Cultures Of Human Epithelial Cells Research Articles

Air-liquid intestinal cell culture allows in situ rheological characterization of intestinal mucus.

Intestinal health heavily depends on establishing a mucus layer within the gut with physical properties that strike a balance between being sufficiently elastic to keep out harmful pathogens yet viscous enough to flow and turnover the contents being digested. Studies investigating dysfunction of the mucus layer in the intestines are largely confined to animal models, which require invasive procedures to collect the mucus fluid. In this work, we develop a nondestructive method to study intestinal mucus. We use an air-liquid interface culture of primary human intestinal epithelial cells that exposes their apical surface to allow in situ analysis of the mucus layer. Mucus collection is not only invasive but also disrupts the mucus microstructure, which plays a crucial role in the interaction between mucus and the gut microbiome. Therefore, we leverage a noninvasive rheology technique that probes the mechanical properties of the mucus without removal from the culture. Finally, to demonstrate biomedical uses for this cell culture system, we characterize the biochemical and biophysical properties of intestinal mucus due to addition of the cytokine IL-13 to recapitulate the gut environment of Nippostrongylus brasiliensis infection.

Isolation and Culture of Primary Human Mammary Epithelial Cells.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

S. aureus biofilm properties correlate with immune B cell subset frequencies and severity of chronic rhinosinusitis

Staphylococcus aureus mucosal biofilms are associated with recalcitrant chronic rhinosinusitis (CRS). However, S. aureus colonisation of sinus mucosa is frequent in the absence of mucosal inflammation. This questions the relevance of S. aureus biofilms in CRS etiopathogenesis. This study aimed to investigate whether strain-level variation in in vitro-grown S. aureus biofilm properties relates to CRS disease severity, in vitro toxicity, and immune B cell responses in sinonasal tissue from CRS patients and non-CRS controls. S. aureus clinical isolates, tissue samples, and matched clinical datasets were collected from CRS patients with nasal polyps (CRSwNP), CRS without nasal polyps (CRSsNP), and controls. B cell responses in tissue samples were characterised by FACS. S. aureus biofilms were established in vitro, followed by measuring their properties of metabolic activity, biomass, colony-forming units, and exoprotein production. S. aureus virulence was evaluated using whole-genome sequencing, mass spectrometry and application of S. aureus biofilm exoproteins to air-liquid interface cultures of primary human nasal epithelial cells (HNEC-ALI). In vitro S. aureus biofilm properties were correlated with increased CRS severity scores, infiltration of antibody-secreting cells and loss of regulatory B cells in tissue samples. Biofilm exoproteins from S. aureus with high biofilm metabolic activity had enriched virulence genes and proteins, and negatively affected the barrier function of HNEC-ALI cultures. These findings support the notion of strain-level variation in S. aureus biofilms to be critical in the pathophysiology of CRS.

Kynurenine 3-monooxygenase limits de novo NAD+ synthesis through dietary tryptophan in renal proximal tubule epithelial cell models.

Nicotinamide adenine dinucleotide (NAD+) is a pivotal coenzyme, essential for cellular reactions, metabolism, and mitochondrial function. Depletion of kidney NAD+ levels and reduced de novo NAD+ synthesis through the tryptophan-kynurenine pathway are linked to acute kidney injury (AKI), whereas augmenting NAD+ shows promise in reducing AKI. We investigated de novo NAD+ biosynthesis using in vitro, ex vivo, and in vivo models to understand its role in AKI. Two-dimensional (2-D) cultures of human primary renal proximal tubule epithelial cells (RPTECs) and HK-2 cells showed limited de novo NAD+ synthesis, likely due to low pathway enzyme gene expression. Using three-dimensional (3-D) spheroid culture model improved the expression of tubular-specific markers and enzymes involved in de novo NAD+ synthesis. However, de novo NAD+ synthesis remained elusive in the 3-D spheroid culture, regardless of injury conditions. Further investigation revealed that 3-D cultured cells could not metabolize tryptophan (Trp) beyond kynurenine (KYN). Intriguingly, supplementation of 3-hydroxyanthranilic acid into RPTEC spheroids was readily incorporated into NAD+. In a human precision-cut kidney slice (PCKS) ex vivo model, de novo NAD+ synthesis was limited due to substantially downregulated kynurenine 3-monooxygenase (KMO), which is responsible for KYN to 3-hydroxykynurenine conversion. KMO overexpression in RPTEC 3-D spheroids successfully reinstated de novo NAD+ synthesis from Trp. In addition, in vivo study demonstrated that de novo NAD+ synthesis is intact in the kidney of the healthy adult mice. Our findings highlight disrupted tryptophan-kynurenine NAD+ synthesis in in vitro cellular models and an ex vivo kidney model, primarily attributed to KMO downregulation.NEW & NOTEWORTHY Nicotinamide adenine dinucleotide (NAD+) is essential in regulating mitochondrial function. Reduced NAD+ synthesis through the de novo pathway is associated with acute kidney injury (AKI). Our study reveals a disruption in de novo NAD+ synthesis in proximal tubular models, but not in vivo, attributed to downregulation of enzyme kynurenine 3-monooxygenase (KMO). These findings highlight a crucial role of KMO in governing de novo NAD+ biosynthesis within the kidney, shedding light on potential AKI interventions.

