Spatial transcriptomic characterization of COVID-19 pneumonitis identifies immune circuits related to tissue injury.

Severe lung damage in COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways and genes present across the spectrum of histopathological damage in COVID-19 lung tissue. We applied correlation network-based approaches to deconvolve gene expression data from 46 areas of interest covering >62,000 cells within well preserved lung samples from three patients. Despite substantial inter-patient heterogeneity, we discovered evidence for a common immune cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines including CXCL9, CXCL10 and CXCL11 which are known to promote the recruitment of CXCR3+ immune cells. The tumour necrosis factor (TNF) superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were found to be consistently upregulated in the areas with severe tissue damage. We used published spatial and single cell SARS-CoV-2 datasets to confirm our findings in the lung tissue from additional cohorts of COVID-19 patients. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies.

Neutrophil Extracellular Traps, Local IL-8 Expression, and Cytotoxic T-Lymphocyte Response in the Lungs of Patients With Fatal COVID-19.

BACKGROUND: Excessive inflammation is pathogenic in the pneumonitis associated with severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defense mechanism against bacteria, but have also been shown to mediate tissue damage in a number of diseases. RESEARCH QUESTION: Could NETs and their tissue-damaging properties inherent to neutrophil-associated functions play a role in the respiratory failure seen in patients with severe COVID-19, and how does this relate to the SARS-CoV-2 viral loads, IL-8 (CXCL8) chemokine expression, and cytotoxic T-lymphocyte infiltrates? STUDY DESIGN AND METHODS: Sixteen lung biopsy samples obtained immediately after death were analyzed methodically as exploratory and validation cohorts. NETs were analyzed quantitatively by multiplexed immunofluorescence and were correlated with local levels of IL-8 messenger RNA (mRNA) and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by reverse-transcriptase polymerase chain reaction and immunohistochemistry analysis. RESULTS: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one of eight liver tissues. NET focal presence correlated negatively with CD8+ T-cell infiltration in the lungs. INTERPRETATION: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, but no correlation was found with viral loads. The strong association between NETs and IL-8 points to this chemokine as a potentially causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered with by the presence of NETs.

Lung Spatial Profiling Reveals a T Cell Signature in COPD Patients with Fatal SARS-CoV-2 Infection.

People with pre-existing lung diseases such as chronic obstructive pulmonary disease (COPD) are more likely to get very sick from SARS-CoV-2 disease 2019 (COVID-19). Still, an interrogation of the immune response to COVID-19 infection, spatially throughout the lung structure, is lacking in patients with COPD. For this study, we characterized the immune microenvironment of the lung parenchyma, airways, and vessels of never- and ever-smokers with or without COPD, all of whom died of COVID-19, using spatial transcriptomic and proteomic profiling. The parenchyma, airways, and vessels of COPD patients, compared to control lungs had (1) significant enrichment for lung-resident CD45RO+ memory CD4+ T cells; (2) downregulation of genes associated with T cell antigen priming and memory T cell differentiation; and (3) higher expression of proteins associated with SARS-CoV-2 entry and primary receptor ubiquitously across the ROIs and in particular the lung parenchyma, despite similar SARS-CoV-2 structural gene expression levels. In conclusion, the lung parenchyma, airways, and vessels of COPD patients have increased T-lymphocytes with a blunted memory CD4 T cell response and a more invasive SARS-CoV-2 infection pattern and may underlie the higher death toll observed with COVID-19.

Evaluation of Macular Retinal Vessels and Histological Changes in Two Cases of COVID-19.

The purpose of this study was to assess vascular and histological alterations in two COVID-19 and three control post-mortem retinas. The macular areas of flat-mounted samples were processed for immunofluorescence. Lectin and collagen IV positive vessels were captured under confocal microscopy, and endothelium loss and tortuosity were analyzed. Expression of ACE2 (angiotensin-converting enzyme 2) (the receptor for SARS-CoV-2), Iba1 (ionized calcium-binding adaptor molecule 1) and GFAP (glial fibrillary acidic protein) were quantified in retinal sections. The number of lectin vessels in COVID-19 retinas decreased by 27% compared to the control (p < 0.01) and the tortuosity increased in COVID-19 retinas (7.3 ± 0.2) vs. control retinas (6.8 ± 0.07) (p < 0.05). Immunofluorescence analysis revealed an increase in ACE2 (2.3 ± 1.3 vs. 1.0 ± 0.1; p < 0.0001) and Iba1 expression (3.06 ± 0.6 vs. 1.0 ± 0.1; p < 0.01) in COVID-19 sections whereas no changes in GFAP were observed. Analysis of the COVID-19 macular retinal tissue suggested that endothelial cells are a preferential target of SARS-CoV-2 with subsequent changes through their ACE2 receptor expression and morphology. Thus, microglial activation was hyperactive when facing an ensuing immunological challenge after SARS-CoV-2 infection.

