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Objective: Immunohistochemistry of post-mortem lung tissue from Covid-19 patients with diffuse alveolar damage demonstrated marked increases in chondroitin sulfate and CHST15 and decline in N-acetylgalactosamine-4-sulfatase. Studies were undertaken to identify the mechanisms involved in these effects. Method(s): Human primary small airway epithelial cells (PCS 301-010;ATCC) were cultured and exposed to the SARSCoV- 2 spike protein receptor binding domain (SPRBD;AA: Lys310-Leu560;Amsbio). Expression of the spike protein receptor, angiotensin converting enzyme 2 (ACE2), was enhanced by treatment with Interferon-beta. Promoter activation, DNA-binding, RNA silencing, QPCR, Western blots, ELISAs, and specific enzyme inhibitors were used to elucidate the underlying molecular mechanisms. Result(s): Treatment of the cultured cells by the SPRBD led to increased CHST15 and CHST11 expression and decline in ARSB expression. Sulfotransferase activity, total chondroitin sulfate, and sulfated glycosaminoglycan (GAG) content were increased. Phospho-T180/T182-p38-MAPK and phospho- S423/S425-Smad3 were required for the activation of the CHST15 and CHST11 promoters. Inhibition by SB203580, a phospho-p38 MAPK inhibitor, and by SIS3, a Smad3 inhibitor, blocked the CHST15 and CHST11 promoter activation. SB203580 reversed the SPRBD-induced decline in ARSB expression, but SIS3 had no effect on ARSB expression or promoter activation. Phospho-p38 MAPK was shown to reduce retinoblastoma protein (RB) S807/S811 phosphorylation and increase RB S249/T252 phosphorylation. E2F-DNA binding declined following exposure to SPRBD, and SB203580 reversed this effect. This indicates a mechanism by which SPRBD, phospho-p38 MAPK, E2F, and RB can regulate ARSB expression and thereby impact on chondroitin 4-sulfate and dermatan sulfate and molecules that bind to these sulfated GAGs, including Interleukin-8, bone morphogenetic protein-4, galectin-3 and SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2). Conclusion(s): The enzyme ARSB is required for the degradation of chondroitin 4-sulfate and dermatan sulfate, and accumulation of these sulfated GAGs can contribute to lung pathophysiology, as evident in Covid-19. Some effects of the SPRBD may be attributable to unopposed Angiotensin II, when Ang1-7 counter effects are diminished due to binding of ACE2 with the SARS-CoV-2 spike protein and reduced production of Ang1-7. Aberrant cell signaling and activation of the phospho-p38 MAPK and Smad3 pathways increase CHST15 and CHST11 production, which can contribute to increased chondroitin sulfate in infected cells. Decline in ARSB may occur as a consequence of effects of phospho-p38 MAPK on RB phosphorylation and E2F1 availability. Decline in ARSB and the resulting impaired degradation of sulfated GAGs have profound consequences on cellular metabolic, signaling, and transcriptional events. Funding is VA Merit Award.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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Respiratory viral infections (RVI) such as influenza and COVID19 impact the host systemic immune system along with causing deleterious chronic inflammatory responses and respiratory distress. While the role of chronic inflammation in cancer is well-established, the role of RVI on tumorigenesis is poorly defined. To study the role of RVI on breast cancer, we first infected murine respiratory epithelial cells (mRES) with murine sendai virus (mSV), an analog for human parainfluenza virus. These infected mRES were co-cultured with 4T1 murine breast cancer cells in 1:1 dilution on a single 2D plate and also in trans-well format. Both in co-culture and transwell culture we saw a 40- 80% (p<0.05) increased proliferation of breast cancer cells. Similarly, when 4T1 cells were treated with the supernatant collected from infected mRES cells in 1:5 dilution, also demonstrated a 2.3 fold increased breast cancer cell proliferation. The cytokine analysis from the supernatant collected from infected mRES cells demonstrated a 17-23 fold enhanced secretion of alpha/beta-defensins. Direct treatment of alpha-defensin (cyptidin-4, 10 pg/mL) and beta-defensin-3 (mBD3, 20 pg/mL) on 4T1 cells demonstrated enhanced expression of chemokine metastatic receptor, CXCR4 (4.3 fold), angiogenic factor, VEGF (12.8 fold) and cell division favoring factor, CDK2 (8.1 fold). Further, analysis of infected mRES cells demonstrated upregulation of toll-like receptor 2 (TLR2) and NODlike receptor protein 3 (NLRP3) expression. Interesting, co-cultured of infected mRES with syngeneic murine CD4 T cells induced exhaustion phenotype (PD1+ and CTLA4+ ) differentiation of CD4 T cells. Taken together, these data suggest that respiratory viral infections through induction of cancer cell proliferation and inhibiting anti-tumor adaptive immune responses promote breast cancer proliferation.