German cockroach extract prevents IL-13-induced CCL26 expression in airway epithelial cells through IL-13 degradation.

Inhaled aeroallergens can directly activate airway epithelial cells (AECs). Exposure to cockroach allergens is a strong risk factor for asthma. Cockroach allergens mediate some of their effects through their serine protease activity; protease activity is also a major contributor to allergenicity. The Th2 cytokine interleukin-13 (IL-13) induces upregulation of the eosinophil chemotactic factor CCL26. CCL26 induces eosinophil migration in allergic inflammation. In this work, we studied the effect of cockroach proteases on IL-13-induced effects. Immersed cultures of the human bronchial epithelial cell line BEAS-2B and air-liquid interface (ALI) cultures of primary normal human bronchial epithelial (NHBE) cells were stimulated with IL-13, Blattella Germanica cockroach extract (CE), or both. IL-13-induced genes were analyzed with qRT-PCR. IL-13 induced upregulation of CCL26, periostin, and IL-13Rα2 in bronchial epithelial cells which were decreased by CE. CE was heat-inactivated (HICE) or pre-incubated with protease inhibitors. HICE and CE preincubated with serine protease inhibitors did not prevent IL-13-induced CCL26 upregulation. CE-degraded IL-13 and specific cleavage sites were identified. CE also decreased IL-4-induced CCL26 upregulation and degraded IL-4. Other serine proteases such as bovine trypsin and house dust mite (HDM) serine proteases did not have the same effects on IL-13-induced CCL26. We conclude that CE serine proteases antagonize IL-13-induced effects in AECs, and this CE effect is mediated primarily through proteolytic cleavage of IL-13. IL-13 cleavage by cockroach serine proteases may modulate CCL26-mediated effects in allergic airway inflammation by interfering directly with the pro-inflammatory effects of IL-13 invivo.

Three Artemisia pollens trigger the onset of allergic rhinitis via TLR4/MyD88 signaling pathway.

