Human Airway Epithelial Cell Cultures Research Articles

Klebsiella pneumoniae peptide hijacks a Streptococcus pneumoniae permease to subvert pneumococcal growth and colonization.

Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a previously uncharacterized peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.

Design and Preclinical Evaluation of a Nanoparticle Vaccine against Respiratory Syncytial Virus Based on the Attachment Protein G.

Human respiratory syncytial virus (RSV) poses a significant human health threat, particularly to infants and the elderly. While efficacious vaccines based on the F protein have recently received market authorization, uncertainties remain regarding the future need for vaccine updates to counteract potential viral drift. The attachment protein G has long been ignored as a vaccine target due to perceived non-essentiality and ineffective neutralization on immortalized cells. Here, we show strong G-based neutralization in fully differentiated human airway epithelial cell (hAEC) cultures that is comparable to F-based neutralization. Next, we designed an RSV vaccine component based on the central conserved domain (CCD) of G fused to self-assembling lumazine synthase (LS) nanoparticles from the thermophile Aquifex aeolicus as a multivalent antigen presentation scaffold. These nanoparticles, characterized by high particle expression and assembly through the introduction of N-linked glycans, showed exceptional thermal and storage stability and elicited potent RSV neutralizing antibodies in a mouse model. In conclusion, our results emphasize the pivotal role of RSV G in the viral lifecycle and culminate in a promising next-generation RSV vaccine candidate characterized by excellent manufacturability and immunogenic properties. This candidate could function independently or synergistically with current F-based vaccines.

SARS-CoV-2-infected human airway epithelial cell cultures uniquely lack interferon and immediate early gene responses caused by other coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of a class of highly pathogenic coronaviruses. The large family of coronaviruses, however, also includes members that cause only mild symptoms, like human coronavirus-229E (HCoV-229E) or OC43 (HCoV-OC43). Unravelling how molecular (and cellular) pathophysiology differs between highly and low pathogenic coronaviruses is important for the development of therapeutic strategies. Here, we analysed the transcriptome of primary human bronchial epithelial cells (PBEC), differentiated at the air-liquid interface (ALI) after infection with SARS-CoV-2, SARS-CoV, Middle East Respiratory Syndrome (MERS)-CoV and HCoV-229E using bulk RNA sequencing. ALI-PBEC were efficiently infected by all viruses, and SARS-CoV, MERS-CoV and HCoV-229E infection resulted in a largely similar transcriptional response. The response to SARS-CoV-2 infection differed markedly as it uniquely lacked the increase in expression of immediate early genes, including FOS, FOSB and NR4A1 that was observed with all other coronaviruses. This finding was further confirmed in publicly available experimental and clinical datasets. Interfering with NR4A1 signalling in Calu-3 lung epithelial cells resulted in a 100-fold reduction in extracellular RNA copies of SARS-CoV-2 and MERS-CoV, suggesting an involvement in virus replication. Furthermore, a lack in induction of interferon-related gene expression characterised the main difference between the highly pathogenic coronaviruses and low pathogenic viruses HCoV-229E and HCoV-OC43. Our results demonstrate a previously unknown suppression of a host response gene set by SARS-CoV-2 and confirm a difference in interferon-related gene expression between highly pathogenic and low pathogenic coronaviruses.

High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity.

Thymic stromal lymphopoietin (TSLP) is a primarily epithelial-derived cytokine that drives type 2 allergic immune responses. Early life viral respiratory infections elicit high TSLP production, which leads to the development of type 2 inflammation and airway hyperreactivity. The goal of this study was to examine in vivo and in vitro the human airway epithelial responses leading to high TSLP production during viral respiratory infections in early infancy. A total of 129 infants (<1-24 m, median age 10 m) with severe viral respiratory infections were enrolled for in vivo (n = 113), and in vitro studies (n = 16). Infants were classified as 'high TSLP' or 'low TSLP' for values above or below the 50th percentile. High versus low TSLP groups were compared in terms of type I-III IFN responses and production of chemokines promoting antiviral (CXCL10), neutrophilic (CXCL1, CXCL5, CXCL8), and type 2 responses (CCL11, CCL17, CCL22). Human infant airway epithelial cell (AEC) cultures were used to define the transcriptomic (RNAseq) profile leading to high versus low TSLP responses in vitro in the absence (baseline) or presence (stimulated) of a viral mimic (poly I:C). Infants in the high TSLP group had greater in vivo type III IFN airway production (median type III IFN in high TSLP 183.2 pg/mL vs. 63.4 pg/mL in low TSLP group, p = 0.007) and increased in vitro type I-III IFN AEC responses after stimulation with a viral mimic (poly I:C). At baseline, our RNAseq data showed that infants in the high TSLP group had significant upregulation of IFN signature genes (e.g., IFIT2, IFI6, MX1) and pro-inflammatory chemokine genes before stimulation. Infants in the high TSLP group also showed a baseline AEC pro-inflammatory state characterized by increased production of all the chemokines assayed (e.g., CXCL10, CXCL8). High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity and increased baseline AEC production of pro-inflammatory chemokines. These findings present a new paradigm underlying the production of TSLP in the human infant airway epithelium following early life viral exposure and shed light on the long-term impact of viral respiratory illnesses during early infancy and beyond childhood.

