Epithelial Calu-3 Cells Research Articles

Inhibition of endocytic uptake of severe acute respiratory syndrome coronavirus 2 and endo-lysosomal acidification by diphenoxylate.

Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.

The effects of fine particulate matter (SRM 2786) on three different 3D lung models exposed at the air-liquid interface – A comparative study

3D cell culture models exposed at the air-liquid interface (ALI) represent a potential alternative to animal experiments for hazard and risk assessment of inhaled compounds. This study compares cocultures composed of either Calu-3, A549 or HBEC3-KT lung epithelial cells, cultured together with THP-1-derived macrophages and EA.hy926 endothelial cells, in terms of barrier capacity and responses to a standard reference sample of fine particulate matter (SRM 2786). High-content imaging analysis revealed a similar cellular composition between the different cell models. The 3D cell cultures with Calu-3 cells showed the greatest barrier capacity, as measured by transepithelial electrical resistance and permeability to Na-fluorescein. Mucus production was detected in 3D cell cultures based on Calu-3 and A549 cells. Exposure to SRM 2786 at ALI increased cytokine release and expression of genes associated with inflammation and xenobiotic metabolism. Moreover, the presence of THP-1-derived macrophages was central to the cytokine responses in all cell models. While the different 3D cell culture models produced qualitatively similar responses, more pronounced pro-inflammatory responses were observed in the basolateral compartment of the A549 and HBEC3-KT models compared to the Calu-3 model, likely due to their reduced barrier capacity and lower retention of secreted mediators in the apical compartment.

Potential Role of Diabetes in Impaired Semaphorin-Mediated Maintenance of Epithelial Barrier Properties

Microvasculature-secreted factors create a local niche for tissue specialization and homeostasis. Diseases like diabetes are considered a microvascular disease. However, the consequences of microvascular alteration in surrounding tissues like epithelial barriers have not been suffciently explored. The objective of this study is to determine whether a cell-cell communication protein, Semaphorin-3E (Sema3E), which we have previously found increased in diabetic mice and in endothelial cells cultured on high glucose, impairs the barrier properties of epithelial cells. We determined by ELISA that HUVEC endothelial cells secreted Sema3E (7.8 ±1.8 pg/sq.cm/day) and while mannitol osmolality control 15mM had no effect, high glucose (25mM) increased Sema3E secretion 6-fold (ANOVA, p<0.01). To test the effect of Sema3E in the tight junction barrier properties of epithelial cells, we measured trans-epithelial electrical resistance (TEER) in Calu-3 epithelial cells cultured in trans-well permeable support. We found that 200ng/ml of recombinant Sema3E inhibited the acquisition of TEER over the course of 2 weeks (vehicle: 803 ± 31 Ω/cm^2 vs. Sema3E: 465 ± 15 Ω/cm^2; t-test p<0.01) The weakening of tight junctions was due to decreased presence of Claudin-2 at the cell surface as measured by surface biotinylation (Surface/intracellular ratio= vehicle: 0.356 ± 0.043 vs. Sema3E: 0.020 ± 0.003; t-test p<0.01). Consequently, the polarity of Calu-3 cells was also impaired as we determined by measuring the polarized distribution of the basolateral protein E-cadherin. We observed a reduction of basolateral E-cadherin from 88 ± 3% (vehicle) to 65 ± 6% (Sema3E), t-test p<0.05. Finally, to determine whether surface Claudin-2 expression was decreased in epithelial cells of diabetic mice, we performed surface biotinylation in freshly isolated kidney renal tubules. We observed that the surface/intracellular ratio of Claudin-2 was decreased by 76% in kidney tubules from type-1 diabetic mice (Akita strain), compared to healthy control mice (control: 4.23 ± 1.44 ratio vs. Akita: 1.03 ± 0.16 ratio, t-test p<0.05). We conclude that glucose stimulates Sema3E secretion from endothelial cells, and this signal protein impairs the epithelial barrier properties of epithelial cells by reducing Claudin-2 presence at tight junctions. Reduced surface Claudin-2 may be a feature of diabetic epithelial cells. American Heart Association NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tobramycin Reduces Pulmonary Toxicity of Polymyxin B via Inhibiting the Megalin-Mediated Drug Uptake in the Human Lung Epithelial Cells.

