Search Results (102)

Air–liquid interface (ALI) cultures recapitulate key features of the airway epithelium by driving basal cell differentiation into ciliated, club, and goblet cells and by generating a functional mucus barrier, thereby representing a highly relevant model of the human respiratory tract. Using a reduced factorial Design of Experiments (DoE) methodology, we simultaneously investigated the effects of seven variables on human coronavirus OC43 (HCoV-OC43) replication in air–liquid interface (ALI)-cultured primary human bronchial epithelial cells (pHBECs) to identify robust conditions that support infection and viral replication. Epithelial differentiation was monitored by measuring transepithelial electrical resistance and determining expression levels of marker genes for basal, goblet, club, and ciliated cells using reverse transcription quantitative PCR (RT-qPCR). HCoV-OC43 replication was monitored by quantifying genomic and subgenomic RNA by RT-qPCR. Viral RNA peaked three days post-infection in cell lysates and four days post-infection in apical washes. Initiation of ALI conditions induced epithelial differentiation, which was complete after 21 days and emerged as the strongest determinant of viral replication. Differentiated pHBEC cultures showed significantly reduced viral RNA compared with undifferentiated cultures, particularly following apical infection. In contrast, basal infection resulted in lower viral RNA levels in undifferentiated cultures than apical infection but was less dependent on epithelial differentiation. However, productive infection following basal exposure was less consistent and more strongly dependent on viral inoculum size. We further demonstrate that repeated mucus washes prior to infection increased HCoV-OC43 replication in mature cultures. In summary, our findings show that epithelial differentiation negatively affects HCoV-OC43 replication and we identify conditions that maximize viral replication in fully differentiated pHBEC cultures. Full article

Background and Objectives: Dysfunction of the airway epithelial barrier is increasingly recognized as an early pathogenic mechanism in allergic respiratory diseases. Although individual aeroallergens such as ragweed (RW) pollen and house dust mite (HDM) are known to impair epithelial integrity, the effects of combined exposure, more reflective of real-world conditions, remain insufficiently characterized. This study aimed to evaluate the impact of single versus combined allergen exposure on airway epithelial barrier function using a multimodal experimental approach. Materials and Methods: Differentiated normal human bronchial epithelial (NHBE) cells were exposed to RW (100 µg/mL), HDM (100 µg/mL), or a combined extract (RW + HDM; total 100 µg/mL). Barrier function under air–liquid interface conditions was assessed by transepithelial electrical resistance (TEER), while real-time cellular responses were evaluated using xCELLigence impedance monitoring. Structural alterations were examined by occludin-based immunofluorescence imaging, and transcriptional changes associated with epithelial stress and inflammation were analyzed by RT-qPCR. Results: Allergen exposure induced time- and concentration-dependent impairment of epithelial barrier function. Combined exposure resulted in the most pronounced and sustained reduction in TEER and impedance measurements. These functional changes were accompanied by disruption of tight junction organization and coordinated transcriptional modulation of genes involved in inflammatory and stress responses. Conclusions: Combined exposure to RW and HDM extracts induced more severe and persistent epithelial barrier dysfunction than individual allergens. These findings support the role of the airway epithelium as a central regulator of allergic airway disease and highlight barrier disruption as an early pathogenic event. The multimodal framework applied in this study provides an integrated platform for investigating epithelial responses to complex environmental exposures. Full article

Cigarette smoke (CS) is the primary risk factor for the development of chronic obstructive pulmonary disease (COPD). Investigating the impact of CS on human airway epithelium is important for understanding COPD development and combating its effects. While some studies show that long exposure to CS activates inflammasome formation in airway epithelium, leading to cytokines’ maturation and release, its acute effect on inflammation regulation requires further elucidation. Due to the importance of acute cellular responses in modulating cell survival and controlling inflammatory outcomes, we examined the effect of acute cigarette smoke extract exposure on human bronchial epithelial cells. Due to the high reactive oxygen species content in CS, we hypothesize that acute CS exposure activates the integrated stress response (ISR) pathway leading to stress granules (SG) formation to facilitate oxidative stress resolution and promote cell survival. Immunostaining, fluorescence confocal imaging, quantitative analyses, and immunoblotting were performed to test our hypothesis. We report here that acute exposure to CS extract triggers canonical SG formation by activating the ISR pathway via the PERK/eIF2α arm in a reactive oxygen species-dependent manner. SG formation is abolished upon inhibiting PERK or eIF2α function, or by scavenging oxidants prior to smoke exposure. Characterizing SG formation in terms of measuring SG size and abundance and the sequestration of the SG marker G3BP1 reveals that SG formation is maximal at 15% CS extract exposure for 2 h and undergoes gradual disassembly at longer exposure times. This is closely dependent on cytoplasmic p-eIF2α levels. These results demonstrate that acute exposure to CS activates the protective ISR pathway to potentially reduce the detrimental effects of CS and promote stress resolution and cell survival. Full article

