Infection Of Bronchial Epithelial Cells Research Articles

Septin-dependent defense mechanisms against Pseudomonas aeruginosa are stalled in cystic fibrosis bronchial epithelial cells

Airway epithelial cells form a physical barrier against inhaled pathogens and coordinate innate immune responses in the lungs. Bronchial cells in people with cystic fibrosis (pwCF) are colonized by Pseudomonas aeruginosa because of the accumulation of mucus in the lower airways and an altered immune response. This leads to chronic inflammation, lung tissue damage, and accelerated decline in lung function. Thus, identifying the molecular factors involved in the host response in the airways is crucial for developing new therapeutic strategies. The septin (SEPT) cytoskeleton is involved in tissue barrier integrity and anti-infective responses. SEPT7 is critical for maintaining SEPT complexes and for sensing pathogenic microbes. In the lungs, SEPT7 may be involved in the epithelial barrier resistance to infection; however, its role in cystic fibrosis (CF) P. aeruginosa infection is unknown. This study aimed to investigate the role of SEPT7 in controlling P. aeruginosa infection in bronchial epithelial cells, particularly in CF. The study findings showed that SEPT7 encages P. aeruginosa in bronchial epithelial cells and its inhibition downregulates the expression of other SEPTs. In addition, P. aeruginosa does not regulate SEPT7 expression. Finally, we found that inhibiting SEPT7 expression in bronchial epithelial cells (BEAS-2B 16HBE14o- and primary cells) resulted in higher levels of internalized P. aeruginosa and decreased IL-6 production during infection, suggesting a crucial role of SEPT7 in the host response against this bacterium. However, these effects were not observed in the CF cells (16HBE14o-/F508del and primary cells) which may explain the persistence of infection in pwCF. The study findings suggest the modification of SEPT7 expression as a potential approach for the anti-infective control of P. aeruginosa, particularly in CF.

Exploring the secrets of virus entry: the first respiratory syncytial virus carrying beta lactamase.

Respiratory Syncytial Virus (RSV) presents a significant health threat, especially to young children. In-depth understanding of RSV entry mechanisms is essential for effective antiviral development. This study introduces an innovative RSV variant, featuring the fusion of the beta-lactamase (BlaM) enzyme with the RSV-P phosphoprotein, providing a versatile tool for dissecting viral entry dynamics. Using the AlphaFold2 algorithm, we modeled the tertiary structure of the P-BlaM chimera, revealing structural similarities with both RSV-P and BlaM. Functional assessments, utilizing flow cytometry, quantified beta-lactamase activity and GFP expression in infected bronchial epithelial cells. Western blot analysis confirmed the integrity of P-BlaM within virions. The modeled P-BlaM chimera exhibited structural parallels with RSV-P and BlaM. Functional assays demonstrated robust beta-lactamase activity in recombinant virions, confirming successful P-BlaM incorporation as a structural protein. Quercetin, known for its antiviral properties, impeded viral entry by affecting virion fusion. Additionally, Ulixertinib, an ERK-1/2 inhibitor, significantly curtailed viral entry, implicating ERK-1/2 pathway signaling. Our engineered RSV-P-BlaM chimera emerges as a valuable tool, illuminating RSV entry mechanisms. Structural and functional analyses unveil potential therapeutic targets. Quercetin and Ulixertinib, identified as distinct stage inhibitors, show promise for targeted antiviral strategies. Time-of-addition assays pinpoint quercetin's specific interference stage, advancing our comprehension of RSV entry and guiding future antiviral developments.

SARS-CoV-2-induced disruption of a vascular bed in a microphysiological system caused by type-I interferon from bronchial organoids.

Blood vessels show various COVID-19-related conditions including thrombosis and cytokine propagation. Existing in vitro blood vessel models cannot represent the consequent changes in the vascular structure or determine the initial infection site, making it difficult to evaluate how epithelial and endothelial tissues are damaged. Here, we developed a microphysiological system (MPS) that co-culture the bronchial organoids and the vascular bed to analyze infection site and interactions. In this system, virus-infected organoids caused damage in vascular structure. However, vasculature was not damaged or infected when the virus was directly introduced to vascular bed. The knockout of interferon-related genes and inhibition of the JAK/STAT pathway reduced the vascular damage, indicating the protective effect of interferon response suppression. The results demonstrate selective infection of bronchial epithelial cells and vascular damage by cytokines and also indicate the applicability of MPS to investigate how the infection influences vascular structure and functions.

