Primary Human Airway Epithelia Research Articles

Identification of a Novel Subset of Human Airway Epithelial Basal Stem Cells.

The basal cell maintains the airway's respiratory epithelium as the putative resident stem cell. Basal cells are known to self-renew and differentiate into airway ciliated and secretory cells. However, it is not clear if every basal cell functions as a stem cell. To address functional heterogeneity amongst the basal cell population, we developed a novel monoclonal antibody, HLO1-6H5, that identifies a subset of KRT5+ (cytokeratin 5) basal cells. We used HLO1-6H5 and other known basal cell-reactive reagents to isolate viable airway subsets from primary human airway epithelium by Fluorescence Activated Cell Sorting. Isolated primary cell subsets were assessed for the stem cell capabilities of self-renewal and differentiation in the bronchosphere assay, which revealed that bipotent stem cells were, at minimum 3-fold enriched in the HLO1-6H5+ cell subset. Crosslinking-mass spectrometry identified the HLO1-6H5 target as a glycosylated TFRC/CD71 (transferrin receptor) proteoform. The HLO1-6H5 antibody provides a valuable new tool for identifying and isolating a subset of primary human airway basal cells that are substantially enriched for bipotent stem/progenitor cells and reveals TFRC as a defining surface marker for this novel cell subset.

A Novel Co-Culture Model Reveals Enhanced CFTR Rescue in Primary Cystic Fibrosis Airway Epithelial Cultures with Persistent Pseudomonas aeruginosa Infection.

People with cystic fibrosis (pwCF) suffer from chronic and recurring bacterial lung infections that begin very early in life and contribute to progressive lung failure. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an ion channel important for maintaining the proper hydration of pulmonary surfaces. When CFTR function is ablated or impaired, airways develop thickened, adherent mucus that contributes to a vicious cycle of infection and inflammation. Therapeutics for pwCF, called CFTR modulators, target the CFTR defect directly, restoring airway surface hydration and mucociliary clearance. However, even with CFTR modulator therapy, bacterial infections persist. To develop a relevant model of diseased airway epithelium, we established a primary human airway epithelium culture system with persistent Pseudomonas aeruginosa infection. We used this model to examine the effects of CFTR modulators on CFTR maturation, CFTR function, and bacterial persistence. We found that the presence of P. aeruginosa increased CFTR mRNA, protein, and function. We also found that CFTR modulators caused a decrease in P. aeruginosa burden. These results demonstrate the importance of including live bacteria to accurately model the CF lung, and that understanding the effects of infection on CFTR rescue by CFTR modulators is critical to evaluating and optimizing drug therapies for all pwCF.

Inhibition of DNA-dependent protein kinase catalytic subunit boosts rAAV transduction of polarized human airway epithelium

Adeno-associated virus 2.5T (AAV2.5T) was selected from the directed evolution of AAV capsid library in human airway epithelia. This study found that recombinant AAV2.5T (rAAV2.5T) transduction of well-differentiated primary human airway epithelia induced a DNA damage response (DDR) characterized by the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all three phosphatidylinositol 3-kinase-related kinases: ataxia telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase (ATR), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). While suppressing the expression of ATR by a specific pharmacological inhibitor or targeted gene silencing inhibited rAAV2.5T transduction, DNA-PKcs inhibition or targeted gene silencing significantly increased rAAV2.5T transgene expression. Notably, DNA-PKcs inhibitors worked as a "booster" to further increase rAAV2.5T transgene expression after treatment with doxorubicin and did not compromise epithelial integrity. Thus, our study provides evidence that DDR is associated with rAAV transduction in well-differentiated human airway epithelia, and DNA-PKcs inhibition has the potential to boost rAAV transduction. These findings highlight that the application of DDR inhibition-associated pharmacological interventions has the potential to increase rAAV transduction and thus to reduce the required vector dose.

Interleukin 13-Induced Inflammation Increases DPP4 Abundance but Does Not Enhance Middle East Respiratory Syndrome Coronavirus Replication in Airway Epithelia.