Brucella infection is frequently acquired by inhalation, but the pathogen disseminates systemically from the lungs. However, little is known about the interaction of Brucella spp. with the airways. Using a 3D air-exposed organotypic human bronchial tissue model (polarized 16HBE14o- bronchial epithelial cells grown over a collagen matrix containing MRC-5 lung fibroblasts), we analyzed Brucella abortus replication, translocation and cytokine responses over prolonged post-infection times. Apically inoculated B. abortus invaded, replicated and persisted during the whole follow-up (16 days) within the bronchial tissue without inducing cytotoxicity. Viable bacteria were also detected in the conditioned medium (CM) since day five post-infection, indicating release from the basolateral side. In parallel experiments, no invasion or bacterial release was detected for Escherichia coli. The levels of IL-6, IL-8 and MCP-1 were increased in CM from Brucella-infected 3D cultures and in monocultures of polarized bronchial epithelial cells or lung fibroblasts. Collagenase/gelatinase activity was increased in 3D cultures and MRC-5 monocultures. Infection transference from bronchial cells to lung fibroblasts was documented using monocultures. An immune cross-talk was detected, as cytokine levels were increased in fibroblasts stimulated with bronchial CM, and vice versa. These results suggest that the bronchial mucosa can sustain B. abortus persistence, replication and dissemination, and that it induces a proinflammatory response to which both epithelial cells and fibroblasts contribute. Full article

Background: Pulmonary neuroendocrine cells (PNECs) are rare airway sensory cells implicated in amplifying allergic inflammation, yet due to their scarcity, the contribution of PNECs to the metabolic programs and responses of the airway epithelium remains poorly defined. Using a newly developed PNEC-enriched human airway epithelial model (ePNEC), we investigated the influence of PNECs on neuroendocrine and immune-modulatory metabolite production in response to the common aeroallergen of the house dust mite (HDM). Methods: Human bronchial epithelial cells (HBECs) and ePNEC cultures were differentiated at the air–liquid interface. Global untargeted metabolomics was performed to quantify metabolite abundance at baseline and following stimulation with HDMs. Differential expression, overlap significance, metabolite class enrichment, and pathway analyses were used to define PNEC-specific metabolic programs. Results: Principal component analysis (PCA) demonstrated strong baseline separation between ePNECs and HBECs, with HDMs inducing additional within-cell-type shifts. ePNECs displayed broader and more pronounced metabolite changes than HBECs. Baseline differences were largely preserved following allergen exposure, with significant overlap in both up- and down-regulated metabolites. ePNECs exhibited enriched neurotransmitter-linked metabolites—including serotonin, L-noradrenaline, dopamine, and histamine—at baseline and after HDM exposure. Amino acid–centered metabolism dominated the dataset, with enhanced histidine and tryptophan pathway activity in ePNECs. Pathway analysis revealed significant enrichment of phenylalanine, tyrosine, tryptophan, glutathione, and arginine–proline metabolism in ePNECs, whereas HBECs showed no significant pathway-level enrichment after HDM exposure. Conclusions: Human ePNECs engage a distinct, neuroactive metabolic program that is amplified upon HDM exposure. These findings provide a metabolic framework for how PNECs shape epithelial and neuroimmune responses to inhaled allergens. Full article

Influenza A viruses remain a major public health threat due to their high mutation rates, antigenic variability, and the emergence of resistance to current antivirals, underscoring the need for novel therapeutic options. Natural compounds rich in polyphenols and flavonoids have attracted increasing attention as potential broad-spectrum antiviral agents. In this study, the activity of Rhus coriaria L. water extract against Influenza A virus in BEAS-2B human bronchial epithelial cells was investigated. Cell viability assay identified non-cytotoxic concentrations, up to 0.1 mg/mL, which were used in infection experiments. Viral replication was assessed at multiple levels by quantitative real-time PCR, western blotting, immunofluorescence and tissue culture infectious dose 50% (TCID₅₀). Treatment with R. coriaria extract resulted in a dose-dependent and statistically significant reduction of viral load. The extract decreased mRNA levels of Hemagglutin (HA), Neuraminidase (NA) and Matrix protein 2 (M2). Consistently, western blot analysis showed a decrease in major viral proteins HA, Nucleoprotein (NP), Matrix protein 1 (M1) and Polymerase Acidic protein (PA). Confocal images revealed a marked reduction in HA and PA signals, results that are statistically significant according to quantitative fluorescence evaluation. The convergence of results obtained through independent methodologies at both the transcriptional and protein levels highlight the robustness of the findings. These data provide the experimental evidence that Rhus coriaria interferes with influenza A virus replication in airway epithelial cells and support its further investigation as a promising phytochemical platform for the development of novel anti-influenza strategies. Full article