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Introduction/Aim: The spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enables it to recognise and bind host receptors. These dynamics have been modelled in various cell types and immortalised lines, but rarely in primary airway epithelial cells (AEC), and especially not in children. Therefore, this study on AEC recapitulated earlier work testing the hypothesis that exposure to the spike protein would induce airway immune responses in airway cells of young children. Method(s): Primary AEC monolayer cultures from healthy children (n = 5, <10 years old, males = 5) were exposed to the spike protein S1 subunit (0.01, 1, and 10 mug/mL) over 48 h. Induced inflammatory cytokines, interleukin (IL) 6 and IL8, and viral-associated chemokines, CCL5 and CXCL10 were measured via ELISA. Basal receptor gene expression (ACE2 and TMPRSS2) was measured in monolayer (n = 5) and terminally differentiated (air-liquid interface [ALI];n = 5) cell models as well as in ex-vivo cells obtained directly from nasal brushings (n = 71). Generalised linear modelling, accounting for individual variability, identified any statistical difference (p < 0.05). Result(s): Exposure to the spike protein resulted in no increase in IL6 and IL8 production, however a significant (p < 0.05) decrease was observed at the highest dose tested (10 mug/mL). CXCL10 was only significantly induced at the highest dose (10 mug/mL) whereas CCL5 was not induced. When compared to ex-vivo samples, baseline expression of ACE2 and TMPRSS2 was significantly lower in monolayer cultures (~57- and ~4- fold respectively, p < 0.05), whereas ALI cultures had similar expression levels. Conclusion(s): The use of recombinant spike protein and monolayer cultures appears to not accurately model SARS-CoV-2 spike protein-host interactions. The lack of inflammatory responses may be attributed to the lower receptor gene expression in monolayer cultures. Future studies should utilise live virus and ALI cultures as a more biologically relevant model to study virus-host interactions.
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Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.
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Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs). The efficiency and robustness of these assays often depends on the quality of primary-isolated cells, but primary cell isolation workflows, with which the user controls the choice of isolation method, cell culture medium, and culture format, may reduce reproducibility. Therefore, an optimized, standardized workflow can enhance and support isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations or particularly sensitive cell populations. We have developed a standardized workflow for isolation and culture of freshly derived airway epithelial cells. Method(s): Briefly, HAECs isolated from primary tissue were expanded in PneumaCult-Ex Plus Medium for 1 week and then seeded into Corning Transwell inserts and expanded until confluency. The cells were then differentiated in PneumaCult-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, aswell as SARS-CoV-2 cell entry targets angiotensin-converting enzyme 2 and transmembrane serine protease 2. Ion transport and barrier function of the ALI culturesand response to CF transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly derived HAECs were seeded into Corning Matrigel domes in the presence of PneumaCult Airway Organoid Seeding Medium. Oneweek later, the mediumwas changed to PneumaCult Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling. Result(s): Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 +/- 14% (n = 1 donor) greater organoid area than in vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolininduced swelling, which could be partially re-established with VX-809 treatment (29 +/- 9%, n = 3). Conclusion(s): In summary, the PneumaCult workflow supports robust, efficient culture of primary-airway epithelial cells that can be used as physiologically relevant models suitable for CF research, CFTR corrector screening, and studying airway biology.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved
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The COVID-19 pandemic caused by the SARS-CoV2 virus poses a global health threat with over 5 million deaths recorded. There is little understanding regarding SARS-CoV2 pathogenesis in the human airways and disease severity increases with age. Neutrophils are white blood cells found in large numbers in the airways of the lungs in severe COVID-19 patients. It is not known whether this influx of neutrophils into the airway has a protective or detrimental effect. We aim to understand the role of neutrophils during COVID-19 pathology, using an experimental infection model of the airway epithelium from the eldelry and children. To do this, we collect nasal airway cells from healthy elderly and children and grow them at air-liquid interface. Once differentiation and ciliation of these cells is reached, we infect the cells with SARS-CoV2 virus and allow neutrophils to migrate from the basolateral (blood) to the apical (air) side of the epithelium, similar to a physiological airway. Using flow cytometric analyses, we measure the expression of activation markers and the number of neutrophils that migrate across the epithelium of different ages in response to SARS-CoV2 infection. Preliminary work shows less viable neutrophils recovered from the elderly epithelium, more activated neutrophils when migrating through the elderly epithelium, as well as increased numbers of neutrophils remaining on the basolateral (blood) side of the elderly epithelium. These findings point to an inflammatory neutrophil phenotype influenced by the damaged elderly epithelium and supports the hypothesis that neutrophils are responsible for the severity of disease.