Polyphyllin II inhibits NLPR3 inflammasome activation and inflammatory response of Mycobacterium tuberculosis-infected human bronchial epithelial cells.

The bronchial infection by Mycobacterium tuberculosis (Mtb) is increasing in prevalence and severity worldwide. Despite appropriate tuberculosis treatment, most patients still develop bronchial stenosis, which often leads to disability. Polyphyllin II (PP2) is a steroidal saponin extracted from Rhizoma Paridis. In this study, we aimed to explore the effect of PP2 on the advancement of Mtb-induced bronchial infection. The effects of PP2 on cell viability were measured by using MTT and lactate dehydrogenase (LDH) kit. The mRNA and protein levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-8 were elucidated by RT-qPCR and ELISA, respectively. The expression of NLR family pyrin domain containing 3 (NLRP3) related inflammasome (NLRP3, IL-1β, and cleaved-caspase-1) and the activated degree of protein kinase B (AKT)/nuclear factor-kappa B (NF-kB; p-AKT and p-NF-κB) were detected by Western blotting. PP2 at 0, 1, 5, and 10 μM had little cytotoxicity on 16HBE cells. PP2 inhibited Mtb-induced cell proliferation and decreased LDH levels. We further found that PP2 could suppress Mtb-induced inflammatory responses and activation of NLPR3 inflammasome. Additionally, the role of PP2 in Mtb is associated with the AKT/NF-kB signaling pathway. PP2 inhibited Mtb infection in bronchial epithelial cells, by inhibiting Mtb-induced inflammatory reactions and activation of NLPR3 inflammasome. These effects may be exerted by suppressing the AKT/NF-kB pathway, which will provide a prospective treatment.

Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner

BackgroundLung infections caused by Streptococcus pneumonia are a global leading cause of death. The reactive oxygen species H2O2 is one of the virulence factors of Streptococcus pneumoniae. The Golgi apparatus is essential for the inflammatory response of a eukaryotic cell. Golgi fragmentation was previously shown to be induced by bacterial pathogens and in response to H2O2 treatment. This led us to investigate whether the Golgi apparatus is actively involved and targeted in host–pathogen interactions during pneumococcal infections.MethodsFollowing in vitro infection of BEAS-2B bronchial epithelial cells with Streptococcus pneumoniae for 16 h, the structure of the Golgi apparatus was assessed by fluorescence staining of the Golgi-associated protein, Golgin-97. To investigate the effect of H2O2 production on Golgi structure, BEAS-2B cells were treated with H2O2 or the H2O2 degrading enzyme Catalase, prior to Golgi staining. Artificial disruption of the Golgi apparatus was induced by treatment of cells with the GBF1 inhibitor, Golgicide A. A proinflammatory cellular response was induced by treatment of cells with the bacterial cell wall component and TLR4 ligand lipoteichoic acid.ResultsIn vitro infection of bronchial epithelial cells with wild type Streptococcus pneumoniae led to a disruption of normal Golgi structure. Golgi fragmentation was not observed after deletion of the pneumococcal H2O2-producing gene, spxB, or neutralization of H2O2 by catalase treatment, but could be induced by H2O2 treatment. Streptococcus pneumoniae infection significantly reduced host cell protein glycosylation and artificial disruption of Golgi structure significantly reduced bacterial adherence, but increased bacterial counts in the supernatant. To understand if this effect depended on cell-contact or soluble factors, pneumococci were treated with cell-supernatant of cells treated with Golgicide A and/or lipoteichoic acid. This approach revealed that lipoteichoic acid conditioned medium inhibits bacterial replication in presence of host cells. In contrast, artificial Golgi fragmentation by Golgicide A treatment prior to lipoteichoic acid treatment rescued bacterial replication. This effect was associated with an increase of IL-6 and IL-8 in the supernatant of lipoteichoic acid treated cells. The increased cytokine release was abolished if cells were treated with Golgicide A prior to lipoteichoic acid treatment.ConclusionStreptococcus pneumoniae disrupts the Golgi apparatus in an H2O2-dependent manner, thereby inhibiting paracrine anti-infective mechanisms.EnamKsCfhZcCbpPUq5E2ztVideo

