Passage of SARS-CoV-2 in cells expressing human and mouse ACE2 selects for mouse-adapted and ACE2-independent viruses

Human ACE2 (hACE2) is required for cell attachment and entry of SARS-CoV-2. Mouse ACE2 (mACE2) does not support infection of early SARS-CoV-2 isolates. Herein we describe a new system for generating mouse-adapted SARS-CoV-2 in vitro by serial passaging virus in co-cultures of cell lines expressing hACE2 and mACE2. Mouse-adapted viruses emerged with a series of spike protein amino acid changes, all of which have been reported in human isolates. Mouse-adapted viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates, and caused severe lung histopathology. Remarkably, one mouse-adapted virus was able to replicate efficiently in ACE2-negative cell lines, a characteristic not described for any SARS-CoV-2 variants. ACE2-independent entry by SARS-CoV-2 represents a new biology for SARS-CoV-2 with potential widespread implications for disease and intervention development.

Modeling SARS-CoV-2 Infection in Mice Using Lentiviral hACE2 Vectors Infers Two Modes of Immune Responses to SARS-CoV-2 Infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a severe global pandemic. Mice models are essential to investigate infection pathology, antiviral drugs, and vaccine development. However, wild-type mice lack the human angiotensin-converting enzyme 2 (hACE2) that mediates SARS-CoV-2 entry into human cells and consequently are not susceptible to SARS-CoV-2 infection. hACE2 transgenic mice could provide an efficient COVID-19 model, but are not always readily available, and practically restricted to specific strains. Therefore, there is a dearth of additional mouse models for SARS-CoV-2 infection. We applied lentiviral vectors to generate hACE2 expression in interferon receptor knock-out (IFNAR1−/−) mice. Lenti-hACE2 transduction supported SARS-CoV-2 replication in vivo, simulating mild acute lung disease. Gene expression analysis revealed two modes of immune responses to SARS-CoV-2 infection: one in response to the exposure of mouse lungs to SARS-CoV-2 particles in the absence of productive viral replication, and the second in response to productive SARS-CoV-2 infection. Our results infer that immune response to immunogenic elements on incoming virus or in productively infected cells stimulate diverse immune effectors, even in absence of type I IFN signaling. Our findings should contribute to a better understanding of the immune response triggered by SARS-CoV-2 and to further elucidate COVID-19.

Estimation of 100 nm particle distribution kinetics in mouse lung using confocal laser scanning microscopy

BACKGROUND: Daily, people inhale airborne viral particles, some of which have a size of about 100 nm, such as particles of SARS-CoV-2. Kinetics of such 100 nm particle distribution in the respiratory tract is important, however, not a properly investigated question. AIM: To estimate the dissemination of inert viral particles based on the analysis of the spatial distribution of fluorescent 100 nm particles in the mouse lungs at different time points after the application. MATHERIALS AND METHODS: Fluorescent particles of 100 nm size were applied to C57BL/6 mice. 6, 24, 48 and 72 hours after, lungs were excised and fixed. Lung lobes were stained with immunohistochemistry as whole-mounts and then underwent optical clearance. Three-dimensional images of whole-mount mouse lung lobes were acquired using confocal laser scanning microscopy. RESULTS: 6 hours after the particle application particles were detected in lungs both as single particles and as particle agglomerates. Particles were both free and internalized by phagocytic cells. 24 hours after the application particles were detected both in bronchial lumen and in the alveolar space. Particles were detected in the mouse lungs up to 72 hours after the application. CONCLUSIONS: Reaching the respiratory tract of mammalian, inert particles which size equal to SARS-CoV-2 particle size distribute both in bronchi and in alveoli and undergoes internalization of phagocytic cells.

BMP1 is Not Required for Lung Fibrosis in Mice

Bone morphogenetic protein 1 (BMP1) belongs to the astacin/BMP1/tolloid-like family of zinc metalloproteinases, which play a fundamental role in the development and formation of extracellular matrix (ECM). BMP1 mediates the cleavage of carboxyl terminal (C-term) propeptides from procollagens, a crucial step in fibrillar collagen fiber formation. Blocking BMP1 by small molecule or antibody inhibitors has been linked to anti-fibrotic activity in the preclinical models of skin, kidney and liver fibrosis. Therefore, we reason that BMP1 may be important for the pathogenesis of lung fibrosis and BMP1 could be a potential therapeutic target for progressive fibrotic disease such as idiopathic pulmonary fibrosis (IPF). Here, we observed the increased expression of BMP1 in both human IPF lungs and mouse fibrotic lungs induced by bleomycin. Furthermore, we developed an inducible Bmp1 conditional knockout (cKO) mouse strain. We found that Bmp1 deletion does not protect mice from lung fibrosis triggered by bleomycin. Moreover, we found no significant impact of BMP1 deficiency upon C-term propeptide of type I procollagen (CICP) production in the fibrotic mouse lungs. Based on these results, we propose that BMP1 is not required for lung fibrosis in mice and BMP1 may not be considered a candidate therapeutic target for IPF.

