ancient viral DNA and an NIAID-NIH CEIRS grant (HHSN272201400008C). Leif Erik Sander reports Berlin Institutes of Health support to the PA-COVID-19 study group. Wolfgang Poller reports that this study was partially funded by Roche Sequencing Solutions, Inc., which also provided material for exome sequencing. Ulf Landmesser reports consulting fees from Abbott, Amgen, Bayer, Cardiac Dimensions, Novartis, Pfizer, and Omeicos; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Novartis, Abott, NovoNordisk, Bayer, Amgen, DaiichiSankyo, Pfizer, Sanofi, Boson Scientific, Astra Zeneca, and Boehringer Ingelheim. All other authors have nothing to declare., (© 2021 The Authors.)

Neutralizing autoantibodies against type I interferons (IFNs) have been found in some patients with critical coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the prevalence of these antibodies, their longitudinal dynamics across the disease severity scale, and their functional effects on circulating leukocytes remain unknown. Here, in 284 patients with COVID-19, we found type I IFN–specific autoantibodies in peripheral blood samples from 19% of patients with critical disease and 6% of patients with severe disease. We found no type I IFN autoantibodies in individuals with moderate disease. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 patients with COVID-19 and 26 non–COVID-19 controls revealed a lack of type I IFN–stimulated gene (ISG-I) responses in myeloid cells from patients with critical disease. This was especially evident in dendritic cell populations isolated from patients with critical disease producing type I IFN–specific autoantibodies. Moreover, we found elevated expression of the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) on the surface of monocytes isolated from patients with critical disease early in the disease course. LAIR1 expression is inversely correlated with ISG-I expression response in patients with COVID-19 but is not expressed in healthy controls. The deficient ISG-I response observed in patients with critical COVID-19 with and without type I IFN–specific autoantibodies supports a unifying model for disease pathogenesis involving ISG-I suppression through convergent mechanisms.

The immunological features that distinguish COVID-19-associated acute respiratory distress syndrome (ARDS) from other causes of ARDS are incompletely understood. Here, we report the results of comparative lower respiratory tract transcriptional profiling of tracheal aspirate from 52 critically ill patients with ARDS from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a "cytokine storm," we observe reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS is characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity. In silico analysis of gene expression identifies several candidate drugs that may modulate gene expression in COVID-19 ARDS, including dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 is characterized by impaired interferon-stimulated gene (ISG) expression. The relationship between SARS-CoV-2 viral load and expression of ISGs is decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients reveals distinct immunological features of COVID-19 ARDS., (© 2021. The Author(s).)

SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2- positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19., Competing Interests: Disclosure statement The authors have no potential conflicts of interest.

Type I interferon (IFN-I) neutralizing autoantibodies have been found in some critical COVID-19 patients; however, their prevalence and longitudinal dynamics across the disease severity scale, and functional effects on circulating leukocytes remain unknown. Here, in 284 COVID-19 patients, we found IFN-I autoantibodies in 19% of critical, 6% of severe and none of the moderate cases. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 COVID-19 patients, 15 non-COVID-19 patients and 11 non-hospitalized healthy controls, revealed a lack of IFN-I stimulated gene (ISG-I) response in myeloid cells from critical cases, including those producing anti-IFN-I autoantibodies. Moreover, surface protein analysis showed an inverse correlation of the inhibitory receptor LAIR-1 with ISG-I expression response early in the disease course. This aberrant ISG-I response in critical patients with and without IFN-I autoantibodies, supports a unifying model for disease pathogenesis involving ISG-I suppression via convergent mechanisms.

IL-13-induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. miRNAs are potent modulators of cellular responses, but their role in mucus regulation is largely unexplored. We hypothesized that airway epithelial miRNAs play roles in IL-13-induced mucus regulation. miR-141 is highly expressed in human and mouse airway epithelium, is altered in bronchial brushings from asthmatic subjects at baseline, and is induced shortly after airway allergen exposure. We established a CRISPR/Cas9-based protocol to target miR-141 in primary human bronchial epithelial cells that were differentiated at air-liquid-interface, and goblet cell hyperplasia was induced by IL-13 stimulation. miR-141 disruption resulted in decreased goblet cell frequency, intracellular MUC5AC, and total secreted mucus. These effects correlated with a reduction in a goblet cell gene expression signature and enrichment of a basal cell gene expression signature defined by single cell RNA sequencing. Furthermore, intranasal administration of a sequence-specific mmu-miR-141-3p inhibitor in mice decreased Aspergillus-induced secreted mucus and mucus-producing cells in the lung and reduced airway hyperresponsiveness without affecting cellular inflammation. In conclusion, we have identified a miRNA that regulates pathological airway mucus production and is amenable to therapeutic manipulation through an inhaled route.