Periodontal pathogens, including Porphyromonas gingivalis (P. gingivalis), are implicated in respiratory inflammatory conditions, and aspirated oral bacterial components may contribute to airway inflammation. This association has prompted the exploration of innovative therapeutic strategies in addition to conventional oral hygiene practices. We evaluated the anti-inflammatory efficacy of an alkaline extract of Sasa senanensis leaves (SE) against heat-inactivated P. gingivalis-induced inflammation in respiratory tissues. In human bronchial epithelial cells (BEAS-2B), SE reduced interleukin (IL)-6 and IL-8 mRNA expression and cytokine secretion in a dose-dependent manner. Moreover, SE attenuated nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs), including p38 and c-Jun N-terminal kinase (JNK), indicating broad anti-inflammatory actions. In mice, SE administration decreased early lung cytokine levels and reduced NF-κB activity following intratracheal challenge with heat-inactivated P. gingivalis. Together, these in vitro and in vivo findings indicate that SE suppresses proinflammatory signaling triggered by P. gingivalis components and may serve as a natural adjunct to mitigate bacteria-associated airway inflammatory responses. Full article

Most respiratory diseases are driven by excessive airway inflammation and oxidative stress, yet current therapies often lack durable efficacy or are unsafe. Host-defense peptides, commonly enriched in animal venoms, offer diverse, target-selective scaffolds for new therapeutics. In this study, we aimed to discover a novel bioactive peptide with therapeutic potential on respiratory tract damage by utilizing Nephila clavata venom gland transcriptome. Using in silico analysis and machine learning-based functional prediction, we designed a peptide, NC-CV, expected to have dual anti-inflammatory and antioxidant activities with low cytotoxicity. In experimental validation, NC-CV improved human bronchial epithelial BEAS-2B cell viability under lipopolysaccharide (LPS) exposure while reducing LPS-induced pro-inflammatory cytokine expression and intracellular reactive oxygen species (ROS) generation. Mechanistic studies and molecular docking simulations indicated that NC-CV prevents toll-like receptor 4 signaling activation, suppressing nuclear factor κB and mitogen-activated protein kinase pathways. Moreover, the antioxidant activity of NC-CV was primarily based on direct intracellular ROS scavenging rather than the induction of endogenous antioxidant enzymes. Collectively, these findings demonstrated that the venom-derived peptide NC-CV disrupts the self-reinforcing cycle involving inflammatory signaling and oxidative stress in airway epithelium, highlighting its promise as a therapeutic candidate for respiratory disease. Full article

Current asthma therapies reduce inflammation and symptoms but there are concerns regarding adverse effects and the long-term treatment burden. The anti-asthmatic potential of Dictamnine (Dic) has not been investigated. The therapeutic effect of Dic on airway inflammation and remodeling was investigated by targeting the tumor growth factor (TGF)-β/Smad2/3 pathway. A murine model of ovalbumin (OVA)-induced asthma was used to evaluate the effects of orally-administered Dic on airway hyperresponsiveness, inflammatory cytokines in bronchoalveolar lavage fluid (BALF), OVA-specific IgE in the serum, and histopathological changes. The expression of TGF-β/Smad2/3 and epithelial markers was assessed. Human bronchial epithelial cells were used in vitro to examine the effects of Dic on TGF-β-induced Smad2/3 phosphorylation. Network pharmacology was conducted to predict Dic-associated targets and pathways. Dic substantially reduced the levels of Th2 cytokines, mucin 5AC in BALF, and OVA-specific IgE in the serum. Histology indicated reduced inflammatory cell infiltration, bronchial wall thickening, and peribronchial fibrosis in Dic-treated mice. Dic downregulated TGF-β and p-Smad2/3 expression and upregulated ZO-1 expression in the lung tissue. Dic downregulated TGF-β-induced Smad2/3 phosphorylation in bronchial epithelial cells. Network pharmacology indicated enrichment of Dic-related genes in the TGF-β pathway. Dic exhibited anti-asthmatic effects and is a potential therapeutic candidate. Full article