Accumulation of polymyxins in the lung epithelial cells can lead to increased mitochondrial oxidative stress and pulmonary toxicity. Aminoglycosides and polymyxins are used, via intravenous and pulmonary delivery, against multidrug-resistant Gram-negative pathogens. Our recent in vitro and animal studies demonstrated that the co-administration of polymyxins with aminoglycosides decreases polymyxin-induced pulmonary toxicity. The aim of this study was to investigate the in vitro transport and uptake of polymyxin B and tobramycin in human lung epithelial Calu-3 cells and the mechanism of reduced pulmonary toxicity resulting from this combination. Transport, intracellular localization, and accumulation of polymyxin B and tobramycin were investigated using doses of 30 mg/L polymyxin B, 70 mg/L tobramycin, and the combination of both. Adding tobramycin significantly (p < 0.05) decreased the polymyxin B-induced cytotoxicity in Calu-3 cells. The combination treatment significantly reduced the transport and uptake of polymyxin B and tobramycin in Calu-3 cells, compared to each drug alone, which supported the reduced pulmonary toxicity. We hypothesized that cellular uptake of polymyxin B and tobramycin shared a common transporter, megalin. We further investigated the megalin expression of Calu-3 cells using confocal microscopy and evaluated megalin activity using a megalin substrate, FITC-BSA, and a megalin inhibitor, sodium maleate. Both polymyxin B and tobramycin significantly inhibited FITC-BSA uptake by Calu-3 cells in a concentration-dependent manner. Sodium maleate substantially inhibited polymyxin B and tobramycin transport and cellular accumulation in the Calu-3 cell monolayer. Our study demonstrated that the significantly reduced uptake of polymyxin B and tobramycin in Calu-3 cells is attributed to the mechanism of action that determines that polymyxin B and tobramycin share a common transporter, megalin.

BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

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).

SARS-CoV-2 infection as a model to study the effect of cinnamaldehyde as adjuvant therapy for viral pneumonia

BackgroundThe recent pandemic outbursts, due to SARS-CoV-2, have highlighted once more the central role of the inflammatory process in the propagation of viral infection. The main consequence of COVID-19 is the induction of a diffuse pro-inflammatory state, also defined as a cytokine storm, which affects different organs, but mostly the lungs. We aimed to prove the efficacy of cinnamaldehyde, the active compound of cinnamon, as an anti-inflammatory compound, able to reduce SARS-CoV-2 induced cytokine storm.ResultsWe enrolled 53 COVID-19 patients hospitalized for respiratory failure. The cohort was composed by 39 males and 13 females, aged 65.0 ± 9.8 years. We reported that COVID-19 patients have significantly higher IL-1β and IL-6 plasma levels compared to non-COVID-19 pneumonia patients. In addition, human mononuclear cells (PBMCs) isolated from SARS-CoV-2 infected patients are significantly more prone to release pro-inflammatory cytokines upon stimuli. We demonstrated, using in vitro cell models, that macrophages are responsible for mediating the pro-inflammatory cytokine storm while lung cells support SARS-CoV-2 replication upon viral infection. In this context, cinnamaldehyde administration significantly reduces SARS-CoV-2-related inflammation by inhibiting NLRP3 mediated IL-1β release in both PBMCs and THP-1 macrophages, as well as viral replication in CaLu-3 epithelial cells. Lastly, aerosol-administered cinnamaldehyde was able to significantly reduce IL-1β release in an in vivo lung-inflammatory model.ConclusionThe obtained results suggest the possible use of cinnamaldehyde as a co-adjuvant preventive treatment for COVID-19 disease together with vaccination, but also as a promising dietary supplement to reduce, more broadly, viral induced inflammation.

Curbside particulate matter and susceptibility to SARS-CoV-2 infection.