The human airway epithelium is essential in homeostasis, and epithelial dysfunction contributes to chronic airway disease. Development of flow-cytometric methods to characterize subsets of airway epithelial cells will enable further dissection of airway epithelial biology. Leveraging single-cell RNA-sequencing data in combination with known cell type-specific markers, we developed panels of antibodies to characterize and isolate the major airway epithelial subsets (basal, ciliated, and secretory cells) from human bronchial epithelial-cell cultures. We also identified molecularly distinct subpopulations of secretory cells and demonstrated cell subset-specific expression of low-abundance transcripts and microRNAs that are challenging to analyze with current single-cell RNA-sequencing methods. These new tools will be valuable for analyzing and separating airway epithelial subsets and interrogating airway epithelial biology.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals 1-3 , many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs) 3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.

Rationale: Asthma is associated with chronic changes in the airway epithelium, a key target of SARS-CoV-2. Many epithelial changes are driven by the type 2 cytokine IL-13, but the effects of IL-13 on SARS-CoV-2 infection are unknown., Objectives: We sought to discover how IL-13 and other cytokines affect expression of genes encoding SARS-CoV-2-associated host proteins in human bronchial epithelial cells (HBECs) and determine whether IL-13 stimulation alters susceptibility to SARS-CoV-2 infection., Methods: We used bulk and single cell RNA-seq to identify cytokine-induced changes in SARS-CoV-2-associated gene expression in HBECs. We related these to gene expression changes in airway epithelium from individuals with mild-moderate asthma and chronic obstructive pulmonary disease (COPD). We analyzed effects of IL-13 on SARS-CoV-2 infection of HBECs., Measurements and Main Results: Transcripts encoding 332 of 342 (97%) SARS-CoV-2-associated proteins were detected in HBECs (≥1 RPM in 50% samples). 41 (12%) of these mRNAs were regulated by IL-13 (>1.5-fold change, FDR < 0.05). Many IL-13-regulated SARS-CoV-2-associated genes were also altered in type 2 high asthma and COPD. IL-13 pretreatment reduced viral RNA recovered from SARS-CoV-2 infected cells and decreased dsRNA, a marker of viral replication, to below the limit of detection in our assay. Mucus also inhibited viral infection., Conclusions: IL-13 markedly reduces susceptibility of HBECs to SARS-CoV-2 infection through mechanisms that likely differ from those activated by type I interferons. Our findings may help explain reports of relatively low prevalence of asthma in patients diagnosed with COVID-19 and could lead to new strategies for reducing SARS-CoV-2 infection.

Rationale: The 17q12-21.1 locus is one of the most highly replicated genetic associations with asthma. Individuals of African descent have lower linkage disequilibrium in this region, which could facilitate identifying causal variants. Objectives: To identify functional variants at 17q12-21.1 associated with early-onset asthma among African American individuals. Methods: We evaluated African American participants from SAPPHIRE (Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-Ethnicity) ( n  = 1,940), SAGE II (Study of African Americans, Asthma, Genes and Environment) ( n  = 885), and GCPD-A (Study of the Genetic Causes of Complex Pediatric Disorders-Asthma) ( n  = 2,805). Associations with asthma onset at ages under 5 years were meta-analyzed across cohorts. The lead signal was reevaluated considering haplotypes informed by genetic ancestry (i.e., African vs. European). Both an expression-quantitative trait locus analysis and a phenome-wide association study were performed on the lead variant. Measurements and Main Results: The meta-analyzed results from SAPPHIRE, SAGE II, and the GCPD-A identified rs11078928 as the top association for early-onset asthma. A haplotype analysis suggested that the asthma association partitioned most closely with the rs11078928 genotype. Genetic ancestry did not appear to influence the effect of this variant. In the expression-quantitative trait locus analysis, rs11078928 was related to alternative splicing of GSDMB (gasdermin-B) transcripts. The phenome-wide association study of rs11078928 suggested that this variant was predominantly associated with asthma and asthma-associated symptoms. Conclusions: A splice-acceptor polymorphism appears to be a causal variant for asthma at the 17q12-21.1 locus. This variant appears to have the same magnitude of effect in individuals of African and European descent.