The development of new approach methodologies that include human cells differentiated into organotypic formats is of high interest due to their structural and functional similarities to tissues in vivo, enabling mechanistic understanding and translation to adverse health outcomes in humans. However, these systems often fail to capture complex intercellular signaling required for processes such as pulmonary inflammation induced by polycyclic aromatic hydrocarbons (PAHs). To investigate airway epithelial–macrophage interactions in response to benzo[a]pyrene and a PAH mixture (Tox Mix), co-culture models utilizing primary human bronchial epithelial cells (HBECs) differentiated at the air–liquid interface were cultured with THP-1 macrophages either directly or indirectly, alongside HBECs alone. After 24 h of exposure, cytokine expression (IL1B, IL6, CXCL8, TNF) as well as PAH biomarkers previously identified for chemical metabolism (CYP1A1, CYP1B1), oxidative stress (ALDH3A1, HMOX1, NQO1), and barrier integrity (TJP2) were evaluated. Cytotoxicity and barrier integrity were also assessed. HBECs alone and direct co-cultures exhibited similar responses after PAH treatment, while indirect co-cultures showed lower sensitivity to induction of inflammatory cytokines and CYP1A1 and CYP1B1 biomarker expression following exposure to PAHs. The expression of other biomarkers, including ALDH3A1, HMOX1, and NQO1, remained largely consistent across all models after treatment. Overall, these findings suggest that direct co-culture systems may provide a more physiologically relevant platform for studies of PAH-induced toxicity and demonstrate that the configuration of co-culture systems can influence cellular responses to chemical exposure. Full article

Cholesterol is an essential plasma membrane component, and altered cholesterol metabolism has been linked to cholesterol accumulation in the airways of COPD and cystic fibrosis patients. However, its role in airway epithelial differentiation is not well understood. Tandem mass spectrometry-based proteomic analysis of differentiating primary human bronchial epithelial cells (phBECs) revealed an overall inhibition of the cholesterol biosynthesis pathway. We hypothesized that excess cholesterol impairs the differentiation of phBECs into a fully functional bronchial epithelium. PhBECs were differentiated in the presence of 80 µM cholesterol for 21 days, the main airway cell type populations monitored using qRT-PCR and immunofluorescent stainings, and epithelial barrier integrity was analyzed via transepithelial electrical resistance measurements. Chronic cholesterol exposure led to a significant increase in CC10⁺ secretory cells at the expense of ciliated cells. Pathway enrichment analysis suggested the tumor protein p53 as a master regulator of genes during normal differentiation of phBECs. Chronic cholesterol exposure drastically impaired the nuclear translocation of p53. Our findings suggest that this inhibition underlies the cholesterol-induced expansion of CC10⁺ secretory cell populations at the expense of ciliated cells. In conclusion, we identify cholesterol as an important regulator of normal bronchial epithelial cell differentiation through inhibition of p53 nuclear translocation. Full article

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continue to demand attention as the virus continues to evolve, demonstrating a remarkable ability to adapt to diverse selective pressures including immune responses, therapeutic treatments, and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic, with new subvariants continuously emerging, many harboring spike protein mutations linked to immune evasion. In this study, we characterized a panel of live SARS-CoV-2 strains, including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the spike protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neutralizing the most recent Omicron subvariants, particularly after the emergence of JN.1 subvariant. Using human airway epithelial cells derived from healthy bronchiolar tissue (hBAEC), we established both monoinfections and coinfections involving SARS-CoV-2, Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Assessments were conducted to compare viral infectivity and the production and release of immune mediators in the apical and basolateral compartments. Notably, Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its parental strain JN.1 and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020, Washington strain). Furthermore, the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead, it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6, IFN-β, and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfection with IFAV_H1N1 and RSV. Taken together, our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants, as well as their interactions with other respiratory viruses, carrying important implications for therapeutic development and public health preparedness. Full article