The prevalence of allergic rhinitis is high, making it a relatively common chronic condition. Countless patients suffer from seasonal Allergic rhinitis (AR). The objective of this investigation is to examine the potential involvement of common pollen allergens in seasonal allergic rhinitis, and study the proposed mechanism of Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response gene 88 (MyD88) signaling pathway in the induction of AR. A mouse AR model (sensitized group) was constructed with pollen extracts and ovalbumin(OVA) of Artemisia annua (An), Artemisia argyi (Ar) and Artemisia Sieversiana (Si), and thereafter, AR symptom score was performed. After successful modeling, mouse serum and nasal mucosa tissues were extracted for subsequent experiments. The expression levels of immunoglobulin E (IgE), Interleukin (IL)-4, IL-5, IL-13 and Tumor Necrosis Factor-α (TNF-α) in serum were detected using Enzyme-linked immunosorbent assay (ELISA); Hematoxylin-eosin (H&E) staining methods were used to observe the pathological changes of the nasal mucosal tissue; Utilizing immunohistochemistry (IHC) staining, the expression levels of TLR4, MyD88 and Nuclear factor kappa B (NF-κB) p65 in mouse nasal mucosa were quantified; The mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of sensitized mice were detected with Quantitative reverse transcription PCR (qRT-PCR) and Western Blot. Finally, the in vitro culture of Human nasal mucosal epithelial cells (HNEpC) cells was conducted, and cells were treated with 200µg/ml Artemisia annua pollen extractand OVA for 24h. Western Blot assay was used to detect the expression level of TLR4, MyD88 and NF-κB p65 proteins before and after HNEpC cells were treated with MyD88 inhibitor ST-2825. On the second day after AR stimulation, the mice showed obvious AR symptoms. H&E results showed that compared to the control group, the nasal mucosal tissue in the sensitized group was significantly more inflamed. Furthermore, ELISA assay showed increased expression levels of IgE, IL-4, IL-5, IL-13 and TNF-α in serum of mice induced by OVA and Artemisia annua pollen, Artemisia argyi pollen and Artemisia Sieversiana pollen than those of the control group. However, the expression level of IL-2 was lower than that of the control group (P < 0.05). Using Immunohistochemistry staining visually observed the expression levels of TLR4, MyD88 and NF-κB p65 in mouse nasal mucosa tissues and quantitatively analyzed. The expression levels of TLR4, MyD88 and NF-κB p65 in the sensitized group were higher than those in the control group, and the differences were statistically significant (P < 0.05). The results from qRT-PCR and Western Blot showed that the mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of the sensitized group were significantly higher than those in the control group (P < 0.05). Finally, HNEpC cells were cultured in vitro and analyzed using Western Blot. The expression levels of TLR4, MyD88 and NF-κB p65 in OVA and An groups were significantly increased (P < 0.05). After ST-2825 treatment, TLR4 protein expression was significantly increased (P < 0.05) and MyD88 and NF-κB p65 protein expression were significantly decreased (P < 0.05). To sum up, the occurrence and development of AR induced by OVA and pollen of Artemisia annua, Artemisia argyi and Artemisia Sieversiana were related to TLR4/MyD88 signal pathway.

In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores.

Multidrug-resistant Pseudomonas aeruginosa is a versatile bacterium that frequently causes lung infections. This pathogen is life-threatening to mechanically-ventilated patients in intensive care units and is a debilitating burden for individuals with cystic fibrosis. However, the role of P. aeruginosa virulence factors and their regulation during infection are not fully understood. Previous murine lung infection studies have demonstrated that the production of siderophores (e.g., pyoverdine and pyochelin) is necessary for full P. aeruginosa virulence. In this report, we provide further mechanistic insight into this phenomenon. We characterize distinct and novel ways these siderophores contribute to virulence using an in vitro human lung epithelial cell culture model.

Artificial Space Weathering to Mimic Solar Wind Enhances the Toxicity of Lunar Dust Simulants in Human Lung Cells.

During NASA's Apollo missions, inhalation of dust particles from lunar regolith was identified as a potential occupational hazard for astronauts. These fine particles adhered tightly to spacesuits and were unavoidably brought into the living areas of the spacecraft. Apollo astronauts reported that exposure to the dust caused intense respiratory and ocular irritation. This problem is a potential challenge for the Artemis Program, which aims to return humans to the Moon for extended stays in this decade. Since lunar dust is "weathered" by space radiation, solar wind, and the incessant bombardment of micrometeorites, we investigated whether treatment of lunar regolith simulants to mimic space weathering enhanced their toxicity. Two such simulants were employed in this research, Lunar Mare Simulant-1 (LMS-1), and Lunar Highlands Simulant-1 (LHS-1), which were added to cultures of human lung epithelial cells (A549) to simulate lung exposure to the dusts. In addition to pulverization, previously shown to increase dust toxicity sharply, the simulants were exposed to hydrogen gas at high temperature as a proxy for solar wind exposure. This treatment further increased the toxicity of both simulants, as measured by the disruption of mitochondrial function, and damage to DNA both in mitochondria and in the nucleus. By testing the effects of supplementing the cells with an antioxidant (N-acetylcysteine), we showed that a substantial component of this toxicity arises from free radicals. It remains to be determined to what extent the radicals arise from the dust itself, as opposed to their active generation by inflammatory processes in the treated cells.

