{"doi":"10.1016/j.chest.2020.10.083","title":"Lower Respiratory Tract Myeloid Cells Harbor SARS-Cov-2 and Display an Inflammatory Phenotype","abstract":"Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) pneumonia may induce an aberrant immune response with brisk recruitment of myeloid cells into the airspaces.1Blanco-Melo D. Nilsson-Payant B.E. Liu W.-C. et al.Imbalanced host response to SARS-CoV-2 drives development of COVID-19.Cell. 2020; 181: 1036-1045.e9Abstract Full Text Full Text PDF PubMed Scopus (2646) Google Scholar Although the clinical implications are unclear, others have suggested that infiltrating myeloid cells may contribute to morbidity and mortality rates during SARS-CoV-2 infection.1Blanco-Melo D. Nilsson-Payant B.E. Liu W.-C. et al.Imbalanced host response to SARS-CoV-2 drives development of COVID-19.Cell. 2020; 181: 1036-1045.e9Abstract Full Text Full Text PDF PubMed Scopus (2646) Google Scholar, 2Vabret N. Britton G.J. Gruber C. et al.Immunology of COVID-19: current state of the science.Immunity. 2020; 52: 910-941Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar, 3Merad M. Martin J.C. Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages.Nat Rev Immunol. 2020; 20: 355-362Crossref PubMed Scopus (1558) Google Scholar However, few reports have characterized myeloid cells from the lower respiratory tract, which appears to be the primary site of viral-induced disease, during severe SARS-CoV-2 pneumonia. Endotracheal aspirate (ETA) samples were collected prospectively from seven patients whose condition required mechanical ventilation for severe pneumonia due to SARS-CoV-2 infection, which was documented by reverse transcriptase polymerase chain reaction from April to June 2020. All patients were enrolled in a University of Pittsburgh lung injury registry and biospecimen repository (IRB# PRO10110387). ETA were fixed in 4% (volume/volume) paraformaldehyde overnight then processed for subsequent imaging. Briefly, ETA samples were washed twice then pelleted at 600g. Samples for electron microscopy were resuspended in 1% (volume/volume) glutaraldehyde, repelleted at 600g, and processed as described in a previous report.4Ladinsky M.S. Khamaikawin W. Jung Y. et al.Mechanisms of virus dissemination in bone marrow of HIV-1-infected humanized BLT mice.Elife. 2019; 8: e46916Crossref PubMed Scopus (19) Google Scholar Samples for light microscopy and immunofluorescence were resuspended in phosphate-buffered saline solution then prepared as cytospins by spinning at 300 rpm onto a Superfrost plus microscope slide (Fisher Scientific). Manual cell counts of ETA samples were performed after Diff-Quick (Siemens; Healthcare Diagnostics, Inc) staining, and representative images were obtained with the use of an Olympus Provis I microscope (Olympus Corporation). For immunofluorescence, cytospin slides were placed in 70% (volume/volume) ethyl alcohol followed by 90% (volume/volume) ethyl alcohol for 10 minutes each, then allowed to air dry. Antibodies that were used include SARS-CoV-2 nucleocapsid protein (NB100-56576; Novus Biologicals), CD14 (#347490; BD Biosciences), CD16 (MA1-84008; Invitrogen), CD142 (Tissue Factor; BD 550252; BD Biosciences), and IL-6 (Novus NBP2-44953; Novus Biologicals). Subsequent staining was performed, and slides were imaged using a Nikon A1 confocal scanning fluorescence microscope (Nikon Inc). Preembed immune-electron microscopy was performed after cytospin preparation with colloidal gold-conjugated secondary antibodies (CD14, 18 nm; SARS-CoV-2, 6 nm nucleocapsid; Jackson ImmunoResearch) using a JEOL JEM 1400 transmission electron microscope (JEOL USA, Inc) at 80 kV with image capture via an Advanced Microscopy Techniques 2K digital camera. RNAscope was performed per manufacturer instructions. Quantitative imaging analysis was performed with the use of object-based area overlap analysis via Nikon Elements software. Statistical comparisons of co-expression of CD14, CD16, IL-6, and tissue factor in cells with or without SARS-CoV-2 nucleocapsid protein were performed with non-parametric te","journal":"CHEST Journal","year":2020,"id":82334,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":13,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9576,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":347792,"name":"Hernán F. Peñaloza","orcid":"0000-0002-1593-678X","position":1,"is_corresponding":false},{"id":228766,"name":"Mark S. Ladinsky","orcid":"0000-0002-1036-3513","position":2,"is_corresponding":false},{"id":347793,"name":"Rick van der Geest","orcid":"0000-0002-1973-5934","position":3,"is_corresponding":false},{"id":339558,"name":"Mara Sullivan","orcid":"0000-0003-3772-1998","position":4,"is_corresponding":false},{"id":129874,"name":"Mark A. Ross","orcid":null,"position":5,"is_corresponding":false},{"id":16243,"name":"Georgios D. Kitsios","orcid":"0000-0002-1018-948X","position":6,"is_corresponding":false},{"id":19811,"name":"Barbara A. Methé","orcid":"0000-0001-8711-1448","position":7,"is_corresponding":false},{"id":3878,"name":"Bryan J. McVerry","orcid":"0000-0002-1175-4874","position":8,"is_corresponding":false},{"id":313765,"name":"Alison Morris","orcid":"0000-0002-7290-6536","position":9,"is_corresponding":false},{"id":52921,"name":"Alan M. 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