Using ex vivo bioengineered lungs to model pathologies and screening therapeutics

Introduction: More than 8 million lives are claimed annually by various respiratory diseases including lung cancer. While therapeutics is the first line of defence, treatment failure always remains challenging and research studies face a lag of transition from preclinical to clinical phase. This is partly due to the inadequate representation of the preclinical models in clinical trials. In this proof-of-concept study, we sought to use an ex-vivo model to identify lung pathologies and therapeutically screen them in a rodent model. Method(s): Briefly, the heart-lung tissues were extracted and decellularized using a detergent-based decellularization technique. Subsequently, lungs were seeded and cultured (6-10 days) with human cell lines: BEAS-2B, A549, and Calu3, demonstrating healthy lung, cancerous state, and congenital pathologies (cystic fibrosis), respectively. By altering the cultural conditions and exploiting the unique characteristics of these cell lines, we were able to model a variety of novel pathological models in ex vivo, such as advanced-stage solid tumours and the primary phase of infection via SARS-COV2. We also validated the above-mentioned observations by histology and immunofluorescence staining. Another novel part of our study includes a qualitative screening of efficacy and impact of important Therapeutics (anti-neoplastic)- Cisplatin and Wogonin, in our cancer models. Result(s): Using A549 and BEAS-2B cells, we were able to model different stages of Non-small cell lung cancer, qualitatively validated the resemblance to clinical samples and monitor the impact of different therapeutics on these models. The qualitative assessment also demonstrated different levels of cell death depending on the efficacy of the drugs. Contribution to research : Collectively this study demonstrates the remarkable versatility and strength of the ex vivo model in representing important lung pathologies and screening therapeutics in the preclinical phase.

Protein Blend Suppress Inflammatory Response from Bronchial Epithelial Cells Induced by SARS-Cov-2

Even after virus elimination, coronavirus disease 2019 (COVID-2019) leaves numerous sequelae. Growing evidence demonstrates that massive release of proinflammatory cytokines, which drives COVID-19 progression, severity, and mortality, remains elevated after acute phase of COVID-2019, playing a central role in the disease' sequelae. In this way, bronchial epithelial cells are the first cells hyperactivated by coronavirus-2 (SARS-Cov-2) leading to massive cytokine release, triggering leukocytes and other cells hyperactivation, mediating COVID-19 sequelae. So, proinflammatory cytokines are initiated by the host. Thus, this in vitro study tested the hypothesis that ImmuneRecovTM, a protein blend, could inhibit the hyperactivation of human bronchial epithelial cells (BEAS-2B) induced by SARS-Cov-2. BEAS-2B (5x104/mL/well) cells were co-cultivated with 1ml of blood of a SARS-Cov-2 infected patient for 4 hours and protein blend (1ug/mL) was added in the first minute of the co-culture. After 4 hours, the cells were recovered and used for analysis of cytotoxicity by MTT and for mRNA expression of IL-1beta, IL-6, IL-10. The supernatant was used to measure cytokines. SARS-Cov-2 incubation resulted in increased levels of IL-1beta and IL-6 by BEAS-2B cells (p<0.001). Treatment with the protein blend resulted in reduced levels of pro-inflammatory IL1beta and IL-6 (p<0.001), and increased the levels of anti-inflammatory IL-10 (p<0.001). Protein blend reduced SARS Cov-2-increased the mRNA expression of IL-1beta and IL-6, and increased the expression of IL-10 and IFN-gamma. In conclusion, protein blend presents important anti-inflammatory effects in the context of SARS-Cov-2 infection.

SARS-CoV-2 spike protein enhanced thrombosis is inhibited by tipiracil in mice

Background: COVID-19 is accompanied by excessive systemic thrombotic events, but the mechanism is unknown. All major COVID-19 vaccines were associated with thrombosis. Thymidine phosphorylase (TYMP) plays an important role in platelet activation, thrombosis, and inflammation. TYMP expression is significantly increased in COVID-19 patients. Aim(s): To test the hypothesis that TYMP mediates SARS-CoV-2 spike protein (SP)-enhanced thrombosis. Method(s): Transfection of plasmid encoding SP or the receptor-binding domain (RBD) with human ACE2 was conducted in COS-7 cells. BEAS-2B cells were treated with SP or RBD containing COS-7 cell lysates, and TYMP expression and activation of NF-kappaB were examined. K18-hACE2 transgenic (ACE2-TG) mice were intraperitoneally treated with SP or RBD containing COS-7 cells lysates, and thrombosis was assessed three days later using the FeCl3 injury-induced carotid artery thrombosis model. Result(s): SP and RBD led to ACE2 shedding, significantly increased TYMP expression, and NF-kappaB activation in BEAS-2B cells. In comparison to wildtype mice, ACE2-TG mice are anti-thrombotic and had significantly prolonged thrombosis time. Treating ACE2-TG mice with COS-7 cells transfected with empty plasmid did not affect the thrombosis. However, treating the ACE2-TG mice with SP-or RBD-containing COS-7 cell lysates dramatically enhanced thrombosis and significantly shortened time to occlusive thrombi formation. SP is more powerful than RBD in enhancing thrombosis. SP-enhanced thrombosis was dramatically inhibited by simultaneously feeding the mice with 1 mg/kg of tipiracil. TYMP is expressed in human type II alveolar epithelial cells and bronchial epithelium. By using the MGH Emergency Department COVID-19 Cohort with Olink Proteomics TYMP data and Receiver Operating Characteristic analysis, we found TYMP is a sensitive and specific marker in diagnosing COVID-19 (AUC 0.8721, p < 0.0001). Conclusion(s): SARS-CoV-2 SP and RBD are pro-inflammatory and pro-thrombotic. SP/RBD-induced thrombosis is inhibited by tipiracil, a TYMP inhibitor. TYMP is a sensitive marker for COVID-19 diagnosis. Targeting TYMP could be a novel effective treatment for COVID-19.

Hypercapnia Increases ACE2 Protein Expression and Pseudo-SARSCoV- 2 Virus Entry in Airway Epithelial Cells by Augmenting Cellular Cholesterol

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.