Tissue proteomic analysis identifies mechanisms and stages of immunopathology in fatal COVID-19

Funding: This work was funded by UK Research and Innovation (UKRI) (Coronavirus Disease [COVID-19] Rapid Response Initiative; MR/V028790/1 to C.D.L., D.A.D., and J.A.H.), LifeArc (through the University of Edinburgh STOPCOVID funding award, to K.D, D.A.D., C.D.L), The Chief Scientist Office (RARC-19 Funding Call, ‘Inflammation in Covid-19: Exploration of Critical Aspects of Pathogenesis; COV/EDI/20/10’ to D.A.D, C.D.L, C.D.R, J.K.B and D.J.H), and Medical Research Scotland (CVG-1722- 2020 to DAD, CDL, CDR, JKB, and DJH). C.D.L is funded by a Wellcome Trust Clinical Career Development Fellowship (206566/Z/17/Z). J.K.B. and C.D.R. are supported by the Medical Research Council (grant MC_PC_19059) as part of the ISARIC Coronavirus Clinical Characterisation Consortium (ISARIC-4C). C.D.R. is supported by an Edinburgh Clinical Academic Track (ECAT)/Wellcome Trust PhD Training Fellowship for Clinicians award (214178/Z/18/Z). J.A.H. is supported by the U.S. Food and Drug Administration (contract 75F40120C00085, Characterization of severe coronavirus infection in humans and model systems for medical countermeasure development and evaluation’). G.C.O is funded by an NRS Clinician award. N.N.G. is funded by a Pathological Society Award. A.R.A. is supported by a Cancer Research UK Clinician Scientist Fellowship award (A24867).Immunopathology occurs in the lung and spleen in fatal COVID-19, involving monocytes/macrophages and plasma cells. Anti-inflammatory therapy reduces mortality but additional therapeutic targets are required. We aimed to gain mechanistic insight into COVID-19 immunopathology by targeted proteomic analysis of pulmonary and splenic tissues. Lung parenchymal and splenic tissue was obtained from 13 post-mortem examinations of patients with fatal COVID-19. Control tissue was obtained from cancer resection samples (lung) and deceased organ donors (spleen). Protein was extracted from tissue by phenol extraction. Olink® multiplex immunoassay panels were used for protein detection and quantification. Proteins with increased abundance in the lung included MCP-3, antiviral TRIM21 and pro-thrombotic TYMP. OSM and EN-RAGE/S100A12 abundance was correlated, and associated with inflammation severity. Unsupervised clustering identified ‘early viral’ and ‘late inflammatory’ clusters with distinct protein abundance profiles, and differences in illness duration prior to death and presence of viral RNA. In the spleen, lymphocyte chemotactic factors and CD8A were decreased in abundance, and pro-apoptotic factors were increased. B-cell receptor signalling pathway components and macrophage colony stimulating factor (CSF-1) were also increased. Additional evidence for a sub-set of host factors (including DDX58, OSM, TYMP, IL-18, MCP-3 and CSF-1) was provided by overlap between (i) differential abundance in spleen and lung tissue, (ii) meta-analysis of existing datasets, and (iii) plasma proteomic data. This proteomic analysis of lung parenchymal and splenic tissue from fatal COVID-19 provides mechanistic insight into tissue anti-viral responses, inflammation and disease stages, macrophage involvement, pulmonary thrombosis, splenic B-cell activation and lymphocyte depletion.Publisher PDFPeer reviewe