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Introduction: Acute respiratory distress syndrome develops in up to 30% of COVID-19 pneumonia patients. Characterizations of cytologic criteria in bronchoalveolar lavage (BAL) specimens are not well-established. This study evaluates the value of applying several cytologic criteria for the diagnosis of COVID-19 infection in BAL samples. Material(s) and Method(s): We performed a retrospective review of 64 BAL samples collected 6/2020 - 8/2021, divided into two groups: COVID-19 positive group (n=30) and negative group (n=34). Median time from COVID-19 diagnosis to BAL collection was 23 days (range;9-208). The type of inflammation and the cytologic features of alveolar macrophages and respiratory epithelial cells were enumerated. The most common features in COVID-19 positive patients were defined as major diagnostic criteria. Blinded review by a second cytopathologist was performed to predict COVID-19 diagnosis using the defined criteria. Result(s): COVID-19 positive group showed more mixedtype (acute and chronic) inflammation (67% vs 21%;P=0.002), fewer pigment-laden macrophages (22% vs 62%;P=0.001), more enlarged macrophages (85% vs 32%;P<0.001), multi-nucleated macrophages (67% vs 21%;P=0.002) and multi-vacuolated cytoplasm of the macrophages (78% vs 29%;P=0.003) [Figures 1&2]. There was insignificant difference for reactive respiratory epithelial Acta Cytologica 2022;66(suppl 1):1-150 DOI: 10.1159/000527858 12 Exfoliative - Fluids (CSF, Pleural, Peritoneal, etc.) cells (37% vs 24%;P=0.4) and macrophages abundance (63% vs 44%;P=0.5). Identification of diagnostic criteria by a second cytopathologist in COVID-19 positive group, predicted COVID- 19 disease in 27% (>3 major criteria present). After exclusion of cases with remote COVID-19 infection (n=4), the mean interval in days between COVID-19 infection and BAL collection was significantly higher in cases with positive disease prediction (30.5 vs 20.7, P=0.04). Conclusion(s): Features associated with macrophage activation (enlarged and multinucleated macrophages with multi-vacuolated cytoplasm) are prominent in COVID-19 BAL samples. These features may become more recognizable with longer time intervals after infection. Observation of these findings suggest consideration of COVID-19 as the etiology of an individual's pneumonia. (Figure Presented).
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SARS-CoV-2 remains a significant public health threat, causing severe respiratory illness in susceptible individuals. Several effective Covid-19 vaccines have been developed but novel SARS-CoV-2 variants continuously emerge that are more transmissible and have potential to evade vaccine immune responses. We are developing a novel therapy that does not depend on an immune response, based on siRNA-mediated silencing of Angiotensin-converting enzyme 2 (ACE2) receptor and Transmembrane Serine Protease 2 (TMPRSS2). SARS-CoV-2 requires these host proteins to enter respiratory epithelial cells at the cell surface, through binding and priming of its Spike protein. As a cell model for SARS-CoV-2 infection, we have utilised primary nasal epithelial cells (NHNE), as well as HEK293T cells overexpressing ACE2 and TMPRSS2. siRNA transfection in NHNE cells led to a 78%-88% knockdown of ACE2 and TMPRSS2, as determined by qRT-PCR and western blot data. TMPRSS2 knockdown in the overexpressing HEK293T cells resulted in an 87% reduction in infectivity from SARS-CoV-2 Spike-pseudotyped lentiviruses expressing a luciferase transgene, indicative of a significant reduction in virus entry (p < 0.0001 by one-way ANOVA). We are now working to confirm these results with live SARS-CoV-2 and to test lipid nanoparticle delivery of the siRNAs to air-liquid interface grown NHNEs to more accurately model the respiratory airway. This siRNA approach could provide a novel therapy for immunocompromised individuals who do not gain sufficient protection from SARS-CoV-2 vaccines. Additionally, by targeting host proteins rather than virus components, our therapy is likely to remain effective in spite of emerging SARS-CoV-2 variants that circumvent pre-existing immune responses.
