Human Air-liquid Interface Cultures Research Articles

Cryptosporidium infection of human small intestinal epithelial cells induces type III interferon and impairs infectivity of Rotavirus

ABSTRACT Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. Cryptosporidium infection induced a strong interferon response from enterocytes, possibly driven, in part, by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.

Inhalable Textile Microplastic Fibers Impair Airway Epithelial Differentiation.

Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 μm nylon 6,6 (nylon) and 15 × 52 μm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.

Spatiotemporally organized immunomodulatory response to SARS-CoV-2 virus in primary human broncho-alveolar epithelia

The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression toward severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4-6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an "early" inflammatory/immune signature preceding a "late" type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

Fluticasone propionate suppresses the SARS-CoV-2 induced increase in respiratory epithelial permeability in vitro.

Disruption of the nasal epithelial barrier is believed to play a role in Coronavirus Disease-2019 (COVID-19) outcomes. Fluticasone propionate has been shown to restore the nasal epithelial barrier in allergic rhinitis to the level of healthy controls. The therapeutic potential of nasal steroid sprays in COVID-19 has recently been reported. However, further insight into the mode of action is warranted. To explore the in vitro mechanisms of the preventive potential of fluticasone propionate in SARS-CoV-2 infection. Human air liquid interface cultures of Calu-3 cells and primary nasal epithelial cells isolated from healthy donors were used to investigate the preventive effect of fluticasone propionate on SARS-CoV-2 induced barrier disruption, virus replication and ACE2 expression. 48 hours pre-treatment with fluticasone propionate prevented the SARS-CoV-2 induced increase in fluorescein isothiocyanate-dextran 4 kDa permeability and reduced infection with SARS-CoV-2. Pre-treatment with fluticasone propionate also decreased ACE2 expression in SARS-CoV-2 infected Calu-3 cells. Fluticasone propionate pre-treatment prevented SARS-CoV-2 increased epithelial permeability, reduced ACE2 expression and SARS-CoV-2 infection, underscoring the therapeutic potential of fluticasone propionate in the context of COVID-19.

In vitro airway models from mice, rhesus macaques, and humans maintain species differences in xenobiotic metabolism and cellular responses to naphthalene.

The translational value of high-throughput toxicity testing will depend on pharmacokinetic validation. Yet, popular in vitro airway epithelia models were optimized for structure and mucociliary function without considering the bioactivation or detoxification capabilities of lung-specific enzymes. This study evaluated xenobiotic metabolism maintenance within differentiated air-liquid interface (ALI) airway epithelial cell cultures (human bronchial; human, rhesus, and mouse tracheal), isolated airway epithelial cells (human, rhesus, and mouse tracheal; rhesus bronchial), and ex vivo microdissected airways (rhesus and mouse) by measuring gene expression, glutathione content, and naphthalene metabolism. Glutathione levels and detoxification gene transcripts were measured after 1-h exposure to 80 µM naphthalene (a bioactivated toxicant) or reactive naphthoquinone metabolites. Glutathione and glutathione-related enzyme transcript levels were maintained in ALI cultures from all species relative to source tissues, while cytochrome P450 monooxygenase gene expression declined. Notable species differences among the models included a 40-fold lower total glutathione content for mouse ALI trachea cells relative to human and rhesus; a higher rate of naphthalene metabolism in mouse ALI cultures for naphthalene-glutathione formation (100-fold over rhesus) and naphthalene-dihydrodiol production (10-fold over human); and opposite effects of 1,2-naphthoquinone exposure in some models-glutathione was depleted in rhesus tissue but rose in mouse ALI samples. The responses of an immortalized bronchial cell line to naphthalene and naphthoquinones were inconsistent with those of human ALI cultures. These findings of preserved species differences and the altered balance of phase I and phase II xenobiotic metabolism among the characterized in vitro models should be considered for future pulmonary toxicity testing.

Transcriptome analysis reveals lung-specific miRNAs associated with impaired mucociliary clearance induced by cigarette smoke in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is strongly associated with impaired mucociliary clearance (MCC), which has been implicated in the pathogenesis of CS-induced respiratory diseases, such as chronic obstructive pulmonary diseases (COPD). In this study, we aimed to identify microRNAs (miRNAs) that are associated with impaired MCC caused by CS in an in vitro human air-liquid-interface (ALI) airway tissue model. ALI cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min of clean air) from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 1week (a total of 3days, 40min/day). Transcriptome analyses of ALI cultures exposed to the high concentration of CS identified 5090 differentially expressed genes and 551 differentially expressed miRNAs after the third exposure. Genes involved in ciliary function and ciliogenesis were significantly perturbed by repeated CS exposures, leading to changes in cilia beating frequency and ciliary protein expression. In particular, a time-dependent decrease in the expression of miR-449a, a conserved miRNA highly enriched in ciliated airway epithelia and implicated in motile ciliogenesis, was observed in CS-exposed cultures. Similar alterations in miR-449a have been reported in smokers with COPD. Network analysis further indicates that downregulation of miR-449a by CS may derepress cell-cycle proteins, which, in turn, interferes with ciliogenesis. Investigating the effects of CS on transcriptome profile in human ALI cultures may provide not only mechanistic insights, but potential early biomarkers for CS exposure and harm.