Chronic pulmonary conditions such as asthma and chronic obstructive pulmonary disease increase the risk of morbidity and mortality during infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). We hypothesized that individuals with such comorbidities are more susceptible to MERS-CoV infection due to increased expression of its receptor, dipeptidyl peptidase 4 (DPP4). We modeled chronic airway disease by treating primary human airway epithelia with the Th2 cytokine interleukin 13 (IL-13), examining how this affected DPP4 protein levels with MERS-CoV entry and replication. IL-13 exposure for 3 days led to greater DPP4 protein abundance, while a 21-day treatment raised DPP4 levels and caused goblet cell metaplasia. Surprisingly, despite this increase in receptor availability, MERS-CoV entry and replication were not significantly affected by IL-13 treatment. Our results suggest that greater DPP4 abundance is likely not the primary mechanism leading to increased MERS severity in the setting of Th2 inflammation. Transcriptional profiling analysis highlighted the complexity of IL-13-induced changes in airway epithelia, including altered expression of genes involved in innate immunity, antiviral responses, and maintenance of the extracellular mucus barrier. These data suggest that additional factors likely interact with DPP4 abundance to determine MERS-CoV infection outcomes.

Bi-directional epithelial-immune communication amplifies inflammasome activation and IL-6 release during SARS-CoV-2 infection

Abstract Increased levels of the pro-inflammatory cytokines IL-1β and IL-6 are linked to severe COVID-19. Here, we investigate the contributions of viral host cells, primary human airway epithelia (HAE), and immune bystanders, peripheral blood mononuclear cells (PBMCs), in the production of these cytokines. Despite having functional inflammasomes and supporting viral replication, HAE do not secrete IL-1β upon SARS-CoV-2 infection. PBMCs do not support productive viral replication, but the SARS-CoV-2 envelope (E) protein primes inflammasomes through stimulation of TLR2. To activate inflammasomes in PBMCs, SARS-CoV-2-infected HAE provide a second signal in the form of damage-associated molecular patterns (DAMPs), which includes mitochondrial and genomic DNA. Co-culture of PBMCs with infected epithelial cells stimulates IL-1β release, while addition of benzonase, STING, or caspase-1 inhibitors, but not NLRP3 inhibitors, abrogates this response. In turn, IL-1β stimulates IL-6 secretion from both populations with a robust response from HAE. Thus, while infection in either cell type in isolation does not yield IL-1β or IL-6, immune bystander detection of SARS-CoV-2 infection in the epithelium stimulates both IL-1β and IL-6 production, amplifying pro-inflammatory cytokine production through cell-cell communication. This circuit is reflected in patient data, as COVID-19 autopsy lungs show hallmarks of inflammasome priming and activation in myeloid populations lacking SARS-CoV-2 antigens. Meta-analysis of single cell RNA sequencing (scRNAseq) data reveals discordant, exacerbated priming of inflammasomes in myeloid cells from severe but not mild COVID-19 patients, indicating this circuit is intensified in severe disease. Supported by grants from NIH R56 AI158314, R01 AI158314, AI029564, AI141333 and CA232109 to J.P.-Y.T; NCI T32CA071341 to K.C.B.; UH3 HL123645, R01 HL136961, P30 DK 065988, P01 HL108808, and from the Cystic Fibrosis Foundation (CFF) BOUCHE19R0 to T.A. and R.C.B; CFF grant BOUCHE19XX0 to R.C.B. and K.O.; CFF grant OKUDA20G0 KO; NC Policy Collaboratory, NIH R01AI157253, and U19 AI100625 to M. T. H.; NSF GRFP 2019255197 to J.F.L. T.A. is a Japan Society for the Promotion of Science Overseas Research Fellow. Y.X. is supported by NSF DMS-191290 and NSF IOS 2107215. Y.X and Y.L.L. are supported by NIH grants R01DE026728 and R01DE031951. Some BSL3 work was performed in the Duke Regional Biocontainment Laboratory (Duke RBL) which received partial support for construction from NIH/NIAID (UC6AI058607).

SARS-CoV-2 infection aggravates cigarette smoke-exposed cell damage in primary human airway epithelia

