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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Rhinovirus infection of bronchial epithelium induces specific bronchial smooth muscle cell migration of severe asthmatic patients.

BACKGROUND: Patients with severe asthma show an increase in both exacerbation frequency and bronchial smooth muscle (BSM) mass. Rhinovirus (RV) infection of the bronchial epithelium (BE) is the main trigger of asthma exacerbations. Histological analysis of biopsies shows that a close connection between BE and hypertrophic BSM is a criterion for severity of asthma. OBJECTIVE: We hypothesized that RV infection of BE specifically increases BSM-cell migration from patients with asthma. METHODS: Serum samples, biopsies, or BSM cells were obtained from 86 patients with severe asthma and 31 subjects without asthma. BE cells from subjects without asthma were cultured in an air-liquid interface and exposed to RV-16. Migration of BSM cells was assessed in response to BE supernatant using chemotaxis assays. Chemokine concentrations were analyzed by transcriptomics and ELISAs. Immunocytochemistry, western blotting, and flow cytometry were used to quantify CXCR3 isoform distribution. CXCR3 downstream signaling pathways were assessed by calcium imaging and western blots. RESULTS: BSM cells from patients with severe asthma specifically migrated toward RV-infected BE, whereas those from subjects without asthma did not. This specific migration is driven by BE C-X-C motif chemokine ligand 10, which was increased in vitro in response to RV infection as well as in vivo in serum from exacerbating patients with severe asthma. The mechanism is related to both decreased expression and activation of the CXCR3-B-specific isoform in BSM cells from those with severe asthma. CONCLUSIONS: We have demonstrated a novel mechanism of BSM remodeling in patients with severe asthma following RV exacerbation. This study highlights the C-X-C motif chemokine ligand 10/CXCR3-A axis as a potential therapeutic target in severe asthma.

Mitochondria are involved in bronchial smooth muscle remodeling in severe preschool wheezers.

BACKGROUND: Bronchial remodeling is a key feature of asthma that is already present in preschoolers with wheezing. Moreover, bronchial smooth muscle (BSM) remodeling at preschool age is predictive of asthma at school age. However, the mechanism responsible for BSM remodeling in preschoolers with wheezing remains totally unknown. In contrast, in adult asthma, BSM remodeling has been associated with an increase in BSM cell proliferation related to increased mitochondrial mass and biogenesis triggered by an altered calcium homeostasis. Indeed, BSM cell proliferation was decreased in vitro by the calcium channel blocker gallopamil. OBJECTIVE: Our aim was to investigate the mechanisms involved in BSM cell proliferation in preschoolers with severe wheezing, with special attention to the role of mitochondria and calcium signaling. METHODS: Bronchial tissue samples obtained from 12 preschool controls without wheezing and 10 preschoolers with severe wheezing were used to measure BSM mass and establish primary BSM cell cultures. BSM cell proliferation was assessed by manual counting and flow cytometry, ATP content was assessed by bioluminescence, mitochondrial respiration was assessed by using either the Seahorse or Oroboros technique, mitochondrial mass and biogenesis were assessed by immunoblotting, and calcium response to carbachol was assessed by confocal microscopy. The effect of gallopamil was also evaluated. RESULTS: BSM mass, cell proliferation, ATP content, mitochondrial respiration, mass and biogenesis, and calcium response were all increased in preschoolers with severe wheezing compared with in the controls. Gallopamil significantly decreased BSM mitochondrial biogenesis and mass, as well as cell proliferation. CONCLUSION: Mitochondria are key players in BSM cell proliferation in preschoolers with severe wheezing and could represent a potential target to treat BSM remodeling at an early stage of the disease.

Crucial role of fatty acid oxidation in asthmatic bronchial smooth muscle remodelling.

BACKGROUND: Bronchial smooth muscle (BSM) remodelling in asthma is related to an increased mitochondrial biogenesis and enhanced BSM cell proliferation in asthma. Since mitochondria produce the highest levels of cellular energy and fatty acid ß-oxidation is the most powerful way to produce ATP, we hypothesised that, in asthmatic BSM cells, energetic metabolism is shifted towards the ß-oxidation of fatty acids. OBJECTIVES: We aimed to characterise BSM cell metabolism in asthma both in vitro and ex vivo to identify a novel target for reducing BSM cell proliferation. METHODS: 21 asthmatic and 31 non-asthmatic patients were enrolled. We used metabolomic and proteomic approaches to study BSM cells. Oxidative stress, ATP synthesis, fatty acid endocytosis, metabolite production, metabolic capabilities, mitochondrial networks, cell proliferation and apoptosis were assessed on BSM cells. Fatty acid content was assessed in vivo using matrix-assisted laser desorption/ionisation spectrometry imaging. RESULTS: Asthmatic BSM cells were characterised by an increased rate of mitochondrial respiration with a stimulated ATP production and mitochondrial ß-oxidation. Fatty acid consumption was increased in asthmatic BSM both in vitro and ex vivo. Proteome remodelling of asthmatic BSM occurred via two canonical mitochondrial pathways. The levels of carnitine palmitoyl transferase (CPT)2 and low-density lipoprotein (LDL) receptor, which internalise fatty acids through mitochondrial and cell membranes, respectively, were both increased in asthmatic BSM cells. Blocking CPT2 or LDL receptor drastically and specifically reduced asthmatic BSM cell proliferation. CONCLUSION: This study demonstrates a metabolic switch towards mitochondrial ß-oxidation in asthmatic BSM and identifies fatty acid metabolism as a new key target to reduce BSM remodelling in asthma.

