A mimetic peptide of ACE2 protects against SARS-CoV-2 infection and decreases pulmonary inflammation related to COVID-19.

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22-46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.

The flavonoid quercetin decreases ACE2 and TMPRSS2 expression but not SARS-CoV-2 infection in cultured human lung cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) on host cells, via its spike protein, and transmembrane protease, serine 2 (TMPRSS2) cleaves the spike-ACE2 complex to facilitate virus entry. As rate-limiting steps for virus entry, modulation of ACE2 and/or TMPRSS2 may decrease SARS-CoV-2 infectivity and COVID-19 severity. In silico modeling suggested the natural bioactive flavonoid quercetin can bind to ACE2 and a recent randomized clinical trial demonstrated that oral supplementation with quercetin increased COVID-19 recovery. A range of cultured human cells were assessed for co-expression of ACE2 and TMPRSS2. Immortalized Calu-3 lung cells, cultured and matured at an air-liquid interface (Calu-3-ALIs), were established as the most appropriate. Primary bronchial epithelial cells (PBECs) were obtained from healthy adult males (N = 6) and cultured under submerged conditions to corroborate the outcomes. Upon maturation or reaching 80% confluence, respectively, the Calu-3-ALIs and PBECs were treated with quercetin, and mRNA and protein expression were assessed by droplet digital PCR and ELISA, respectively. SARS-CoV-2 infectivity, and the effects of pre- and co-treatment with quercetin, was assessed by median tissue culture infectious dose assay. Quercetin dose-dependently decreased ACE2 and TMPRSS2 mRNA and protein in both Calu-3-ALIs and PBECs after 4 h, while TMPRSS2 remained suppressed in response to prolonged treatment with lower doses (twice daily for 3 days). Quercetin also acutely decreased ADAM17 mRNA, but not ACE, in Calu-3-ALIs, and this warrants further investigation. Calu-3-ALIs, but not PBECs, were successfully infected with SARS-CoV-2; however, quercetin had no antiviral effect, neither directly nor indirectly through downregulation of ACE2 and TMPRSS2. Calu-3-ALIs were reaffirmed to be an optimal cell model for research into the regulation of ACE2 and TMPRSS2, without the need for prior genetic modification, and will prove valuable in future coronavirus and respiratory infectious disease work. However, our data demonstrate that a significant decrease in the expression of ACE2 and TMPRSS2 by a promising prophylactic candidate may not translate to infection prevention.

MicroRNA expression profile of alveolar epithelial cells infected with Aspergillus fumigatus.

There are limited number of studies investigating the role of microRNAs (miRNAs) in Aspergillus infections. In this study, we designed an in vitro aspergillosis model to identify differentially expressed Aspergillus-related miRNAs. For this purpose, carcinoma cell lines "A549" and "Calu-3" were infected with Aspergillus fumigatus. Total miRNA was isolated at 0, 1, 6, and 24 h post-infection. Quantitative real-time PCR assay was conducted to screen 31 human miRNAs that were possibly related to aspergillosis. Up- and downregulated miRNAs were detected in the infected cells. Highest level of miRNA expression was detected at 6 h post-infection. miR-21, hsa-miR-186-5p, hsa-miR-490-5p, miR-26a-5p, miR-26b-5p, hsa-miR-424-5p, hsa-miR-548d-3p, hsa-miR-196a-5p, miR-150-5p, miR-17-5p, and hsa-miR-99b-5p were found to be significantly upregulated (p < 0.001) at 6 h after A. fumigatus infection compared with the controls. Among the screened miRNAs, hsa-miR-145-5p (p < 0.001); hsa-miR-583 and hsa-miR-3978 (p < 0.01); and miR-21-5p, hsa-miR-4488, and hsa-miR-4454 (p < 0.05) were found to be downregulated compared with the controls. In conclusion, screening the identified miRNAs may reveal the personal predisposition to aspergillosis, which might be valuable from the perspective of personalized medicine.

Upregulation of mRNA Expression of ADGRD1/GPR133 and ADGRG7/GPR128 in SARS-CoV-2-Infected Lung Adenocarcinoma Calu-3 Cells.