High-fat diet and palmitic acid amplify airway type 2 inflammation.

Metabolic dysfunction such as elevated levels of saturated fatty acids (SFA) may play a role in obese asthma, but its contribution to airway inflammation remains unclear. We sought to determine the role of high-fat diet (HFD) and palmitic acid (PA), a major form of SFA, in regulating type 2 inflammation. Airway samples from asthma patients with or without obesity, mouse models and human airway epithelial cell culture were utilized to test if SFA amplify type 2 inflammation. Asthma patients with obesity had higher levels of airway PA than asthma patients without obesity. HFD increased the levels of PA in mice, and subsequently enhanced IL-13-induced airway eosinophilic inflammation. PA treatment amplified airway eosinophilic inflammation in mice that were previously exposed to IL-13 or house dust mite. IL-13 alone or in combination with PA increased dipeptidyl peptidase 4 (DPP4) release (soluble DPP4) and/or activity in mouse airways and human airway epithelial cells. Inhibition of DPP4 activity by linagliptin in mice pre-exposed to IL-13 or both IL-13 and PA increased airway eosinophilic and neutrophilic inflammation. Our results demonstrated the exaggerating effect of obesity or PA on airway type 2 inflammation. Up-regulation of soluble DPP4 by IL-13 and/or PA may serve as a mechanism to prevent excessive type 2 inflammation. Soluble DPP4 may have the therapeutic potential in asthma patients with obesity who have an endotype with mixed airway eosinophilic and neutrophilic inflammation.

Globoside and the mucosal pH mediate parvovirus B19 entry through the epithelial barrier.

Parvovirus B19 (B19V) is transmitted primarily via the respiratory route, however, the mechanism involved remains unknown. B19V targets a restricted receptor expressed in erythroid progenitor cells in the bone marrow. However, B19V shifts the receptor under acidic conditions and targets the widely expressed globoside. The pH-dependent interaction with globoside may allow virus entry through the naturally acidic nasal mucosa. To test this hypothesis, MDCK II cells and well-differentiated human airway epithelial cell (hAEC) cultures were grown on porous membranes and used as models to study the interaction of B19V with the epithelial barrier. Globoside expression was detected in polarized MDCK II cells and the ciliated cell population of well-differentiated hAEC cultures. Under the acidic conditions of the nasal mucosa, virus attachment and transcytosis occurred without productive infection. Neither virus attachment nor transcytosis was observed under neutral pH conditions or in globoside knockout cells, demonstrating the concerted role of globoside and acidic pH in the transcellular transport of B19V. Globoside-dependent virus uptake involved VP2 and occurred by a clathrin-independent pathway that is cholesterol and dynamin-dependent. This study provides mechanistic insight into the transmission of B19V through the respiratory route and reveals novel vulnerability factors of the epithelial barrier to viruses.

Calcitriol modifies tight junctions, improves barrier function, and reduces TNF-α-induced barrier leak in the human lung-derived epithelial cell culture model, 16HBE 14o.

