Human Respiratory Epithelial Cells Research Articles

Virus-like structures for combination antigen protein mRNA vaccination.

Improved vaccination requires better delivery of antigens and activation of the natural immune response. Here we report a lipid nanoparticle system with the capacity to carry antigens, including mRNA and proteins, which is formed into a virus-like structure by surface decoration with spike proteins, demonstrating application against SARS-CoV-2 variants. The strategy uses S1 protein from Omicron BA.1 on the surface to deliver mRNA of S1 protein from XBB.1. The virus-like particle enables specific augmentation of mRNAs expressed in human respiratory epithelial cells and macrophages via the interaction the surface S1 protein with ACE2 or DC-SIGN receptors. Activation of macrophages and dendritic cells is demonstrated by the same receptor binding. The combination of protein and mRNA increases the antibody response in BALB/c mice compared with mRNA and protein vaccines alone. Our exploration of the mechanism of this robust immunity suggests it might involve cross-presentation to diverse subsets of dendritic cells ranging from activated innate immune signals to adaptive immune signals.

Lytic bacteriophages induce the secretion of antiviral and proinflammatory cytokines from human respiratory epithelial cells.

Phage therapy is a therapeutic approach to treat multidrug-resistant (MDR) infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. Using a panel of Pseudomonas aeruginosa phages and human airway epithelial cells (AECs) derived from a person with cystic fibrosis (CF), we determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.

Emissions from plastic incineration induce inflammation, oxidative stress, and impaired bioenergetics in primary human respiratory epithelial cells.

Inhalation exposure to plastic incineration emissions (PIEs) is a problem of increasing human relevance, as plastic production and waste creation have drastically increased since mainstream integration during the 20th century. We investigated the effects of PIEs on human nasal epithelial cells (HNECs) to understand if such exposures cause damage and dysfunction to respiratory epithelia. Primary HNECs from male and female donors were cultured at air-liquid interface (ALI), and 16HBE cells were cultured on coverslips. Smoke condensates were generated from incineration of plastic at flaming (640 °C) and smoldering (500 °C) temperatures, and cells were subsequently exposed to these materials at 5-50 μg/cm2 concentrations. HNECs were assessed for mitochondrial dysfunction and 16HBE cells for glutathione oxidation in real-time analyses. HNEC culture supernatants and total RNA were collected at 4 hours post exposure for cytokine and gene expression analysis, and results show that PIEs can acutely induce inflammation, oxidative stress, and mitochondrial dysfunction in HNECs, and that incineration temperature modifies biological responses. Specifically, condensates from flaming and smoldering PIEs significantly increased HNEC secretion of cytokines IL-8, IL-1β, and IL-13, as well as expression of xenobiotic metabolism pathways and genes such as CYP1A1 and CYP1B1 at 5 and 20 μg/cm2 concentrations. Only 50 μg/cm2 flaming PIEs significantly increased glutathione oxidation in 16HBEs, and decreased respiration and ATP production in HNEC mitochondria. Impact Statement: Our data reveal the impact of incineration temperatures on biological outcomes associated with PIE exposures, emphasizing the importance of temperature as a factor when evaluating respiratory disease associated with PIEs exposure.

Multidimensional assessment of the biological effects of electronic cigarettes on lung bronchial epithelial cells

Cigarette smoke (CS) exposure is known to cause injury to respiratory tract epithelial cells and is a contributing factor in the development of chronic obstructive pulmonary disease and lung cancer. Electronic cigarettes (e-cigarettes) are gaining popularity as a potential substitute for conventional cigarettes due to their potential for aiding smoking cessation. However, the safety of e-cigarettes remains uncertain, and scientific evidence on this topic is still limited. In this study, we aimed to investigate the effects of CS and e-cigarette smoke (ECS) of different flavors on human lung bronchial epithelial cells. Real-time smoke exposure was carried out using an air–liquid interface system, and cell viability was assessed. RNA-Seq transcriptome analysis was performed to compare the differences between CS and ECS. The transcriptome analysis revealed a significantly higher number of differentially expressed genes in CS than in ECS. Moreover, the impact of mint-flavored e-cigarettes on cells was found to be greater than that of tobacco-flavored e-cigarettes, as evidenced by the greater number of differentially expressed genes. These findings provide a reference for future safety research on traditional cigarettes and e-cigarettes, particularly those of different flavors. The use of omics-scale methodologies has improved our ability to understand the biological effects of CS and ECS on human respiratory tract epithelial cells, which can aid in the development of novel approaches for smoking cessation and lung disease prevention.