Adhesion G protein-coupled receptors (aGPCRs) play an important role in neurodevelopment, immune defence and cancer; however, their role throughout viral infections is mostly unexplored. We have been searching for specific aGPCRs involved in SARS-CoV-2 infection of mammalian cells. In the present study, we infected human epithelial cell lines derived from lung adenocarcinoma (Calu-3) and colorectal carcinoma (Caco-2) with SARS-CoV-2 in order to analyse changes in the level of mRNA encoding individual aGPCRs at 6 and 12 h post infection. Based on significantly altered mRNA levels, we identified four aGPCR candidates-ADGRB3/BAI3, ADGRD1/GPR133, ADGRG7/GPR128 and ADGRV1/GPR98. Of these receptors, ADGRD1/GPR133 and ADGRG7/GPR128 showed the largest increase in mRNA levels in SARS-CoV-2-infected Calu-3 cells, whereas no increase was observed with heat-inactivated SARS-CoV-2 and virus-cleared conditioned media. Next, using specific siRNA, we downregulated the aGPCR candidates and analysed SARS-CoV-2 entry, replication and infectivity in both cell lines. We observed a significant decrease in the amount of SARS-CoV-2 newly released into the culture media by cells with downregulated ADGRD1/GPR133 and ADGRG7/GPR128. In addition, using a plaque assay, we observed a reduction in SARS-CoV-2 infectivity in Calu-3 cells. In summary, our data suggest that selected aGPCRs might play a role during SARS-CoV-2 infection of mammalian cells.

Mathematical model explains differences in Omicron and Delta SARS-CoV-2 dynamics in Caco-2 and Calu-3 cells.

Within-host infection dynamics of Omicron dramatically differs from previous variants of SARS-CoV-2. However, little is still known about which parameters of virus-cell interplay contribute to the observed attenuated replication and pathogenicity of Omicron. Mathematical models, often expressed as systems of differential equations, are frequently employed to study the infection dynamics of various viruses. Adopting such models for results of in vitro experiments can be beneficial in a number of aspects, such as model simplification (e.g., the absence of adaptive immune response and innate immunity cells), better measurement accuracy, and the possibility to measure additional data types in comparison with in vivo case. In this study, we consider a refinement of our previously developed and validated model based on a system of integro-differential equations. We fit the model to the experimental data of Omicron and Delta infections in Caco-2 (human intestinal epithelium model) and Calu-3 (lung epithelium model) cell lines. The data include known information on initial conditions, infectious virus titers, and intracellular viral RNA measurements at several time points post-infection. The model accurately explains the experimental data for both variants in both cell lines using only three variant- and cell-line-specific parameters. Namely, the cell entry rate is significantly lower for Omicron, and Omicron triggers a stronger cytokine production rate (i.e., innate immune response) in infected cells, ultimately making uninfected cells resistant to the virus. Notably, differences in only a single parameter (e.g., cell entry rate) are insufficient to obtain a reliable model fit for the experimental data.

In vitro evaluation of physicochemical-dependent effects of polymeric nanoparticles on their cellular uptake and co-localization using pulmonary calu-3 cell lines.

OBJECTIVE: The study evaluated physicochemical properties of eight different polymeric nanoparticles (NPs) and their interaction with lung barrier and their suitability for pulmonary drug delivery. METHODS: Eight physiochemically different NPs were fabricated from Poly lactic-co-glycolic acid (PLGA, PL) and Poly glycerol adipate-co-ω-pentadecalactone (PGA-co-PDL, PG) via emulsification-solvent evaporation. Pulmonary barrier integrity was investigated in vitro using Calu-3 under air-liquid interface. NPs internalization was investigated using a group of pharmacological inhibitors with subsequent microscopic visual confirmation. RESULTS: Eight NPs were successfully formulated from two polymers using emulsion-solvent evaporation; 200, 500 and 800 nm, negatively-charged and positively-charged. All different NPs did not alter tight junctions and PG NPs showed similar behavior to PL NPs, indicating its suitability for pulmonary drug delivery. Active endocytosis uptake mechanisms with physicochemical dependent manner were observed. In addition, NPs internalization and co-localization with lysosomes were visually confirmed indicating their vesicular transport. CONCLUSION: PG and PL NPs had shown no or low harmful effects on the barrier integrity, and with effective internalization and vesicular transport, thus, prospectively can be designed for pulmonary delivery applications.