BackgroundThe coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide pandemic with over 627 million cases and over 6.5 million deaths. It was reported that smoking-related chronic obstructive pulmonary disease (COPD) might be a crucial risk for COVID-19 patients to develop severe condition. As cigarette smoke (CS) is the major risk factor for COPD, we hypothesize that barrier dysfunction and an altered cytokine response in CS-exposed airway epithelial cells may contribute to increased SARS-CoV-2-induced immune response that may result in increased susceptibility to severe disease. The aim of this study was to evaluate the role of CS on SARS-CoV-2-induced immune and inflammatory responses, and epithelial barrier integrity leading to airway epithelial damage.MethodsPrimary human airway epithelial cells were differentiated under air-liquid interface culture. Cells were then exposed to cigarette smoke medium (CSM) before infection with SARS-CoV-2 isolated from a local patient. The infection susceptibility, morphology, and the expression of genes related to host immune response, airway inflammation and damages were evaluated.ResultsCells pre-treated with CSM significantly caused higher replication of SARS-CoV-2 and more severe SARS-CoV-2-induced cellular morphological alteration. CSM exposure caused significant upregulation of long form angiotensin converting enzyme (ACE)2, a functional receptor for SARS-CoV-2 viral entry, transmembrane serine protease (TMPRSS)2 and TMPRSS4, which cleave the spike protein of SARS-CoV-2 to allow viral entry, leading to an aggravated immune response via inhibition of type I interferon pathway. In addition, CSM worsened SARS-CoV-2-induced airway epithelial cell damage, resulting in severe motile ciliary disorder, junctional disruption and mucus hypersecretion.ConclusionSmoking led to dysregulation of host immune response and cell damage as seen in SARS-CoV-2-infected primary human airway epithelia. These findings may contribute to increased disease susceptibility with severe condition and provide a better understanding of the pathogenesis of SARS-CoV-2 infection in smokers.

Regulation of angiotensin-converting enzyme 2 isoforms by type 2 inflammation and viral infection in human airway epithelium

SARS-CoV-2 enters human cells through its main receptor, angiotensin-converting enzyme 2 (ACE2), which constitutes a limiting factor of infection. Recent findings demonstrating novel ACE2 isoforms implicate that this receptor is regulated in a more complex way than previously anticipated. However, it remains unknown how various inflammatory conditions influence the abundance of these ACE2 variants. Hence, we studied expression of ACE2 messenger RNA (mRNA) and protein isoforms, together with its glycosylation and spatial localization in primary human airway epithelium upon allergic inflammation and viral infection. We found that interleukin-13, the main type 2 cytokine, decreased expression of long ACE2 mRNA and reduced glycosylation of full-length ACE2 protein via alteration of N-linked glycosylation process, limiting its availability on the apical side of ciliated cells. House dust mite allergen did not affect the expression of ACE2. Rhinovirus infection increased short ACE2 mRNA, but it did not influence its protein expression. In addition, by screening other SARS-CoV-2 related host molecules, we found that interleukin-13 and rhinovirus significantly regulated mRNA, but not protein of transmembrane serine protease 2 and neuropilin 1. Regulation of ACE2 and other host proteins was comparable in healthy and asthmatic epithelium, underlining the lack of intrinsic differences but dependence on the inflammatory milieu in the airways.

An epithelial-immune circuit amplifies inflammasome and IL-6 responses to SARS-CoV-2

Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.

The interplay between the airway epithelium and tissue macrophages during the SARS-CoV-2 infection.

The first line of antiviral immune response in the lungs is secured by the innate immunity. Several cell types take part in this process, but airway macrophages (AMs) are among the most relevant ones. The AMs can phagocyte infected cells and activate the immune response through antigen presentation and cytokine release. However, the precise role of macrophages in the course of SARS-CoV-2 infection is still largely unknown. In this study, we aimed to evaluate the role of AMs during the SARS-CoV-2 infection using a co-culture of fully differentiated primary human airway epithelium (HAE) and human monocyte-derived macrophages (hMDMs). Our results confirmed abortive SARS-CoV-2 infection in hMDMs, and their inability to transfer the virus to epithelial cells. However, we demonstrated a striking delay in viral replication in the HAEs when hMDMs were added apically after the epithelial infection, but not when added before the inoculation or on the basolateral side of the culture. Moreover, SARS-CoV-2 inhibition by hMDMs seems to be driven by cell-to-cell contact and not by cytokine production. Together, our results show, for the first time, that the recruitment of macrophages may play an important role during the SARS-CoV-2 infection, limiting the virus replication and its spread.

