Single cell gene expression profiling of nasal ciliated cells reveals distinctive biological processes related to epigenetic mechanisms in patients with severe COVID-19

Objective: To explore the molecular processes associated with cellular regulatory programs in patients with COVID-19, including gene activation or repression mediated by epigenetic mechanisms. We hypothesized that a comprehensive gene expression profiling of nasopharyngeal epithelial cells might expand understanding of the pathogenic mechanisms of severe COVID-19. Methods: We used single-cell RNA sequencing (scRNAseq) profiling of ciliated cells (n = 12725) from healthy controls (SARS-CoV-2 negative n =13) and patients with mild/moderate (n =13) and severe (n =14) COVID-19. ScRNAseq data at the patient level were used to perform gene set and pathway enrichment analyses. We prioritized candidate miRNA-target interactions and epigenetic mechanisms. Results: Pathways linked to mitochondrial function, misfolded proteins, and membrane permeability were upregulated in patients with mild/moderate disease compared to healthy controls. Besides, we noted that compared to mild/moderate disease, cells derived from severe COVID-19 patients had downregulation of sub-networks associated with epigenetic mechanisms, including DNA and histone H3K4 methylation and chromatin remodeling regulation. We found 11-ranked miRNAs that may explain miRNA-dependent regulation of histone methylation, some of which share seed sequences with SARS-CoV-2 miRNAs. Conclusion: Our results may provide novel insights into the epigenetic mechanisms mediating the clinical course of SARS-CoV-2 infection.

Nonspecific binding of common anti-CFTR antibodies in ciliated cells of human airway epithelium

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR.

Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR

Glutamine amidotransferase-1 domain-containing AraC-family transcriptional regulators (GATRs) are present in the genomes of many bacteria, including all Pseudomonas species. The involvement of several characterized GATRs in amine-containing compound metabolism has been determined, but the full scope of GATR ligands and regulatory networks are still unknown. Here, we characterize Pseudomonas putida's detection of the animal-derived amine compound, creatine, a compound particularly enriched in muscle and ciliated cells by a creatine-specific GATR, PP_3665, here named CahR (Creatine amidohydrolase Regulator). cahR is necessary for transcription of the gene encoding creatinase (PP_3667/creA) in the presence of creatine and is critical for P. putida's ability to utilize creatine as a sole source of nitrogen. The CahR/creatine regulon is small and electrophoretic mobility shift demonstrates strong and specific CahR binding only at the creA promoter, supporting the conclusion that much of the regulon is dependent on downstream metabolites. Phylogenetic analysis of creA orthologs associated with cahR orthologs highlights a strain distribution and organization supporting likely horizontal gene transfer, particularly evident within the genus Acinetobacter. This study identifies and characterizes the GATR that transcriptionally controls P. putida metabolism of creatine, broadening the scope of known GATR ligands and suggesting GATR diversification during evolution of metabolism for aliphatic nitrogen compounds.

Computational Modeling of Aerosol Particle Transport through Lung Mucosa

Delivery of aerosols to the lungs have great potential for the treatment of various lung diseases. However, the lungs are coated by a protective mucus layer whose complex properties make this form of delivery difficult. Mucus is non-Newtonian and cleared from the lungs over time by ciliated cells. Further, its gel-like structure hinders the diffusion of particles through it. Any aerosolized lung disease treatment must have certain properties to circumvent this barrier, and these properties may vary between diseases, drugs, and patients. Using Computational Fluid Dynamics (CFD) modeling, a model of this mucus layer was constructed such that the diffusion of an impacted aerosol might be studied. The model predicts what amount of a particle of a certain size might be expected to penetrate the mucus and reach the underlying tissue, as well as the distance downstream of the dosage site where concentration is maximized. Using this information, a personalized treatment plan may be designed. The model maintains modularity so that various lung regions and patient health states may be simulated.

Changes in the cellular composition of guinea pig’s distal airways epithelium in the dynamics of experimental ovalbumin-induced allergic inflammation

The problem of studying the processes of restructuring of airways epithelium of humans and animals of adaptive nature under the influence of various factors on the body remains the subject of scientific discussions. The aim of this work is to study morphometric changes in the cellular composition guinea pig’s distal airways epithelium in the dynamics of experimental ovalbumin-induced allergic inflammation. We studied lung of 48 male guinea pigs, using histological, morphometric and statistical methods, under conditions of experimental ovalbumin-induced allergic inflammation, simulated by subcutaneous sensitization and subsequent intranasal inhalation with ovalbumin. The average number of epithelial cells of small bronchi and terminal bronchioles was determined: basal epithelium cells, ciliated cells, goblet cells and exocrine bronchiolar cells per unit area of 10000 μm2. We have shown the most significant reactive morphometric changes on the 23rd and 30th days of the experiment. We demonstrated a decrease of the number of basal cells (by 1.5 times compared to the control, p*/**<0.01) and ciliated cells (by 1.6 times compared to the control, p*/**<0.001) and an elevation of the average number of goblet cells (by 2.6 times compared to the control, p*/**<0.01) in bronchioles, a decrease of the average number of exocrine bronchiolar cells (by 1.6 times compared to the control, p*/**<0.01) in terminal bronchioles. These changes are the morphological confirmation of the development of bronchial hyperreactivity as a result of the action of the allergen. Sensitization and allergization with ovalbumin cause statistically significant morphological changes in the cellular composition of small bronchi and terminal bronchioles of an alterative nature in the early period and adaptive – in the late period of allergic inflammation, corresponding to the main morphological manifestations of allergic inflammation.