Using the 16HBE 14o- human airway epithelial cell culture model, calcitriol (Vitamin D) was shown to improve barrier function by two independent metrics - increased transepithelial electrical resistance (TER) and reduced transepithelial diffusion of 14 C-D-mannitol (Jm ). Both effects were concentration dependent and active out to 168 h post-treatment. Barrier improvement associated with changes in the abundance of specific tight junctional (TJ) proteins in detergent-soluble fractions, most notably decreased claudin-2. TNF-α-induced compromise of barrier function could be attenuated by calcitriol with a concentration dependence similar to that observed for improvement of control barrier function. TNF-α-induced increases in claudin-2 were partially reversed by calcitriol. The ERK 1,2 inhibitor, U0126, itself improved 16HBE barrier function indicating MAPK pathway regulation of 16HBE barrier function. Calcitriol's action was additive to the effect of U0126 in reducing TNF- α -induced barrier compromise, suggesting that calcitriol may be acting through a non-ERK pathway in its blunting of TNF- α - induced barrier compromise. This was supported by calcitriol being without effect on pERK levels elevated by the action of TNF-α. Lack of effect of TNF- α on the death marker, caspase-3, and the inability of calcitriol to decrease the elevated LC3B II level caused by TNF-α, suggest that calcitriol's barrier improvement does not involve a cell death pathway. Calcitriol's improvement of control barrier function was not additive to barrier improvement induced by retinoic acid (Vitamin A). Calcitriol improvement and protection of airway barrier function could in part explain Vitamin D's reported clinical efficacy in COVID-19 and other airway diseases.

Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials.

Cystic fibrosis (CF) is a muco-obstructive lung disease where inflammatory responses due to chronic infection result in the accumulation of neutrophil extracellular traps (NETs) in the airways. NETs are web-like complexes comprised mainly of decondensed chromatin that function to capture and kill bacteria. Prior studies have established excess release of NETs in CF airways increases viscoelasticity of mucus secretions and reduces mucociliary clearance. Despite the pivotal role of NETs in CF disease pathogenesis, current in vitro models of this disease do not account for their contribution. Motivated by this, we developed a new approach to study the pathobiological effects of NETs in CF by combining synthetic NET-like biomaterials, composed of DNA and histones, with an in vitro human airway epithelial cell culture model. To determine the impact of synthetic NETs on airway clearance function, we incorporated synthetic NETs into mucin hydrogels and cell culture derived airway mucus to assess their rheological and transport properties. We found that the addition of synthetic NETs significantly increases mucin hydrogel viscoelasticity. As a result, mucociliary transport in vitro was significantly reduced with the addition of mucus containing synthetic NETs. Given the prevalence of bacterial infection in the CF lung, we also evaluated the growth of Pseudomonas aeruginosa in mucus with or without synthetic NETs. We found mucus containing synthetic NETs promoted microcolony growth and prolonged bacterial survival. Together, this work establishes a new biomaterial enabled approach to study innate immunity mediated airway dysfunction in CF.

Comprehensive single cell analysis of pandemic influenza A virus infection in the human airways uncovers cell-type specific host transcriptional signatures relevant for disease progression and pathogenesis.

The respiratory epithelium constitutes the first line of defense against invading respiratory pathogens, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), and plays a crucial role in the host antiviral response to infection. Despite its importance, however, it remains unknown how individual cell types within the respiratory epithelium respond to IAV infection or how the latter may influence IAV disease progression and pathogenesis. Here, we used single cell RNA sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to influenza virus infection in its natural target cells – namely, the human respiratory epithelium.

New Microfluidic System for Electrochemical Impedance Spectroscopy Assessment of Cell Culture Performance: Design and Development of New Electrode Material.

Electrochemical impedance spectroscopy (EIS) is widely accepted as an effective and non-destructive method to assess cell health during cell-culture. However, there is a lack of compact devices compatible with microfluidic integration and microscopy that could provide the real-time and non-invasive monitoring of cell-cultures using EIS. In this paper, we reported the design and characterization of a modular EIS testing system based on a patented technology. This device was fabricated using easily processable methodologies including screen-printing of the impedance electrodes and molding or micromachining of the cell culture chamber with an easy assembly procedure. Accordingly, to obtain processable, biocompatible and sterilizable electrode materials that lower the impact of interfacial impedance on TEER (Transepithelial electrical resistance) measurements, and to enable concomitant microscopy observations, we optimized the formulation of the electrode inks and the design of the EIS electrodes, respectively. First, electrode materials were based on carbon biocompatible inks enriched with IrOx particles to obtain low interfacial impedance electrodes approaching the performances of classical non-biocompatible Ag/AgCl second-species electrodes. Secondly, we proposed three original electrode designs, which were compared to classical disk electrodes that were optically compatible with microscopy. We assessed the impact of the electrode design on the response of the impedance sensor using COMSOL Multiphysics. Finally, the performance of the impedance spectroscopy devices was assessed in vitro using human airway epithelial cell cultures.