Interleukin 13 (IL-13) alters hypoxia-associated genes and upregulates CD73.

Interleukin 13 (IL-13) is a pleiotropic cytokine that has been shown to be important in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) and other type 2 inflammation-related diseases. Increased IL-13 expression can elicit several pro-inflammatory effects, including eosinophilia, and pathology such as increased mucus secretion. Polypogenesis in chronic rhinosinusitis (CRS) can be caused by hypoxia, which can also lead to hyperpermeability of airway epithelium and epithelium-to-mesenchymal translation through the upregulation of hypoxia-associated genes, such as HIF1. Whether T-helper 2 (Th2) inflammatory cytokines, such as IL-13, can also induce sinonasal epithelial hypoxia-associated genes is currently unknown. Human air-liquid interface (ALI) sinonasal epithelial cell cultures treated with recombinant IL-13 were analyzed by real-time polymerase chain reaction (PCR) and flow cytometry to determine the effect on epithelial cells. Whole tissue from CRSwNP subjects showed increased HIF1A gene expression. Treatment of fully differentiated human ALI cultures with IL-13 resulted in a concurrent increase in HIF1A and ARNT messenger RNA (mRNA) expression. However, the level of EPAS1 expression was significantly reduced. IL-13 also had a dose-dependent response on the expression of HIF genes and the time course experiment showed peak expression of HIF1A and ARNT at 5 to 7 days poststimulation. Remarkably, CD73 surface expression also peaked at day 5 poststimulation. Our data suggests that IL-13 can induce hypoxia signaling pathway genes leading to surface expression of CD73, which has an anti-inflammatory effect.

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene...

534. Preparation for a First-in-Man Lentivirus Trial in Cystic Fibrosis Patients

Background: We have recently shown that non-viral gene therapy can stabilise the decline of lung function in cystic fibrosis (CF) patients. However, the effect was modest, and it is important to develop more potent gene transfer agents in parallel. F/HN-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in pre-clinical models. In preparation for a first-in-man CF trial using the lentiviral vector we have undertaken key translational pre-clinical studies. Methods: Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air liquid interface cultures to select the lead candidate; CFTR expression and function were assessed in CF models (knockout mice and human intestinal organoids) using this lead candidate vector. Toxicity was assessed and “benchmarked” against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. Results: A hybrid promoter consisting of the elongation factor 1α promoter and the CMV enhancer was most efficacious in both murine lungs and human air liquid interface cultures. The efficacy, toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector is stable in clinically relevant delivery devices. Conclusions: The data support progression of the F/HN pseudotyped lentiviral vector into a first-in-man CF trial due to start in Q2 2017. Regulatory-compliant toxicology studies are currently being performed.

Assessment of F/HN-pseudotyped lentivirus as a clinically relevant vector for lung gene therapy.

Ongoing efforts to improve pulmonary gene transfer thereby enabling gene therapy for the treatment of lung diseases, such as cystic fibrosis (CF), has led to the assessment of a lentiviral vector (simian immunodeficiency virus [SIV]) pseudotyped with the Sendai virus envelope proteins F and HN. To place this vector onto a translational pathway to the clinic by addressing some key milestones that have to be achieved. F/HN-SIV transduction efficiency, duration of expression, and toxicity were assessed in mice. In addition, F/HN-SIV was assessed in differentiated human air-liquid interface cultures, primary human nasal epithelial cells, and human and sheep lung slices. A single dose produces lung expression for the lifetime of the mouse (~2 yr). Only brief contact time is needed to achieve transduction. Repeated daily administration leads to a dose-related increase in gene expression. Repeated monthly administration to mouse lower airways is feasible without loss of gene expression. There is no evidence of chronic toxicity during a 2-year study period. F/HN-SIV leads to persistent gene expression in human differentiated airway cultures and human lung slices and transduces freshly obtained primary human airway epithelial cells. The data support F/HN-pseudotyped SIV as a promising vector for pulmonary gene therapy for several diseases including CF. We are now undertaking the necessary refinements to progress this vector into clinical trials.

Assessment of the nuclear pore dilating agent trans‐cyclohexane‐1,2‐diol in differentiated airway epithelium

The nuclear membrane of differentiated airway epithelial cells is a significant barrier for nonviral vectors. Trans-cyclohexane-1,2-diol (TCHD) is an amphipathic alcohol that has been shown to collapse nuclear pore cores and allow the uptake of macromolecules that would otherwise be too large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro. We aimed to reproduce these in vitro studies using the cationic lipid GL67A, which we are currently assessing in cystic fibrosis trials and, more importantly, we assessed the effects of TCHD on transfection efficiency in differentiated airway epithelium ex vivo and in mouse lung in vivo using three different drug delivery protocols (nebulisation and bolus administration of TCHD to the mouse lung, as well as perfusion of TCHD to the nasal epithelium, which prolongs contact time between the airway epithelium and drug). TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells by up to 2 logs. Encouragingly, exposure to 8% TCHD (but not 0.5% or 2.0%) increased gene expression in fully differentiated human air liquid interface cultures by approximately 20-fold, although this was accompanied by significant cell damage. However, none of the TCHD treated mice in any of the three protocols had higher gene expression compared to no TCHD controls. Although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights that promising in vitro findings often cannot be translated into in vivo applications.

Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer

The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.

Species-specific differences in mouse and human airway epithelial biology of recombinant adeno-associated virus transduction.

Differences in airway epithelial biology between mice and humans have presented challenges to evaluating gene therapies for cystic fibrosis (CF) using murine models. In this context, recombinant adeno-associated virus (rAAV) type 2 and rAAV5 vectors have very different transduction efficiencies in human air-liquid interface (ALI) airway epithelia (rAAV2 approximately = rAAV5) as compared with mouse lung (rAAV5 >> rAAV2). It is unclear if these differences are due to species-specific airway biology or limitations of ALI cultures to reproduce in vivo airway biology. To this end, we compared rAAV2 and rAAV5 transduction biology in mouse and human ALI cultures, and investigated the utility of murine deltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) ALI epithelia to study CFTR complementation. Our results demonstrate that mouse ALI epithelia retain in vivo preferences for rAAV serotype transduction from the apical membrane (rAAV5 >> rAAV2) not seen in human epithelia (rAAV2 approximately = rAAV5). Viral binding of rAAV2 and rAAV5 to the apical surface of mouse ALI airway epithelia was not significantly different, and proteasome-modulating agents significantly enhanced rAAV2 transduction to a level equivalent to that of rAAV5 in the presence of these agents, suggesting that the ubiquitin/proteasome pathway represents a more significant intracellular block for rAAV2 transduction of mouse airway epithelia. Interestingly, cAMP-inducible chloride currents were enhanced in deltaF508CFTR mouse ALI cultures, making this model incompatible with CFTR complementation studies. These studies emphasize species-specific differences in airway biology between mice and humans that significantly influence the use of mice as surrogate models for rAAV transduction and gene therapy for CF.

499. Species-Specific Differences in the Polarity of rAAV5 and rAAV2 Transduction of Mouse and Human Airway Epithelia

Top of pageAbstract Species-specific differences in the tropism of viral vectors between mice and humans have made it difficult to anticipate the most appropriate adeno-associated virus (AAV) serotypes for clinical trials. In the context of developing gene therapies for cystic fibrosis lung disease, human air-liquid interface (ALI) models of airway epithelium have been useful as an intermediate surrogate for evaluating the human tropisms of various rAAV serotypes. In this model, we have previously shown that apical infection with rAAV2 and rAAV5 vectors have very similar transduction efficiencies. Additionally, in the presence of proteasome inhibitors, transduction is enhanced |[sim]|1000-fold with both serotypes following apical infection. In contrast to these findings in the human ALI model, in vivo rAAV5 transduction is 50-100-fold greater than that of rAAV2 in mouse lung. These differences could be the result of model system dependencies or species-specific differences in the tropism of AAV2 and AAV5 for airway epithelium in mice and humans. To resolve whether species-specific differences (human vs. mouse) or model system-specific differences (in vitro ALI culture vs. in vivo lung) are the cause of the discrepancies in rAAV5 and rAAV2 transduction in these various studies, we have compared transduction of these rAAV serotypes in mouse and human airway epithelia in ALI culture using pseudotyped luciferase rAAV vectors. Differentiated mouse airway epithelia in ALI culture gave electrophysiologic characteristics similar to those previously reported for murine tracheal epithelium, but distinct from that of human ALI cultures. Following apical infection of mouse airway epithelia in ALI culture, rAAV2/5 gave |[sim]|100-fold greater levels of transduction than infection with rAAV2/2. These results were similar to the differences in transduction between these two serotypes seen in vivo in mouse lung. These findings demonstrate that ALI cultures of mouse airway epithelium retain species-specific differences in comparison to human airway epithelium, which is reflected in a greater apical tropism for rAAV5 as compared to rAAV2 capsid serotypes. Interestingly, basolateral infection of mouse ALI cultures also retained a greater transduction efficiency (10-fold) with rAAV2/5, as compared to rAAV2/2. These differences in basolateral tropisms were reversed for human ALI cultures. Viral transduction for both rAAV2/2 and rAAV2/5 from the apical membrane of both mouse and human ALI cultures was enhanced 2-3 logs by the addition of proteasome modulating agents Doxorubicin and LLnL at the time of infection. Basolateral infection in the absence of proteasome modulating was always more effective than apical infection regardless of the species. However, the addition of proteasome modulating agents appeared to normalize transduction from the apical membrane to maximal levels regardless of the serotype or species. These findings suggest that rAAV2 and rAAV5 vectors have different transduction efficiencies for mouse and human airway epithelium due to serotype-specific differences in post-entry blocks modulated by the proteasome pathway.

Capital Appreciation of World Equity Markets 1971–1994

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air–liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and ‘benchmarked’ against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90–100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.