Pericytes may facilitate SARS-CoV-2 entry into the nervous system

Objective: Although the neurological and olfactory symptoms of coronavirus disease 2019 have been identified, the neurotropic properties of the causative virus, severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), remain unknown. We sought to identify the susceptible cell types and potential routes of SARS-CoV-2 entry into the central nervous system, olfactory system, and respiratory system. Method(s): We collected single-cell RNA data from normal brain and nasal epithelium specimens, along with bronchial, tracheal, and lung specimens in public datasets. The susceptible cell types that express SARS-CoV-2 entry genes were identified using single-cell RNA sequencing and the expression of the key genes at protein levels was verified by immunohistochemistry. We compared the coexpression patterns of the entry receptor angiotensin-converting enzyme 2 (ACE2) and the spike protein priming enzyme transmembrane serine protease (TMPRSS)/cathepsin L among the specimens. Result(s): The SARS-CoV-2 entry receptor ACE2 and the spike protein priming enzyme TMPRSS/cathepsin L were coexpressed by pericytes in brain tissue;this coexpression was confirmed by immunohistochemistry. In the nasal epithelium, ciliated cells and sustentacular cells exhibited strong coexpression of ACE2 and TMPRSS. Neurons and glia in the brain and nasal epithelium did not exhibit coexpression of ACE2 and TMPRSS. However, coexpression was present in ciliated cells, vascular smooth muscle cells, and fibroblasts in tracheal tissue;ciliated cells and goblet cells in bronchial tissue;and alveolar epithelium type 1 cells, AT2 cells, and ciliated cells in lung tissue. Conclusion(s): Neurological symptoms in patients with coronavirus disease 2019 could be associated with SARS-CoV-2 invasion across the blood-brain barrier via pericytes. Additionally, SARS-CoV-2-induced olfactory disorders could be the result of localized cell damage in the nasal epithelium.Copyright © Wolters Kluwer Health, Inc. All rights reserved.

Robust, efficient workflow to establish, culture, and functionally assess primary-isolated airway epithelial cells

Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs). The efficiency and robustness of these assays often depends on the quality of primary-isolated cells, but primary cell isolation workflows, with which the user controls the choice of isolation method, cell culture medium, and culture format, may reduce reproducibility. Therefore, an optimized, standardized workflow can enhance and support isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations or particularly sensitive cell populations. We have developed a standardized workflow for isolation and culture of freshly derived airway epithelial cells. Method(s): Briefly, HAECs isolated from primary tissue were expanded in PneumaCult-Ex Plus Medium for 1 week and then seeded into Corning Transwell inserts and expanded until confluency. The cells were then differentiated in PneumaCult-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, aswell as SARS-CoV-2 cell entry targets angiotensin-converting enzyme 2 and transmembrane serine protease 2. Ion transport and barrier function of the ALI culturesand response to CF transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly derived HAECs were seeded into Corning Matrigel domes in the presence of PneumaCult Airway Organoid Seeding Medium. Oneweek later, the mediumwas changed to PneumaCult Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling. Result(s): Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 +/- 14% (n = 1 donor) greater organoid area than in vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolininduced swelling, which could be partially re-established with VX-809 treatment (29 +/- 9%, n = 3). Conclusion(s): In summary, the PneumaCult workflow supports robust, efficient culture of primary-airway epithelial cells that can be used as physiologically relevant models suitable for CF research, CFTR corrector screening, and studying airway biology.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Single cell mRNA sequencing of nasal swabs indicate an impaired ciliated cell function in COVID-19 convalescents with persisting dyspnea