Global quantitative proteomic analysis profiles of host protein expression in response to enterovirus A71 infection in bronchial epithelial cells based on tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS uncovers the key role of proteasome in virus replication

Enterovirus A71 (EV-A71) is a neurotropic human pathogen which mainly caused hand, foot and mouth disease (HFMD) mostly in children under 5 years-old. Generally, EV-A71-associated HFMD is a relatively self-limiting febrile disease, but there will still be a small percentage of patients with rapid disease progression and severe neurological complications. To date, the underlying mechanism of EV-A71 inducing pathological injury of central nervous system (CNS) remains largely unclear. It has been investigated and discussed the changes of mRNA, miRNA and circRNA expression profile during infection by EV-A71 in our previous studies. However, these studies were only analyzed at the RNA level, not at the protein level. It's the protein levels that ultimately do the work in the body. Here, to address this, we performed a tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS approach to quantitatively identify cellular proteome changes at 24 h post-infection (hpi) in EV-A71-infected 16HBE cells. In total, 6615 proteins were identified by using TMT coupled with LC-MS/MS in this study. In the EV-A71- and mock-infected groups, 210 differentially expressed proteins were found, including 86 upregulated and 124 downregulated proteins, at 24 hpi. To ensure the validity and reliability of the proteomics data, 3 randomly selected proteins were verified by Western blot and Immunofluorescence analysis, and the results were consistent with the TMT results. Subsequently, functional enrichment analysis indicated that the up-regulated and down-regulated proteins were individually involved in various biological processes and signaling pathways, including metabolic process, AMPK signaling pathway, Neurotrophin signaling pathway, Viral myocarditis, GABAergic synapse, and so on. Moreover, among these enriched functional analysis, the “Proteasome” pathway was up-regulated, which has caught our attention. Inhibition of proteasome was found to obviously suppress the EV-A71 replication. Finally, further in-depth analysis revealed that these differentially expressed proteins contained distinct domains and localized in different subcellular components. Taken together, our data provided a comprehensive view of host cell response to EV-A71 and identified host proteins may lead to better understanding of the pathogenic mechanisms and host responses to EV-A71 infection, and also to the identification of new therapeutic targets for EV-A71 infection.

Lung Epithelial Cells from Obese Patients Have Impaired Control of SARS-CoV-2 Infection.

Obesity is known to increase the complications of the COVID-19 coronavirus disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the exact mechanisms of SARS-CoV-2 infection in obese patients have not been clearly elucidated. This study aims to better understand the effect of obesity on the course of SARS-CoV-2 infection and identify candidate molecular pathways involved in the progression of the disease, using an in vitro live infection model and RNA sequencing. Results from this study revealed the enhancement of viral load and replication in bronchial epithelial cells (NHBE) from obese subjects at 24 h of infection (MOI = 0.5) as compared to non-obese subjects. Transcriptomic profiling via RNA-Seq highlighted the enrichment of lipid metabolism-related pathways along with LPIN2, an inflammasome regulator, as a unique differentially expressed gene (DEG) in infected bronchial epithelial cells from obese subjects. Such findings correlated with altered cytokine and angiotensin-converting enzyme-2 (ACE2) expression during infection of bronchial cells. These findings provide a novel insight on the molecular interplay between obesity and SARS-CoV-2 infection. In conclusion, this study demonstrates the increased SARS-CoV-2 infection of bronchial epithelial cells from obese subjects and highlights the impaired immunity which may explain the increased severity among obese COVID-19 patients.