Liposomal Formulation Reduces Transport and Cell Uptake of Colistin in Human Lung Epithelial Calu-3 Cell and 3D Human Lung Primary Tissue Models.

Respiratory tract infections caused by multi-drug-resistant (MDR) bacteria have been a severe risk to human health. Colistin is often used to treat the MDR Gram-negative bacterial infections as a last-line therapy. Inhaled colistin can achieve a high concentration in the lung but none of aerosolized colistin products has been approved in the USA. Liposome has been reported as an advantageous formulation strategy for antibiotics due to its controlled release profile and biocompatibility. We have developed colistin liposomal formulations in our previous study. In the present study, the cellular uptake and transport of colistin in colistin liposomes were examined in two human lung epithelium in vitro models, Calu-3 monolayer and EpiAirway 3D tissue models. In both models, cellular uptake (p < 0.05) and cellular transport (p < 0.01) of colistin were significantly reduced by the colistin liposome compared to the colistin solution. Our findings indicate that inhaled colistin liposomes could be a promising treatment for extracellular bacterial lung infections caused by MDR Pseudomonas aeruginosa (P. aeruginosa).

Multitask learning for predicting pulmonary absorption of chemicals.

Pulmonary absorption is an important route for drug delivery and chemical exposure. To streamline the chemical assessment process for the reduction of animal experiments, several animal-free models were developed for pulmonary absorption research. While Calu-3 and Caco-2 cells and their derived computational models were used in estimating pulmonary permeability, the ex vivo isolated perfused lung (IPL) models are considered more clinically relevant measurements. However, the IPL experiments are resource-consuming making it infeasible for the large-scale screening of potential inhaled toxicants and drugs. In silico models are desirable for estimating pulmonary absorption. This study presented a novel machine learning method that employed an extratrees-based multitask learning approach to predict the IPL absorption rate constant (kaIPL) of various chemicals. The shared permeability knowledge was extracted by simultaneously learning three relevant tasks of Caco-2 and Calu-3 cell permeability and IPL absorption rate. Seven informative physicochemical descriptors were identified. A rigorous evaluation of the developed prediction model showed good performance with a high correlation between predictions and observations (r = 0.84) in the independent test dataset. Two case studies of inhalation drugs and respiratory sensitizers revealed the potential application of this model, which may serve as a valuable tool for predicting pulmonary absorption of chemicals.

Enterovirus D68 vRNA induces type III IFN production via MDA5.

Enterovirus D68 (EV-D68) primarily spreads through the respiratory tract and causes respiratory symptoms in children and acute flaccid myelitis (AFM). Type III interferons (IFNs) play a critical role in inhibiting viral growth in respiratory epithelial cells. However, the mechanism by which EV-D68 induces type III IFN production is not yet fully understood. In this study, we show that EV-D68 infection stimulates Calu-3 cells to secrete IFN-λ. The transfection of EV-D68 viral RNA (vRNA) stimulated IFN-λ via MDA5. Furthermore, our findings provide evidence that EV-D68 infection also induces MDA5-IRF3/IRF7-mediated IFN-λ. In addition, we discovered that EV-D68 infection downregulated MDA5 expression. Knockdown of MDA5 increased EV-D68 replication in Calu-3 cells. Finally, we demonstrated that the IFN-λ1 and IFN-λ2/3 proteins effectively inhibit EV-D68 infection in respiratory epithelial cells. In summary, our study shows that EV-D68 induces type III IFN production via the activated MDA5-IRF3/IRF7 pathway and that type III IFNs inhibit EV-D68 replication in Calu-3 cells.