Non-typeable Haemophilus influenzae biofilm formation on primary human airway epithelia cultured at air-liquid interface

Introduction Primary ciliary dyskinesia (PCD) is an inherited heterogeneous disorder associated with defective motile cilia on airway epithelia, resulting in impaired mucociliary function and ultimately, bronchiectasis. Non-typeable Haemophilus influenzae (NTHi) is frequently isolated from PCD airways where it resides in biofilms. It is unclear if biological pathways in response to NTHi infection differ in PCD, contributing to increased susceptibility to biofilm-associated infection. Aim The development of a biologically representative co-culture model to investigate the interactions between PCD airway epithelia and NTHi biofilms. Methods Air-liquid interface (ALI)-cultured non-PCD primary nasal cells were grown in transwells. Barrier properties were assessed by trans-epithelial electrical resistance (TEER), FITC-dextran passage and tight junction staining. Ciliary function was assessed by high-speed video microscopy at 37°C. ALI cultures were infected with a PCD NTHi isolate at multiplicity of infections (MOIs) 10, 50 or 100 and co-cultured to form biofilms over 3 days. TEER was measured pre- and post-infection, NTHi recoverability assessed by conventional culture, and biofilm formation confirmed by scanning electron microscopy (SEM). Results Epithelial culture at ALI was successful, with tight barriers being formed and normal ciliary beat pattern and frequency recorded (mean 14Hz). Following co-culture, TEER increased relative to pre-infection at all MOIs but maximum NTHi recoverability was at MOI 50 (median colony forming units, 6.8x106per cm2). SEM confirmed NTHi biofilm formation at MOI 50. Conclusions A successful NTHi biofilm co-culture model on human primary epithelia was established. This will be used with PCD epithelia to investigate host-pathogen interactions during NTHi biofilm colonisation.

Drug-Free Nasal Spray as a Barrier against SARS-CoV-2 and Its Delta Variant: In Vitro Study of Safety and Efficacy in Human Nasal Airway Epithelia.

The nasal epithelium is a key portal for infection by respiratory viruses such as SARS-CoV-2 and represents an important target for prophylactic and therapeutic interventions. In the present study, we test the safety and efficacy of a newly developed nasal spray (AM-301, marketed as Bentrio) against infection by SARS-CoV-2 and its Delta variant on an in vitro 3D-model of the primary human nasal airway epithelium. Safety was assessed in assays for tight junction integrity, cytotoxicity and cilia beating frequency. Efficacy against SARS-CoV-2 infection was evaluated in pre-viral load and post-viral load application on airway epithelium. No toxic effects of AM-301 on the nasal epithelium were found. Prophylactic treatment with AM-301 significantly reduced viral titer vs. controls over 4 days, reaching a maximum reduction of 99% in case of infection from the wild-type SARS-CoV-2 variant and more than 83% in case of the Delta variant. When AM-301 administration was started 24 h after infection, viral titer was reduced by about 12-folds and 3-folds on Day 4. The results suggest that AM-301 is safe and significantly decelerates SARS-CoV-2 replication in cell culture inhibition assays of prophylaxis (pre-viral load application) and mitigation (post-viral load application). Its physical (non-pharmaceutical) mechanism of action, safety and efficacy warrant additional investigations both in vitro and in vivo for safety and efficacy against a broad spectrum of airborne viruses and allergens.

Bordetella pertussis-infected innate immune cells drive the anti-pertussis response of human airway epithelium

Pertussis is a severe respiratory tract infection caused by Bordetella pertussis. This bacterium infects the ciliated epithelium of the human airways. We investigated the epithelial cell response to B. pertussis infection in primary human airway epithelium (HAE) differentiated at air–liquid interface. Infection of the HAE cells mimicked several hallmarks of B. pertussis infection such as reduced epithelial barrier integrity and abrogation of mucociliary transport. Our data suggests mild immunological activation of HAE by B. pertussis indicated by secretion of IL-6 and CXCL8 and the enrichment of genes involved in bacterial recognition and innate immune processes. We identified IL-1β and IFNγ, present in conditioned media derived from B. pertussis-infected macrophage and NK cells, as essential immunological factors for inducing robust chemokine secretion by HAE in response to B. pertussis. In transwell migration assays, the chemokine-containing supernatants derived from this HAE induced monocyte migration. Our data suggests that the airway epithelium on its own has a limited immunological response to B. pertussis and that for a broad immune response communication with local innate immune cells is necessary. This highlights the importance of intercellular communication in the defense against B. pertussis infection and may assist in the rational design of improved pertussis vaccines.

