Serial Histological Changes in the Cartilaginous Eustachian Tube in the Rat Following Balloon Dilation

Although balloon dilation has shown promising results in the treatment of dilatory Eustachian tube (ET) dysfunction, the histological effects of ET balloon dilation (ETBD) remain unclear. We evaluated this in a rat model. The left ET of 20 Wistar rats was dilated with a balloon catheter and the right ET was used as a control. Five rats were sacrificed immediately after ETBD, at 1, 4 and 12 weeks after the procedure for histological examination. The epithelial cells, presence of squamous metaplasia, and the proportion of the goblet cells in the epithelium; the vascular structures and dimensions of the submucosa; and presence of cartilage fracture and the area of the ET lumen were evaluated and compared between the groups. Desquamation of nearly all epithelial cells and the fracture of tubal cartilages were observed immediately after ETBD. At 1-week post-ETBD, the ciliated epithelial cells started to recover with squamous metaplasia and epithelial hyperplasia. The goblet cells recovered by 4 weeks post-ETBD and epithelial hyperplasia decreased but was still present at 12 weeks post-ETBD. The depth of the submucosa increased and neovascularization in this region was observed at 1-week post-ETBD and persisted up to 12 weeks post-ETBD. The lumen of the cartilaginous ET increased immediately after ETBD but decreased at 1-week post-ETBD. The cartilaginous ET lumen recovered to the normal value at 4 weeks post-ETBD. This study is the first to describe the serial histological changes to the cartilaginous ET after ETBD and helps our understanding of the histological changes that occur after an ETBD intervention for intractable ET dysfunction.

Bronchoalveolar Lavage Fluid Cytology in Healthy Cynomolgus Macaques

Bronchoalveolar lavage, or BAL, is a minimally invasive procedure frequently used for clinical and non-clinical research, allowing studies of the respiratory system. Macaques are the most widely used non-human primate models in biomedical research. However, very little information is available in the literature concerning BAL cytology in macaques. The purpose of this study was to establish BAL reference values and document an atlas of BAL cytology from healthy cynomolgus macaques. BALs were obtained from 30 macaques and BAL fluid differential cell counts based on 400 nucleated cells/BAL sample were performed by a board-certified clinical pathologist. Results were analyzed using Reference Value Advisor macroinstructions and the effect of blood and oropharyngeal contaminations was investigated. Overall, nucleated cells interval percentages in BAL fluids were 55.8 to 93.7 for macrophages, 1.8 to 37.1 for lymphocytes, 0.4 to 8.7 for neutrophils, and 0.4 to 9.8 for eosinophils. Mild oropharyngeal contamination did not affect BAL differential cell counts, whilst a slight but significant increase of the percentage of lymphocytes was observed in samples with mild blood contamination. Mucus and variable numbers of ciliated epithelial cells were commonly present. Rarely, multinucleated macrophages and mastocytes were also observed. The reference intervals established in this study provide a useful baseline for the assessment of BAL cytological data in cynomolgus macaques.

Single cell RNA sequencing identifies IGFBP5 and QKI as ciliated epithelial cell genes associated with severe COPD

Background Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. Methods To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. Results T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. Conclusions scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

Lung gene expression and single cell analyses reveal two subsets of idiopathic pulmonary fibrosis (IPF) patients associated with different pathogenic mechanisms

Idiopathic pulmonary fibrosis is a progressive and debilitating lung disease with large unmet medical need and few treatment options. We describe an analysis connecting single cell gene expression with bulk gene expression-based subsetting of patient cohorts to identify IPF patient subsets with different underlying pathogenesis and cellular changes. We reproduced earlier findings indicating the existence of two major subsets in IPF and showed that these subsets display different alterations in cellular composition of the lung. We developed classifiers based on the cellular changes in disease to distinguish subsets. Specifically, we showed that one subset of IPF patients had significant increases in gene signature scores for myeloid cells versus a second subset that had significantly increased gene signature scores for ciliated epithelial cells, suggesting a differential pathogenesis among IPF subsets. Ligand-receptor analyses suggested there was a monocyte-macrophage chemoattractant axis (including potentially CCL2-CCR2 and CCL17-CCR4) among the myeloid-enriched IPF subset and a ciliated epithelium-derived chemokine axis (e.g. CCL15) among the ciliated epithelium-enriched IPF subset. We also found that these IPF subsets had differential expression of pirfenidone-responsive genes suggesting that our findings may provide an approach to identify patients with differential responses to pirfenidone and other drugs. We believe this work is an important step towards targeted therapies and biomarkers of response.