Sperm induction of somatic cell-cell fusion as a novel functional test.

The fusion of mammalian gametes requires the interaction between IZUMO1 on the sperm and JUNO on the oocyte. We have recently shown that ectopic expression of mouse IZUMO1 induces cell-cell fusion and that sperm can fuse to fibroblasts expressing JUNO. Here, we found that the incubation of mouse sperm with hamster fibroblasts or human epithelial cells in culture induces the fusion between these somatic cells and the formation of syncytia, a pattern previously observed with some animal viruses. This sperm-induced cell-cell fusion requires a species-matching JUNO on both fusing cells, can be blocked by an antibody against IZUMO1, and does not rely on the synthesis of new proteins. The fusion is dependent on the sperm's fusogenic capacity, making this a reliable, fast, and simple method for predicting sperm function during the diagnosis of male infertility.

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air–liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

Standalone cell culture microfluidic device-based microphysiological system for automated cell observation and application in nephrotoxicity tests.

Microphysiological systems (MPS) offer an alternative method for culturing cells on microfluidic platforms to model organ functions in pharmaceutical and medical sciences. Although MPS hardware has been proposed to maintain physiological organ function through perfusion culture, no existing MPS can automatically assess cell morphology and conditions online to observe cellular dynamics in detail. Thus, with this study, we aimed to establish a practical strategy for automating cell observation and improving cell evaluation functions with low temporal resolution and throughput in MPS experiments. We developed a versatile standalone cell culture microfluidic device (SCCMD) that integrates microfluidic chips and their peripherals. This device is compliant with the ANSI/SLAS standards and has been seamlessly integrated into an existing automatic cell imaging system. This integration enables automatic cell observation with high temporal resolution in MPS experiments. Perfusion culture of human kidney proximal tubule epithelial cells using the SCCMD improves cell function. By combining the proximal tubule MPS with an existing cell imaging system, nephrotoxicity studies were successfully performed to automate morphological and material permeability evaluation. We believe that the concept of building the ANSI/SLAS-compliant-sized MPS device proposed herein and integrating it into an existing automatic cell imaging system for the online measurement of detailed cell dynamics information and improvement of throughput by automating observation operations is a novel potential research direction for MPS research.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Farnesol Inhibits PI3 Kinase Signaling and Inflammatory Gene Expression in Primary Human Renal Epithelial Cells.

Chronic inflammation and elevated cytokine levels are closely associated with the progression of chronic kidney disease (CKD), which is responsible for the manifestation of numerous complications and mortality. In addition to conventional CKD therapies, the possibility of using natural compounds with anti-inflammatory potential has attracted widespread attention in scientific research. This study aimed to study the potential anti-inflammatory effects of a natural oil compound, farnesol, in primary human renal proximal tubule epithelial cell (RPTEC) culture. Farnesol was encapsulated in lipid-based small unilamellar vesicles (SUVs) to overcome its insolubility in cell culture medium. The cell attachment of empty vesicles (SUVs) and farnesol-loaded vesicles (farnesol-SUVs) was examined using BODIPY, a fluorescent dye with hydrophobic properties. Next, we used multiple protein, RNA, and protein phosphorylation arrays to investigate the impact of farnesol on inflammatory signaling in RPTECs. The results indicated that farnesol inhibits TNF-α/IL-1β-induced phosphorylation of the PI3 kinase p85 subunit and subsequent transcriptional activation of the inflammatory genes TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2. Therefore, farnesol may be a promising natural compound for treating CKD.

Recirculating Regulatory T Cells Mediate Thymic Regeneration in a ZFP36L1 and Amphiregulin Dependent Manner