Immunological Biomarkers of Fatal COVID-19: A Study of 868 Patients.

Information on the immunopathobiology of coronavirus disease 2019 (COVID-19) is rapidly increasing; however, there remains a need to identify immune features predictive of fatal outcome. This large-scale study characterized immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection using multidimensional flow cytometry, with the aim of identifying high-risk immune biomarkers. Holistic and unbiased analyses of 17 immune cell-types were conducted on 1,075 peripheral blood samples obtained from 868 COVID-19 patients and on samples from 24 patients presenting with non-SARS-CoV-2 infections and 36 healthy donors. Immune profiles of COVID-19 patients were significantly different from those of age-matched healthy donors but generally similar to those of patients with non-SARS-CoV-2 infections. Unsupervised clustering analysis revealed three immunotypes during SARS-CoV-2 infection; immunotype 1 (14% of patients) was characterized by significantly lower percentages of all immune cell-types except neutrophils and circulating plasma cells, and was significantly associated with severe disease. Reduced B-cell percentage was most strongly associated with risk of death. On multivariate analysis incorporating age and comorbidities, B-cell and non-classical monocyte percentages were independent prognostic factors for survival in training (n=513) and validation (n=355) cohorts. Therefore, reduced percentages of B-cells and non-classical monocytes are high-risk immune biomarkers for risk-stratification of COVID-19 patients.

Persistent Retinal Microvascular Impairment in COVID-19 Bilateral Pneumonia at 6-Months Follow-Up Assessed by Optical Coherence Tomography Angiography.

The purpose of this study was to evaluate the long-term evolution of retinal changes in COVID-19 patients with bilateral pneumonia. A total of 17 COVID-19 patients underwent retinal imaging 6 months after hospital discharge with structural optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA). The parafoveal retinal nerve fiber layer (RNFL) and ganglion cell layer (GCL) were significantly thinner in COVID-19 patients at 6 months compared to 0 months (p = <0.001 in both cases). In the optic nerve analysis, a significantly thinner RNFL was observed (p = 0.006) but persisted significantly thickened, compared to controls (p = 0.02). The vascular density (VD) at 6 months persisted significantly decreased when compared to the control group, and no significant differences were found with the 0 months evaluation; in addition, when analyzed separately, women showed a worsening in the VD. Moreover, a significantly greater foveal area zone (FAZ) (p = 0.003) was observed in COVID-19 patients at 6 months, compared to 0 months. The cotton wool spots (CWSs) observed at baseline were no longer present at 6 months, except for one patient that developed new ones. This study demonstrates that some of the previously known microvascular alterations resulting from COVID-19, persist over time and are still evident 6 months after hospital discharge in patients who have suffered from bilateral pneumonia.

Retinal Microvascular Impairment in COVID-19 Bilateral Pneumonia Assessed by Optical Coherence Tomography Angiography.

The purpose of this study was to evaluate the presence of retinal and microvascular alterations in COVID-19 patients with bilateral pneumonia due to SARS-COV-2 that required hospital admission and compare this with a cohort of age- and sex-matched controls. COVID-19 bilateral pneumonia patients underwent retinal imaging 14 days after hospital discharge with structural optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) measurements. Vessel density (VD) and foveal avascular zone (FAZ) area were evaluated in the superficial, deep capillary plexus (SCP, DCP), and choriocapillaris (CC). After exclusion criteria, only one eye per patient was selected, and 50 eyes (25 patients and 25 controls) were included in the analysis. COVID-19 patients presented significantly thinner ganglion cell layer (GCL) (p = 0.003) and thicker retinal nerve fiber layer (RNFL) compared to controls (p = 0.048), and this RNFL thickening was greater in COVID-19 cases with cotton wool spots (CWS), when compared with patients without CWS (p = 0.032). In both SCP and DCP, COVID-19 patients presented lower VD in the foveal region (p < 0.001) and a greater FAZ area than controls (p = 0.007). These findings suggest that thrombotic and inflammatory phenomena could be happening in the retina of COVID-19 patients. Further research is warranted to analyze the longitudinal evolution of these changes over time as well as their correlation with disease severity.

Immunologic characterization of COVID-19 patients with hematological cancer.

Not available.

Histopathological findings in fatal COVID-19 severe acute respiratory syndrome: preliminary experience from a series of 10 Spanish patients.

In December 2019, an outbreak of severe acute respiratory syndrome associated to SARS-CoV2 was reported in Wuhan, China. To date, little is known on histopathological findings in patients infected with the new SARS-CoV2. Lung histopathology shows features of acute and organising diffuse alveolar damage. Subtle cellular inflammatory infiltrate has been found in line with the cytokine storm theory. Medium-size vessel thrombi were frequent, but capillary thrombi were not present. Despite the elevation of biochemical markers of cardiac injury, little histopathological damage could be confirmed. Viral RNA from paraffin sections was detected at least in one organ in 90% patients.