Biologic plausibility for the association between exposure to particulate matter (PM) less than 10 μm in aerodynamic diameter (PM10) and coronavirus disease 2019 (COVID-19) morbidity in epidemiologic studies has not been determined. The upregulation of angiotensin-converting enzyme 2 (ACE2), the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) entry receptor on host cells, by PM10 is a putative mechanism. We sought to assess the effect of PM10 on SARS-CoV-2 infection of cells invitro. PM10 from the curbside of London's Marylebone Road and from exhaust emissions was collected by cyclone. A549 cells, human primary nasal epithelial cells (HPNEpCs), SARS-CoV-2-susceptible Vero-E6 and Calu3 cells were cultured with PM10. ACE2 expression (as determined by median fluorescent intensity) was assessed by flow cytometry, and ACE2 mRNA transcript level was assessed by PCR. The role of oxidative stress was determined by N-acetyl cysteine. The cytopathic effect of SARS-CoV-2 (percentage of infection enhancement) and expression of SARS-CoV-2 genes' open reading frame (ORF) 1ab, S protein, and N protein (focus-forming units/mL) were assessed in Vero-E6 cells. Data were analyzed by either the Mann-Whitney U test or Kruskal-Wallis test with the Dunn multiple comparisons test. Curbside PM10 at concentrations of 10 μg/mL or more increased ACE2 expression in A549 cells (P= .0021). Both diesel PM10 and curbside PM10 in a concentration of 10 μg/mL increased ACE2 expression in HPNEpCs (P= .0022 and P= .0072, respectively). ACE2 expression simulated by curbside PM10 was attenuated by N-acetyl cysteine in HPNEpCs (P= .0464). Curbside PM10 increased ACE2 expression in Calu3 cells (P= .0256). In Vero-E6 cells, curbside PM10 increased ACE2 expression (P= .0079), ACE2 transcript level (P= .0079), SARS-CoV-2 cytopathic effect (P= .0002), and expression of the SARS-CoV-2 genes' ORF1ab, S protein, and N protein (P= .0079). Curbside PM10 increases susceptibility to SARS-COV-2 infection invitro.

Discovery of Fungus-Derived Nornidulin as a Novel TMEM16A Inhibitor: A Potential Therapy to Inhibit Mucus Secretion in Asthma.

Inhibition of Ca2+-activated transmembrane protein 16A (TMEM16A) Cl- channels has been proposed to alleviate mucus secretion in asthma. In this study, we identified a novel class of TMEM16A inhibitors from natural sources in airway epithelial Calu-3 cells and determine anti-asthmatic efficacy of the most potent candidate in a mouse model of asthma. For electrophysiological analyses, IL-4-primed Calu-3 cell monolayers were mounted in Ussing chamber and treated with various fungus-derived depsidones prior to the addition of UTP, ionomycin, thapsigargin, or Eact to stimulate TMEM16A Cl- current. Ca2+-induced mucus secretion in Calu-3 cell monolayers was assessed by determining MUC5AC protein remaining in the cells using immunofluorescence staining. OVA-induced female BALB/c mice was used as an animal model of asthma. After the course of induction, cellular and mucus components in bronchoalveolar lavage were analyzed. Lungs were fixed and undergone with H&E and PAS staining for the evaluation of airway inflammation and mucus production, respectively. The screening of fungus-derived depsidones revealed that nornidulin completely abolished the UTP-activated TMEM16A current in Calu-3 cell monolayers with the IC50 and a maximal effect being at ~0.8 µM and 10 µM, respectively. Neither cell viability nor barrier function was affected by nornidulin. Mechanistically, nornidulin (10 µM) suppressed Cl- currents induced by ionomycin (a Ca2+-specific ionophore), thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ ATPase), and Eact (a putative TMEM16A activator) without interfering with intracellular Ca2+ ([Ca2+]i) levels. These results suggest that nornidulin exerts its effect without changing [Ca2+]i, possibly through direct effect on TMEM16A. Interestingly, nornidulin (at 10 µM) reduced Ca2+-dependent mucus release in the Calu-3 cell monolayers. In addition, nornidulin (20 mg/kg) inhibited bronchoalveolar mucus secretion without impeding airway inflammation in ovalbumin-induced asthmatic mice. Our study revealed that nornidulin is a novel TMEM16A inhibitor that suppresses mucus secretion without compromising immunologic activity. Further development of nornidulin may provide a new remedy for asthma or other diseases associated with allergic mucus hypersecretion without causing opportunistic infections.

Airborne transmission of human-isolated avian H3N8 influenza virus between ferrets

H3N8 avian influenza viruses (AIVs) in China caused two confirmed human infections in 2022, followed by a fatal case reported in 2023. H3N8 viruses are widespread in chicken flocks; however, the zoonotic features of H3N8 viruses are poorly understood. Here, we demonstrate that H3N8 viruses were able to infect and replicate efficiently in organotypic normal human bronchial epithelial (NHBE) cells and lung epithelial (Calu-3) cells. Human isolates of H3N8 virus were more virulent and caused severe pathology in mice andferrets, relative to chicken isolates. Importantly, H3N8 virus isolated from a patient with severe pneumonia was transmissible between ferrets through respiratory droplets; it had acquired human-receptor-binding preference and amino acid substitution PB2-E627K necessary for airborne transmission. Human populations, even when vaccinated against human H3N2 virus, appear immunologically naive to emerging mammalian-adapted H3N8 AIVs and could be vulnerable to infection at epidemic or pandemic proportion.

Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface.

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.

Ivy Leaf Dry Extract EA 575® Has an Inhibitory Effect on the Signalling Cascade of Adenosine Receptor A2B.

Ivy leaf dry extract EA 575® is used to improve complaints of chronic inflammatory bronchial diseases and acute inflammation of the respiratory tract accompanied by coughing. Its mechanism of action has so far been explained by influencing β2-adrenergic signal transduction. In the present study, we investigated a possible influence on adenosine receptor A2B (A2BAR) signalling, as it has been described to play a significant and detrimental role in chronic inflammatory airway diseases. The influence of EA 575® on A2BAR signalling was assessed with measurements of dynamic mass redistribution. Subsequently, the effects on A2BAR-mediated second messenger cAMP levels, β-arrestin 2 recruitment, and cAMP response element (CRE) activation were examined using luciferase-based HEK293 reporter cell lines. Lastly, the impact on A2BAR-mediated IL-6 release in Calu-3 epithelial lung cells was investigated via the Lumit™ Immunoassay. Additionally, the adenosine receptor subtype mediating these effects was specified, and A2BAR was found to be responsible. The present study demonstrates an inhibitory influence of EA 575® on A2BAR-mediated general cellular response, cAMP levels, β-arrestin 2 recruitment, CRE activation, and IL-6 release. Since these EA 575®-mediated effects occur within a time frame of several hours of incubation, its mode of action can be described as indirect. The present data are the first to describe an inhibitory effect of EA 575® on A2BAR signalling. This may offer an explanation for the beneficial clinical effects of the extract in adjuvant asthma therapy.

SARS-CoV-2 ORF6 protein does not antagonize interferon signaling in respiratory epithelial Calu-3 cells during infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths, posing a substantial threat to global public health. Viruses evolve different strategies to antagonize or evade host immune responses. While ectopic expression of SARS-CoV-2 accessory protein ORF6 blocks interferon (IFN) production and downstream IFN signaling, the role of ORF6 in IFN signaling during bona fide viral infection of respiratory cells is unclear. By comparing wild-type (WT) and ORF6-deleted (ΔORF6) SARS-CoV-2 infection and IFN signaling in respiratory cells, we found that ΔORF6 SARS-CoV-2 replicates more efficiently than WT virus and, thus, stimulates more robust immune signaling. Loss of ORF6 does not alter innate signaling in infected cells: both WT and ΔORF6 virus induce delayed IFN responses only in bystander cells. Moreover, expression of ORF6 in the context of SARS-CoV-2 infection has no effect on Sendai virus-stimulated IFN induction: robust translocation of IRF3 is observed in both SARS-CoV-2 infected and bystander cells. Furthermore, IFN pretreatment potently blocks WT and ΔORF6 virus replication similarly, and both viruses fail to suppress the induction of interferon-stimulated genes (ISGs) upon IFN-β treatment. However, upon treatment with IFN-β, only bystander cells induce STAT1 translocation during infection with WT virus, whereas ΔORF6 virus-infected cells now show translocation. This suggests that under conditions of high IFN activation, ORF6 can attenuate STAT1 activation. These data provide evidence that ORF6 is not sufficient to antagonize IFN production or IFN signaling in SARS-CoV-2-infected respiratory cells but may impact the efficacy of therapeutics that stimulate innate immune pathways. IMPORTANCE Previous studies identified several SARS-CoV-2 proteins, including ORF6, that antagonize host innate immune responses in the context of overexpression of viral proteins in non-respiratory cells. We set out to determine the role of ORF6 in IFN responses during SARS-CoV-2 infection of respiratory cells. Using a deletion strain, we observed no reduction of infection and no difference in evasion of IFN signaling, with responses limited to bystander cells. Moreover, stimulation of Sendai virus-induced IFN production or IFN-β-stimulated ISG expression was comparable between SARS-CoV-2 virus and SARS-CoV-2 lacking ORF6 virus, suggesting that ORF6 is not sufficient to counteract IFN induction or IFN signaling during viral infection.

IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting early ACE2-mediated events

Abstract Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) infection is mediated by binding of viral spike (S) protein to membrane ACE2 on host epithelial cells, preferentially in the airway. Asthmatics with type 2 (T2) inflammation are reportedly less susceptible to disease, but the underlying mechanisms are unclear. We recently showed that ACE2expression is decreased in airway epithelial cells from T2 asthmatics and is consistently suppressed in human primary epithelial cells cultured with IL-13 (PMID: 32422146). To dissect IL-13/SARS-CoV-2 interactions, we assessed the effects of IL-13 on discrete events in SARS-CoV-2 infection. S1 protein binding to green monkey kidney Vero E6, human lung Calu-3 epithelial cells and HEK293T cells stably transfected with human ACE2(hACE2-HEK293T) was measured by flow cytometry with His-tagged S1 protein. S protein-mediated viral entry into Vero E6 and hACE2-HEK293T cells was assessed using S protein-pseudotyped lentiviral particles carrying a GFP reporter. ACE2transcription in Vero E6, Calu-3 and primary human airway epithelial cells was measured by RT-qPCR. ACE2 expression on Vero E6, Calu-3 and hACE2-HTK293T cells was evaluated by flow cytometry. USA-WA1/2020 SARS-CoV-2 was used to infect Calu-3 cells. IL-13 strongly inhibited S protein binding, cell entry, and infection of epithelial cells by SARS-CoV-2. These effects resulted at least in part from IL-13-dependent inhibition of ACE2expression because IL-13 did not affect S protein binding to, and S protein-pseudotyped lentivirus entry into, hACE2-HEK293T cells that do not downregulate ACE2 in response to IL-13. We suggest that IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting early ACE2-mediated events. Supported by grant from NIAID: U19 AI125357

The Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) Is under Post-Transcriptional Control of microRNAs: Analysis of the Effects of agomiRNAs Mimicking miR-145-5p, miR-101-3p, and miR-335-5p.

(1) Background: MicroRNAs are involved in the expression of the gene encoding the chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator); the objective of this short report is to study the effects of the treatment of bronchial epithelial Calu-3 cells with molecules mimicking the activity of pre-miR-145-5p, pre-miR-335-5p, and pre-miR-101-3p, and to discuss possible translational applications of these molecules in pre-clinical studies focusing on the development of protocols of possible interest in therapy; (2) Methods: CFTR mRNA was quantified by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). The production of the CFTR protein was assessed by Western blotting; (3) Results: The treatment of Calu-3 cells with agomiR-145-5p caused the highest inhibition of CFTR mRNA accumulation and CFTR production; (4) Conclusions: The treatment of target cells with the agomiR pre-miR-145-5p should be considered when CFTR gene expression should be inhibited in pathological conditions, such as polycystic kidney disease (PKD), some types of cancer, cholera, and SARS-CoV-2 infection.

Antiviral Activity of Micafungin and Its Derivatives against SARS-CoV-2 RNA Replication.

Echinocandin antifungal drugs, including micafungin, anidulafungin, and caspofungin, have been recently reported to exhibit antiviral effects against various viruses such as flavivirus, alphavirus, and coronavirus. In this study, we focused on micafungin and its derivatives and analyzed their antiviral activities against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The micafungin derivatives Mi-2 and Mi-5 showed higher antiviral activity than micafungin, with 50% maximal inhibitory concentration (IC50) of 5.25 and 6.51 µM, respectively (3.8 to 4.7-fold stronger than micafungin) and 50% cytotoxic concentration (CC50) of >64 µM in VeroE6/TMPRSS2 cells. This high anti-SARS-CoV-2 activity was also conserved in human lung epithelial cell-derived Calu-3 cells. Micafungin, Mi-2, and Mi-5 were suggested to inhibit the intracellular virus replication process; additionally, these compounds were active against SARS-CoV-2 variants, including Delta (AY.122, hCoV-19/Japan/TY11-927/2021), Omicron (BA.1.18, hCoV-19/Japan/TY38-873/2021), a variant resistant to remdesivir (R10/E796G C799F), and a variant resistant to casirivimab/imdevimab antibody cocktail (E406W); thus, our results provide basic evidence for the potential use of micafungin derivatives for developing antiviral agents.

Construction of Human Three-Dimensional Lung Model Using Layer-by-Layer Method.