Isoglycyrrhizin protects mouse lungs against acute respiratory distress syndrome via regulation of AMPK/Nrf2/ARE pathway

Purpose: To study the effect of isoglycyrrhizin on LPS-mediated acute respiratory distress syndrome (ARDS) in a mouse model, as well as the associated mechanism of action.Methods: Ninety (90) wild-type C57BL/6 male mice were randomly assigned to 3 groups, viz, control, ARDS and isoglycyrrhizin groups. Pathological lesions in mice lungs were determined using H&E staining. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS), heme oxygenase (HO-1), cyclooxygenase-2 (COX-2), AMP- dependent protein kinase (AMPK), serine/threonine proteinkinase (Akt), glycogen synthase kinase 3 (GSK3), nucleotide-binding domain-like receptor protein 3 (NLRP3), and Nrf2 were assayed using quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting, respectively.Results: The levels of mRNA and protein expressions of INO) and COX-2 were significantly upregulated in ARDS, when compared to control, but were markedly down-regulated by isoglycyrrhizin (p < 0.05). Similarly, exposure of ARDS mice to isoglycyrrhizin led to upregulations of mRNA and proteinlevels of Nrf2, NQO1, HO-1, GCLM, GCLC, p-GSK3, GSK3, p-AMPK, AMPK, p-Akt and AKT (p < 0.05). Moreover, isoglycyrrhizin significantly downregulated p-IκB and Nucl-p65 with respect to protein and mRNA levels, but upregulated IκBα expression. Histopathological examination revealed that pretreatment of ARDS mice with isoglycyrrhizin significantly reduced the number of infiltrating inflammatory cells, edema and ARDS score (p < 0.05).Conclusion: Isoglycyrrhizin protects mouse lungs against ARDS via regulation of AMPK/Nrf2/ARE pathway. Thus, this compound has potential for use in the treatment of ARDS.

Nickel nanoparticle-induced cell transformation: involvement of DNA damage and DNA repair defect through HIF-1α/miR-210/Rad52 pathway

Background Nickel nanoparticles (Nano-Ni) are increasingly used in industry and biomedicine with the development of nanotechnology. However, the genotoxic and carcinogenic effects of Nano-Ni and the underlying mechanisms are still unclear. Methods At first, dose–response (0, 10, 20, and 30 μg/mL) and time-response (0, 3, 6, 12, and 24 h) studies were performed in immortalized normal human bronchial epithelial cells BEAS-2B to observe the effects of Nano-Ni on DNA damage response (DDR)-associated proteins and the HIF-1α/miR-210/Rad52 pathway by real-time PCR or Western blot. Then, a Hsp90 inhibitor (1 µM of 17-AAG, an indirect HIF-1α inhibitor), HIF-1α knock-out (KO) cells, and a miR-210 inhibitor (20 nM) were used to determine whether Nano-Ni-induced Rad52 down-regulation was through HIF-1α nuclear accumulation and miR-210 up-regulation. In the long-term experiments, cells were treated with 0.25 and 0.5 µg/mL of Nano-Ni for 21 cycles (~ 150 days), and the level of anchorage-independent growth was determined by plating the cells in soft agar. Transduction of lentiviral particles containing human Rad52 ORF into BEAS-2B cells was used to observe the role of Rad52 in Nano-Ni-induced cell transformation. Nano-Ni-induced DNA damage and dysregulation of HIF-1α/miR-210/Rad52 pathway were also investigated in vivo by intratracheal instillation of 50 µg per mouse of Nano-Ni. gpt delta transgenic mice were used to analyze mutant frequency and mutation spectrum in mouse lungs after Nano-Ni exposure. Results Nano-Ni exposure caused DNA damage at both in vitro and in vivo settings, which was reflected by increased phosphorylation of DDR-associated proteins such as ATM at Ser1981, p53 at Ser15, and H2AX. Nano-Ni exposure also induced HIF-1α nuclear accumulation, miR-210 up-regulation, and down-regulation of homologous recombination repair (HRR) gene Rad52. Inhibition of or knocking-out HIF-1α or miR-210 ameliorated Nano-Ni-induced Rad52 down-regulation. Long-term low-dose Nano-Ni exposure led to cell malignant transformation, and augmentation of Rad52 expression significantly reduced Nano-Ni-induced cell transformation. In addition, increased immunostaining of cell proliferation markers, Ki-67 and PCNA, was observed in bronchiolar epithelial cells and hyperplastic pneumocytes in mouse lungs at day 7 and day 42 after Nano-Ni exposure. Finally, using gpt delta transgenic mice revealed that Nano-Ni exposure did not cause increased gpt mutant frequency and certain DNA mutations, such as base substitution and small base insertions/deletions, are not the main types of Nano-Ni-induced DNA damage. Conclusions This study unraveled the mechanisms underlying Nano-Ni-induced cell malignant transformation; the combined effects of Nano-Ni-induced DNA damage and DNA repair defects through HIF-1α/miR-210/Rad52 pathway likely contribute to Nano-Ni-induced genomic instability and ultimately cell transformation. Our findings will provide information to further elucidate the molecular mechanisms of Nano-Ni-induced genotoxicity and carcinogenicity. Graphical Abstract