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Introduction: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many monoclonal antibody therapies illustrates the need for anti-viral treatments with low susceptibility to evolutionary escape. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly. This compound has minimal host toxicity, including once daily oral dosing in rats that achieves >200-fold of the 90% effective concentration (EC90) in blood. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low suceptability to evolutionary escape. We hypothesized that PAV-104 would be active against SARSCoV- 2 variants in human airway epithelial cells. Methods: Airway epithelial cells were differentiated from lung transplant tissue at air-liquid interface (ALI) for four weeks prior to challenge with Alpha (Pango lineage designation B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) SARS-CoV-2 variants. Viral replication was determined by quantitative PCR measurement of the SARS-CoV-2 nucleocapsid (N) gene. Dose-dependent virus inhibition and cytotoxicity of PAV-104 in the Calu-3 airway epithelial cell line was determined by PCR and MTT assay. Student's t-tests were used to evaluate statistical significance. Results: Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 showed comparable infectivity in human primary airway epithelial cells at ALI (N=3 donors), 47- to 550-fold higher than the parent (USA-WA1/2020) strain. PAV-104 reached 50% cytotoxicity in Calu-3 cells at 240 nM (Fig. 1A). Dose-response studies in Calu-3 cells demonstrated PAV-104 has a 6 nM 50% inhibitory concentration (IC50) for blocking replication of SARS-CoV-2 (USA-WA1/2020) (Fig.1B). In primary cells at ALI from 3 donors tested, there was >99% inhibition of infection by SARS-CoV-2 Gamma variant (N=3, MOI 0.1, P <0.01) with 100 nM PAV-104 (Fig. 1C). Addition of 100 nM PAV-104 2-hours post-infection, but not pre-infection, resulted in >99% suppression of viral replication, indicating a post-entry drug mechanism. PAV-104 bound a small subset of the known allosteric modulator 14-3-3, itself implicated in the interactome of SARS-CoV-2. Conclusion: PAV-104 is a host-targeted, orally bioavailable, pan-viral small molecule inhibitor with promising activity against SARS-CoV-2 variants in human primary airway epithelial cells. (Figure Presented).
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Rationale. Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the third leading cause of death in the United States. While many risk factors for severe COVID-19 are emerging, the effects by which other inhalational exposures affect susceptibility are not well defined. Patients with COVID-19 demonstrate high rates of co-infection with respiratory viruses, including influenza A (IAV). When infected with IAV, human small airway epithelial cells (SAEC) exhibit increased abundance of angiotensin-converting enzyme 2 (ACE2), the primary receptor for SARS-CoV-2. However, it remains unknown if this effect increases the risk for COVID-19. Similarly, there are conflicting reports of the effect of e-cigarette (E-cig) vaping on COVID-19 manifestations. We hypothesized that exposures to IAV or E-cig increase the severity of SARS-CoV-2 infection. Methods. Golden Syrian hamsters (male and female) were exposed to E-cig vapor via nebulization for 5d. IAV was administered intranasally once on day 6 (A/California/07/2009 H1N1, 106 PFU/hamster). On day 3 post-IAV infection, SARSCoV- 2 was administered intranasally (WA01;104 PFU/hamster). On day 7 post-SARS-CoV-2 infection animals were sacrificed, bronchoalveolar lavage fluid (BALF) cell differentials were obtained, and inflated lung sections were stained and scored for immunohistology. Lung RNA was quantified for ACE2, TMPRSS2, STAT1, CXCL10, IFN-gamma, gene expression using RT-qPCR. Results: SARS-CoV-2 infection caused progressive weight loss that was less pronounced in animals pre-infected with IAV. SARS-CoV-2 titers from nasal swabs peaked at day 2 in both groups. IAV pre-infection reduced PMN and eosinophils in the BALF, and the overall inflammatory cell infiltration in the lung parenchyma of SARS-CoV-2-infected animals. IAV pre-infection reduced lung levels of STAT1, CXCL10 (2.5-fold;p<0.01), CCL5, and IFN-gamma in SARS-CoV-2-infected animals compared to animals that were only infected with SARS-CoV-2. Pre-exposure to E-cig worsened the SARS-CoV-2-induced weight loss in female animals only. E-cig pre-exposure increased lymphocytes and decreased PMN and eosinophils in the BALF compared to animals that were only infected with SARS-CoV-2. E-cig pre-exposure increased lung levels of STAT1, CXCL10 (2.5-fold;p<0.05), CCL5, and IFN-gamma in SARS-CoV-2-infected animals compared to animals that were only infected with SARS-CoV-2. Conclusion: Pre-infection with IAV resulted in decreased inflammatory response to SARS-CoV-2 infection. In contrast, pre-exposure to E-cig vaping increased the severity of the inflammatory response to SARS-CoV-2 with notable differences between sexes. Whereas anti-viral priming effects of prior viral infection are well described, the mechanisms that explain the worsening effects of E-cig on SARS-CoV-2 outcomes remain unknown.