COVID-19 convalescents often experience persistent symptoms such as fatigue, neurologic complications or dyspnea, often referred to as "long COVID". To elucidate molecular mechanisms underlying ongoing dyspnea in COVID-19 convalescents, we analyzed single-cell RNA sequencing data from nasal swabs collected during acute infection, and three, six and twelve months post infection together with matching healthy controls. Patients with and without persisting symptoms and with varying severity during the acute phase were included. Post infection, we observed a time-dependent decrease in immune cells. Transcriptional analysis of nasal epithelial cells provided evidence of an impaired cilia assembly, organization and function in COVID-19 convalescents with dyspnea compared to healthy controls and convalescents without ongoing respiratory symptoms. Moreover, differences in the differentiation trajectories of ciliated cells were evident between patients with and without dyspnea, with less diverse differentiation endpoints in the dyspnea patients than in healthy controls or convalescents without respiratory impairment. Overall, our analyses revealed a potential deficiency of ciliated cells in COVID-19 convalescents with dyspnea compared to convalescents without ongoing respiratory symptoms or compared with healthy controls. Ciliated cells clear the lung from particles and mucus. If these cells are functionally impaired, pathogens remain in the airways, causing respiratory problems and infections. Thus, it is reasonable to assume, that impaired ciliated cell function contributes to the persistent respiratory symptoms seen in COVID-19 convalescents.

Hypercoagulability in COVID-19 and its putative association with the downregulation of protein C signalling

Background: Hematological complications associated with prothrombotic events with extrapulmonary manifestations have been demonstrated in autopsies of patients affected by coronavirus disease 2019 (COVID-19). Based on the close relationship of coagulation and immune response, we hypothesized that hypercoagulability in COVID-19 could result from the activation of tissue factor (F3) and subsequent alterations in Activated Protein C (APC) signaling (Figure 1). Aim(s): We aimed to identify changes in the expression of APC signaling network in liver, peripheral blood and nasal epithelium of COVID-19 patients that may contribute to local and systemic disarrangement of hemostasis. Method(s): For the expression of PROC and receptor genes public single-cell- RNA- sequencing datasets were analyzed from COVID-19 patients and healthy individuals, using the toolkit Scanpy 1.7.2 in Phyton. Result(s): The key compounds of Protein C (PC) activation and signaling;PROCR, F2R, THBD, S1PR1 and PROC were downregulated in COVID-19 patients;a greater expression of F3 in all COVID-19 tissues analyzed and upregulation of AGTR1, NFKB1, PTPN1, THBS1, PTGS2, PLAU, SERPINE1 and F5 pro-inflammatory and procoagulant genes in the liver of COVID-19 patients compared to control (Figure 2B, E and G). The hepatocyte PROC expression was changed in COVID-19 patients from hepatocyte 4 ADH1B+ PCK1+ in healthy liver (Figure 2F) to hepatocyte 3 CYP2A6+ in the liver of COVID-19 patients (Figure 2A). The ACE2 expression was increased in all COVID-19 tissues (Figure 2B, E and G) overlapping the PROC expression in the epithelium (Figure 2D) and liver tissues (Figure 2A). There was a co-expression of ACE2, PROC, PROS1, RHOA, and RAC1 in ciliated cells of COVID-19 patients (Figure 2C-D). Conclusion(s): The results provide evidence indicating a deficient synthesis and activation of PC and its receptors in COVID-19 patients that might contribute to a pronounced hypercoagulable state in response to endothelial COVID-19- related injury.

Oseltamivir and Remdesivir as a Targeted Combination Therapy for a Young and Previously Healthy Adult with Acute Respiratory Distress Syndrome Secondary to Flurona