The respiratory tract is one of the frontline barriers for biological defense. Lung epithelial intercellular adhesions provide protection from bacterial and viral infections and prevent invasion into deep tissues by pathogens. Dysfunction of lung epithelial intercellular adhesion caused by pathogens is associated with development of several diseases, such as acute respiratory distress syndrome, pneumonia, and asthma. To elucidate the pathological mechanism of respiratory infections, two-dimensional cell cultures and animal models are commonly used, although are not useful for evaluating host specificity or human biological response. With the rapid progression and worldwide spread of severe acute respiratory syndrome coronavirus-2, there is increasing interest in the development of a three-dimensional (3D) in vitro lung model for analyzing interactions between pathogens and hosts. However, some models possess unclear epithelial polarity or insufficient barrier functions and need the use of complex technologies, have high cost, and long cultivation terms. We previously reported about the fabrication of 3D cellular multilayers using a layer-by-layer (LbL) cell coating technique with extracellular matrix protein, fibronectin (FN), and gelatin (G). In the present study, such a LbL cell coating technique was utilized to construct a human 3D lung model in which a monolayer of the human lower airway epithelial adenocarcinoma cell line Calu-3 cells was placed on 3D-cellular multilayers composed of FN-G-coated human primary pulmonary fibroblast cells. The 3D lung model thus constructed demonstrated an epithelial-fibroblast layer that maintained uniform thickness until 7 days of incubation. Moreover, expressions of E-cadherin, ZO-1, and mucin in the epithelial layer were observed by immunohistochemical staining. Epithelial barrier integrity was evaluated using transepithelial electrical resistance values. The results indicate that the present constructed human 3D lung model is similar to human lung tissues and also features epithelial polarity and a barrier function, thus is considered useful for evaluating infection and pathological mechanisms related to pneumonia and several pathogens. Impact statement A novel in vitro model of lung tissue was established. Using a layer-by-layer cell coating technique, a three-dimensional cultured lung model was constructed. The present novel model was shown to have epithelial polarity and chemical barrier functions. This model may be useful for investigating interaction pathogens and human biology.

Antiviral effects and tissue exposure of tetrandrine against SARS-CoV-2 infection and COVID-19.

Tetrandrine (TET) has been used to treat silicosis in China for decades. The aim of this study was to facilitate rational repurposing of TET against SARS-CoV-2 infection. In this study, we confirmed that TET exhibited antiviral potency against SARS-CoV-2 in the African green monkey kidney (Vero E6), human hepatocarcinoma (Huh7), and human lung adenocarcinoma epithelial (Calu-3) cell lines. TET functioned during the early-entry stage of SARS-CoV-2 and impeded intracellular trafficking of the virus from early endosomes to endolysosomes. An in vivo study that used adenovirus (AdV) 5-human angiotensin-converting enzyme 2 (hACE2)-transduced mice showed that although TET did not reduce pulmonary viral load, it significantly alleviated pathological damage in SARS-CoV-2-infected murine lungs. The systemic preclinical pharmacokinetics were investigated based on in vivo and in vitro models, and the route-dependent biodistribution of TET was explored. TET had a large volume of distribution, which contributed to its high tissue accumulation. Inhaled administration helped TET target the lung and reduced its exposure to other tissues, which mitigated its off-target toxicity. Based on the available human pharmacokinetic data, it appeared feasible to achieve an unbound TET 90% maximal effective concentration (EC90) in human lungs. This study provides insights into the route-dependent pulmonary biodistribution of TET associated with its efficacy.

Comparison of viral inactivation methods on the characteristics of extracellular vesicles from SARS-CoV-2 infected human lung epithelial cells.

The interaction of SARS-CoV-2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS-CoV-2 contaminated-EV isolates in instruments located outside of the biosafety level-3 (BSL-3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet-C (UVC; 1350 mJ/cm2 ), β-propiolactone (BPL; 0.005%), heat (56°C, 45min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS-CoV-2-infected human lung epithelial Calu-3 cells by stepwise centrifugation, ultrafiltration and qEV size-exclusion chromatography. The EV isolates contained SARS-CoV-2. UVC, BPL and heat completely abolished SARS-CoV-2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS-CoV-2 spike detectability by quantitative RT-PCR and slightly altered EV-derived β-actin detection. Fibroblast migration-wound healing activity of the SARS-CoV-2 contaminated-EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS-CoV-2 contaminated-EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS-CoV-2 infected cells.