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Rationale: Individuals with COPD who develop COVID-19 are at increased risk of hospitalization, ICU admission and death. COPD is associated with increased airway epithelial expression of ACE2, the receptor mediating SARS-CoV-2 entry into cells. Hypercapnia commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary infections. We previously reported that hypercapnia increases viral replication, lung injury and mortality in mice infected with influenza A virus. Also, global gene expression profiling of primary human bronchial epithelial (HBE) cells showed that elevated CO2 upregulates expression of cholesterol biosynthesis genes, including HMGCS1, and downregulates ATP-binding cassette (ABC) transporters that promote cholesterol efflux. Given that cellular cholesterol is important for entry of viruses into cells, in the current study we assessed the impact of hypercapnia on regulation of cellular cholesterol levels, and resultant effects on expression of ACE2 and entry of Pseudo-SARS-CoV-2 in cultured HBE, BEAS-2B and VERO cells, and airway epithelium of mice. Methods: Differentiated HBE, BEAS-2B or VERO cells were pre-incubated in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia (15% CO2, PCO2 108 mmHg), both with normoxia, for 4 days. Expression of ACE2 and sterol regulatory element binding protein 2 (SREPB2), the master regulator of cholesterol synthesis, was assessed by immunoblot or immunofluorescence. Cholesterol was measured in cell lysates by Amplex red assay. Cells cultured in normocapnia or hypercapnia were also infected with Pseudo SARS-CoV-2, a Neon Green reporter baculovirus. For in vivo studies, C57BL/6 mice were exposed to normoxic hypercapnia (10% CO2/21% O2) for 7 days, or air as control, and airway epithelial expression of ACE2, SREBP2, ABCA1, ABCG1 and HMGCS1 was assessed by immunofluorescence. SREBP2 was blocked using the small molecules betulin or AM580, and cellular cholesterol was disrupted using MβCD. Results: Hypercapnia increased expression and activation of SREBP2 and decreased expression of ABC transporters, thereby augmenting epithelial cholesterol levels. Elevated CO2 also augmented ACE2 expression and Pseudo-SARSCoV- 2 entry into epithelial cells in vitro and in vivo. These effects were all reversed by blocking SREBP2 or disrupting cellular cholesterol. Conclusion: Hypercapnia augments cellular cholesterol levels by altering expression of cholesterol biosynthetic enzymes and efflux transporters, leading to increased epithelial expression of ACE2 and entry of Pseudo-SARS-CoV-2 into cells. These findings suggest that ventilatory support to limit hypercapnia or pharmacologic interventions to decrease cellular cholesterol might reduce viral burden and improve clinical outcomes of SARSCoV- 2 infection in advanced COPD and other severe lung diseases.
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Rationale The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs, the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits. Thus, new antivirals targeting SARS-CoV-2 are urgently needed. Methods Artificial intelligence algorithm MediKanren was used to query FDA-approved and late-stage drug compounds for potential interactions with SARS-CoV-2 proteins, coronaviruses, and host cell networks for possible antiviral activity. From this, 157 compounds were further tested in an antiviral screen against live SARS-CoV-2 for reduction in viral growth. Select compounds were further assessed for synergistic activity with remdesivir. Both in vitro and cell free systems identified tocopherol succinate compounds that inhibited the RNA-dependent RNA polymerase (RdRp). Validation of antiviral and synergistic activity was performed in primary human airway epithelial cell cultures against multiple SARS-CoV-2 variants.Results Here we show that water-soluble derivatives of α-tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D-α-tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and β-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of α- tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV- 2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp. Conclusion In summary, solubilizing modifications to α-tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection.