Streptococcus suis Induces Expression of Cyclooxygenase-2 in Porcine Lung Tissue

Streptococcus suis is a common pathogen colonising the respiratory tract of pigs. It can cause meningitis, sepsis and pneumonia leading to economic losses in the pig industry worldwide. Cyclooxygenase-2 (COX-2) and its metabolites play an important regulatory role in different biological processes like inflammation modulation and immune activation. In this report we analysed the induction of COX-2 and the production of its metabolite prostaglandin E2 (PGE2) in a porcine precision-cut lung slice (PCLS) model. Using Western blot analysis, we found a time-dependent induction of COX-2 in the infected tissue resulting in increased PGE2 levels. Immunohistological analysis revealed a strong COX-2 expression in the proximity of the bronchioles between the ciliated epithelial cells and the adjacent alveolar tissue. The morphology, location and vimentin staining suggested that these cells are subepithelial bronchial fibroblasts. Furthermore, we showed that COX-2 expression as well as PGE2 production was detected following infection with two prevalent S. suis serotypes and that the pore-forming toxin suilysin played an important role in this process. Therefore, this study provides new insights in the response of porcine lung cells to S. suis infections and serves as a basis for further studies to define the role of COX-2 and its metabolites in the inflammatory response in porcine lung tissue during infections with S. suis.

Fibrinolysis influences SARS-CoV-2 infection in ciliated cells

Rapid spread of COVID-19 has caused an unprecedented pandemic worldwide, and an inserted furin site in SARS-CoV-2 spike protein (S) may account for increased transmissibility. Plasmin, and other host proteases, may cleave the furin site of SARS-CoV-2 S protein and γ subunits of epithelial sodium channels (γ ENaC), resulting in an increment in virus infectivity and channel activity. As for the importance of ENaC in the regulation of airway surface and alveolar fluid homeostasis, whether SARS-CoV-2 will share and strengthen the cleavage network with ENaC proteins at the single-cell level is urgently worthy of consideration. To address this issue, we analyzed single-cell RNA sequence (scRNA-seq) datasets, and found the PLAU (encoding urokinase plasminogen activator), SCNN1G (γENaC), and ACE2 (SARS-CoV-2 receptor) were co-expressed in alveolar epithelial, basal, club, and ciliated epithelial cells. The relative expression level of PLAU, TMPRSS2, and ACE2 were significantly upregulated in severe COVID-19 patients and SARS-CoV-2 infected cell lines using Seurat and DESeq2 R packages. Moreover, the increments in PLAU, FURIN, TMPRSS2, and ACE2 were predominately observed in different epithelial cells and leukocytes. Accordingly, SARS-CoV-2 may share and strengthen the ENaC fibrinolytic proteases network in ACE2 positive airway and alveolar epithelial cells, which may expedite virus infusion into the susceptible cells and bring about ENaC associated edematous respiratory condition. Keywords: SARS-CoV-2; plasmin; ENaC; COVID-19; furin

Recombinant ACE2 Expression Is Required for SARS-CoV-2 To Infect Primary Human Endothelial Cells and Induce Inflammatory and Procoagulative Responses