Tissue-specific immunopathology in fatal COVID-19

Funding: Inflammation in COVID-19: Exploration of Critical Aspects of Pathogenesis (ICECAP) receives funding and support from the Chief Scientist Office (RapidResearch in COVID-19 programme [RARC-19] funding call, “Inflammation in Covid-19: Exploration of Critical Aspects of Pathogenesis; COV/EDI/20/10” to D.A.D., C.D.L., C.D.R., J.K.B., and D.J.H.), LifeArc (through the University of Edinburgh STOPCOVID funding award to K.D., D.A.D., and C.D.L.), UK Research and Innovation (UKRI) (Coronavirus Disease [COVID-19] Rapid Response Initiative; MR/V028790/1 to C.D.L., D.A.D., and J.A.H.), and Medical Research Scotland (CVG-1722-2020 to D.A.D., C.D.L., C.D.R., J.K.B., and D.J.H.). C.D.L. is funded by a Wellcome Trust Clinical Career Development Fellowship(206566/Z/17/Z). J.K.B. and C.D.R. are supported by the Medical Research Council (grant MC_PC_19059) as part of the International Severe AcuteRespiratory Infection Consortium Coronavirus Clinical Characterisation Consortium (ISARIC-4C). D.J.H., I.H.U., and M.E. are supported by the Industrial Centre for Artificial Intelligence Research in Digital Diagnostics. S.P. is supported by Kidney Research UK, and G.T. is supported by the Melville Trust for the Cure and Care of Cancer. Identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and sequencing work was supported by theU.S. Food and Drug Administration grant HHSF223201510104C (“Ebola Virus Disease: correlates of protection, determinants of outcome and clinicalmanagement”; amended to incorporate urgent COVID-19 studies) and contract 75F40120C00085 (“Characterization of severe coronavirus infection inhumans and model systems for medical countermeasure development and evaluation”; awarded to J.A.H.). J.A.H. is also funded by the Centre of Excellence in Infectious Diseases Research and the Alder Hey Charity. R.P.-R. is directly supported by the Medical Research Council Discovery Medicine North Doctoral Training Partnership. The group of J.A.H. is supported by the National Institute for Health Research Health Protection Research Unit in Emerging and Zoonotic Infections at the University of Liverpool in partnership with Public Health England and in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.Rationale: In life-threatening Covid-19, corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of SARS-CoV-2 or an independent immunopathologic process is unknown. Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses, and the relationships between viral presence, inflammation, and organ injury. Methods: Tissue was acquired from eleven detailed post-mortem examinations. SARS-CoV-2 organotropism was mapped by multiplex PCR and sequencing, with cellular resolution achieved by in situ viral spike protein detection. Histological evidence of inflammation was quantified from 37 anatomical sites, and the pulmonary immune response characterized by multiplex immunofluorescence. Measurements and main results: Multiple aberrant immune responses in fatal Covid-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein, both between and within tissues. An arteritis was identified in the lung, which was further characterised as a monocyte/myeloid-rich vasculitis, and occurred along with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticulo-endothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues. Conclusions: Tissue-specific immunopathology occurs in Covid-19, implicating a significant component of immune-mediated, virus-independent immunopathology as a primary mechanism in severe disease. Our data highlight novel immunopathological mechanisms, and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma cell responses as well as promoting pathogen tolerance in Covid-19.Publisher PDFPeer reviewe

Epithelial Cells and Inflammation in Pulmonary Wound Repair

Respiratory diseases are frequently characterised by epithelial injury, airway inflammation, defective tissue repair, and airway remodelling. This may occur in a subacute or chronic context, such as asthma and chronic obstructive pulmonary disease, or occur acutely as in pathogen challenge and acute respiratory distress syndrome (ARDS). Despite the frequent challenge of lung homeostasis, not all pulmonary insults lead to disease. Traditionally thought of as a quiescent organ, emerging evidence highlights that the lung has significant capacity to respond to injury by repairing and replacing damaged cells. This occurs with the appropriate and timely resolution of inflammation and concurrent initiation of tissue repair programmes. Airway epithelial cells are key effectors in lung homeostasis and host defence; continual exposure to pathogens, toxins, and particulate matter challenge homeostasis, requiring robust defence and repair mechanisms. As such, the epithelium is critically involved in the return to homeostasis, orchestrating the resolution of inflammation and initiating tissue repair. This review examines the pivotal role of pulmonary airway epithelial cells in initiating and moderating tissue repair and restitution. We discuss emerging evidence of the interactions between airway epithelial cells and candidate stem or progenitor cells to initiate tissue repair as well as with cells of the innate and adaptive immune systems in driving successful tissue regeneration. Understanding the mechanisms of intercellular communication is rapidly increasing, and a major focus of this review includes the various mediators involved, including growth factors, extracellular vesicles, soluble lipid mediators, cytokines, and chemokines. Understanding these areas will ultimately identify potential cells, mediators, and interactions for therapeutic targeting