Neutrophils provide a critical line of defense in immune responses to various pathogens, but also inflict self-damage upon transition to a hyperactivated, procoagulant state. Recent work has highlighted proinflammatory neutrophil phenotypes contributing to lung injury and acute respiratory distress syndrome (ARDS) in patients suffering from COVID-19. Here, we utilize state-of-the art mass spectrometry-based proteomics, transcriptomic and correlative analyses as well as functional in vitro and in vivo studies to dissect how neutrophils contribute to the progression to severe COVID-19. We identify a reinforcing loop of both systemic and neutrophil intrinsic interleukin-8 (CXCL8/IL-8) dysregulation, which initiates and perpetuates neutrophil-driven immunopathology. This positive feedback loop of systemic and neutrophil autocrine IL-8 production leads to an activated, prothrombotic neutrophil phenotype characterized by degranulation and neutrophil extracellular trap (NET) formation. In severe COVID-19, neutrophils directly initiate the coagulation and complement cascade, highlighting a link to the immunothrombotic state observed in these patients. Targeting the IL-8-CXCR-1/-2 axis interferes with this vicious cycle and attenuates neutrophil activation, degranulation, NETosis, and IL-8 release. Finally, we show that blocking IL-8-like signaling reduces SARS-CoV-2 spike protein-induced, hACE2-dependent pulmonary microthrombosis in mice. In summary, our data provide comprehensive insights into the activation mechanisms of neutrophils in COVID-19 and uncover a self-sustaining neutrophil-IL-8-axis as promising therapeutic target in severe SARS-CoV-2 infection.
Rainer Kaiser, Alexander Leunig, Kami Pekayvaz, Oliver Popp, Markus Joppich, Vivien Polewka, Raphael Escaig, Afra Anjum, Marie-Louise Hoffknecht, Christoph Gold, Sophia Brambs, Anouk Engel, Sven Stockhausen, Viktoria Knottenberg, Anna Titova, Mohamed Haji, Clemens Scherer, Maximilian Muenchhoff, Johannes C. Hellmuth, Kathrin Saar, Benjamin Schubert, Anne Hilgendorff, Christian Schulz, Stefan Kääb, Ralf Zimmer, Norbert Hübner, Steffen Massberg, Philipp Mertins, Leo Nicolai, Konstantin Stark
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the pandemic Coronavirus Disease 2019 (COVID-19) and now many face the burden of prolonged symptoms—long-lasting COVID-19 symptoms or “long-COVID”. Long-COVID is thought to be linked to immune dysregulation due to harmful inflammation, with the exact causes being unknown. Given the role of the microbiome in mediating inflammation, we aimed to examine the relationship between the oral microbiome and the duration of long-COVID symptoms. Tongue swabs were collected from patients presenting with symptoms concerning for COVID-19. Confirmed infections were followed until resolution of all symptoms. Bacterial composition was determined by metagenomic sequencing. We used random forest modeling to identify microbiota and clinical covariates that associated with long-COVID symptoms. Of the patients followed, 63% (17/27) developed ongoing symptomatic COVID-19 and 37% (10/27) went on to long-COVID. Patients with prolonged symptoms had significantly higher abundances of microbiota that induce inflammation, such as members of the genera Prevotella and Veillonella. Of note are species that produce lipopolysaccharides and the similarity of long-COVID patients’ oral microbiome to those of patients with chronic fatigue syndrome. All together, we our findings suggest an association with the oral microbiome and long-COVID revealing the possibility that dysfunction of the oral microbiome may contribute to this draining disease.
John P. Haran, Evan Bradley, Abigail L. Zeamer, Lindsey Cincotta, Marie-Claire Salive, Protiva Dutta, Shafik Mutaawe, Otuwe Anya, Mario Meza-Segura, Ann M. Moormann, Doyle V. Ward, Beth A. McCormick, Vanni Bucci
Cell lines are the mainstay in understanding the biology of COVID-19 infection, but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue allows for the ex vivo study of the inter-individual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 21 donors, many of whom had clinical risk factors for severe COVID-19. Cryopreserved tissues preserved 90% cell viability and contained heterogenous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infectable with HCoV-OC43 and SARS-CoV-2 coronaviruses, and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL6, CXCL8 and IFNB1 in response to SARS-CoV2 infection. Treatment of tissues with dexamethasone and the experimental drug, n-hydroxycytidine, suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted but unproven antiviral activity, each suppressed viral replication in tissues from a subset of donors. In summary, we developed a novel system for the ex vivo study of human SARS-CoV- 2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.