Introduction/Background COVID-19 and influenza typically present in a very similar clinical picture. The co-infection of influenza among COVID-19 patients (i.e., flurona) can occur in the fall and winter of the year. The prevalence of flurona was estimated to be 0.4% and 4.5% in America and Asia, respectively. The damage of respiratory ciliated cells by the influenza virus can facilitate COVID-19 infection. Few studies reported COVID-19 co-infection with influenza virus. The majority of flurona cases affected older patients with co-morbidities. The co-infection of influenza among COVID-19 patients was associated with more severe disease, especially among older patients with co-morbidities. Young and healthy adults are less likely to develop severe COVID-19 leading to ARDS even with co-infection. However, severe COVID-19 can still occur regardless of age and co-morbidities. Herein, we report a case of severe ARDS in a young and previously healthy adult secondary to flurona that was successfully treated with targeted combination therapy with oseltamivir and remdesivir. Objective(s) A 21-year-old Caucasian male patient without significant past medical history presented the ED with a chief complaint of fatigue, cough, and generalized body aches. The patient mentioned that symptoms started a few days before his presentation. He suspected it was the flu, so he did not seek medical care initially. However, his symptoms continued to worsen, to the point that he could not move without getting severely out of breath. He was tachycardic, tachypneic in the emergency department (ED). His COVID-19 swab returned positive, and a respiratory pathogen panel was also positive for influenza A infection. Initial CTA was negative for PE but showed extensive multifocal bilateral infiltrates consistent with viral pneumonia. He was started on a high-flow nasal cannula. Still, his oxygen was peaking around 85% with increased work of breathing. The patient also did not tolerate BiPAP. Therefore, the patient was intubated in the ED and admitted to the intensive care unit (ICU). He was started on a five-day course of oseltamivir, remdesivir, and intravenous methylprednisolone. The patient remained intubated and mechanically ventilated on the next day, and PaO2/FIO2 ratio was 100. He was started on ARDS treatment protocol, and daily prone positioning was initiated. Gradually the patient started to improve. On day nine, he successfully passed a CPAP trial and was extubated. His ICU stay was complicated by the development of a small segmental PE that was treated with IV heparin. He also had upper GI bleeding, and esophagogastroduodenoscopy revealed a bleeding gastric ulcer, which was successfully managed with endoscopic clipping. The patient gradually improved, and his oxygen requirements decreased significantly over the next few days. He was discharged home with no supplemental oxygen on apixaban and pantoprazole. Methods Our study highlights the importance of screening for co-infecting influenza virus in COVID-19 patients, which could be the leading cause of disease severity. Early detection of flurona can play an important role in managing these patients, especially if they develop ARDS. Targeted combined therapy against influenza and COVID-19 with oseltamivir and remdesivir may effectively mitigate the morbidity and mortality of these patients. Improving compliance with flu vaccination is highly recommended to reduce influenza virus transmission during this long COVID-19 pandemic and reduce the risk of COVID-19 severity.

Electrophysiological approaches to study cystic fibrosis transmembrane conductance regulator function from human induced pluripotent stem cell-derived airway basal cells

Background: Induced pluripotent stem cells (iPSCs), self-renewable and reprogrammed from somatic cells using different transcription factors, are considered an ideal resource for regenerative medicine to replace diseased or damaged tissues. Airway basal cells not only serve as precursors for secretory and multiciliated cells, but also contribute to maintenance and regeneration of the airway epithelial barrier. Recently, it was reported that induced basal cells (iBCs) from human iPSCs recapitulate molecular and functional features from human iPSCs of airway basal cells, including selfrenewal and multilineage differentiation [1]. iBCs as in vitro model can be used in research on diseases affecting the airway, including COVID-19, influenza, asthma, and cystic fibrosis, although despite these advantages in generating iBCs, this is insufficient to support electrophysiological evidence. Our goal in this study is to define CFTR function in iPSCderived iBCs using electrophysiological methods. Method(s): An iPSC line containing dual reporter NKX2.1GFP and TP63tdTOMATO were used to generate iBCs according to a previously published protocol [1]). iBCs were differentiated into ciliated cells using air-liquid interface (ALI) culture. Short-circuit current measurements were taken on the cells cultured in ALI culture using an Ussing chamber using a previously described protocol [2]). To measure CFTR current using electrophysiological studies, fully differentiated monolayers on filters were dissociated into single cells, which were fixed onto a collagencoated cover glass using cytospin. Whole-cell patch-clamp recordingswere performed according to a previous published protocol [3]. Result(s): We generated proximal airway iBCs from iPSCs with the dualfluorescent reporter system of green fluorescent protein (marks lung progenitors) and tdTomato (marks subsequent airway progenitor) (Figure 1a). These cells on ALI culture demonstrated CFTR function using short-circuit current measurements (Figure 1b). We also measured CFTRdependent currents in iPSC-airway basal cells using whole-cell patchclamp recording (Figure 2). Conclusion(s): We identified CFTR function in electrophysiological experiments using airway iBCs in vitro from iPSCs. Therefore, our study helps advance the field of regenerative medicine, benefiting airway and lung diseases. This may ultimately allowfor development of individual, diseasespecific airway basal stem cells, leading to drug development and a platform on which targeted drug approaches can be tested. Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