Quercetin improves and protects Calu-3 airway epithelial barrier function.

Introduction: In light of the impact of airway barrier leaks in COVID-19 and the significance of vitamin D in COVID-19 outcomes, including airway barrier protection, we investigated whether the very common dietary flavonoid quercetin could also be efficacious in supporting airway barrier function. Methods: To address this question, we utilized the widely used airway epithelial cell culture model, Calu-3. Results: We observed that treating Calu-3 cell layers with quercetin increased transepithelial electrical resistance while simultaneously reducing transepithelial leaks of 14C-D-mannitol (Jm) and 14C-inulin. The effects of quercetin were concentration-dependent and exhibited a biphasic time course. These effects of quercetin occurred with changes in tight junctional protein composition as well as a partial inhibition of cell replication that resulted in decreased linear junctional density. Both of these effects potentially contribute to improved barrier function. Quercetin was equally effective in reducing the barrier compromise caused by the pro-inflammatory cytokine TNF-α, an action that seemed to derive, in part, from reducing the elevation of ERK 1/2 caused by TNF-α. Discussion: Quercetin improved Calu-3 barrier function and reduced TNF-α-induced barrier compromise, mediated in part by changes in the tight junctional complex.

Transcriptional Profiling of SARS-CoV-2-Infected Calu-3 Cells Reveals Immune-Related Signaling Pathways.

The COVID-19 disease, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in late 2019 and rapidly spread worldwide, becoming a pandemic that infected millions of people and caused significant deaths. COVID-19 continues to be a major threat, and there is a need to deepen our understanding of the virus and its mechanisms of infection. To study the cellular responses to SARS-CoV-2 infection, we performed an RNA sequencing of infected vs. uninfected Calu-3 cells. Total RNA was extracted from infected (0.5 MOI) and control Calu-3 cells and converted to cDNA. Sequencing was performed, and the obtained reads were quality-analyzed and pre-processed. Differential expression was assessed with the EdgeR package, and functional enrichment was performed in EnrichR for Gene Ontology, KEGG pathways, and WikiPathways. A total of 1040 differentially expressed genes were found in infected vs. uninfected Calu-3 cells, of which 695 were up-regulated and 345 were down-regulated. Functional enrichment analyses revealed the predominant up-regulation of genes related to innate immune response, response to virus, inflammation, cell proliferation, and apoptosis. These transcriptional changes following SARS-CoV-2 infection may reflect a cellular response to the infection and help to elucidate COVID-19 pathogenesis, in addition to revealing potential biomarkers and drug targets.

Toxicological assessment of E-cigarette flavored E-liquids aerosols using Calu-3 cells: A 3D lung model approach.

Scientific progress and ethical considerations are increasingly shifting the toxicological focus from in vivo animal models to in vitro studies utilizing physiologically relevant cell cultures. Consequently, we evaluated and validated a three-dimensional (3D) model of the human lung using Calu-3 cells cultured at an air-liquid interface (ALI) for 28 days. Assessment of seven essential genes of differentiation and transepithelial electrical resistance (TEER) measurements, in conjunction with mucin (MUC5AC) staining, validated the model. We observed a time-dependent increase in TEER, genetic markers of mucus-producing cells (muc5ac, muc5b), basal cells (trp63), ciliated cells (foxj1), and tight junctions (tjp1). A decrease in basal cell marker krt5 levels was observed. Subsequently, we utilized this validated ALI-cultured Calu-3 model to investigate the adversity of the aerosols generated from three flavored electronic cigarette (EC) e-liquids: cinnamon, vanilla tobacco, and hazelnut. These aerosols were compared against traditional cigarette smoke (3R4F) to assess their relative toxicity. The aerosols generated from PG/VG vehicle control, hazelnut and cinnamon e-liquids, but not vanilla tobacco, significantly decreased TEER and increased lactate dehydrogenase (LDH) release compared to the incubator and air-only controls. Compared to 3R4F, there were no significant differences in TEER or LDH with the tested flavored EC aerosols other than vanilla tobacco. This starkly contrasted our expectations, given the common perception of e-liquids as a safer alternative to cigarettes. Our study suggests that these results depend on flavor type. Therefore, we strongly advocate for further research, increased user awareness regarding flavors in ECs, and rigorous regulatory scrutiny to protect public health.