Comprehensive profiling and characterization of cellular microRNAs in response to coxsackievirus A10 infection in bronchial epithelial cells

Coxsackievirus A10 (CV-A10), the causative agent of hand, foot, and mouth disease (HFMD), caused a series of outbreaks in recent years and often leads to neurological impairment, but a clear understanding of the disease pathogenesis and host response remains elusive. Cellular microRNAs (miRNAs), a large family of non-coding RNA molecules, have been reported to be key regulators in viral pathogenesis and virus-host interactions. However, the role of host cellular miRNAs defensing against CV-A10 infection is still obscure. To address this issue, we systematically analyzed miRNA expression profiles in CV-A10-infected 16HBE cells by high-throughput sequencing methods in this study. It allowed us to successfully identify 312 and 278 miRNAs with differential expression at 12 h and 24 h post-CV-A10 infection, respectively. Among these, 4 miRNAs and their target genes were analyzed by RT-qPCR, which confirmed the sequencing data. Gene target prediction and enrichment analysis revealed that the predicted targets of these miRNAs were significantly enriched in numerous cellular processes, especially in regulation of basic physical process, host immune response and neurological impairment. And the integrated network was built to further indicate the regulatory roles of miRNAs in host-CV-A10 interactions. Consequently, our findings could provide a beneficial basis for further studies on the regulatory roles of miRNAs relevant to the host immune responses and neuropathogenesis caused by CV-A10 infection.

IL-25 blockade augments antiviral immunity during respiratory virus infection

IL-25 is implicated in the pathogenesis of viral asthma exacerbations. However, the effect of IL-25 on antiviral immunity has yet to be elucidated. We observed abundant expression and colocalization of IL-25 and IL-25 receptor at the apical surface of uninfected airway epithelial cells and rhinovirus infection increased IL-25 expression. Analysis of immune transcriptome of rhinovirus-infected differentiated asthmatic bronchial epithelial cells (BECs) treated with an anti-IL-25 monoclonal antibody (LNR125) revealed a re-calibrated response defined by increased type I/III IFN and reduced expression of type-2 immune genes CCL26, IL1RL1 and IL-25 receptor. LNR125 treatment also increased type I/III IFN expression by coronavirus infected BECs. Exogenous IL-25 treatment increased viral load with suppressed innate immunity. In vivo LNR125 treatment reduced IL-25/type 2 cytokine expression and increased IFN-β expression and reduced lung viral load. We define a new immune-regulatory role for IL-25 that directly inhibits virus induced airway epithelial cell innate anti-viral immunity.

Asthmatic bronchial smooth muscle decreases epithelial response to rhinovirus infection through a CCL-20/EIF2AK2 dependent pathway

Asthma is a very frequent airway disease affecting 6 to 20% of the population of western European countries. The disease is characterized by an increase of bronchial smooth muscle (BSM) mass correlated with the severity of asthma. Recent clinical investigations have shown a link between BSM mass and the exacerbations rate. Exacerbations are mainly the consequence from inhalation of aero-contaminant acting on bronchial epithelium cells with the most important trigger being the human rhinovirus (RV). However, the role of the BSM in infections of the epithelium by the RV is still unknown. Asthmatic and non-asthmatic BSM cells and bronchial epithelial cells (BEC) were obtained from patient biopsies (COBRA cohort). BEC were cultured and differentiated in Air-Liquide- Interface. RV infection was measured using qPCR. Cytokines concentrations were assessed using ELISA assays. Proteomic and transcriptomic data were analyzed using IPA (Qiagen). Using a co-culture model between BEC and BSM cells, we found an increased RV replication in BEC induced by asthmatic BSM cells. These findings were related to an increased production of CCL20 by asthmatic BSM cells. Moreover, we demonstrated a down regulation of the epithelial PKR/eIF2α antiviral pathway through this BSM-related CCL20 overproduction. Using both in vitro and ex vivo experiments, we demonstrated, for the first time, a direct bottom-up effect of BSM cells in severe asthmatic patients on BE susceptibility to RV infection. Asthmatic BSM cells are able to increase RV infection in BEC. Proteomic analysis highlight modified expression of CCL20 in BSM cells, showing that asthmatic BSM cells increase RV replication in BE through CCL-20 production.

The role of type I interferon in the treatment of COVID-19.