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Background: A novel coronavirus (SARS-CoV-2) has led to the worldwide spread of pandemic proportions and currently no effective therapy is available. The minor pulmonary surfactant lipids, palmitoyl-oleoyl-phosphatidylglycerol (POPG), and phosphatidylinositol (PI), are potent regulators of inflammatory processes, and are effective as anti-viral agents for multiple respiratory viruses including Respiratory syncytial virus (RSV), Influenza A virus (IAV) and Rhinoviruses (RVs). Objective: The primary objectives of this study are to determine whether POPG or PI are potent against SARS-CoV-2 in vitro, using human airway epithelial cells, and examine the potency of PI against SARS-CoV-2 in vivo, in a hamster model. Methods: We examined efficacies of POPG or PI against SARS-CoV-2 (USA WA/2020) in human bronchial epithelial cells, and nasal epithelial cells from healthy control subjects differentiated by ALI cultures. We quantified SARS-CoV-2 replication by quantitative plaque assays and qRT-PCR. We determined the potency of PI against SARS-CoV- 2 in golden Syrian hamster as in vivo model for SARS-CoV-2 infection. Results: We examined the efficacies of POPG and PI using primary human tracheal and nasal epithelial cells, differentiated in ALI culture. Cells were treated with POPG (10mg/ml) and PI (4mg/ml) added to apical media alone for 16hrs. Subsequently, cells were infected with SARS-CoV-2 at m.o.i = 0.02, for 48hrs, harvested for RNA extraction and qRT-PCR. SARS-CoV-2 replicated in tracheal cells with a 106-fold increase in mRNA. POPG and PI reduced viral mRNA expression by 70% and 85%, respectively (subject numbers n=3). In nasal epithelia, SARS-CoV-2 mRNA expression increased 105 -fold compared to sham infected cultures. Both POPG and PI attenuated the increase in viral mRNA expression by 70% - 82% (subject numbers n=6). We determined the PI effect in an in vivo study in hamsters. Hamsters were challenged with 103 pfu of SARS-CoV-2, either with, or without PI (2mg/hamster) administered intranasally. Hamsters were harvested at Day 3, and lungs were processed for histopathology. Pharyngeal swabs were used to examine viral burden by plaque assays. PI reduced plaque numbers compared to viral infection alone groups at day1 (Virus alone: 2.4±2.7(X104pfu/ml), Virus+PI: 0.9±2.1(X106pfu/ml), p<0.05). PI reduced lung histopathology score at day 3 (Virus alone: 28.0±15.6, Virus+PI: 6.7±7.0, p<0.05). Conclusions: POPG and PI significantly reduced SARS-CoV2 replication in human differentiated airway epithelial cells. PI inhibited SARS-CoV-2 infection in vivo in hamsters. These findings suggest that inhalation of POPG, or PI might be effective as novel anti-viral compounds for treating and preventing SARSCoV- 2 infection.
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Rationale We have previously reported blocking the IL-25 receptor (IL-17RB) prevented viral increased allergic airways inflammation and this was associated with reduced lung viral load. To investigate IL-25 regulation of airway anti-viral immunity we hypothesised that IL-25 directly inhibits airway epithelial cell (AEC) type I/III interferon expression and antibody blockade of IL-25 in vivo boosts lung interferon expression and reduces lung viral load in parallel with reduced type 2 airway inflammation. Methods In vitro Immunofluorescence was used to visualise epithelial IL-25 and IL- 17RB proteins in endobronchial biopsies from patients with asthma and healthy subjects and in AEC differentiated at ALI. AEC from n = 14 donors with asthma were differentiated at the air-liquid interface (ALI) and infected with RV-A1, MOI=0.1. A subset of AECs was treated with anti-IL-25 mAb (LNR125) before infecting with RV-A1 or human coronavirus 229E. Differentiated AEC from healthy donors were treated with recombinant IL-25 protein and infected with RV-A1. Nanostring immune transcriptomic data expressed as digital mRNA counts for exact copy number or was expressed as log2 fold change ratio against -log10 Bejamini-Yekutieli-corrected p-values. In vivo 6- 8-week-old, BALB/c mice sensitised and intranasally challenged daily for 3 days with ovalbumin to induced allergic airways disease. A single subcutaneous injection of 250 μg LNR125 was administered during ovalbumin challenge. Mice were then infected i.n. with RV-A1, 6 hours after final allergen challenge. On day 1 and day 7 post-infection, BAL were collected, lung lobe tissue was collected for viral RNA and cytokine expression. Results IL-25 and IL-17RB were constitutively expressed at the apical surface of airway epithelium in biopsies and AEC cultures. RV infection increased IL-25 expression by AEC from asthmatic donors. LNR125 treatment reduced IL-25 mRNA and significantly increased RV induced IFN-β a and IFN-λ protein expression and this was confirmed by Nanostring transcriptomic analyses which also identified down-regulated type-2 immune genes CCL26 (eotaxin 3) and IL1RL1(IL-33 receptor). LN125 treatment also increased IFN-λ expression by 229E-infected differentiated AECs. IL-25 treatment increased viral load associated with 50% reduced expression of IFN-β and CXCL10 and 75% reduced IFN-λ. Allergen challenged, RV-infected mice treated with LNR125 had significantly increased BAL IFN-β protein and 60% reduction in lung viral load associated with reduced IL-25, IL-4, IL-5 and IL-13 BAL proteins compared to controls. Conclusion IL-25-induced inflammation combined with suppression of AEC anti-viral immunity identify IL-25 as a central mediator of viral asthma exacerbations and therefore a target for mAb-based treatment.