T cell reconstitution correlates with outcomes in patients undergoing stem cell transplantation (HCT). A functional thymus is needed for the maturation of T cells, but unfortunately, the thymus is highly sensitive to acute injury, such as cytoreductive conditioning given to HCT patients. To enhance T cell reconstitution, it is therefore imperative to explore therapies that can boost thymic regeneration and T cell output. Regulatory T cells (Tregs) are known to mediate homeostasis and tissue regeneration in a variety of tissues, but their role in thymic injury is not known. This study aims to shed light on the role of regulatory T cells (Tregs) in thymic regeneration. Using a model of thymic injury and regeneration in mice receiving sublethal total body irradiation (SL-TBI, 550cGy), we observed a significant increase in Tregs ( Fig. 1A), located in both the medullary and cortical areas of the thymus on multiparameter immunohistochemistry. Employing a targeted ablation of Tregs at the time of injury using the FOXP3DTR mouse model, we could observe significant delay in thymic regeneration in mice depleted of Tregs (day 7 post SL-TBI, p=&amp;lt;0.0001, n=10, Fig. 1B). RAG2GFP mice, where GFP is expressed under the RAG2 promoter, have been used to discern recirculating and de-novo produced Tregs. To characterize the nature of the Treg expansion, we treated RAG2GFP mice with SL-TBI and analyzed their thymus at multiple time points after injury. Interestingly, the increase in Tregs was mainly due to recirculating RAG2-GFP- Tregs but not de-novo produced RAG2+GFP+ Tregs after injury. Brdu administration after injury indicated proliferation of these RAG2-GFP- Tregs. Parabiotic experiments linking RAG2GFP mice expressing either the congenic marker CD45.1 or CD45.2 confirmed the recirculating nature of these cells, both at steady state and after injury. Furthermore, when adoptively transferring sorted Thymic Tregs from FOXP3RFP mice to mice receiving SL-TBI, we significantly enhanced thymic cellularity after injury and homing of these Tregs to the thymus. To discern the transcriptional profile of these cells, we used single-cell sequencing of CD45+ RAG2- cells in 2- and 18-month-old RAG2GFP mice before and at day 1, 4, and 7 after sublethal irradiation. This enabled us to discover the age- and injury-associated changes in recirculating T cells. While the aged thymus had a similar surge of Treg numbers after injury, this response was blunted compared to their young counterparts. Factor normalization methods enabled us to find two distinct gene regulatory networks highly specific to the RAG2-GFP-Tregs. One gene network was highly enriched for Zfp36l1, a poised RNA known to mediate cell stability. Selectively ablating Zfp36l1 in Tregs using the Foxp3Cre ZFP36L1 flox/flox model impaired regeneration significantly after SL-TBI. The other distinct RAG2-GFP- Treg specific gene network was enriched for the regenerative factor Amphiregulin ( Areg). Amphiregulin was seen to be increased in thymic supernatants, and its receptor Egfr was seen to be transcriptionally upregulated in thymic epithelial cells after injury. In parallel, the most significantly upregulated pathway using cell-to-cell interaction prediction models on single-cell data indicated a strong likelihood of Treg to thymic epithelial cell interactions through amphiregulin to Egfr. Functional inhibition of amphiregulin with an antibody enabled us to observe delayed thymic regeneration, indicating amphiregulin as a factor in the regenerative capacity of thymic recirculating Tregs. Mapping human gene orthologs of the gene regulatory program provided by factor normalization in murine RAG2GFP- Tregs on single cell data of human pediatric thymic samples from children undergoing open heart surgeries enabled us to distinguish a putatively analogous human population of recirculating thymic Tregs. This population was found to be amphiregulin positive. And human thymic epithelial cell cultures with recombinant amphiregulin enhanced the growth of human thymic epithelial cells, as measured by life imaging, and promoted signalling through MAPK ERK1/2. Taken together, our findings reveal a novel role for injury-resistant recirculating thymic regulatory T cells existing in both the human and murine thymus and may enable the development of new therapeutic targets for thymic regeneration for patients undergoing cytoreductive therapies.

Reciprocal mutations of lung-tropic AAV capsids lead to improved transduction properties.