Although significant research has been done to find effective drugs against coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), no definite effective drug exists. Thus, research has now shifted towards immunomodulatory agents other than antivirals. In this review, we aim to describe the latest findings on the role of type I interferon (IFN)‐mediated innate antiviral response against SARS‐CoV‐2 and discuss the use of IFNs as a medication for COVID‐19. A growing body of evidence has indicated a promoting active but delayed IFNs response to SARS‐CoV‐2 and Middle East respiratory syndrome coronavirus in infected bronchial epithelial cells. Studies have demonstrated that IFNs' administration before the viral peak and the inflammatory phase of disease could offer a highly protective effect. However, IFNs' treatment during the inflammatory and severe stages of the disease causes immunopathology and long‐lasting harm for patients. Therefore, it is critical to note the best time window for IFNs' administration. Further investigation of the clinical effectiveness of interferon for patients with mild to severe COVID‐19 and its optimal timing and route of administration can be beneficial in finding a safe and effective antiviral therapy for the COVID‐19 disease.

Infection of Bronchial Epithelial Cells by the Human Adenoviruses A12, B3, and C2 Differently Regulates the Innate Antiviral Effector APOBEC3B.

Human adenoviruses (HAdVs) are a large family of DNA viruses that include more than 100 genotypes divided into seven species (A to G) and induce respiratory tract infections, gastroenteritis, and conjunctivitis. Genetically modified adenoviruses are also used as vaccines, gene therapies, and anticancer treatments. The APOBEC3s are a family of cytidine deaminases that restrict viruses by introducing mutations in their genomes. Viruses developed different strategies to cope with the APOBEC3 selection pressure, but nothing is known on the interplay between the APOBEC3s and the HAdVs. In this study, we focused on three HAdV strains: the B3 and C2 strains, as they are very frequent, and the A12 strain, which is less common but is oncogenic in animal models. We demonstrated that the three HAdV strains induce a similar APOBEC3B upregulation at the transcriptional level. At the protein level, however, APOBEC3B is abundantly expressed during HAdV-A12 and -C2 infection and shows a nuclear distribution. On the contrary, APOBEC3B is barely detectable in HAdV-B3-infected cells. APOBEC3B deaminase activity is detected in total protein extracts upon HAdV-A12 and -C2 infection. Bioinformatic analysis demonstrates that the HAdV-A12 genome bears a stronger APOBEC3 evolutionary footprint than that of the HAdV-C2 and HAdV-B3 genomes. Our results show that HAdV infection triggers the transcriptional upregulation of the antiviral innate effector APOBEC3B. The discrepancies between the APOBEC3B mRNA and protein levels might reflect the ability of some HAdV strains to antagonize the APOBEC3B protein. These findings point toward an involvement of APOBEC3B in HAdV restriction and evolution. IMPORTANCE The APOBEC3 family of cytosine deaminases has important roles in antiviral innate immunity and cancer. Notably, APOBEC3A and APOBEC3B are actively upregulated by several DNA tumor viruses and contribute to transformation by introducing mutations in the cellular genome. Human adenoviruses (HAdVs) are a large family of DNA viruses that cause generally asymptomatic infections in immunocompetent adults. HAdVs encode several oncogenes, and some HAdV strains, like HAdV-A12, induce tumors in hamsters and mice. Here, we show that HAdV infection specifically promotes the expression of the APOBEC3B gene. We report that infection with the A12 strain induces a strong expression of an enzymatically active APOBEC3B protein in bronchial epithelial cells. We provide bioinformatic evidence that HAdVs' genomes and notably the A12 genome are under APOBEC3 selection pressure. Thus, APOBEC3B might contribute to adenoviral restriction, diversification, and oncogenic potential of particular strains.

Associations between COVID-19 and skin conditions identified through epidemiology and genomic studies