Heme sequestration by hemophilin from Haemophilus haemolyticus reduces respiratory tract colonization and infection with non-typeable Haemophilus influenzae.

The microbiome provides a critical layer of protection against infection with bacterial pathogens. This protection is accomplished through a variety of mechanisms, including interference with pathogen growth and adherence to host cells. In terms of immune defense, another way to prevent pathogens from establishing infections is by limiting the availability of nutrients, referred to as nutritional immunity. Restricting pathogen access to iron is a central component of this approach. Here, we uncovered an example where these two strategies intersect to impede infection with the respiratory tract bacterial pathogen Haemophilus influenzae. Specifically, we find that a non-pathogenic (commensal) bacterium closely related to H. influenzae called Haemophilus haemolyticus improves protection against H. influenzae by limiting the ability of this pathogen to access iron. These findings suggest that beneficial members of the microbiome improve protection against pathogen infection by effectively contributing to host nutritional immunity.

How do deer respiratory epithelial cells weather the initial storm of SARS-CoV-2 WA1/2020 strain?

The potential infectivity of severe acute respiratory syndrome associated coronavirus-2 (SARS-CoV-2) in animals raises a public health and economic concern, particularly the high susceptibility of white-tailed deer (WTD) to SARS-CoV-2. The disparity in the disease outcome between humans and WTD is very intriguing, as the latter are often asymptomatic, subclinical carriers of SARS-CoV-2. To date, no studies have evaluated the innate immune factors responsible for the contrasting SARS-CoV-2-associated disease outcomes in these mammalian species. A comparative transcriptomic analysis in primary respiratory epithelial cells of human (HRECs) and WTD (Deer-RECs) infected with the SARS-CoV-2 WA1/2020 strain was assessed throughout 48 h post inoculation (hpi). Both HRECs and Deer-RECs were susceptible to virus infection, with significantly (P < 0.001) lower virus replication in Deer-RECs. The number of differentially expressed genes (DEG) gradually increased in Deer-RECs but decreased in HRECs throughout the infection. The ingenuity pathway analysis of DEGs further identified that genes commonly altered during SARS-CoV-2 infection mainly belong to cytokine and chemokine response pathways mediated via interleukin-17 (IL-17) and nuclear factor-κB (NF-κB) signaling pathways. Inhibition of the NF-κB signaling in the Deer-RECs pathway was predicted as early as 6 hpi. The findings from this study could explain the lack of clinical signs reported in WTD in response to SARS-CoV-2 infection as opposed to the severe clinical outcomes reported in humans.IMPORTANCEThis study demonstrated that human and white-tailed deer primary respiratory epithelial cells are susceptible to the SARS-CoV-2 WA1/2020 strain infection. However, the comparative transcriptomic analysis revealed that deer cells could limit viral replication without causing hypercytokinemia by downregulating IL-17 and NF-κB signaling pathways. Identifying differentially expressed genes in human and deer cells that modulate key innate immunity pathways during the early infection will lead to developing targeted therapies toward preventing or mitigating the "cytokine storm" often associated with severe cases of coronavirus disease 19 (COVID-19). Moreover, results from this study will aid in identifying novel prognostic biomarkers in predicting SARS-CoV-2 adaption and transmission in deer and associated cervids.

Inhalation of gaseous hypochlorous acid and its effect on human respiratory epithelial cells in laboratory model systems.

During the disinfection of indoor spaces using gaseous hypochlorous acid (HOCl(g)), inhalation is the most common route of exposure for humans. In this study, an artificial human respiratory tract model was exposed to 12-140 ppb HOCl(g) at an aspiration flow rate of 800 mL/s for 15 h in a 1 m3 chamber. The respiratory tract model was equipped with 5th order bronchi and all gas-contact parts were made of silicone rubber with no other chlorine-consuming substances. The concentration of HOCl(g) reaching the lung pseudo-space was approximately 47.4% of the HOCl(g) concentrations in the chamber and was calculated to be very close to zero when the chamber concentration was less than 20.5 ppb. The disappearance of HOCl(g) during inhalation is likely due to the adsorption of HOCl(g) on the gas-contact silicone rubber surfaces. The cytotoxicity of HOCl(g) on respiratory epithelial cells was also examined using human air-liquid-interface airway tissue models. Human nasal epithelium and bronchiolar epithelium were exposed to 100 ppb and 500 ppb HOCl(g) for 8 h and 5 d, respectively. No significant effects of HOCl(g) on cell viability and ciliary activity were observed in any cell type, indicating that low concentrations of HOCl(g）, less than 500 ppb, had no cytotoxic effect.