Antiviral Effect of 5'-Arylchalcogeno-3-aminothymidine Derivatives in SARS-CoV-2 Infection.

The understanding that zidovudine (ZDV or azidothymidine, AZT) inhibits the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 and that chalcogen atoms can increase the bioactivity and reduce the toxicity of AZT has directed our search for the discovery of novel potential anti-coronavirus compounds. Here, the antiviral activity of selenium and tellurium containing AZT derivatives in human type II pneumocytes cell model (Calu-3) and monkey kidney cells (Vero E6) infected with SARS-CoV-2, and their toxic effects on these cells, was evaluated. Cell viability analysis revealed that organoselenium (R3a-R3e) showed lower cytotoxicity than organotellurium (R3f, R3n-R3q), with CC50 ≥ 100 µM. The R3b and R3e were particularly noteworthy for inhibiting viral replication in both cell models and showed better selectivity index. In Vero E6, the EC50 values for R3b and R3e were 2.97 ± 0.62 µM and 1.99 ± 0.42 µM, respectively, while in Calu-3, concentrations of 3.82 ± 1.42 µM and 1.92 ± 0.43 µM (24 h treatment) and 1.33 ± 0.35 µM and 2.31 ± 0.54 µM (48 h) were observed, respectively. The molecular docking calculations were carried out to main protease (Mpro), papain-like protease (PLpro), and RdRp following non-competitive, competitive, and allosteric inhibitory approaches. The in silico results suggested that the organoselenium is a potential non-competitive inhibitor of RdRp, interacting in the allosteric cavity located in the palm region. Overall, the cell-based results indicated that the chalcogen-zidovudine derivatives were more potent than AZT in inhibiting SARS-CoV-2 replication and that the compounds R3b and R3e play an important inhibitory role, expanding the knowledge about the promising therapeutic capacity of organoselenium against COVID-19.

3D model of the bronchial epithelial barrier to study repeated exposure to xenobiotics: Application to silver nanoparticles.

There is still a lack of in vitro human models to evaluate the chronic toxicity of drugs and environmental pollutants. Here, we used a 3D model of the human bronchial epithelium to assess repeated exposures to xenobiotics. The Calu-3 human bronchial cell line was exposed to silver nanoparticles (AgNP) 5 times during 12 days, at the air-liquid interface, to mimic single and repeated exposure to inhaled particles. Repeated exposures induced a stronger induction of the metal stress response and a steady oxidative stress over time. A sustained translocation of silver was observed after each exposure without any loss of the epithelial barrier integrity. The proteomic analysis of the mucus revealed changes in the secreted protein profiles associated with the epithelial immune response after repeated exposures only. These results demonstrate that advanced in vitro models are efficient to investigate the adaptive response of human cells submitted to repeated xenobiotic exposures.

Impact of mucus modulation by N-acetylcysteine on nanoparticle toxicity.

Human respiratory mucus is a biological hydrogel that forms a protective barrier for the underlying epithelium. Modulation of the mucus layer has been employed as a strategy to enhance transmucosal drug carrier transport. However, a drawback of this strategy is a potential reduction of the mucus barrier properties, in particular in situations with an increased exposure to particles. In this study, we investigated the impact of mucus modulation on its protective role. In vitro mucus was produced by Calu-3 cells, cultivated at the air-liquid interface for 21 days and used for further testing as formed on top of the cells. Analysis of confocal 3D imaging data revealed that after 21 days Calu-3 cells secrete a mucus layer with a thickness of 24 ± 6 µm. Mucus appeared to restrict penetration of 500 nm carboxyl-modified polystyrene particles to the upper 5-10 µm of the layer. Furthermore, a mucus modulation protocol using aerosolized N-acetylcysteine (NAC) was developed. This treatment enhanced the penetration of particles through the mucus down to deeper layers by means of the mucolytic action of NAC. These findings were supported by cytotoxicity data, indicating that intact mucus protects the underlying epithelium from particle-induced effects on membrane integrity. The impact of NAC treatment on the protective properties of mucus was probed by using 50 and 100 nm amine-modified and 50 nm carboxyl-modified polystyrene nanoparticles, respectively. Cytotoxicity was only induced by the amine-modified particles in combination with NAC treatment, implying a reduced protective function of modulated mucus. Overall, our data emphasize the importance of integrating an assessment of the protective function of mucus into the development of therapy approaches involving mucus modulation.