BackgroundCoronavirus disease 2019 (COVID-19) is commonly associated with skin manifestations, and may also exacerbate existing skin diseases, yet the relationship between COVID-19 and skin diseases remains unclear.ObjectiveBy investigating this relationship through a multiomics approach, we sought to ascertain whether patients with skin conditions are more susceptible to COVID-19.MethodsWe conducted an epidemiological study and then compared gene expression across 9 different inflammatory skin conditions and severe acute respiratory syndrome coronavirus 2–infected bronchial epithelial cell lines, and then performed a genome-wide association study transdisease meta-analysis between COVID-19 susceptibility and 2 skin diseases (psoriasis and atopic dermatitis).ResultsSkin conditions, including psoriasis and atopic dermatitis, increase the risk of COVID-19 (odds ratio, 1.55; P = 1.4 × 10−9) but decrease the risk of mechanical ventilation (odds ratio, 0.22; P = 8.5 × 10−5). We observed significant overlap in gene expression between the infected normal bronchial epithelial cells and inflammatory skin diseases, such as psoriasis and atopic dermatitis. For genes that are commonly induced in both the severe acute respiratory syndrome coronavirus 2 infection and skin diseases, there are 4 S100 family members located in the epidermal differentiation complex, and we also identified the “IL-17 signaling pathway” (P = 4.9 × 10−77) as one of the most significantly enriched pathways. Furthermore, a shared genome-wide significant locus in the epidermal differentiation complex was identified between psoriasis and severe acute respiratory syndrome coronavirus 2 infection, with the lead marker being a significant expression quantitative trait locus for S100A12 (P = 3.3 × 10−7).ConclusionsTogether our findings suggest association between inflammatory skin conditions and higher risk of COVID-19, but with less severe course, and highlight shared components involved in anti–COVID-19 immune response.

The Cell Tropism of Porcine Respiratory Coronavirus for Airway Epithelial Cells Is Determined by the Expression of Porcine Aminopeptidase N.

Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air–liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.

The Small RNA ErsA Plays a Role in the Regulatory Network of Pseudomonas aeruginosa Pathogenicity in Airway Infections.

Bacterial small RNAs play a remarkable role in the regulation of functions involved in host-pathogen interaction. ErsA is a small RNA of Pseudomonas aeruginosa that contributes to the regulation of bacterial virulence traits such as biofilm formation and motility. Shown to take part in a regulatory circuit under the control of the envelope stress response sigma factor σ22, ErsA targets posttranscriptionally the key virulence-associated gene algC Moreover, ErsA contributes to biofilm development and motility through the posttranscriptional modulation of the transcription factor AmrZ. Intending to evaluate the regulatory relevance of ErsA in the pathogenesis of respiratory infections, we analyzed the impact of ErsA-mediated regulation on the virulence potential of P. aeruginosa and the stimulation of the inflammatory response during the infection of bronchial epithelial cells and a murine model. Furthermore, we assessed ErsA expression in a collection of P. aeruginosa clinical pulmonary isolates and investigated the link of ErsA with acquired antibiotic resistance by generating an ersA gene deletion mutant in a multidrug-resistant P. aeruginosa strain which has long been adapted in the airways of a cystic fibrosis (CF) patient. Our results show that the ErsA-mediated regulation is relevant for the P. aeruginosa pathogenicity during acute infection and contributes to the stimulation of the host inflammatory response. Besides, ErsA was able to be subjected to selective pressure for P. aeruginosa pathoadaptation and acquirement of resistance to antibiotics commonly used in clinical practice during chronic CF infections. Our findings establish the role of ErsA as an important regulatory element in the host-pathogen interaction.IMPORTANCEPseudomonas aeruginosa is one of the most critical multidrug-resistant opportunistic pathogens in humans, able to cause both lethal acute and chronic lung infections. Thorough knowledge of the regulatory mechanisms involved in the establishment and persistence of the airways infections by P. aeruginosa remains elusive. Emerging candidates as molecular regulators of pathogenesis in P. aeruginosa are small RNAs, which act posttranscriptionally as signal transducers of host cues. Known for being involved in the regulation of biofilm formation and responsive to envelope stress response, we show that the small RNA ErsA can play regulatory roles in acute infection, stimulation of host inflammatory response, and mechanisms of acquirement of antibiotic resistance and adaptation during the chronic lung infections of cystic fibrosis patients. Elucidating the complexity of the networks regulating host-pathogen interactions is crucial to identify novel targets for future therapeutic applications.

Protein Coding and Long Noncoding RNA (lncRNA) Transcriptional Landscape in SARS-CoV-2 Infected Bronchial Epithelial Cells Highlight a Role for Interferon and Inflammatory Response.