Considerable effort has been devoted to developing adeno-associated virus (AAV)-based vectors for gene therapy in cystic fibrosis (CF). As a result of directed evolution and capsid shuffling technology, AAV capsids are available with widespread tropism for airway epithelial cells. For example, AAV2.5T and AAV6.2 are two evolved capsids with improved airway epithelial cell transduction properties over their parental serotypes. However, limited research has been focused on identifying their specific cellular tropism. Restoring cystic fibrosis transmembrane conductance regulator (CFTR) expression in surface columnar epithelial cells is necessary for the correction of the CF airway phenotype. Basal cells are a progenitor population of the conducting airways responsible for replenishing surface epithelial cells (including secretory cells and ionocytes), making correction of this cell population vital for a long-lived gene therapy strategy. In this study, we investigate the tropism of AAV capsids for three cell types in primary cultures of well-differentiated human airway epithelial (HAE) cells and primary human airway basal cells. We observed that AAV2.5T transduced surface epithelial cells better than AAV6.2, while AAV6.2 transduced airway basal cells better than AAV2.5T. We also investigated a recently developed capsid, AAV6.2FF, which has two surface tyrosines converted to phenylalanines. Next, we incorporated reciprocal mutations to create AAV capsids with further improved surface and basal cell transduction characteristics. Lastly, we successfully employed a split-intein approach using AAV to deliver an adenine base editor (ABE) to repair the CFTR R553X mutation. Our results suggest that rational incorporation of AAV capsid mutations improves AAV transduction of the airway surface and progenitor cells and may ultimately lead to improved pulmonary function in people with CF.

Exposure to urban nanoparticles at low PM_1 concentrations as a source of oxidative stress and inflammation

Exposures to fine particulate matter (PM_1) have been associated with health impacts, but the understanding of the PM_1 concentration-response (PM_1-CR) relationships, especially at low PM_1, remains incomplete. Here, we present novel data using a methodology to mimic lung exposure to ambient air (2<PM_1< 60 upmug m^{-3}), with minimized sampling artifacts for nanoparticles. A reference model (Air Liquid Interface cultures of human bronchial epithelial cells, BEAS-2B) was used for aerosol exposure. Non-linearities observed in PM_1-CR curves are interpreted as a result of the interplay between the aerosol total oxidative potential (OP_t) and its distribution across particle size (d_p). A d_p-dependent condensation sink (CS) is assessed together with the distribution with d_p of reactive species . Urban ambient aerosol high in OP_t, as indicated by the DTT assay, with (possibly copper-containing) nanoparticles, shows higher pro-inflammatory and oxidative responses, this occurring at lower PM_1 concentrations (< 5 upmug m^{-3}). Among the implications of this work, there are recommendations for global efforts to go toward the refinement of actual air quality standards with metrics considering the distribution of OP_t with d_p also at relatively low PM_1.

A Novel Sandwich Method for Serial Block Face SEM Imaging of Airway Multiciliated Epithelium.

Volume electron microscopy technologies such as serial block face scanning electron microscopy (SBF-SEM) allow the characterization of tissue organization and cellular content in three dimensions at nanoscale resolution. Here, we describe the procedure to process and image an air-liquid interface culture of human or mouse airway epithelial cells for visualization of the multiciliated epithelium by SBF-SEM in vertical or horizontal cross section.

A potent MAPK13-14 inhibitor prevents airway inflammation and mucus production.

Common respiratory diseases continue to represent a major public health problem, and much of the morbidity and mortality is due to airway inflammation and mucus production. Previous studies indicated a role for mitogen-activated protein kinase 14 (MAPK14) in this type of disease, but clinical trials are unsuccessful to date. Our previous work identified a related but distinct kinase known as MAPK13 that is activated in respiratory airway diseases and is required for mucus production in human cell-culture models. Support for MAPK13 function in these models came from effectiveness of MAPK13 versus MAPK14 gene-knockdown and from first-generation MAPK13-14 inhibitors. However, these first-generation inhibitors were incompletely optimized for blocking activity and were untested in vivo. Here we report the next generation and selection of a potent MAPK13-14 inhibitor (designated NuP-3) that more effectively downregulates type-2 cytokine-stimulated mucus production in air-liquid interface and organoid cultures of human airway epithelial cells. We also show that NuP-3 treatment prevents respiratory airway inflammation and mucus production in new minipig models of airway disease triggered by type-2 cytokine challenge or respiratory viral infection. The results thereby provide the next advance in developing a small-molecule kinase inhibitor to address key features of respiratory disease.NEW & NOTEWORTHY This study describes the discovery of a potent mitogen-activated protein kinase 13-14 (MAPK13-14) inhibitor and its effectiveness in models of respiratory airway disease. The findings thereby provide a scheme for pathogenesis and therapy of lung diseases [e.g., asthma, chronic obstructive pulmonary disease (COPD), Covid-19, postviral, and allergic respiratory disease] and related conditions that implicate MAPK13-14 function. The findings also refine a hypothesis for epithelial and immune cell functions in respiratory disease that features MAPK13 as a possible component of this disease process.