Putting bicarbonate on the spot: pharmacological insights for CFTR correction in the airway epithelium.

Introduction: Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) proteins. CFTR controls chloride (Cl-) and bicarbonate (HCO3 -) transport into the Airway Surface Liquid (ASL). We investigated the impact of F508del-CFTR correction on HCO3 - secretion by studying transepithelial HCO3 - fluxes. Methods: HCO3 - secretion was measured by pH-stat technique in primary human respiratory epithelial cells from healthy subjects (WT) and people with CF (pwCF) carrying at least one F508del variant. Its changes after CFTR modulation by the triple combination VX445/661/770 and in the context of TNF-α+IL-17 induced inflammation were correlated to ASL pH and transcriptional levels of CFTR and other HCO3 - transporters of airway epithelia such as SLC26A4 (Pendrin), SLC26A9 and NBCe1. Results: CFTR-mediated HCO3 - secretion was not detected in F508del primary human respiratory epithelial cells. It was rescued up to ∼ 80% of the WT level by VX-445/661/770. In contrast, TNF-α+IL-17 normalized transepithelial HCO3 - transport and increased ASL pH. This was related to an increase in SLC26A4 and CFTR transcript levels. VX-445/661/770 induced an increase in pH only in the context of inflammation. Effects on HCO3 - transport were not different between F508del homozygous and F508del compound heterozygous CF airway epithelia. Conclusion: Our studies show that correction of F508del-CFTR HCO3 - is not sufficient to buffer acidic ASL and inflammation is a key regulator of HCO3 - secretion in CF airways. Prediction of the response to CFTR modulators by theratyping should take into account airway inflammation.

Apoptosis as a mechanism of human respiratory cell death upon exposure to carbon nanotubes

Introduction. Carbon nanotubes (CNTs) are a group of promising nanomaterials for industrial and biomedical applications. There has been shown influence of the physicochemical characteristics of CNTs on the toxic effects, including the ability to cause DNA damage and induce apoptosis. In this study, there was carried out a comparative assessment of pro-apoptotic effects under exposure to single-walled and multi-walled CNTs produced in Russia on human respiratory cells.&#x0D; Materials and methods. Human bronchial epithelial cells BEAS-2B, alveolar epithelial cells A549, and lung fibroblasts MRC5-SV40 were exposed to pristine and purified TUBALLTM SWCNTs and Taunit-M MWCNTs. In cells exposed to 4 concentrations (100, 50, 0.03, 0.0006 μg/ml) of all types of CNTs for 72 hours, the level of mRNA of the P53, BAX and BCL2 genes, as well as the level of reactive oxygen species were assessed.&#x0D; Results. All types of CNTs initiated apoptosis in human respiratory epithelial cells BEAS-2B and A549, but not in MRC5-SV40 lung fibroblasts. BEAS-2B were more sensitive to the effects of MWCNTs, while A549 were more sensitive to pristine SWCNTs. Apoptosis was initiated at low concentrations, including those corresponding to industrial exposures. The mechanism of oxidative stress could act as a factor in triggering apoptosis in lung epithelial cells.&#x0D; Limitations. Relatively short (72 hours) cell incubation time and the use of 2D cell models that do not consider real cell interactions.&#x0D; Conclusion. There were revealed differences in the mechanisms of initiation of the internal pathway of apoptosis and sensitivity to different types of CNTs depending on the type of epithelial cells. Comparative analysis of the initiation of apoptosis by different types of CNTs has shown that there are differences in potential target cells and toxic mechanisms, which should be considered in further studies.