We performed comparative lower respiratory tract transcriptional profiling of 52 critically ill patients with the acute respiratory distress syndrome (ARDS) from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a cytokine storm, we observed reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS was characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity that were predicted to be modulated by dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 was characterized by impaired interferon-stimulated gene expression (ISG). We found that the relationship between SARS-CoV-2 viral load and expression of ISGs was decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients with COVID-19 ARDS did not demonstrate cytokine storm but instead revealed a unique and dysregulated host response predicted to be modified by dexamethasone.

SARS-CoV-2 infection has led to a global health crisis, and yet our understanding of the disease and potential treatment options remains limited. The infection occurs through binding of the virus with angiotensin converting enzyme 2 (ACE2) on the cell membrane. Here, we established a screening strategy to identify drugs that reduce ACE2 levels in human embryonic stem cell (hESC)-derived cardiac cells and lung organoids. Target analysis of hit compounds revealed androgen signaling as a key modulator of ACE2 levels. Treatment with antiandrogenic drugs reduced ACE2 expression and protected hESC-derived lung organoids against SARS-CoV-2 infection. Finally, clinical data on COVID-19 patients demonstrated that prostate diseases, which are linked to elevated androgen, are significant risk factors and that genetic variants that increase androgen levels are associated with higher disease severity. These findings offer insights on the mechanism of disproportionate disease susceptibility in men and identify antiandrogenic drugs as candidate therapeutics for COVID-19., Competing Interests: Declaration of Interests P.N. receives grants from Apple, Amgen, and Boston Scientific, and personal fees from Apple and Blackstone Life Sciences, all outside the submitted work. F.F. receives grants from Takeda Pharmaceuticals on projects outside of the submitted work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Mitochondria support critical cellular functions, such as energy production through oxidative phosphorylation, regulation of reactive oxygen species, apoptosis, and calcium homeostasis., Objective: Given the heightened level of cellular activity in patients with asthma, we sought to determine whether mitochondrial DNA (mtDNA) copy number measured in peripheral blood differed between individuals with and without asthma., Methods: Whole genome sequence data was generated as part of the Trans-Omics for Precision Medicine (TOPMed) Program on participants from the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-ethnicity (SAPPHIRE) and the Study of African Americans, Asthma, Genes, & Environment II (SAGE II). We restricted our analysis to individuals who self-identified as African American (3,651 asthma cases and 1,344 controls). Mitochondrial copy number was estimated using the sequencing read depth ratio for the mitochondrial and nuclear genomes. Respiratory complex expression was assessed using RNA-sequencing., Results: Average mitochondrial copy number was significantly higher among individuals with asthma when compared with controls (SAPPHIRE: 218.60 vs. 200.47, P<0.001; SAGE II: 235.99 vs. 223.07, P<0.001). Asthma status was significantly associated with mitochondrial copy number after accounting for potential explanatory variables, such as participant age, sex, leukocyte counts, and mitochondrial haplogroup. Despite the consistent relationship between asthma status and mitochondrial copy number, the latter was not associated with time-to-exacerbation or patient-reported asthma control. Mitochondrial respiratory complex gene expression was disproportionately lower in individuals with asthma when compared with individuals without asthma and other protein-encoding genes., Conclusions: We observed a robust association between asthma and higher mitochondrial copy number. Asthma having an effect on mitochondria function was also supported by lower respiratory complex gene expression in this group., Competing Interests: Dr. Abecasis reported receiving personal fees and salary support from Regeneron Pharmaceuticals. This support was outside of the submitted work and did not alter our adherence to PLOS ONE policies regarding the sharing of data and/or research materials. The remaining authors reported no competing interests.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense., One Sentence Summary: In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF , which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.

Given the increasing interest in their use as disease biomarkers, the establishment of reproducible, accurate, sensitive, and specific platforms for microRNA (miRNA) quantification in biofluids is of high priority. We compare four platforms for these characteristics: small RNA sequencing (RNA-seq), FirePlex, EdgeSeq, and nCounter. For a pool of synthetic miRNAs, coefficients of variation for technical replicates are lower for EdgeSeq (6.9%) and RNA-seq (8.2%) than for FirePlex (22.4%); nCounter replicates are not performed. Receiver operating characteristic analysis for distinguishing present versus absent miRNAs shows small RNA-seq (area under curve 0.99) is superior to EdgeSeq (0.97), nCounter (0.94), and FirePlex (0.81). Expected differences in expression of placenta-associated miRNAs in plasma from pregnant and non-pregnant women are observed with RNA-seq and EdgeSeq, but not FirePlex or nCounter. These results indicate that differences in performance among miRNA profiling platforms impact ability to detect biological differences among samples and thus their relative utility for research and clinical use., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