Hydrophobic Optimization of Functional poly(TPAE-co-suberoyl chloride) for Extrahepatic mRNA Delivery following Intravenous Administration

Messenger RNA (mRNA) has generated great attention due to its broad potential therapeutic applications, including vaccines, protein replacement therapy, and immunotherapy. Compared to other nucleic acids (e.g., siRNA and pDNA), there are more opportunities to improve the delivery efficacy of mRNA through systematic optimization. In this report, we studied a high-throughput library of 1200 functional polyesters for systemic mRNA delivery. We focused on the chemical investigation of hydrophobic optimization as a method to adjust mRNA polyplex stability, diameter, pKa, and efficacy. Focusing on a region of the library heatmap (PE4K-A17), we further explored the delivery of luciferase mRNA to IGROV1 ovarian cancer cells in vitro and to C57BL/6 mice in vivo following intravenous administration. PE4K-A17-0.2C8 was identified as an efficacious carrier for delivering mRNA to mouse lungs. The delivery selectivity between organs (lungs versus spleen) was found to be tunable through chemical modification of polyesters (both alkyl chain length and molar ratio in the formulation). Cre recombinase mRNA was delivered to the Lox-stop-lox tdTomato mouse model to study potential application in gene editing. Overall, we identified a series of polymer-mRNA polyplexes stabilized with Pluronic F-127 for safe and effective delivery to mouse lungs and spleens. Structure–activity relationships between alkyl side chains and in vivo delivery were elucidated, which may be informative for the continued development of polymer-based mRNA delivery.

The effect of Java Plum Fruit (Zyzygium cumini) extract on leucocyte and lung histopathology of mouse exposed cigarette smoke

Java Plum fruit can decrease free radical activities. The overwhelmed production of free radicals will reduce the immunity system in the body. This research aims to study the effects of java plum fruit extracts on leukocytes and histopathology of mouse lungs that have been exposed to cigarette smoke for 30 days. Frozen-dried java plum fruit is macerated with water. The mice are divided into Control groups (K-). The mice of the control group were given only water orally. (K+) are mice that are exposed to commercial cigarette smoke once a day. (RJ) were the mice exposed to cigarette smoke once a day as java plum fruit extract orally of 180 mg/kg body weight. (RF) were the mice exposed to cigarette smoke given with java plum fruit extract with 180 mg/kg of body weight that is applied on its filter. The results showed a significantly lower number of leukocytes (p < 0.05) in the K-,RF, and RJ groups compared to K+. Meanwhile, there was a significant increase in the number of lung pathological cells (p < 0.05) in the K+, RF, and RJ compared to the control group (K-). The results conclude that java plum fruit can reduce free radicals in animals affected by inflammation due to cigarette smoke particles in the lungs.

Recombinant BCG-Prime and DNA-Boost Immunization Confers Mice with Enhanced Protection against Mycobacterium kansasii

The incidence of infections with nontuberculous mycobacteria (NTM) has been increasing worldwide. The emergence of multidrug-resistant NTM is a serious clinical concern, and a vaccine for NTM has not yet been developed. We previously developed a new recombinant Bacillus Calmette–Guérin (rBCG) vaccine encoding the antigen 85B (Ag85B) protein of Mycobacterium kansasii—termed rBCG-Mkan85B—which was used together with a booster immunization with plasmid DNA expressing the same M. kansasii Ag85B gene (DNA-Mkan85B). We reported that rBCG-Mkan85B/DNA-Mkan85B prime–boost immunization elicited various NTM strain-specific CD4+ and CD8+ T cells and induced Mycobacterium tuberculosis-specific immunity. In this study, to investigate the protective effect against M. kansasii infection, we challenged mice vaccinated with a rBCG-Mkan85B or rBCG-Mkan85B/DNA-Mkan85B prime–boost strategy with virulent M. kansasii. Although BCG and rBCG-Mkan85B immunization each suppressed the growth of M. kansasii in the mouse lungs, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination reduced the bacterial burden more significantly. Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination induced antigen-specific CD4+ and CD8+ T cells. Our data suggest that rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination effectively enhances antigen-specific T cells. Our novel rBCG could be a potential alternative to clinical BCG for preventing various NTM infections.