Establishment of well-differentiated camelid airway cultures to study Middle East respiratory syndrome coronavirus

In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in Saudi Arabia and was mostly associated with severe respiratory illness in humans. Dromedary camels are the zoonotic reservoir for MERS-CoV. To investigate the biology of MERS-CoV in camelids, we developed a well-differentiated airway epithelial cell (AEC) culture model for Llama glama and Camelus bactrianus. Histological characterization revealed progressive epithelial cellular differentiation with well-resemblance to autologous ex vivo tissues. We demonstrate that MERS-CoV displays a divergent cell tropism and replication kinetics profile in both AEC models. Furthermore, we observed that in the camelid AEC models MERS-CoV replication can be inhibited by both type I and III interferons (IFNs). In conclusion, we successfully established camelid AEC cultures that recapitulate the in vivo airway epithelium and reflect MERS-CoV infection in vivo. In combination with human AEC cultures, this system allows detailed characterization of the molecular basis of MERS-CoV cross-species transmission in respiratory epithelium.

Immunofluorescence-Mediated Detection of Respiratory Virus Infections in Human Airway Epithelial Cultures.

A diverse collection of viral pathogens target airway epithelial cells for infection, with effects ranging from mild upper respiratory tract symptoms to death of the infected individual. Among these pathogens are recently discovered and/or emergent viruses that sometimes fail to infect commonly used, immortalized cell lines and for which infection phenotypes in the respiratory tract remain unknown. Human airway epithelial cultures have been developed over the past several decades and have proven to be a useful model system in culturing hard‐to‐grow viruses and assaying various features of infection in a physiologically relevant setting. This article includes methods for the generation of well‐differentiated human airway epithelial cell cultures at air‐liquid interface that recapitulate the mucosal epithelium of the trachea/bronchus in vivo. We further detail inoculation of these cultures with respiratory viruses—specifically rhinovirus, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)—and provide a protocol for the detection of double‐stranded RNA or viral antigen–positive cells by immunofluorescence microscopy. These techniques, together with a post‐imaging analysis, can be applied to characterize the efficiency of infection and kinetics of spread within the airway epithelium. Furthermore, these methods can be utilized in conjunction with antibodies against cellular targets to determine cell tropism and colocalization with specific host factors during infection. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human airway epithelial cultures at air‐liquid interface (HAE‐ALI) Basic Protocol 2: Viral inoculation of HAE‐ALI Basic Protocol 3: Immunofluorescence (IF)‐based detection of infected cells in HAE‐ALI

SARS-CoV-2 Virion Infectivity and Cytokine Production in Primary Human Airway Epithelial Cells

The emergence of new SARS-CoV-2 variants and the replacement of preceding isolates have been observed through B.1.1.7, B.1.351, B.1.617.2, and B.1.1.529 lineages (corresponding to alpha, beta, delta, and omicron variants of concern (VoC), respectively). However, there is still a lack of biological evidence to which extent those VoC differ from the ancestral lineages. By exploiting human airway epithelial cell (HAEC) cultures, which closely resemble the human airway architecture and physiology, we report distinctive SARS-CoV-2 tropism in different respiratory tissues. In general, SARS-CoV-2 VoC predominantly infect and replicate in HAEC better than the progenitor USA-WA1 isolate or the BavPat1 isolate, which contains the D614G mutation, even though there is little to no difference between variants regarding their infectivity (i.e., virion-per-vRNA copy ratio). We also observe differential tissue-specific innate immunity activation between the upper and lower respiratory tissues in the presence of the virus. Our study provides better comprehension of the behavior of the different VoC in this physiologically relevant ex vivo model.

Airway mucins promote immunopathology in virus-exacerbated chronic obstructive pulmonary disease.