The global spread of COVID-19, caused by pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for an imminent response from medical research communities to better understand this rapidly spreading infection. Employing multiple bioinformatics and computational pipelines on transcriptome data from primary normal human bronchial epithelial cells (NHBE) during SARS-CoV-2 infection revealed activation of several mechanistic networks, including those involved in immunoglobulin G (IgG) and interferon lambda (IFNL) in host cells. Induction of acute inflammatory response and activation of tumor necrosis factor (TNF) was prominent in SARS-CoV-2 infected NHBE cells. Additionally, disease and functional analysis employing ingenuity pathway analysis (IPA) revealed activation of functional categories related to cell death, while those associated with viral infection and replication were suppressed. Several interferon (IFN) responsive gene targets (IRF9, IFIT1, IFIT2, IFIT3, IFITM1, MX1, OAS2, OAS3, IFI44 and IFI44L) were highly upregulated in SARS-CoV-2 infected NBHE cell, implying activation of antiviral IFN innate response. Gene ontology and functional annotation of differently expressed genes in patient lung tissues with COVID-19 revealed activation of antiviral response as the hallmark. Mechanistic network analysis in IPA identified 14 common activated, and 9 common suppressed networks in patient tissue, as well as in the NHBE cell model, suggesting a plausible role for these upstream regulator networks in the pathogenesis of COVID-19. Our data revealed expression of several viral proteins in vitro and in patient-derived tissue, while several host-derived long noncoding RNAs (lncRNAs) were identified. Our data highlights activation of IFN response as the main hallmark associated with SARS-CoV-2 infection in vitro and in human, and identified several differentially expressed lncRNAs during the course of infection, which could serve as disease biomarkers, while their precise role in the host response to SARS-CoV-2 remains to be investigated.

Airway Epithelial Cells Generate Pro-inflammatory Tenascin-C and Small Extracellular Vesicles in Response to TLR3 Stimuli and Rhinovirus Infection.

Viral infections are a common cause of asthma exacerbations, with human rhinoviruses (RV) the most common trigger. RV signals through a number of different receptors, including toll-like receptor (TLR)3. Tenascin-C (TN-C) is an immunomodulatory extracellular matrix protein present in high quantities in the airway of people with asthma, and expression is also upregulated in nasal lavage fluid in response to RV infection. Respiratory viral infection has been demonstrated to induce the release of small extracellular vesicles (sEV) such as exosomes, whilst exosomal cargo can also be modified in the bronchoalveolar lavage fluid of people with asthma. These sEVs may potentiate airway inflammation and regulate the immune response to infection. This study characterizes the relationship between RV infection of bronchial epithelial cells and the release of TN-C, and the release of sEVs following stimulation with the TLR3 agonist and synthetic viral mimic, poly(I:C), as well as the function of the released protein/vesicles. The BEAS-2B airway epithelial cell line and primary human bronchial epithelial cells (PBECs) from asthmatic and non-asthmatic donors were infected with RV or treated with poly(I:C). TN-C expression, release and localization to sEVs was quantified. TN-C expression was also assessed following intra-nasal challenge of C57BL/6 mice with poly(I:C). BEAS-2B cells and macrophages were subsequently challenged with TN-C, or with sEVs generated from BEAS-2B cells pre-treated with siRNA targeted to TN-C or control. The results revealed that poly(I:C) stimulation induced TN-C release in vivo, whilst both poly(I:C) stimulation and RV infection promoted release in vitro, with elevated TN-C release from PBECs obtained from people with asthma. Poly(I:C) also induced the release of TN-C-rich sEVs from BEAS-2B cells. TN-C, and sEVs from poly(I:C) challenged cells, induced cytokine synthesis in macrophages and BEAS-2B cells, whilst sEVs from control cells did not. Moreover, sEVs with ~75% reduced TN-C content did not alter the capacity of sEVs to induce inflammation. This study identifies two novel components of the inflammatory pathway that regulates the immune response following RV infection and TLR3 stimulation, highlighting TN-C release and pro-inflammatory sEVs in the airway as relevant to the biology of virally induced exacerbations of asthma.