Matthew A. Schaller, Yamini Sharma, Zadia Dupee, Duy T. Nguyen, Juan M. Urueña, Ryan A. Smolchek, Julia C. Loeb, Tiago N. Machuca, John A. Lednicky, David J. Odde, Robert F. Campbell, W. Gregory Sawyer, Borna Mehrad
Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbate disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in COVID-19 patients and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, two signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in COVID-19 patients. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation leading to production of pathological HA fragments which are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.
Kimberly A. Queisser, Rebecca A. Mellema, Elizabeth A. Middleton, Irina Portier, Bhanu Kanth Manne, Frederik Denorme, Ellen J. Beswick, Matthew T. Rondina, Robert A. Campbell, Aaron C. Petrey
Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infects epithelial cells of the human gastrointestinal (GI) tract and causes related symptoms. HIV infection impairs gut homeostasis and is associated with an increased risk of COVID-19 fatality. To investigate the potential link between these observations, we analysed single cell transcriptional profiles and SARS-CoV-2 entry receptor expression across lymphoid and mucosal human tissue from chronically HIV infected individuals and uninfected controls. Absorptive gut enterocytes displayed the highest co-expression of SARS-CoV-2 receptors ACE2, TMPRSS2 and TMPRSS4, of which ACE2 expression was associated with canonical interferon response and antiviral genes. Chronic treated HIV infection was associated with a clear antiviral response in gut enterocytes and, unexpectedly, with a significant reduction of ACE2 and TMPRSS2 target cells. Gut tissue from SARS-CoV-2 infected individuals, however, showed abundant SARS-CoV-2 nucleocapsid protein in both the large and small intestine, including an HIV co-infected individual. Thus, upregulation of antiviral response genes and downregulation of ACE2 and TMPRSS2 in the GI tract of HIV infected individuals, does not prevent SARS-CoV-2 infection in this compartment. The impact of these HIV-associated intestinal mucosal changes on SARS-CoV-2 infection dynamics, disease severity and vaccine responses remains unclear and require further investigation.
Rabiah Fardoos, Osaretin E. Asowata, Nicholas Herbert, Sarah K. Nyquist, Yenzekile Zungu, Alveera Singh, Abigail Ngoepe, Ian M. Mbano, Ntombifuthi Mthabela, Dirhona Ramjit, Farina Karim, Warren Kuhn, Fusi G. Madela, Vukani T. Manzini, Frank Anderson, Bonnie Berger, Tune H. Pers, Alex K. Shalek, Alasdair Leslie, Henrik Kløverpris
The SARS-CoV-2 Receptor Binding Domain (RBD) is both the principal target of neutralizing antibodies, and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralising capacity of antibodies at the ACE2-RBD interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P and N501Y to the ACE2 receptor, and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research; informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.
Ester Lopez, Ebene R. Haycroft, Amy Adair, Francesca L. Mordant, Matthew T. O'Neill, Phillip Pymm, Samuel J. Redmond, Wen Shi Lee, Nicholas A. Gherardin, Adam K. Wheatley, Jennifer A. Juno, Kevin John Selva, Samantha K. Davis, Samantha L. Grimley, Leigh Harty, Damian F.J. Purcell, Kanta Subbarao, Dale I. Godfrey, Stephen J. Kent, Wai-Hong Tham, Amy W. Chung
Evidence suggests an association between severe acute respiratory syndrome-cornavirus-2 (SARS-CoV-2) infection and the occurrence of new-onset diabetes. We examined pancreatic expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), the cell entry factors for SARS-CoV-2, using public single cell RNA sequencing datasets, and pancreas tissue from control male and female non-human primates (NHPs) and humans. We also examined SARS-CoV-2 immunolocalization in pancreas cells of SARS-CoV-2-infected NHPs, and patients deceased from coronavirus disease 2019 (COVID-19). We report expression of ACE2 in pancreatic islet, ductal, and endothelial cells in NHPs and humans. In pancreata from SARS-CoV-2-infected NHPs and COVID-19 patients, SARS-CoV-2 infected ductal, endothelial and islet cells. These pancreata also exhibited generalized fibrosis associated with multiple vascular thrombi. Two out of eight NHPs developed new onset diabetes following SARS-CoV-2 infection. Two out of five COVID-19 patients exhibited new onset diabetes at admission. These results suggest that SARS-CoV-2 infection of the pancreas may promote acute and especially chronic pancreatic dysfunction that could potentially lead to new-onset diabetes.