The respiratory tract surface is protected from inhaled pathogens by a secreted layer of mucus rich in mucin glycoproteins. Abnormal mucus accumulation is a cardinal feature of chronic respiratory diseases, but the relationship between mucus and pathogens during exacerbations is poorly understood. We identified elevations in airway mucin 5AC (MUC5AC) and MUC5B concentrations during spontaneous and experimentally induced chronic obstructive pulmonary disease (COPD) exacerbations. MUC5AC was more sensitive to changes in expression during exacerbation and was therefore more predictably associated with viral load, inflammation, symptom severity, decrements in lung function, and secondary bacterial infections. MUC5AC was functionally related to inflammation, as Muc5ac-deficient (Muc5ac–/–) mice had attenuated RV-induced (RV-induced) airway inflammation, and exogenous MUC5AC glycoprotein administration augmented inflammatory responses and increased the release of extracellular adenosine triphosphate (ATP) in mice and human airway epithelial cell cultures. Hydrolysis of ATP suppressed MUC5AC augmentation of RV-induced inflammation in mice. Therapeutic suppression of mucin production using an EGFR antagonist ameliorated immunopathology in a mouse COPD exacerbation model. The coordinated virus induction of MUC5AC and MUC5B expression suggests that non-Th2 mechanisms trigger mucin hypersecretion during exacerbations. Our data identified a proinflammatory role for MUC5AC during viral infection and suggest that MUC5AC inhibition may ameliorate COPD exacerbations.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host–virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13–treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Disparate temperature-dependent virus-host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium.

Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus-host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.

In adults exposed to COVID-19, hydroxychloroquine did not reduce confirmed or probable COVID-19; trial stopped for futility.

Boulware DR, Pullen MF, Bangdiwala AS, et al. A randomized trial of hydroxychloroquine as postexposure prophylaxis for COVID-19. N Engl J Med. 2020;383:517-25. 32492293.

Inhaled corticosteroids downregulate the SARS-CoV-2 receptor ACE2 in COPD through suppression of type I interferon

BackgroundThe mechanisms underlying altered susceptibility and propensity to severe Coronavirus disease 2019 (COVID-19) disease in at-risk groups such as patients with chronic obstructive pulmonary disease (COPD) are poorly understood. Inhaled corticosteroids (ICSs) are widely used in COPD, but the extent to which these therapies protect or expose patients to risk of severe COVID-19 is unknown.ObjectiveThe aim of this study was to evaluate the effect of ICSs following pulmonary expression of the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme-2 (ACE2).MethodsWe evaluated the effect of ICS administration on pulmonary ACE2 expression in vitro in human airway epithelial cell cultures and in vivo in mouse models of ICS administration. Mice deficient in the type I IFN-α/β receptor (Ifnar1−/−) and administration of exogenous IFN-β were used to study the functional role of type-I interferon signaling in ACE2 expression. We compared sputum ACE2 expression in patients with COPD stratified according to use or nonuse of ICS.ResultsICS administration attenuated ACE2 expression in mice, an effect that was reversed by exogenous IFN-β administration, and Ifnar1−/− mice had reduced ACE2 expression, indicating that type I interferon contributes mechanistically to this effect. ICS administration attenuated expression of ACE2 in airway epithelial cell cultures from patients with COPD and in mice with elastase-induced COPD-like changes. Compared with ICS nonusers, patients with COPD who were taking ICSs also had reduced sputum expression of ACE2.ConclusionICS therapies in COPD reduce expression of the SARS-CoV-2 entry receptor ACE2. This effect may thus contribute to altered susceptibility to COVID-19 in patients with COPD.

Type I and Type III Interferons Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures.

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.

Treatment Options for Coronavirus Disease 2019 in Patients With Reduced or Absent Kidney Function.

Coronavirus disease 2019, the disease caused by the severe acute respiratory syndrome coronavirus 2 virus, was first identified in the Hubei Province of China in late 2019. Currently, the only role for therapy is treatment of the disease, as opposed to postexposure prophylaxis, however multiple clinical trials are currently ongoing for both treatment and prophylaxis. Treating coronavirus disease 2019 relies on two components; the first is inhibition of the viral entrance and replication within the body and the second is inhibition of an exacerbated immune response which can be seen in patients with severe disease. Many drugs have shown in vitro antiviral activity; however, clinical trials have not been as promising. This review summarizes the current data for the most commonly used drugs for coronavirus disease 2019 and will cover the unique factors that may affect the dosing of these medications in patients with CKD. While clinical trials are ongoing, most are in patients with normal kidney function. During a pandemic, when patients with CKD are at higher risk of both infection and death, it is imperative to include patients these patients in the clinical trials.