Inter-individual Variation in Receptor Expression Influences MERS-CoV Infection and Immune Responses in Airway Epithelia.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory infection in humans, with symptom severity that ranges from asymptomatic to severe pneumonia. Known risk factors for severe MERS include male sex, older age, and the presence of various comorbidities. MERS-CoV gains entry into cells by binding its receptor, dipeptidyl peptidase 4 (DPP4), on the surface of airway epithelia. We hypothesized that expression of this receptor might be an additional determinant of outcomes in different individuals during MERS-CoV infection. To learn more about the role of DPP4 in facilitating MERS-CoV infection and spread, we used ELISA and immunofluorescent staining to characterize DPP4 expression in well-differentiated primary human airway epithelia (HAE). We noted wide inter-individual variation in DPP4 abundance, varying by as much as 1000-fold between HAE donors. This variability appears to influence multiple aspects of MERS-CoV infection and pathogenesis, with greater DPP4 abundance correlating with early, robust virus replication and increased cell sloughing. We also observed increased induction of interferon and some interferon-stimulated genes in response to MERS-CoV infection in epithelia with the greatest DPP4 abundance. Overall, our results indicate that inter-individual differences in DPP4 abundance are one host factor contributing to MERS-CoV replication and host defense responses, and highlight how HAE may serve as a useful model for identifying risk factors associated with heightened susceptibility to serious respiratory pathogens.

Antiviral Potential of the Antimicrobial Drug Atovaquone against SARS-CoV-2 and Emerging Variants of Concern.

The antimicrobial medication malarone (atovaquone/proguanil) is used as a fixed-dose combination for treating children and adults with uncomplicated malaria or as chemoprophylaxis for preventing malaria in travelers. It is an inexpensive, efficacious, and safe drug frequently prescribed around the world. Following anecdotal evidence from 17 patients in the provinces of Quebec and Ontario, Canada, suggesting that malarone/atovaquone may present some benefits in protecting against COVID-19, we sought to examine its antiviral potential in limiting the replication of SARS-CoV-2 in cellular models of infection. In VeroE6 expressing human TMPRSS2 and human lung Calu-3 epithelial cells, we show that the active compound atovaquone at micromolar concentrations potently inhibits the replication of SARS-CoV-2 and other variants of concern including the alpha, beta, and delta variants. Importantly, atovaquone retained its full antiviral activity in a primary human airway epithelium cell culture model. Mechanistically, we demonstrate that the atovaquone antiviral activity against SARS-CoV-2 is partially dependent on the expression of TMPRSS2 and that the drug can disrupt the interaction of the spike protein with the viral receptor, ACE2. Additionally, spike-mediated membrane fusion was also reduced in the presence of atovaquone. In the United States, two clinical trials of atovaquone administered alone or in combination with azithromycin were initiated in 2020. While we await the results of these trials, our findings in cellular infection models demonstrate that atovaquone is a potent antiviral FDA-approved drug against SARS-CoV-2 and other variants of concern in vitro.

Influenza A induces lactate formation to inhibit type I IFN in primary human airway epithelium

SummaryPathogenic viruses induce metabolic changes in host cells to secure the availability of biomolecules and energy to propagate. Influenza A virus (IAV) and severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) both infect the human airway epithelium and are important human pathogens. The metabolic changes induced by these viruses in a physiologically relevant human model and how this affects innate immune responses to limit viral propagation are not well known. Using an ex vivo model of pseudostratified primary human airway epithelium, we here demonstrate that infection with both IAV and SARS-CoV-2 resulted in distinct metabolic changes including increases in lactate dehydrogenase A (LDHA) expression and LDHA-mediated lactate formation. Interestingly, LDHA regulated both basal and induced mitochondrial anti-viral signaling protein (MAVS)-dependent type I interferon (IFN) responses to promote IAV, but not SARS-CoV-2, replication. Our data demonstrate that LDHA and lactate promote IAV but not SARS-CoV-2 replication by inhibiting MAVS-dependent induction of type I IFN in primary human airway epithelium.

The TMPRSS2 Inhibitor Nafamostat Reduces SARS-CoV-2 Pulmonary Infection in Mouse Models of COVID-19.