ABSTRACT
Introduction: SARS-CoV-2 respiratory infection is pandemic and continues to cause significant mortality and morbidity worldwide. Respiratory viral infections in general are a leading cause of hospital admissions and mortality throughout the world as well. Most respiratory viral infections require an acidic intracellular and endosomal environment in order to enter host cells, replicate, and cause illness. We study the beneficial effects of airway alkalinization by an inhaled drug, Optate, that we currently have demonstrated is safe to inhale by healthy subjects and those with stable airways disease. We have recently shown that treatment with 4.5 mg/ml Optate safely inhibits SARS-CoV-2 infection in primary human airway epithelial cells (HAECs). We hypothesized that this inhibition would be dose dependent and that Optate would also inhibit other viral infections in a dosedependent manner. Methods: HAECs were infected with respiratory syncytial virus with green fluorescent protein (RSV-GFP) or SARS-CoV-2 virus. A dose-response curve of Optate was performed in each infection model and compared to a control group. Viral infection was quantified using fluorescence microscopy, plaque assays, and viral protein quantification. Optate pH was measured at each dose and a corresponding dose/pH curve was calculated to compare pH to dose-response. Results: SARS-CoV-2 infection was significantly inhibited by doses of Optate > 2.25 mg/ml, corresponding with an Optate pH > 9.2 (n = 4, p < 0.001). RSV infection was significantly inhibited by doses of Optate > 2 mg/ml, corresponding with an Optate pH > 9 (n = 3, p < 0.001). No significant difference was noted between control and Optate treated HAECs at lower concentrations of Optate. Conclusions: Optate inhibits SARS-CoV-2 and RSV viral infections in a dose-dependent manner that correlates with Optate pH. These findings suggest that Optate may be an inhaled therapeutic for patients with respiratory viral infections. (Table Presented).
ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a strain of coronavirus that causes COVID-19 (coronavirus disease 2019), has been responsible for a respiratory tract disease which has taken the proportion of the pandemic (COVID-19) ravaging the planet for last 2 and half year. The COVID-19 pandemic has been responsible for about sixty-one million deaths and about 500 million infections worldwide. The medication of infected individuals has been mainly cantered around repurposing of available known antiviral drugs, immunosuppressor/ immunomodulator drugs, monoclonal antibody concoctions and various vaccines as prophylaxis measures. Use phytometabolites in studies have been found to very effective in counter the SARS-CoV-2 spike protein binding sites, the main step to virus attack on the respiratory epithelial cells. Lichen secondary metabolites are well known for their antimicrobial, immunomodulator and antiviral activities. Current study was done to assess the spike protein binding capabilities of some lichen secondary metabolites of SARS-CoV-2 (Wuhan strain) spike protein binding sites using in-silico docking modelling. The study found that some of the lichen metabolites such as Cryptostictic acid and Quaesitic acid were effective in blocking the target cell recognising regions of the SARS-CoV-2 spike protein and can be effectively developed as therapeutic medicine.