Background: Cell-penetrating peptides cross cell membrane barriers while carrying cargoes in a functional form. Our work identified two novel lung-targeting peptides, S7A and R11A. Here, we present studies on biodistribution, the cell types targeted, and an in vitro proof of application. Methods: Studies were performed in human bronchial epithelial cells (HBECs) with and without various endocytic inhibitors, and coincubation with fluorescently labeled transferrin or endocytic markers. Cyclic R11A (cR11A) was conjugated to siRNA duplexes and anti-viral activity against SARS-CoV-2 was tested. Biodistribution studies were performed by injecting wild-type mice with fluorescently labeled peptides, and various circulation times were allowed for, as well as cross-staining of lung sections or isolated single cells with various cellular markers, followed by fluorescence-activated cell sorting or confocal microscopy. Results: cR11A showed peak uptake in 15 min, with the highest uptake in airway epithelial type II (ATII) cells, followed by p63+ basal cells and ionocytes. Cyclization increased transduction efficiencies ~100-fold. Endocytosis studies showed a decrease in peptide uptake by pre-treatment with Pitstop2 but not Amiloride or Nystatin. Endocytic marker Lamp1 showed colocalization at the earliest time point, with the escape of the peptide from endocytic vesicles later. cR11A conjugated to ant-spike and anti-envelop proteins showed anti-viral effects with an EC₉₀ of 0.6 μM and 1.0 µM, respectively. Conclusions: We have identified a novel peptide, cR11A, that targets ATII, basal cells, and ionocytes, the cyclization of which increased transduction efficiency in vitro and in vivo. The uptake mechanism appears to be via clathrin-mediated endocytosis with escape from endocytic vesicles. cR11A can act as a vector to deliver anti-viral siRNA to epithelial cells. Full article

Delta-9-tetrahydrocannabinol (Δ-9-THC or THC), the primary psychoactive constituent of cannabis, can lead to adverse health conditions, including mental health issues, brain impairment, and cardiac and respiratory problems. The amount of THC in cannabis has steadily climbed over the past few decades, with today’s cannabis having three times the concentration of THC compared to 25 years ago. Inhalation is a major route of exposure, allowing substances to enter the body via the respiratory tract. THC exposure causes cell death in the airway epithelium; however, the molecular underpinning of THC exposure-induced bronchial epithelial cell death is not clearly understood. To address the mechanisms involved in this process, the present study examined the cell viability, oxidative stress, lipid peroxidation, and transcriptional alterations caused by various concentrations of Δ-9-THC (0, 800, 1000, 1200, and 1500 ng/mL) in a human bronchial epithelial cell line (BEAS-2B) in vitro. Δ-9-THC exposure caused a significant dose-dependent decrease in cell viability after 24 h exposure. Transcriptome analysis showed a distinct dose-dependent response. HIF-1 signaling, ferroptosis, AMPK signaling, and immunogenic pathways were activated by Δ-9-THC-upregulated genes. Glutathione and fatty acid metabolic pathways were significantly altered by Δ-9-THC-dependent downregulated genes. Ingenuity Pathway Analysis (IPA) revealed several top canonical pathways altered by Δ-9-THC exposure, including ferroptosis, NRF-2-mediated oxidative stress response, caveolar-mediated endocytosis (loss of cell adhesion to the substrate), tumor microenvironment, HIF1alpha signaling, and the unfolded protein response pathway. Δ-9-THC-induced cell death was ameliorated by inhibiting the ferroptosis pathway, whereas treatments with ferroptosis agonist exacerbated the cell death process, suggesting that Δ-9-THC-induced bronchial epithelial cell death potentially involves the ferroptosis pathway. Full article

With the concurrent circulations of SARS-CoV-2 omicron and influenza A viruses in the community, there is evidence showing co-infection with both viruses. However, disease severity may vary due to the complex immunity landscape of the patients and the neutralizing antibody waning status. The intrinsic dynamic relationship and pathological significance for such co-infections remain largely unknown. The replication kinetics and innate immune responses from the co-infections of SARS-CoV-2 (Omicron BA.1 and D614G variant) and influenza A viruses (pandemic H1N1, seasonal H3N2 and highly pathogenic avian H5N1) were characterized in human respiratory tissue explants, human airway, and alveolar epithelial cells. SARS-CoV-2 reduced the replication of influenza A viruses, but not vice versa, during co-infections in human bronchial tissues and airway epithelial cells. In lung tissues, the co-infections showed minimal effects on each other, but the viral replications of the two viruses were mutually reduced except for H1N1pdm in the alveolar epithelial cells irrespective of the enhancement of the ACE2 receptor. Notably, the co-infections showed a significant upregulation of the innate immune responses of SARS-CoV-2 in comparison to single infections in both respiratory epithelial cells, suggesting that co-infections of influenza A viruses potentially lead to more severe damage to the host than SARS-CoV-2 single infections. Full article