Pathogenesis of Achromobacter xylosoxidans respiratory infections: colonization, persistence, and transcriptome profiling in synthetic cystic fibrosis sputum medium

ABSTRACT Cystic fibrosis (CF) is a genetic disease affecting epithelial ion transport, resulting in thickened mucus and impaired mucociliary clearance. Persons with CF (pwCF) experience life-long infections of the respiratory mucosa caused by a diverse array of opportunists, which are leading causes of morbidity and mortality. In recent years, there has been increased appreciation for the range and diversity of microbes causing CF-related respiratory infections. The introduction of new therapeutics and improved detection methodology has revealed CF-related opportunists such as Achromobacter xylosoxidans ( Ax ). Ax is a Gram-negative bacterial species which is widely distributed in environmental sources and has been increasingly observed in sputa and other samples from pwCF, typically in patients in later stages of CF disease. In this study, we characterized CF clinical isolates of Ax and tested colonization and persistence of Ax in respiratory infection using immortalized human CF respiratory epithelial cells and BALB/c mice. Genomic analyses of clinical Ax isolates showed homologs for factors including flagellar synthesis, antibiotic resistance, and toxin secretion systems. Ax isolates adhered to polarized cultures of CFBE41o- human immortalized CF bronchial epithelial cells and caused significant cytotoxicity and depolarization of cell layers. Ax colonized and persisted in mouse lungs for up to 72 h post infection, with inflammatory consequences that include increased neutrophil influx in the lung, lung damage, cytokine production, and mortality. We also identified genes that are differentially expressed in synthetic CF sputum media. Based on these results, we conclude that Ax is an opportunistic pathogen of significance in CF.

Bacterial respiratory inhibition triggers dispersal of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa grows as a biofilm under many environmental conditions, and the bacterium can disperse from biofilms via highly regulated, dynamic processes. However, physiologic triggers of biofilm dispersal remain poorly understood. Based on prior literature describing dispersal triggered by forms of starvation, we tested bacterial respiratory inhibitors for biofilm dispersal in two models resembling chronic airway infections. Our underlying hypothesis was that respiratory inhibitors could serve as a model for the downstream effects of starvation. We used two experimental conditions. In the first condition, biofilms were grown and dispersed from the surface of airway epithelial cells, and the second condition was a model where biofilms were grown on glass in cell culture media supplemented with host-relevant iron sources. In both biofilm models, the respiratory inhibitors potassium cyanide and sodium azide each triggered biofilm dispersal. We hypothesized that cyanide-induced dispersal was due to respiratory inhibition rather than signaling via an alternative mechanism, and, indeed, if respiration was supported by overexpression of cyanide-insensitive oxidase, dispersal was prevented. Dispersal required the activity of the cyclic-di-GMP regulated protease LapG, reinforcing the role of matrix degradation in dispersal. Finally, we examined the roles of individual phosphodiesterases, previously implicated in dispersal to specific triggers, and found signaling to be highly redundant. Combined deletion of the phosphodiesterases dipA, bifA, and rbdA was required to attenuate the dispersal phenotype. In summary, this work adds insight into the physiology of biofilm dispersal under environmental conditions in which bacterial respiration is abruptly limited. IMPORTANCE The bacterium Pseudomonas aeruginosa grows in biofilm communities that are very difficult to treat in human infections. Growing as a biofilm can protect bacteria from antibiotics and the immune system. Bacteria can leave a biofilm through a process called "dispersal." Dispersed bacteria seed new growth areas and are more susceptible to killing by antibiotics. The triggers for biofilm dispersal are not well understood, and if we understood dispersal better it might lead to the development of new treatments for infection. In this paper, we find that inhibiting P. aeurginosa's ability to respire (generate energy) can trigger dispersal from a biofilm grown in association with human respiratory epithelial cells in culture. The dispersal process requires a protease which is previously known to degrade the biofilm matrix. These findings give us a better understanding of how the biofilm dispersal process works so that future research can discover better ways of clearing bacteria growing in biofilms.

SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SCV2), which has resulted in higher morbidity and mortality rate than other respiratory viral infections, such as Influenza A virus (IAV) infection. Investigating the molecular mechanisms of SCV2-host infection vs IAV is vital in exploring antiviral drug targets against SCV2. We assessed differential gene expression in human nasal cells upon SCV2 or IAV infection using RNA sequencing. Compared to IAV, we observed alterations in both metabolic and cytoskeletal pathways suggestive of epithelial remodeling in the SCV2-infected cells, reminiscent of pathways activated as a response to chronic injury. We found that spike protein interaction with the epithelium was sufficient to instigate these epithelial responses using a SCV2 spike pseudovirus. Specifically, we found downregulation of the mitochondrial markers SIRT3 and TOMM22. Moreover, SCV2 spike infection increased extracellular acidification and decreased oxygen consumption rate in the epithelium. In addition, we observed cytoskeletal rearrangements with a reduction in the actin-severing protein cofilin-1 and an increase in polymerized actin, indicating epithelial cytoskeletal rearrangements. This study revealed distinct epithelial responses to SCV2 infection, with early mitochondrial dysfunction in the host cells and evidence of cytoskeletal remodeling that could contribute to the worsened outcome in COVID-19 patients compared to IAV patients. These changes in cell structure and energetics could contribute to cellular resilience early during infection, allowing for prolonged cell survival and potentially paving the way for more chronic symptoms. IMPORTANCE COVID-19 has caused a global pandemic affecting millions of people worldwide, resulting in a higher mortality rate and concerns of more persistent symptoms compared to influenza A. To study this, we compare lung epithelial responses to both viruses. Interestingly, we found that in response to SARS-CoV-2 infection, the cellular energetics changed and there were cell structural rearrangements. These changes in cell structure could lead to prolonged epithelial cell survival, even in the face of not working well, potentially contributing to the development of chronic symptoms. In summary, these findings represent strategies utilized by the cell to survive the infection but result in a fundamental shift in the epithelial phenotype, with potential long-term consequences, which could set the stage for the development of chronic lung disease or long COVID-19.

Host mRNA deadenylation machinery selectively targets interferon mRNAs, regulating antiviral immunity

Abstract Elucidating host-mediated regulation of viral infection is critical in the development of antivirals and in understanding viral pathogenesis. Here, through high-throughput RNA interference screening, we determine that CNOT2 promotes SARS-CoV-2 infection in human respiratory epithelial cells. CNOT2 is a non-enzymatic subunit of the CCR4-NOT complex, which plays conserved roles in the control of gene expression by deadenylating the poly(A) tails of host mRNAs, thus targeting them for decay. CCR4-NOT can selectively degrade cohorts of mRNAs that are delivered by RNA Binding Proteins. Tristetraprolin (TTP) is among the best characterized of such cargo adapters and is known to recruit mRNAs containing AU-rich elements in their 3′ UTR to CCR4-NOT for deadenylation and decay. We found that in addition to CNOT2, another non-catalytic subunit of CCR4-NOT, CNOT1, in addition to the selectivity factor, TTP, also promote SARS-CoV-2 infection. Global transcriptomics revealed that CNOT2, CNOT1, and TTP specifically regulate interferons (IFNs) and interferon stimulated genes (ISGs) and that depletion of these mRNA decay factors leads to increased levels of IFNs and ISGs. Moreover, the pro-viral activity of CCR4-NOT components and TTP is dependent on IFN signaling: the CCR4-NOT complex has no effect on SARS-CoV-2 infection when IFN signaling is blocked. This led us to discover that CNOT2 promotes the selective deadenylation of IFN mRNAs, and that depletion of CNOT2 leads to increased poly(A) tail lengths of IFN mRNAs. Therefore, we found that the basal levels of IFNs, and thus IFN signaling, are post-transcriptionally regulated by deadenylation machinery, which in turn impacts susceptibility to viral infections including SARS-CoV-2. Supported by grants from NIH (T32-AI-007324)

Human airway and nasal organoids reveal escalating replicative fitness of SARS-CoV-2 emerging variants.

The high transmissibility of SARS-CoV-2 Omicron subvariants was generally ascribed to immune escape. It remained unclear whether the emerging variants have gradually acquired replicative fitness in human respiratory epithelial cells. We sought to evaluate the replicative fitness of BA.5 and earlier variants in physiologically active respiratory organoids. BA.5 exhibited a dramatically increased replicative capacity and infectivity than B.1.1.529 and an ancestral strain wildtype (WT) in human nasal and airway organoids. BA.5 spike pseudovirus showed a significantly higher entry efficiency than that carrying WT or B.1.1.529 spike. Notably, we observed prominent syncytium formation in BA.5-infected nasal and airway organoids, albeit elusive in WT- and B.1.1.529-infected organoids. BA.5 spike-triggered syncytium formation was verified by lentiviral overexpression of spike in nasal organoids. Moreover, BA.5 replicated modestly in alveolar organoids, with a significantly lower titer than B.1.1.529 and WT. Collectively, the higher entry efficiency and fusogenic activity of BA.5 spike potentiated viral spread through syncytium formation in the human airway epithelium, leading to enhanced replicative fitness and immune evasion, whereas the attenuated replicative capacity of BA.5 in the alveolar organoids may account for its benign clinical manifestation.