People with cystic fibrosis do not show an increased interferonresponse transcriptomic signature in nasal epithelial cells

Objectives: People with CF (PwCF) are at increased risk of respiratory infections and chronic inflammation.We sought to determine whether the inflammatory response is different in nasal epithelium of PwCF compared to healthy volunteers (HV). Since Interferons can increase ACE2 expression, a protein required for SARS-CoV-2 entry,we focused our analysis on the the focusing on the interferon-response signature. Methods: We reanalysed nasal curettage sample bulk RNA-seq signatures of pilot and validation datasets for which the study methods and demographics of the recruited cohort have already been reported. For this analysis, we performed in-silico deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data from nasal epitheliumas a reference to determine the abundance of the specific cell types in each sample. Results: Hierarchical clustering of the pilot and validation cohorts revealed 3 clusters. Analysis of the larger validation cohort revealed that Cluster A included HV (11 out of 11 subjects) and both homozygous (7 out of 13 subjects) and heterozygous (3 out of 10 subjects) PwCF. Subject cluster A was characterised by increased expression of genes related to secretory and ciliated epithelial cells, whereas Clusters B and C contained both homozygous and heterozygous PwCF only and were characterised by genes restricted to neutrophils and involved in immune responses. We then compared samples from cluster A that contained samples from HV (n = 11), PwCF homozygous (n = 7) and heterozygous (n = 3) for F508del. This analysis identified 379 genes upregulated in HV and 146 genes upregulated in PwCF homozygous for F508del and only 44 and 6 genes upregulated in HV and PwCF heterozygous for F508del, respectively (FDR q < 0.05). ACE2, TMPRS2 or other interferon-response genes were not deferentially expressed in either comparison of cluster A. Conclusion: PwCF do not have higher expression of interferon-response genes in nasal epithelial cells

Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and Type-III interferon infectivity restriction

Bronchi of the upper respiratory tract are considered the site of SARSCoV- 2 infection initiation from where a possible spread to the lower respiratory tract can cause acute respiratory distress syndrome with a high degree of mortality in elderly patients. Here we established functional reconstituted primary bronchial epithelia (BE) derived from donors including both adults and children to study SARS-CoV-2 infection dynamics in a physiologically relevant model. We identified multi-ciliated cells as the primary target cells for SARS-CoV-2 in our reconstituted BE. We further observed rapid viral spread throughout the entire BE within 24-48hours. Within 3-4 days, we observed syncytia formation between ciliated cells and basal cells which accumulate at the apical side of the BE. We show that infected cells including syncytia are released into the apical lumen and contribute to the transmittable virus dose. Interestingly, some BE mainly reconstituted from young donor, showed an intrinsic resistance to infection and virus spread. This restricted infection phenotype correlated with a faster release of type-III interferon secretion. Moreover, exogenous type-III interferon treatment to permissive epithelia installed infection restriction while interferon gene knockout promoted infection. Taken together our data uncover syncytia formation as possible contribution to tissue or environmental SARS-CoV-2 dissemination and the type-III IFN response as a central contributor to SARS-CoV-2 resistance in BE, which may explain epidemiological observations that SARS-CoV-2 fatality is age dependent.