T helper 17 cells (Th17) are critical for fighting infections at mucosal surfaces; however, they have also been found to contribute to the pathogenesis of multiple autoimmune diseases and have been targeted therapeutically. Due to the role of Th17 cells in autoimmune pathogenesis, it is important to understand the factors that control Th17 development. Here we identify the activating transcription factor 7 interacting protein (ATF7ip) as a critical regulator of Th17 differentiation. Mice with T cell-specific deletion of Atf7ip have impaired Th17 differentiation secondary to the aberrant overproduction of IL-2 with T cell receptor (TCR) stimulation and are resistant to colitis in vivo. ChIP-seq studies identified ATF7ip as an inhibitor of Il2 gene expression through the deposition of the repressive histone mark H3K9me3 in the Il2-Il21 intergenic region. These results demonstrate a new epigenetic pathway by which IL-2 production is constrained, and this may open up new avenues for modulating its production., (© 2019 Sin et al.)

Compared to coding sequences, untranslated regions of the transcriptome are not well conserved, and functional annotation of these sequences is challenging. Global relationships between nucleotide composition of 3' UTR sequences and their sequence conservation have been appreciated since mammalian genomes were first sequenced, but the functional relevance of these patterns remain unknown. We systematically measured the effect on gene expression of the sequences of more than 25,000 RNA-binding protein (RBP) binding sites in primary mouse T cells using a massively parallel reporter assay. GC-rich sequences were destabilizing of reporter mRNAs and come from more rapidly evolving regions of the genome. These sequences were more likely to be folded in vivo and contain a number of structural motifs that reduced accumulation of a heterologous reporter protein. Comparison of full-length 3' UTR sequences across vertebrate phylogeny revealed that strictly conserved 3' UTRs were GC-poor and enriched in genes associated with organismal development. In contrast, rapidly evolving 3' UTRs tended to be GC-rich and derived from genes involved in metabolism and immune responses. Cell-essential genes had lower GC content in their 3' UTRs, suggesting a connection between unstructured mRNA noncoding sequences and optimal protein production. By reducing gene expression, GC-rich RBP-occupied sequences act as a rapidly evolving substrate for gene regulatory interactions., (© 2019 Litterman et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Although inhaled corticosteroid (ICS) medication is considered the cornerstone treatment for patients with persistent asthma, few ICS pharmacogenomic studies have involved nonwhite populations., Objective: We sought to identify genetic predictors of ICS response in multiple population groups with asthma., Methods: The discovery group comprised African American participants from the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-Ethnicity (SAPPHIRE) who underwent 6 weeks of monitored ICS therapy (n = 244). A genome-wide scan was performed to identify single nucleotide polymorphism (SNP) variants jointly associated (ie, the combined effect of the SNP and SNP × ICS treatment interaction) with changes in asthma control. Top associations were validated by assessing the joint association with asthma exacerbations in 3 additional groups: African Americans (n = 803 and n = 563) and Latinos (n = 1461). RNA sequencing data from 408 asthmatic patients and 405 control subjects were used to examine whether genotype was associated with gene expression., Results: One variant, rs3827907, was significantly associated with ICS-mediated changes in asthma control in the discovery set (P = 7.79 × 10 -8 ) and was jointly associated with asthma exacerbations in 3 validation cohorts (P = .023, P = .029, and P = .041). RNA sequencing analysis found the rs3827907 C-allele to be associated with lower RNASE2 expression (P = 6.10 × 10 -4 ). RNASE2 encodes eosinophil-derived neurotoxin, and the rs3827907 C-allele appeared to particularly influence ICS treatment response in the presence of eosinophilic inflammation (ie, high pretreatment eosinophil-derived neurotoxin levels or blood eosinophil counts)., Conclusion: We identified a variant, rs3827907, that appears to influence response to ICS treatment in multiple population groups and likely mediates its effect through eosinophils., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Eosinophilic esophagitis (EoE) is a recently described disease in which exposure to specific foods and allergens leads to type 2 inflammation, epithelial barrier dysfunction, and difficulty in swallowing. In the current issue of the JCI, Wen and colleagues investigate tissue T cell heterogeneity in patients with EoE using single-cell RNA sequencing (scRNA-seq). Esophageal epithelium from individuals with EoE convtained a prominent population of Th2 cells not seen in controls. The short-chain fatty acid (SCFA) receptor FFAR3 was found to be highly expressed in EoE Th2 cells. Experiments presented here provide evidence that SCFAs may promote type 2 inflammation in allergic diseases such as EoE and asthma. This study provides an early example of scRNA-seq for identifying relevant cell populations and mechanisms underlying allergic diseases.