Asthmatic bronchial smooth muscle increases rhinovirus replication within the bronchial epithelium.

Rhinovirus (RV) infection of the bronchial epithelium is implicated in the vast majority of severe asthma exacerbations. Interestingly, the susceptibility of bronchial epithelium to RV infection is increased in persons with asthma. Bronchial smooth muscle (BSM) remodeling is an important feature of severe asthma pathophysiology, and its reduction using bronchial thermoplasty has been associated with a significant decrease in the exacerbation rate. We hypothesized that asthmatic BSM can play a role in RV infection of the bronchial epithelium. Using an original co-culture model between bronchial epithelium and BSM cells, we show that asthmatic BSM cells increase RV replication in bronchial epithelium following RV infection. These findings are related to the increased production of CCL20 by asthmatic BSM cells. Moreover, we demonstrate an original downregulation of the activity of the epithelial protein kinase RNA-activated (PKR) antiviral pathway. Finally, we identify a direct bottom-up effect of asthmatic BSM cells on bronchial epithelium susceptibility to RV infection.

Asthmatic Bronchial Smooth Muscle Increases CCL5-Dependent Monocyte Migration in Response to Rhinovirus-Infected Epithelium.

Asthma exacerbations, a major concern in therapeutic strategies, are most commonly triggered by viral respiratory infections, particularly with human rhinovirus (HRV). Infection of bronchial epithelial (BE) cells by HRV triggers inflammation, notably monocyte recruitment. The increase of bronchial smooth muscle (BSM) mass in asthma, a hallmark of bronchial remodeling, is associated with the annual rate of exacerbations. The aim of the present study was to assess whether or not BSM could increase monocyte migration induced by HRV-infected BE. We used an advanced in vitro model of co-culture of human BE cells in air-liquid interface with human BSM cells from control and asthmatic patients. Inflammation triggered by HRV infection (HRV-16, MOI 0.1, 1 h) was assessed at 24 h with transcriptomic analysis and multiplex ELISA. In vitro CD14+ monocyte migration was evaluated with modified Boyden chamber. Results showed that HRV-induced monocyte migration was substantially increased in the co-culture model with asthmatic BSM, compared with control BSM. Furthermore, the well-known monocyte migration chemokine, CCL2, was not involved in this increased migration. However, we demonstrated that CCL5 was further increased in the asthmatic BSM co-culture and that anti-CCL5 blocking antibody significantly decreased monocyte migration induced by HRV-infected BE. Taken together, our findings highlight a new role of BSM cells in HRV-induced inflammation and provide new insights in mucosal immunology which may open new opportunities for prevention and/or treatment of asthma exacerbation.

Design and characterization of a chitosan-enriched fibrin hydrogel for human dental pulp regeneration.

OBJECTIVE: Regenerating a functional dental pulp in the pulpectomized root canal has been recently proposed as a novel therapeutic strategy in dentistry. To reach this goal, designing an appropriate scaffold able to prevent the growth of residual endodontic bacteria, while supporting dental pulp tissue neoformation, is needed. Our aim was to create an innovative cellularized fibrin hydrogel supplemented with chitosan to confer this hydrogel antibacterial property. METHODS: Several fibrin-chitosan formulations were first screened by rheological analyses, and the most appropriate for clinical use was then studied in terms of microstructure (by scanning electron microscopy), antimicrobial effect (analysis of Enterococcus fæcalis growth), dental pulp-mesenchymal stem/stromal cell (DP-MSC) viability and spreading after 7 days of culture (LiveDead® test), DP-MSC ultrastructure and extracellular matrix deposition (transmission electron microscopy), and DP-MSC proliferation and collagen production (RT-qPCR and immunohistochemistry). RESULTS: A formulation associating 10mg/mL fibrinogen and 0.5% (w/w), 40% degree of acetylation, medium molar mass chitosan was found to be relevant in order to forming a fibrin-chitosan hydrogel at cytocompatible pH (# 7.2). Comparative analysis of fibrin-alone and fibrin-chitosan hydrogels revealed a potent antibacterial effect of the chitosan in the fibrin network, and similar DP-MSC viability, fibroblast-like morphology, proliferation rate and type I/III collagen production capacity. SIGNIFICANCE: These results indicate that incorporating chitosan within a fibrin hydrogel would be beneficial to promote human DP tissue neoformation thanks to chitosan antibacterial effect and the absence of significant detrimental effect of chitosan on dental pulp cell morphology, viability, proliferation and collagenous matrix production.

Current challenges in human tooth revitalization.

Tooth vitality and health are related to the presence of a living connective tissue, the dental pulp (DP), in the center of the dental organ. The DP contains the tooth immune defence system that is activated against invading oral cariogenic bacteria during the caries process and the tissue repair/regeneration machinery involved following microorganisms' eradication. However, penetration of oral bacteria into the DP often leads to complete tissue destruction and colonization of the endodontic space by microorganisms. Classical endodontic therapies consist of disinfecting then sealing the endodontic space with a gutta percha-based material. However, re-infections of the endodontic space by oral bacteria can occur, owing to the lack of tightness of the material. Recent findings suggest that regenerating a fully functional pulp tissue may be an ideal therapeutic solution to maintain a tooth defence system that will detect and help manage future injuries. The objective of this paper was to explain the different revascularization and regeneration strategies that have been proposed to reconstitute a living DP tissue and to discuss the main challenges that have to be resolved to improve these therapeutic strategies.