Cigarette smoke promotes HIV infection of primary bronchial epithelium and additively suppresses CFTR function

Recurrent lung infections are a common cause of morbidity and mortality in people living with HIV and this is exacerbated in smokers even when administered combination antiretroviral therapy (cART). The incidence of pneumonia is increased with smoking and treatment interruption and is directly dependent on viral load in patients when adjusted for CD4 counts. CFTR dysfunction plays an important role in aberrant airway innate immunity as it is pivotal in regulating mucociliary clearance (MCC) rates and other antibacterial mechanisms of the airway. In our earlier work, we have demonstrated that bronchial epithelium expresses canonical HIV receptors CD4, CCR5 and CXCR4 and can be infected with HIV. HIV Tat suppresses CFTR mRNA and function via TGF-β signaling. In the present study, we demonstrate that cigarette smoke (CS) potentiates HIV infection of bronchial epithelial cells by upregulating CD4 and CCR5 expression. HIV and CS individually and additively suppress CFTR biogenesis and function, possibly explaining the increased incidence of lung infections in HIV patients and its exacerbation in HIV smokers.

Interferon response to respiratory syncytial virus by bronchial epithelium from children with asthma is inversely correlated with pulmonary function

Respiratory viral infection in early childhood, including that from respiratory syncytial virus (RSV), has been previously associated with the development of asthma. We aimed to determine whether exvivo RSV infection of bronchial epithelial cells (BECs) from children with asthma would induce specific gene expression patterns and whether such patterns were associated with lung function among BEC donors. Primary BECs from carefully characterized children with asthma (n=18) and matched healthy children without asthma (n=8) were differentiated at an air-liquid interface for 21days. Air-liquid interface cultures were infected with RSV for 96hours and RNA was subsequently isolated from BECs. In each case, we analyzed gene expression using RNA sequencing and assessed differences between conditions by linear modeling of the data. BEC donors completed spirometry to measure lung function. RSV infection of BECs from subjects with asthma, compared with uninfected BECs from subjects with asthma, led to a significant increase in expression of 6199 genes. There was significantly greater expression of 195 genes in BECs from children with asthma and airway obstruction (FEV1/forced vital capacity<0.85 and FEV1<100% predicted) than in BECs from children with asthma without obstruction, or in BECs from healthy children. These specific genes were found to be highly enriched for viral response genes induced in parallel with types I and III interferons. BECs from children with asthma and with obstructive physiology exhibit greater expression of types I and III interferons and interferon-stimulated genes than do cells from children with normal lung function, and expression of interferon-associated genes correlates with the degree of airway obstruction. These findings suggest that an exaggerated interferon response to viral infection by airway epithelial cells may be a mechanism leading to lung function decline in a subset of children with asthma.

Upregulation of miRNA-4776 in Influenza Virus Infected Bronchial Epithelial Cells Is Associated with Downregulation of NFKBIB and Increased Viral Survival.

Influenza A virus (IAV) infection remains a significant cause of morbidity and mortality worldwide. One key transcription factor that is activated upon IAV infection is nuclear factor Kappa B (NF-κB). NF-κB regulation involves the inhibitor proteins NF-κB inhibitor beta (NFKBIB), (also known as IκB β), which form complexes with NF-κB to sequester it in the cytoplasm. In this study, microarray data showed differential expression of several microRNAs (miRNAs) on exposure to IAV. Target scan analysis revealed that miR-4776, miR-4514 and miR-4742 potentially target NFKBIB messenger RNA (mRNA). Time-course analysis of primary bronchial epithelial cells (HBEpCs) showed that miR-4776 expression is increased within 1 h of infection, followed by its downregulation 4 h post-exposure to IAV. NFKBIB upregulation of miR-4776 correlated with a decrease in NFKBIB expression within 1 h of infection and a subsequent increase in NFKBIB expression 4 h post-infection. In addition, miRNA ago-immunoprecipitation studies and the three prime untranslated region (3’ UTR) luciferase assay confirmed that miR-4776 targets NFKBIB mRNA. Furthermore, uninfected HBEpCs transfected with miR-4776 mimic showed decreased expression of NFKBIB mRNA. Overexpression of NFKBIB protein in IAV infected cells led to lower levels of IAV. Taken together, our data suggest that miRNA-4776 modulates IAV production in infected cells through NFKBIB expression, possibly through the modulation of NF-κB.