ABSTRACT
Background: SARS-CoV-2 infection in immunocompromised individuals has been associated with prolonged virus shedding and the development of novel viral variants. Rapamycin and rapamycin analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved for use as mTOR inhibitors in multiple clinical settings, including cancer and autoimmunity, but a common side effect of these drugs is immunosuppression and increased susceptibility to infection. Immune impairment caused by rapalog use is traditionally attributed to their impacts on T cell signaling and cytokine production. Methods: We used replication-competent SARS-CoV-2 and HIV pseudotyped with betacoronavirus Spike proteins to assess how rapalog pretreatment of cells ex vivo and rodent animals in vivo impacts susceptibility to Spike-mediated infection. Results: We show that exposure to rapalogs increases cellular susceptibility to SARS-CoV-2 infection by antagonizing components of the constitutive and interferon-induced cell-intrinsic immune response. Pre-treatment of cells (including human lung epithelial cells and primary human small airway epithelial cells) with rapalogs promoted the early stages of SARS-CoV-2 infection by facilitating Spike-mediated virus entry. Rapalogs also boosted infection mediated by Spike from SARS-CoV and MERS-CoV in addition to hemagglutinin of influenza A virus and glycoprotein from vesicular stomatitis virus, suggesting that rapalogs downmodulate antiviral defenses that pose a common barrier to these viral fusion proteins. By identifying one rapalog (ridaforolimus) that lacks this function, we demonstrate that the extent to which rapalogs promote virus entry is linked to their capacity to trigger the lysosomal degradation of IFITM2 and IFITM3, intrinsic inhibitors of virus-cell membrane fusion. Mechanistically, rapalogs that promote virus entry inhibit the mTOR-mediated phosphorylation of TFEB, a transcription factor controlling lysosome biogenesis and lysosomal degradation pathways such as autophagy. In contrast, TFEB phosphorylation by mTOR was not inhibited by ridaforolimus. In the hamster model of SARS-CoV-2 infection, injection of rapamycin four hours prior to virus exposure resulted in elevated virus titers in lungs, accelerated weight loss, and decreased survival. Conclusion: Our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating a lysosome-mediated suppression of intrinsic immunity.
ABSTRACT
Background: Galectin-9 (Gal-9) is a β-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis. Multiple recent reports demonstrate that plasma levels of Gal-9 are elevated in the setting of severe COVID-19 disease. However, a causal role of Gal-9 in SARS-CoV-2 pathology remains to be elucidated. Here, we determined the impact of Gal-9 on SARS-CoV-2 replication and pro-inflammatory signaling in immortalized and primary human airway epithelial cells (AECs). Methods: Dose-dependent cytotoxicity of recombinant human Gal-9 in the Calu-3 AEC line was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 lineage P.1 (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the nucleocapsid (N) gene, immunofluorescence assay (IFA) of N protein, and titration of supernatant (TCID50). Viral entry was measured using luciferase activity of VSV-SARS-CoV-2 S-ΔG-Luciferase reporter pseudovirus. ACE2 and TMPRSS2 cell-surface expression were measured by flow cytometry. Pro-inflammatory factors (IL-6, IL-8, and TNFα) were detected by RT-PCR. Total RNA-seq was used to evaluate Gal-9 effects on the host transcriptome. Groups were compared by Student's t-test, and differential expression analyses were performed using DESeq2. Results: Gal-9 reached 50% cytotoxicity in Calu-3 cells at 597 nM. Gal-9 significantly increased SARS-CoV-2 expression (8.1 to 25.5 fold;p<0.0001) and infectious virus release (1.9 to 17.8 fold;p<0.038) in a dose-dependent manner in Calu-3 cells. Pseudovirus entry into Calu-3 cells was enhanced by Gal-9 (2.4 to 5.6 fold;p<0.0016), and the enhanced entry was inhibited by anti-ACE2 antibody (p<0.0027). Cell surface ACE2 and TMPRSS2 expression were unaffected by Gal-9. Gal-9 treatment accelerated virus-induced expression of IL-6, IL-8, and TNFα (p<0.018) in Calu-3 cells. Gal-9 increased SARS-CoV-2 production (p=0.03) and pro-inflammatory factor expression (p<0.05) in primary AECs (N=5 donors). RNA-seq data revealed that Gal-9 significantly induced IL-17, EIF2, IL-8 and IL-6 signaling pathways in the setting of SARS-CoV-2 infection. Conclusion: Gal-9 facilitates SARS-CoV-2 entry, replication, and virus-induced pro-inflammatory signaling in AECs ex vivo. Our data suggest that pharmacologic manipulation of Gal-9 should be explored as a SARS-CoV-2 therapeutic strategy.