ABSTRACT SARS-CoV-2 causes COVID-19, an acute respiratory distress syndrome (ARDS) characterized by pulmonary edema, viral pneumonia, multiorgan dysfunction, coagulopathy, and inflammation. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) receptors to infect and damage ciliated epithelial cells in the upper respiratory tract. In alveoli, gas exchange occurs across an epithelial-endothelial barrier that ties respiration to endothelial cell (EC) regulation of edema, coagulation, and inflammation. How SARS-CoV-2 dysregulates vascular functions to cause ARDS in COVID-19 patients remains an enigma focused on dysregulated EC responses. Whether SARS-CoV-2 directly or indirectly affects functions of the endothelium remains to be resolved and is critical to understanding SARS-CoV-2 pathogenesis and therapeutic targets. We demonstrate that primary human ECs lack ACE2 receptors at protein and RNA levels and that SARS-CoV-2 is incapable of directly infecting ECs derived from pulmonary, cardiac, brain, umbilical vein, or kidney tissues. In contrast, pulmonary ECs transduced with recombinant ACE2 receptors are infected by SARS-CoV-2 and result in high viral titers (∼1 × 107/ml), multinucleate syncytia, and EC lysis. SARS-CoV-2 infection of ACE2-expressing ECs elicits procoagulative and inflammatory responses observed in COVID-19 patients. The inability of SARS-CoV-2 to directly infect and lyse ECs without ACE2 expression explains the lack of vascular hemorrhage in COVID-19 patients and indicates that the endothelium is not a primary target of SARS-CoV-2 infection. These findings are consistent with SARS-CoV-2 indirectly activating EC programs that regulate thrombosis and endotheliitis in COVID-19 patients and focus strategies on therapeutically targeting epithelial and inflammatory responses that activate the endothelium or initiate limited ACE2-independent EC infection. IMPORTANCE SARS-CoV-2 infects pulmonary epithelial cells through ACE2 receptors and causes ARDS. COVID-19 causes progressive respiratory failure resulting from diffuse alveolar damage and systemic coagulopathy, thrombosis, and capillary inflammation that tie alveolar responses to EC dysfunction. This has prompted theories that SARS-CoV-2 directly infects ECs through ACE2 receptors, yet SARS-CoV-2 antigen has not been colocalized with ECs and prior studies indicate that ACE2 colocalizes with alveolar epithelial cells and vascular smooth muscle cells, not ECs. Here, we demonstrate that primary human ECs derived from lung, kidney, heart, brain, and umbilical veins require expression of recombinant ACE2 receptors in order to be infected by SARS-CoV-2. However, SARS-CoV-2 lytically infects ACE2-ECs and elicits procoagulative and inflammatory responses observed in COVID-19 patients. These findings suggest a novel mechanism of COVID-19 pathogenesis resulting from indirect EC activation, or infection of a small subset of ECs by an ACE2-independent mechanism, that transforms rationales and targets for therapeutic intervention.

Recombinant ACE2 Expression is Required for SARS-CoV-2 to Infect Primary Human Endothelial Cells and Induce Inflammatory and Procoagulative Responses

SARS-CoV-2 causes COVID-19, an acute respiratory distress syndrome (ARDS) characterized by pulmonary edema, viral pneumonia, multiorgan dysfunction, coagulopathy and inflammation. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) receptors to infect and damage ciliated epithelial cells in the upper respiratory tract. In alveoli, gas exchange occurs across an epithelial-endothelial barrier that ties respiration to endothelial cell (EC) regulation of edema, coagulation and inflammation. How SARS-CoV-2 dysregulates vascular functions to cause ARDS in COVID-19 patients remains an enigma focused on dysregulated EC responses. Whether SARS-CoV-2 directly or indirectly affects functions of the endothelium remains to be resolved and critical to understanding SARS-CoV-2 pathogenesis and therapeutic targets. We demonstrate that primary human ECs lack ACE2 receptors at protein and RNA levels, and that SARS-CoV-2 is incapable of directly infecting ECs derived from pulmonary, cardiac, brain, umbilical vein or kidney tissues. In contrast, pulmonary ECs transduced with recombinant ACE2 receptors are infected by SARS-CoV-2 and result in high viral titers (∼1×107/ml), multinucleate syncytia and EC lysis. SARS-CoV-2 infection of ACE2-expressing ECs elicits procoagulative and inflammatory responses observed in COVID-19 patients. The inability of SARS-CoV-2 to directly infect and lyse ECs without ACE2 expression explains the lack of vascular hemorrhage in COVID-19 patients and indicates that the endothelium is not a primary target of SARS-CoV-2 infection. These findings are consistent with SARS-CoV-2 indirectly activating EC programs that regulate thrombosis and endotheliitis in COVID-19 patients, and focus strategies on therapeutically targeting epithelial and inflammatory responses that activate the endothelium or initiate limited ACE2 independent EC infection.ImportanceSARS-CoV-2 infects pulmonary epithelial cells through ACE2 receptors and causes ARDS. COVID-19 causes progressive respiratory failure resulting from diffuse alveolar damage and systemic coagulopathy, thrombosis and capillary inflammation that tie alveolar responses to EC dysfunction. This has prompted theories that SARS-CoV-2 directly infects ECs through ACE2 receptors, yet SARS-CoV-2 antigen has not been co-localized with ECs and prior studies indicate that ACE2 co-localizes with alveolar epithelial cells and vascular smooth muscle cells, not ECs. Here we demonstrate that primary human ECs derived from lung, kidney, heart, brain and umbilical veins require expression of recombinant ACE2 receptors in order to be infected by SARS-CoV-2. However, SARS-CoV-2 lytically infects ACE2-ECs and elicits procoagulative and inflammatory responses observed in COVID-19 patients. These findings suggest a novel mechanism of COVID-19 pathogenesis resulting from indirect EC activation, or infection of a small subset of ECs by an ACE2 independent mechanism, that transform rationales and targets for therapeutic intervention.