ABSTRACTThe coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality on a global scale. The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiates host cell entry when its spike protein (S) binds to its receptor, angiotensin-converting enzyme 2 (ACE2). In airway epithelia, the spike protein is cleaved by the cell surface protease TMPRSS2, facilitating membrane fusion and entry at the cell surface. This dependence on TMPRSS2 and related proteases suggests that protease inhibitors might limit SARS-CoV-2 infection in the respiratory tract. Here, we tested two serine protease inhibitors, camostat mesylate and nafamostat mesylate, for their ability to inhibit entry of SARS-CoV-2 and that of a second pathogenic coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). Both camostat and nafamostat reduced infection in primary human airway epithelia and in the Calu-3 2B4 cell line, with nafamostat exhibiting greater potency. We then assessed whether nafamostat was protective against SARS-CoV-2 in vivo using two mouse models. In mice sensitized to SARS-CoV-2 infection by transduction with human ACE2, intranasal nafamostat treatment prior to or shortly after SARS-CoV-2 infection significantly reduced weight loss and lung tissue titers. Similarly, prophylactic intranasal treatment with nafamostat reduced weight loss, viral burden, and mortality in K18-hACE2 transgenic mice. These findings establish nafamostat as a candidate for the prevention or treatment of SARS-CoV-2 infection and disease pathogenesis.

The SARS-CoV-2 Transcriptome and the Dynamics of the S Gene Furin Cleavage Site in Primary Human Airway Epithelia.

ABSTRACTThe spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686. Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent.

C5aR inhibition of nonimmune cells suppresses inflammation and maintains epithelial integrity in SARS-CoV-2–infected primary human airway epithelia

BackgroundExcessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and is associated with enhanced pathogenicity and mortality.ObjectiveComplement hyperactivation promotes lung injury and was observed in patients suffering from Middle East respiratory syndrome-related coronavirus, SARS-CoV-1, and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells on exposure to SARS-CoV-2 in terms of complement component 3 (C3)-mediated effects.MethodsFor this, we used highly differentiated primary human 3-dimensional tissue models infected with SARS-CoV-2 patient isolates. On infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms, and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses, and transepithelial electrical resistance measurements.ResultsHere, we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2–infected 3-dimensional cultures secreted significantly higher levels of C3a and the proinflammatory cytokines IL-6, monocyte chemoattractant protein 1, IL-1α, and RANTES.ConclusionsCrucially, we illustrate here for the first time that targeting the anaphylotoxin receptors C3a receptor and C5a receptor in nonimmune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.

SARS-CoV-2 triggers an MDA-5-dependent interferon response which is unable to control replication in lung epithelial cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to acute respiratory distress syndrome, and death in the most severe cases. Immune dysregulation with altered innate cytokine responses is thought to contribute to disease severity. Here, we characterized in depth host cell responses against SARS-CoV-2 in primary human airway epithelia (HAE) and immortalized cell lines. Our results demonstrate that primary HAE and model cells elicit a robust induction of type I and III interferons (IFNs). Importantly, we show for the first time that melanoma differentiation associated gene (MDA)-5 is the main sensor of SARS-CoV-2 in lung cells. IFN exposure strongly inhibited viral replication and de novo production of infectious virions. However, despite high levels of IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication in lung cells, contrary to what was previously reported in intestinal epithelial cells. Altogether, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.IMPORTANCE Mammalian cells express sensors able to detect specific features of pathogens and induce the interferon response, which is one of the first line of defenses against viruses and help controlling viral replication. The mechanisms and impact of SARS-CoV-2 sensing in lung epithelial cells remained to be deciphered. In this study, we report that despite a high production of type I and III interferons specifically induced by MDA-5-mediated sensing of SARS-CoV-2, primary and immortalized lung epithelial cells are unable to control viral replication. However, exogenous interferons potently inhibited replication, if provided early upon viral exposure. A better understanding of the ambiguous interplay between the interferon response and SARS-CoV-2 replication is essential to guide future therapeutical interventions.

RNA demethylation by FTO stabilizes the FOXJ1 mRNA for proper motile ciliogenesis.

Adenosine N6-methylation (m6A) is one of the most pervasive mRNA modifications, and yet the physiological significance of m6A removal (demethylation) remains elusive. Here, we report that the m6A demethylase FTO functions as a conserved regulator of motile ciliogenesis. Mechanistically, FTO demethylates and thereby stabilizes the mRNA that encodes the master ciliary transcription factor FOXJ1. Depletion of Fto in Xenopus laevis embryos caused widespread motile cilia defects, and Foxj1 was identified as one of the major phenocritical targets. In primary human airway epithelium, FTO depletion also led to FOXJ1 mRNA destabilization and a severe loss of ciliated cells with an increase of neighboring goblet cells. Consistently, Fto knockout mice showed strong asthma-like phenotypes upon allergen challenge, a result owing to defective ciliated cells in the airway epithelium. Altogether, our study reveals a conserved role of the FTO-FOXJ1 axis in embryonic and homeostatic motile ciliogenesis.