Sars-Cov-2-Specific CD4+ and CD8+ T Cell Responses Can Originate from Cross-Reactive CMV-Specific T Cells

Introduction: SARS-CoV-2-specific CD4+ and CD8+ T cells play an important role in COVID-19 pathogenesis. In addition to de novo SARS-CoV-2-specific T cell responses in infected individuals, SARS-CoV-2-specific T cells have also been identified in samples frozen down before the pandemic. So far, pre-existing SARS-CoV-2 specific T cell responses has mostly been ascribed to cross-reactive T cells that were initially primed by other coronaviruses. The presence of these cross-reactive T cells is not surprising considering these human coronaviruses share a relatively high sequence homology. However, T cells are also able to cross-react towards dissimilar pathogens, called heterologous immunity. Given the high prevalence and magnitudes of pre-pandemic SARS-CoV-2-specific T cells, we hypothesized that pre-pandemic SARS-CoV-2 T cell immunity could be explained by cross-reactive cytomegalovirus (CMV)-specific T cells. Methods & Results: PBMCs from CMV seropositive and seronegative donors that were cryopreserved before the pandemic were incubated with SARS-CoV-2 peptide pools. To detect SARS-CoV-2-specific T cells using flow cytometry, CD4+ T cell responses were identified by upregulation of CD154 and CD137 whereas CD8+ T cell responses were identified by upregulation of CD137 and IFN-γ production. In the CMV seropositive group, clear CD4+ T cell responses against SARS-CoV-2 membrane and CD8+ T cell responses against SARS-CoV-2 spike were detected whilst this was not observed in CMV seronegative donors. To investigate whether these T cells were reactive against both SARS-CoV-2 and CMV, the SARS-CoV-2 reactive T cells were single cell sorted, clonally expanded, and tested in co-culture assays in which stimulator cells expressing the relevant human leukocyte antigen (HLA) alleles were pulsed with overlapping peptide pools, peptide libraries or single peptides. The results demonstrated that the pre-pandemic SARS-CoV-2 membrane-specific CD4+ T cells recognized both SARS-CoV-2 membrane and CMV-pp65 peptide pools in HLA-DRB3*02:02, and specifically recognized AGILRNLVPM peptide of CMV-pp65. The pre-pandemic SARS-CoV-2 spike-specific CD8+ T cells recognized spike peptide FVSNGTHWF (FVS) and, interestingly, dissimilar CMV-pp65 peptide IPSINVHHY (IPS) both presented by HLA-B*35:01. Importantly, these FVS/IPS-cross-reactive T cells displayed a higher avidity for CMV compared to SARS-CoV-2, which illustrates that these cells were originally primed against CMV since repeated antigen exposure results in a selection for high avidity T cell clonotypes. This selection cannot have occurred in response to SARS-CoV-2-specific peptides in SARS-CoV-2-unexposed individuals. PBMCs from pre-pandemic CMV-seropositive donors that express HLA-B*35:01 were screened for the presence of these dual IPS/FVS-specific CD8+ T cells by peptide-HLA tetramer technology. In multiple donors these cross-reactive T cells were detected, and isolation followed by direct T cell receptor (TCR) sequencing of the IPS/FVS-tetramer positive cells showed that these T cells express an amino acid-identical TCR, illustrating that this SARS-CoV-2/CMV cross-reactivity of the IPS/FVS T cells is caused by a public TCR. To investigate the potential of the IPS/FVS-specific T cells to kill virus-infected cells, and thereby reducing spreading of SARS-CoV-2, the T cells were incubated with SARS-CoV-2-infected airway epithelial Calu-3 cells. Intracellular SARS-CoV-2 RNA was reduced at low virus titers in IPS/FVS-specific T cell co-cultures, indicating that the cross-reactive T cells are killing SARS-CoV-2-infected cells and thereby reduce spreading of SARS-CoV-2 during initial infection. Circulating IPS/FVS-specific T cells were not phenotypically activated in two patients with advanced COVID-19, as measured by flow cytometry analysis of CD38 and HLA-DR expression, indicating that cross-reactive T cells may more likely play a role during early stage of infection. Conclusion: CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-pandemic immunity against SARS-CoV-2. Pre-print link: https://www.biorxiv.org/content/10.1101/2022.07.31.502203v1