Cross-talk of inflammatory mediators and airway epithelium reveals CFTR as a major target

Airway inflammation, mucus hyperproduction and epithelial remodelling are hallmarks of many chronic airway diseases, including asthma, Chronic Obstructive Pulmonary Disease and Cystic Fibrosis. While several cytokines are dysregulated in these diseases, most studies focus on the response of airways to IL-4 and IL-13, which were shown to induce mucus hyperproduction and shift the airway epithelium towards a hypersecretory phenotype.We hypothesised that other cytokines might induce the expression of chloride (Cl−) channels/transporters, regulate epithelial differentiation and mucus production. To this end, fully-differentiated human airway basal cells (BCi-NS1.1) were treated with cytokines identified as dysregulated in those diseases, namely interleukins-8, 1β, 4, 17A, 10, 22, and tumour necrosis factor-α (TNF-α).Our results show that CFTR is the main Cl− channel modulated by inflammation, in contrast to TMEM16A, whose levels only changed with IL-4. Furthermore, we identified novel roles for IL-10 and IL-22 by influencing epithelial differentiation towards ciliated cells and away from pulmonary ionocytes. Contrarily, IL-1β and IL-4 reduced the number of ciliated cells while increasing club cells. Interestingly, while IL-1β, IL-4 and IL-10 upregulated CFTR expression, IL-4 was the only cytokine that increased both its function and the number of CFTR-expressing club cells, suggesting that this cell-type may be the main contributor for CFTR function. Additionally, all cytokines assessed increased mucus production through a differential upregulation of MUC5AC and MUC5B transcript levels.Altogether, this study reveals a novel insight into differentiation resulting from the cross-talk of inflammatory mediators and airway epithelial cells, which is particularly relevant for chronic airway diseases.

Estrogen and Progesterone Receptor Immunoexpression in Fallopian Tubes among Postmenopausal Women Based on Time since the Last Menstrual Period

Existing data on the expression of estrogen receptor (ERα) and progesterone receptor (PR) in fallopian tubes in postmenopausal women are mostly inconclusive. Therefore, we assessed ERα and PR immunoexpression in the oviducts of these women. One hundred postmenopausal women were divided into three groups based on time elapsed since the last menstrual period: (A) 1–5 years, (B) 6–10 years, and (C) ≥11 years. In all groups, both in the glandular epithelium and stroma of the ampulla and isthmus of the oviduct, immunolocalization of ERα and PR were noted. The glandular epithelium of the ampulla showed a higher percentage of PR-positive cells than the isthmus in each group. Regarding ERα, there were no significant differences. In the glandular epithelium in both the ampulla and isthmus, the percentage of ERα- and PR-positive cells was significantly higher than that in the stroma in each study group and higher in the A group than in the C group. In conclusion, in postmenopausal women, time elapsed since the last menstrual period in the fallopian tubes was positively correlated with the following: (1) the epithelium showed vacuolation of cytoplasm with greater frequency, (2) the proportion of ciliated cells decreased, and (3) the percentage of ERα- and PR-positive cells also decreased. The obtained results indicate a significant decrease in ERα and PR expression depending on the time that has elapsed since the last menstruation, which is undoubtedly related to the loss of the reproductive function of the patients.

The Primary Cilium on Cells of Developing Skeletal Rudiments; Distribution, Characteristics and Response to Mechanical Stimulation

Embryo movement is important for tissue differentiation and the formation of functional skeletal elements during embryonic development: reduced mechanical stimulation results in fused joints and misshapen skeletal rudiments with concomitant changes in the signaling environment and gene expression profiles in both mouse and chick immobile embryos. Despite the clear relationship between movement and skeletogenesis, the precise mechanisms by which mechanical stimuli influence gene regulatory processes are not clear. The primary cilium enables cells to sense mechanical stimuli in the cellular environment, playing a crucial mechanosensory role during kidney development and in articular cartilage and bone but little is known about cilia on developing skeletal tissues. Here, we examine the occurrence, length, position, and orientation of primary cilia across developing skeletal rudiments in mouse embryos during a period of pronounced mechanosensitivity and we report differences and similarities between wildtype and muscle-less mutant (Pax3Spd/Spd) rudiments. Strikingly, joint regions tend to have cilia positioned and oriented away from the joint, while there was a less obvious, but still significant, preferred position on the posterior aspect of cells within the proliferative and hypertrophic zones. Regions of the developing rudiments have characteristic proportions of ciliated cells, with more cilia in the resting and joint zones. Comparing wildtype to muscle-less mutant embryos, cilia are shorter in the mutant with no significant difference in the proportion of ciliated cells. Cilia at the mutant joint were also oriented away from the joint line.