New insights into how popular electronic cigarette aerosols and aerosol constituents affect SARS-CoV-2 infection of human bronchial epithelial cells

The relationship between the use of tobacco products and SARS-CoV-2 infection is poorly understood and controversial. Few studies have examined the effect of electronic cigarettes (ECs) on SARS-CoV-2 infection. We tested the hypothesis that EC fluids and aerosols with nicotine promote SARS-COV-2 infection by increasing viral entry into human respiratory epithelial cells. Responses of BEAS-2B cells to JUUL aerosols or their individual constituents were compared using three exposure platforms: submerged culture, air–liquid-interface (ALI) exposure in a cloud chamber, and ALI exposure in a Cultex system, which produces authentic heated EC aerosols. In general, nicotine and nicotine + propylene glycol/vegetable glycerin aerosols increased ACE2 (angiotensin converting enzyme 2) levels, the SARS-CoV-2 receptor; and increased the activity of TMPRSS2 (transmembrane serine protease 2), an enzyme essential for viral entry. Lentivirus pseudoparticles with spike protein were used to test viral penetration. Exposure to nicotine, EC fluids, or aerosols altered the infection machinery and increased viral entry into cells. While most data were in good agreement across the three exposure platforms, cells were more responsive to treatments when exposed at the ALI in the Cultex system, even though the exposures were brief and intermittent. While both nicotine and JUUL aerosols increased SARS-CoV-2 infection, JUUL significantly decreased the effect of nicotine alone. These data support the idea that vaping can increase the likelihood of contracting COVID-19 and that e-liquid composition may modulate this effect.

Influence of glycan structure on the colonization of Streptococcus pneumoniae on human respiratory epithelial cells

Virtually all living cells are encased in glycans. They perform key cellular functions such as immunomodulation and cell-cell recognition. Yet, how their composition and configuration affect their functions remains enigmatic. Here, we constructed isogenic capsule-switch mutants harboring 84 types of capsular polysaccharides (CPSs) in Streptococcus pneumoniae. This collection enables us to systematically measure the affinity of structurally related CPSs to primary human nasal and bronchial epithelial cells. Contrary to the paradigm, the surface charge does not appreciably affect epithelial cell binding. Factors that affect adhesion to respiratory cells include the number of rhamnose residues and the presence of human-like glycomotifs in CPS. Besides, pneumococcal colonization stimulated the production of interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractantprotein-1 (MCP-1) in nasal epithelial cells, which also appears to be dependent on the serotype. Together, our results reveal glycomotifs of surface polysaccharides that are likely to be important for colonization and survival in the human airway.

Adaptation of Nontypeable Haemophilus influenzae in Human Airways in COPD: Genome Rearrangements and Modulation of Expression of HMW1 and HMW2.

