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 regulation of macrophage function within the pulmonary micro-environment

The lungs play a major role in gas exchange, yet the continuous and direct exposure of pulmonary epithelium to infections, toxins, and airborne pollutants requires a robust response to maintain homeostasis and prevent extensive damage that could impact tissue function. Lung epithelium protects against infections and injury by functioning as a physical barrier to pathogens and potentially harmful particles. It can also secrete mucus which traps dust, irritants and pathogens and is then cleared to prevent noxious material from damaging tissue; airway mucus has also been found to have antibacterial and antiviral properties. Unregulated acute pulmonary inflammation, including conditions such as acute respiratory distress syndrome (ARDS) and pneumonitis, are associated with epithelial damage and frequently associated with poor patient outcomes and impacts upon morbidity and mortality. Exacerbated inflammation can also lead to chronic conditions such as airway allergy and chronic obstructive pulmonary disease (COPD) and can impact upon pulmonary fibrosis and irreparable tissue damage. Apoptosis is a form of programmed cell death to remove unneeded or damaged cells and tissue and thus is necessary for normal embryonic development, maintenance of tissue homeostasis, ageing, and many diseases. Damage to the epithelium, neighbouring stromal cells and recruited immune cells frequently results in apoptosis. Apoptosis is considered an anti-inflammatory form of cell death, preventing damage to surrounding cells for example by the accumulation of DNA which leads to inflammation and autoimmunity. During apoptosis, intracellular components are dismantled in a controlled manner which is regulated by the activation of selected proteases known as caspases. Hallmark characteristics of apoptosis include nuclear chromatin condensation and significant reduction in cell size. The dying cells release “find me” signals to recruit phagocytes such as the small nucleotides ATP and UTP. The release of these signals is mediated by various channels, for example, pannexin channels of which pannexin 1 (Panx1) is the most extensively studied. Panx1 channels are activated by caspases during apoptosis and Panx1 has been found to regulate epithelial proliferation and efficient epithelial repair post tissue injury as well as regulating inflammation. Following the release of “find me” signals, structural and molecular changes also occur called “eat me” signals that promote engulfment of the apoptotic cells by macrophages. Efferocytosis is the process of quick and efficient engulfment of apoptotic cells by phagocytes, particularly macrophages and is therefore indispensable for the normal functioning of tissue. It is required for organ development, maintaining tissue homeostasis, resolution of inflammation and regeneration following injury. Defects in efferocytosis are linked to various autoimmune diseases, chronic inflammatory conditions and even cancer. Failure to eliminate damaged cells by efferocytosis is undesirable as the dying cells can release pro-inflammatory and immunogenic cell components which are harmful to the tissue microenvironment. The process of efferocytosis involves four stages i) attraction of the macrophage towards the apoptotic cell due to the released “find-me” signals, ii) recognition and tethering of the apoptotic cells by receptors on the macrophages iii) engulfment of apoptotic cells and finally, iv) degradation of apoptotic cells and induction of an anti-inflammatory response by these macrophages. Efferocytosis is one of the major functions of macrophages and engulfment of these apoptotic cells alters macrophage phenotype to be more anti-inflammatory. These anti-inflammatory macrophages secrete anti-inflammatory cytokines, pro-resolving mediators and reduce recruitment of other immune cells. Little is known about the molecular mechanisms and pathways that regulate the communication between epithelial cells and nearby inflammatory cells at homeostasis. Furthermore, pulmonary epithelium is known to produce various signals that influence the inflammatory response after injury, but less is known about the impact of this communication on immune cell phenotype and function. The central hypothesis for this thesis is that pulmonary epithelial cells can regulate macrophage function and phenotype to maintain homeostasis and promote tissue repair after injury. To investigate this hypothesis, we measured: i. the effect of pulmonary epithelium secreted mediators on macrophage phagocytosis of apoptotic cells at homeostasis, ii. changes to pulmonary macrophage phenotype after epithelial injury and iii. the effect of disrupting Panx1 on the development of fibrosis. Epithelial cell supernatant was used to determine the role epithelial cells play in the regulation of macrophage phenotype and phagocytic clearance of apoptotic cells during homeostasis. Human monocyte-derived macrophages were cultured, and an increase in efferocytosis was measured following incubation with lung and colonic epithelial cell supernatant. Further experiments showed the contributing factors in the secretome are likely involved in apoptotic corpse internalisation through cytoskeletal rearrangement. This highlights one communication network between epithelial cells and macrophages and its influence on efferocytosis at homeostasis. In addition, pulmonary macrophages were isolated and phenotypes assessed using single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) which synchronously quantifies cell surface protein and transcriptomic data in an in vivo model of acute airway epithelial injury induced by naphthalene. Naphthalene leads to early airway epithelial cell death and disruption of normal airway epithelial architecture. Following injury, macrophages are known to be required for epithelial regeneration, but their specific roles are yet to be elucidated. Macrophage phenotype after epithelial injury was altered to promote wound healing, regeneration and engulfment by the upregulation of genes related to growth factors and phagocytosis receptors. Both bleomycin and silica are known to cause epithelial cell death by inducing double strand breaks in the cell’s DNA and epithelial regeneration is modified to lead to fibrosis. As the Panx1 channel has been shown to regulate epithelial repair post tissue injury, Panx1 regulation of fibrogenesis was investigated as its effects on tissue fibrosis have not yet been studied. Lack of Panx1 expression had no effect on immune cell infiltration into the lungs. However, Panx1 knockout mice had exacerbated lung fibrosis after the initial inflammatory phase and ongoing experiments are underway to understand the molecular mechanism by which Panx1 regulates tissue fibrosis. The data in this thesis demonstrates epithelial cell regulation of macrophage function (efferocytosis) and phenotype (transcriptome) both at homeostasis and following epithelial injury to promote a pro-reparative environment

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

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

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

Tissue-specific immunopathology in fatal COVID-19

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.</p