Nicotine transport across calu-3 cell monolayer: effect of nicotine salts and flavored e-liquids.

OBJECTIVE: This study aimed to investigate the transport capability of nicotine across Calu-3 cell monolayer in various nicotine forms, including nicotine freebase, nicotine salts, and flavored e-liquids with nicotine benzoate. SIGNIFICANCE: Nicotine is rapidly absorbed from the respiratory system into systemic circulation during e-cigarettes use. However, the mechanism of nicotine transport in the lung has not been well understood yet. This study may offer critical biological evidence and have implications for the use and regulation of e-cigarettes. METHODS: The viability of Calu-3 cells after administration of nicotine freebase, nicotine salts and representative e-liquid were evaluated using the MTT assay, and the integrity of the Calu-3 cell monolayer was evaluated by transepithelial electrical resistance measurement and morphological analysis. Further, the nicotine transport capacity across the Calu-3 cell monolayer in various formulations of nicotine was investigated by analysis of nicotine transport amount. RESULTS: The findings indicated that nicotine transport occurred passively and was time-dependent across the Calu-3cell monolayer. In addition, the nicotine transport was influenced by the type of nicotine salts and their respective pH value. The nicotine benzoate exhibited the highest apparent permeability coefficient (Papp), and higher nicotine-to-benzoic acid ratios led to higher Papp values. The addition of flavors to e-liquid resulted in increased Papp values, with the most significant increment being observed in tobacco-flavored e-liquid. CONCLUSIONS: In summary, the transport capability of nicotine across the Calu-3 cell monolayer was influenced by the pH values of nicotine salts and flavor additives in e-liquids.

Drug-induced phospholipidosis is not correlated with the inhibition of SARS-CoV-2 - inhibition of SARS-CoV-2 is cell line-specific.

Recently, Tummino et al. reported that 34 compounds, including Chloroquine and Fluoxetine, inhibit SARS-CoV-2 replication by inducing phospholipidosis, although Chloroquine failed to suppress viral replication in Calu-3 cells and patients. In contrast, Fluoxetine represses viral replication in human precision-cut lung slices (PCLS) and Calu-3 cells. Thus, it is unlikely that these compounds have similar mechanisms of action. Here, we analysed a subset of these compounds in the viral replication and phospholipidosis assays using the Calu-3 cells and PCLS as the patient-near system. Trimipramine and Chloroquine induced phospholipidosis but failed to inhibit SARS-CoV-2 replication in Calu-3 cells, which contradicts the reported findings and the proposed mechanism. Fluoxetine, only slightly induced phospholipidosis in Calu-3 cells but reduced viral replication by 2.7 orders of magnitude. Tilorone suppressed viral replication by 1.9 orders of magnitude in Calu-3 cells without causing phospholipidosis. Thus, induction of phospholipidosis is not correlated with the inhibition of SARS-CoV-2, and the compounds act via other mechanisms. However, we show that compounds, such as Amiodarone, Tamoxifen and Tilorone, with antiviral activity on Calu-3 cells, also inhibited viral replication in human PCLS. Our results indicate that antiviral assays against SARS-CoV-2 are cell-line specific. Data from Vero E6 can lead to non-transferable results, underlining the importance of an appropriate cell system for analysing antiviral compounds against SARS-CoV-2. We observed a correlation between the active compounds in Calu-3 cells and PCLS.

Three-Dimensional Bioprinting of Organoid-Based Scaffolds (OBST) for Long-Term Nanoparticle Toxicology Investigation.