Chronic obstructive pulmonary disease (COPD) is a common debilitating disorder that is the third most common cause of death globally. Chronic lower airway infection by nontypeable Haemophilus influenzae (NTHi) in adults with COPD increases airway inflammation, causes increased symptoms, and accelerates progressive loss of lung function. Little is known about the mechanisms by which NTHi survives in COPD airways. To explore this question, the present study analyzes, in detail, 14 prospectively collected, serial isolates of a strain that persisted for 543 days in a patient with COPD, including analysis of four gap-free complete genomes. The NTHi genome underwent inversion of a ~400-kb segment three times during persistence. This inversion event resulted in switching of expression of the HMW1A and HMW2A adhesins as the inversion sites are in the promoter regions of HMW1 and HMW2. Regulation of the level of expression of HMW 1 and HMW2 in the human airways was controlled by the ~400-kb inversion and by 7-bp repeats in the HMW promoters. Analysis of knockout mutants of the persistent strain demonstrated that HMW1 and HMW2 proteins both function in the adherence of NTHi to human respiratory epithelial cells during persistence and that HMW1 also facilitates invasion of epithelial cells. An inverse relationship between biofilm formation and HMW1 expression was observed during persistence. This work advances understanding of the mechanisms of persistence of NTHi in COPD airways, which can inform the development of novel interventions to treat and prevent chronic NTHi infection in COPD. IMPORTANCE Nontypeable Haemophilus influenzae (NTHi) persists in the lower airways of adults with chronic obstructive pulmonary disease (COPD) for months to years, increasing airway inflammation that accelerates the progressive loss of lung function. Understanding the mechanisms of persistence in human airways by NTHi is critical in developing novel interventions. Here, in detail, we studied longitudinally collected sequential isolates of a strain of NTHi that persisted in an adult with COPD, including analysis of four gap-free genomes and knockout mutants to elucidate how the genome adapts in human airways. The NTHi genome underwent a genome rearrangement during persistence and this inversion impacted regulation of expression of key virulence phenotypes, including adherence to respiratory epithelial cells, invasion of epithelial cells and biofilm formation. These novel observations advance our understanding of the mechanisms of persistence of NTHi in the airways of adults with COPD.

SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.

Mucociliary clearance augmenting drugs block SARS-CoV-2 replication in human airway epithelial cells.

The coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2 coronavirus, is devastatingly impacting human health. A prominent component of COVID-19 is the infection and destruction of the ciliated respiratory cells, which perpetuates dissemination and disrupts protective mucociliary transport (MCT) function, an innate defense of the respiratory tract. Thus, drugs that augment MCT could improve the barrier function of the airway epithelium and reduce viral replication and, ultimately, COVID-19 outcomes. We tested five agents known to increase MCT through distinct mechanisms for activity against SARS-CoV-2 infection using a model of human respiratory epithelial cells terminally differentiated in an air/liquid interphase. Three of the five mucoactive compounds tested showed significant inhibitory activity against SARS-CoV-2 replication. An archetype mucoactive agent, ARINA-1, blocked viral replication and therefore epithelial cell injury; thus, it was further studied using biochemical, genetic, and biophysical methods to ascertain the mechanism of action via the improvement of MCT. ARINA-1 antiviral activity was dependent on enhancing the MCT cellular response, since terminal differentiation, intact ciliary expression, and motion were required for ARINA-1-mediated anti-SARS-CoV2 protection. Ultimately, we showed that the improvement of cilia movement was caused by ARINA-1-mediated regulation of the redox state of the intracellular environment, which benefited MCT. Our study indicates that intact MCT reduces SARS-CoV-2 infection, and its pharmacologic activation may be effective as an anti-COVID-19 treatment.

Approaches in Hydroxytyrosol Supplementation on Epithelial-Mesenchymal Transition in TGFβ1-Induced Human Respiratory Epithelial Cells.

Hydroxytyrosol (HT) is an olive polyphenol with anti-inflammatory and antioxidant properties. This study aimed to investigate the effect of HT treatment on epithelial-mesenchymal transition (EMT) in primary human respiratory epithelial cells (RECs) isolated from human nasal turbinate. HT dose-response study and growth kinetic study on RECs was performed. Several approaches on HT treatment and TGFβ1 induction with varying durations and methods was studied. RECs morphology and migration ability were evaluated. Vimentin and E-cadherin immunofluorescence staining and Western blotting [E-cadherin, vimentin, SNAIL/SLUG, AKT, phosphorylated (p)AKT, SMAD2/3 and pSMAD2/3] were performed after 72-h treatment. In silico analysis (molecular docking) of HT was performed to evaluate the potential of HT to bind with the TGFβ receptor. The viability of the HT-treated RECs was concentration-dependent, where the median effective concentration (EC50) was 19.04 μg/mL. Testing of the effects of 1 and 10 µg/mL HT revealed that HT suppressed expression of the protein markers vimentin and SNAIL/SLUG while preserving E-cadherin protein expression. Supplementation with HT protected against SMAD and AKT pathway activation in the TGFβ1-induced RECs. Furthermore, HT demonstrated the potential to bind with ALK5 (a TGFβ receptor component) in comparison to oleuropein. TGFβ1-induced EMT in RECs and HT exerted a positive effect in modulating the effects of EMT.