Household laundry detergents disrupt barrier integrity and induce inflammation in mouse and human skin.

Epithelial barrier impairment is associated with many skin and mucosal inflammatory disorders. Laundry detergents have been demonstrated to affect epithelial barrier function in vitro using air-liquid interface cultures of human epithelial cells. Back skin of C57BL/6 mice was treated with two household laundry detergents at several dilutions. Barrier function was assessed by electric impedance spectroscopy (EIS) and transepidermal water loss (TEWL) measurements after the 4 h of treatments with detergents. RNA sequencing (RNA-seq) and targeted multiplex proteomics analyses in skin biopsy samples were performed. The 6-h treatment effect of laundry detergent and sodium dodecyl sulfate (SDS) was investigated on ex vivo human skin. Detergent-treated skin showed a significant EIS reduction and TEWL increase compared to untreated skin, with a relatively higher sensitivity and dose-response in EIS. The RNA-seq showed the reduction of the expression of several genes essential for skin barrier integrity, such as tight junctions and adherens junction proteins. In contrast, keratinization, lipid metabolic processes, and epidermal cell differentiation were upregulated. Proteomics analysis showed that the detergents treatment generally downregulated cell adhesion-related proteins, such as epithelial cell adhesion molecule and contactin-1, and upregulated proinflammatory proteins, such as interleukin 6 and interleukin 1 beta. Both detergent and SDS led to a significant decrease in EIS values in the ex vivo human skin model. The present study demonstrated that laundry detergents and its main component, SDS impaired the epidermal barrier in vivo and ex vivo human skin. Daily detergent exposure may cause skin barrier disruption and may contribute to the development of atopic diseases.

Interferon-independent processes constrain measles virus cell-to-cell spread in primary human airway epithelial cells.

Amplification of measles virus (MeV) in human airway epithelia may contribute to its extremely high contagious nature. We use well-differentiated primary cultures of human airway epithelial cells (HAE) to model ex vivo how MeV spreads in human airways. In HAE, MeV spreads cell-to-cell for 3-5 days, but then, infectious center growth is arrested. What stops MeV spread in HAE is not understood, but interferon (IFN) is known to slow MeV spread in other in vitro and in vivo models. Here, we assessed the role of type I and type III IFN in arresting MeV spread in HAE. The addition of IFN-β or IFN-λ1 to the medium of infected HAE slowed MeV infectious center growth, but when IFN receptor signaling was blocked, infectious center size was not affected. In contrast, blocking type-I IFN receptor signaling enhanced respiratory syncytial virus spread. HAE were also infected with MeV mutants defective for the V protein. The V protein has been demonstrated to interact with both MDA5 and STAT2 to inhibit activation of innate immunity; however, innate immune reactions were unexpectedly muted against the V-defective MeV in HAE. Minimal innate immunity activation was confirmed by deep sequencing, quantitative RT-PCR, and single-cell RNA-seq analyses of the transcription of IFN and IFN-stimulated genes. We conclude that in HAE, IFN-signaling can contribute to slowing infectious center growth; however, IFN-independent processes are most important for limiting cell-to-cell spread. IMPORTANCE Fundamental biological questions remain about the highly contagious measles virus (MeV). MeV amplifies within airway epithelial cells before spreading to the next host. This final step likely contributes to the ability of MeV to spread host-to-host. Over the course of 3-5 days post-infection of airway epithelial cells, MeV spreads directly cell-to-cell and forms infectious centers. Infectious center formation is unique to MeV. In this study, we show that interferon (IFN) signaling does not explain why MeV cell-to-cell spread is ultimately impeded within the cell layer. The ability of MeV to spread cell-to-cell in airway cells without appreciable IFN induction may contribute to its highly contagious nature. This study contributes to the understanding of a significant global health concern by demonstrating that infectious center formation occurs independent of the simplest explanation for limiting viral transmission within a host.