The toxicity of nanoparticles absorbed through contact or inhalation is one of the major concerns for public health. It is mandatory to continually evaluate the toxicity of nanomaterials. In vitro nanotoxicological studies are conventionally limited by the two dimensions. Although 3D bioprinting has been recently adopted for three-dimensional culture in the context of drug release and tissue regeneration, little is known regarding its use for nanotoxicology investigation. Therefore, aiming to simulate the exposure of lung cells to nanoparticles, we developed organoid-based scaffolds for long-term studies in immortalized cell lines. We printed the viscous cell-laden material via a customized 3D bioprinter and subsequently exposed the scaffold to either 40 nm latex-fluorescent or 11-14 nm silver nanoparticles. The number of cells significantly increased on the 14th day in the 3D environment, from 5 × 105 to 1.27 × 106, showing a 91% lipid peroxidation reduction over time and minimal cell death observed throughout 21 days. Administered fluorescent nanoparticles can diffuse throughout the 3D-printed scaffolds while this was not the case for the unprinted ones. A significant increment in cell viability from 3D vs. 2D cultures exposed to silver nanoparticles has been demonstrated. This shows toxicology responses that recapitulate in vivo experiments, such as inhaled silver nanoparticles. The results open a new perspective in 3D protocols for nanotoxicology investigation supporting 3Rs.

Effects of brake wear nanoparticles on the protection and repair functions of the airway epithelium.

Long term exposure to particulate air pollution is known to increase respiratory morbidity and mortality. In urban areas with dense traffic most of these particles are generated by vehicles, via engine exhaust or wear processes. Non-exhaust particles come from wear processes such as those concerning brakes and their toxicity is little studied. To improve our understanding of the lung toxicity mechanisms of the nanometric fraction of brake wear nanoparticles (BWNPs), we studied whether these particles affect the barrier properties of the respiratory epithelium considering particle translocation, mucus production and repair efficiency. The Calu-3 cell line grown in two-compartment chambers was used to mimic the bronchial epithelial barrier. BWNPs detected by single-particle ICP-MS were shown to cross the epithelial tissue in small amounts without affecting the barrier integrity properties, because the permeability to Lucifer yellow was not increased and there was no cytotoxicity as assessed by the release of lactate-dehydrogenase. The interaction of BWNPs with the barrier did not induce a pro-inflammatory response, but increased the expression and production of MU5AC, a mucin, by a mechanism involving the epidermal growth factor receptor pathway. During a wound healing assay, BWNP-loaded cells exhibited the same ability to migrate, but those at the edge of the wound showed higher 5-ethynyl-2'-deoxyuridine incorporation, suggesting a higher proliferation rate. Altogether these results showed that BW. NPs do not exert overt cytotoxicity and inflammation but can translocate through the epithelial barrier in small amounts and increase mucus production, a key feature of acute inflammatory and chronic obstructive pulmonary diseases. Their loading in epithelial cells may impair the repair process through increased proliferation.

Application of the PHENotype SIMulator for rapid identification of potential candidates in effective COVID-19 drug repurposing.

The current, rapidly diversifying pandemic has accelerated the need for efficient and effective identification of potential drug candidates for COVID-19. Knowledge on host-immune response to SARS-CoV-2 infection, however, remains limited with few drugs approved to date. Viable strategies and tools are rapidly arising to address this, especially with repurposing of existing drugs offering significant promise. Here we introduce a systems biology tool, the PHENotype SIMulator, which -by leveraging available transcriptomic and proteomic databases-allows modeling of SARS-CoV-2 infection in host cells in silico to i) determine with high sensitivity and specificity (both>96%) the viral effects on cellular host-immune response, resulting in specific cellular SARS-CoV-2 signatures and ii) utilize these cell-specific signatures to identify promising repurposable therapeutics. Powered by this tool, coupled with domain expertise, we identify several potential COVID-19 drugs including methylprednisolone and metformin, and further discern key cellular SARS-CoV-2-affected pathways as potential druggable targets in COVID-19 pathogenesis.