Mirza Muhammad Fahd Qadir, Manika Bhondeley, Wandy Beatty, Dina D. Gaupp, Lara A. Doyle-Meyers, Tracy Fischer, Ishitri Bandyopadhyay, Robert V. Blair, Rudolf Bohm, Jay Rappaport, Eric Lazartigues, Richard S. Vander Heide, Jay K. Kolls, Xuebin Qin, Franck Mauvais-Jarvis
Some studies suggest that recent common coronavirus (CCV) infections are associated with reduced COVID-19 severity upon SARS-CoV-2 infection. We completed serological assays using samples collected from health care workers to identify antibody types associated with SARS-CoV-2 protection and COVID-19 symptom duration. Rare SARS-CoV-2 cross-reactive antibodies elicited by past CCV infections were not associated with protection; however, the duration of symptoms following SARS-CoV-2 infections was significantly reduced in individuals with higher common betacoronavirus (βCoV) antibody titers. Since antibody titers decline over time after CCV infections, individuals in our cohort with higher βCoV antibody titers were more likely recently infected with common βCoVs compared to individuals with lower antibody titers. Therefore, our data suggest that recent βCoV infections potentially limit the duration of symptoms following SARS-CoV-2 infections through mechanisms that do not involve cross-reactive antibodies. Our data are consistent with the emerging hypothesis that cellular immune responses elicited by recent common βCoV infections transiently reduce symptom duration following SARS-CoV-2 infections.
Sigrid Gouma, Madison E. Weirick, Marcus J. Bolton, Claudia P. Arevalo, Eileen C. Goodwin, Elizabeth M. Anderson, Christopher M. McAllister, Shannon R. Christensen, Debora Dunbar, Danielle Fiore, Amanda Brock, JoEllen Weaver, John S. Millar, Stephanie DerOhannessian, Ian Frank, Daniel J. Rader, E. John Wherry, Scott E. Hensley
Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here we report that elevated interleukin-13 (IL-13) was associated with the need for mechanical ventilation in two independent patient cohorts. In addition, patients who acquired COVID-19 while prescribed Dupilumab, a mAb that blocks IL-13 and IL-4 signaling, had less severe disease. In SARS-CoV-2 infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, hyaluronan synthase 1 (Has1) was the most downregulated gene and accumulation of the hyaluronan polysaccharide was decreased in the lung. In patients with COVID-19, hyaluronan was increased in the lungs and plasma. Blockade of the hyaluronan receptor, CD44, reduced mortality in infected mice, supporting the importance of hyaluronan as a pathogenic mediator. Finally, hyaluronan was directly induced in the lungs of mice by administration of IL-13, indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and hyaluronan has important implications for therapy of COVID-19 and potentially other pulmonary diseases.
Alexandra N. Donlan, Tara E. Sutherland, Chelsea Marie, Saskia Preissner, Benjamin T. Bradley, Rebecca M. Carpenter, Jeffrey M. Sturek, Jennie Z. Ma, G. Brett Moreau, Jeffrey R. Donowitz, Gregory A. Buck, Myrna G. Serrano, Stacey L. Burgess, Mayuresh M. Abhyankar, Cameron Mura, Philip E. Bourne, Robert Preissner, Mary K. Young, Genevieve R. Lyons, Johanna J. Loomba, Sarah J. Ratcliffe, Melinda D. Poulter, Amy J. Mathers, Anthony J. Day, Barbara J Mann, Judith E. Allen, William A. Petri Jr.
The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils is recognized to play an important role in the thromboinflammatory milieu inherent to severe presentations of COVID-19. At the same time, a variety of functional autoantibodies have been observed in individuals with severe COVID-19 where they likely contribute to immunopathology. Here, we aimed to determine the extent to which autoantibodies might target NETs in COVID-19 and, if detected, to elucidate their potential functions and clinical associations. We measured anti-NET antibodies in 328 individuals hospitalized with COVID-19 alongside 48 healthy controls. We found high anti-NET activity in the IgG and IgM fractions of 27% and 60% of patients, respectively. There was a strong correlation between anti-NET IgG and anti-NET IgM (r=0.4, p<0.0001). Both anti-NET IgG and IgM tracked with high levels of circulating NETs, impaired oxygenation efficiency, and high circulating D-dimer. Furthermore, patients who required mechanical ventilation had a greater burden of anti-NET antibodies than did those not requiring oxygen supplementation. Levels of anti-NET IgG (and to a lesser extent anti-NET IgM) demonstrated an inverse correlation with the efficiency of NET degradation by COVID sera. Furthermore, purified IgG from COVID sera with high levels of anti-NET antibodies impaired the ability of healthy control serum to degrade NETs. In summary, many individuals hospitalized with COVID-19 have anti-NET antibodies, which likely impair NET clearance and may potentiate SARS-CoV-2-mediated thromboinflammation.
Yu Zuo, Srilakshmi Yalavarthi, Sherwin A. Navaz, Claire K. Hoy, Alyssa Harbaugh, Kelsey Gockman, Melanie Zuo, Jacqueline A. Madison, Hui Shi, Yogendra Kanthi, Jason S. Knight
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