The preventive effect and mechanism of Dayuanyin in hypoxic pulmonary hypertension through NF-kappaB signaling pathway.

Dayuanyin (DYY) has been shown to reduce lung inflammation in both coronavirus disease 2019 (COVID-19) and lung injury. This experiment was designed to investigate the efficacy and mechanism of action of DYY against hypoxic pulmonary hypertension (HPH) and to evaluate the effect of DYY on the protection of lung function. Animal welfare and experimental procedures are approved and in accordance with the provision of the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Science. Male C57/BL6J mice were randomly divided into 4 groups: control group, model group, DYY group (800 mg.kg-1), and positive control sildenafil group (100 mg.kg-1). The animals were given control solvents or drugs by gavage three days in advance. On day 4, the animals in the model group, DYY group and sildenafil group were kept in a hypoxic chamber containing 10% +/- 0.5% oxygen, and the animals in the control group were kept in a normal environment, and the control solvent or drugs continued to be given continuously for 14 days. The right ventricular systolic pressure, right ventricular hypertrophy index, organ indices and other metrics were measured in the experimental endpoints. Meantime, the expression levels of the inflammatory factors in mice lung tissues were measured. The potential therapeutic targets of DYY on pulmonary hypertension were predicted using network pharmacology, the expression of nuclear factor kappa B (NF- kappaB) signaling pathway-related proteins were measured by Western blot assay. It was found that DYY significantly reduced the right ventricular systolic pressure, attenuated lung injury and decreased the expression of inflammatory factors in mice. It can also inhibit hypoxia-induced activation of NF- kappaB signaling pathway. DYY has a protective effect on lung function, as demonstrated by DYY has good efficacy in HPH, and preventive administration can slow down the disease progression, and its mechanism may be related to inhibit the activation of NF-kappaB and signal transducer and activator of transcription 3 (STAT3) by DYY.Copyright © 2023, Chinese Pharmaceutical Association. All rights reserved.

Co-exposure to urban particulate matter and aircraft noise adversely impacts the cerebro-pulmonary-cardiovascular axis in mice

Worldwide, up to 8.8 million excess deaths/year have been attributed to air pollution, mainly due to the exposure to fine particulate matter (PM). Traffic-related noise is an additional contributor to global mortality and morbidity. Both health risk factors substantially contribute to cardiovascular, metabolic and neuropsychiatric sequelae. Studies on the combined exposure are rare and urgently needed because of frequent co-occurrence of both risk factors in urban and industrial settings. To study the synergistic effects of PM and noise, we used an exposure system equipped with aerosol generator and loud-speakers, where C57BL/6 mice were acutely exposed for 3d to either ambient PM (NIST particles) and/or noise (aircraft landing and take-off events). The combination of both stressors caused endothelial dysfunction, increased blood pressure, oxidative stress and inflammation. An additive impairment of endothelial function was observed in isolated aortic rings and even more pronounced in cerebral and retinal arterioles. The increase in oxidative stress and inflammation markers together with RNA sequencing data indicate that noise particularly affects the brain and PM particularly affects the lungs. Noise also increased levels of circulating stress hormones adrenaline and noradrenaline, while PM increased levels of circulating cytokines CD68 and MCP-1. The combination of both stressors has additive adverse effects on the cardiovascular system that are based on PM-induced systemic inflammation and noise-triggered stress hormone signaling. We demonstrate an additive upregulation of ACE-2 in the lung, suggesting that there may be an increased vulnerability to COVID-19 infection. The data warrant further mechanistic studies to characterize the propagation of primary target tissue damage (lung, brain) to remote organs such as aorta and heart by combined noise and PM exposure.Copyright © 2023

The preventive effect and mechanism of Dayuanyin in hypoxic pulmonary hypertension through NF-kappaB signaling pathway

Dayuanyin (DYY) has been shown to reduce lung inflammation in both coronavirus disease 2019 (COVID-19) and lung injury. This experiment was designed to investigate the efficacy and mechanism of action of DYY against hypoxic pulmonary hypertension (HPH) and to evaluate the effect of DYY on the protection of lung function. Animal welfare and experimental procedures are approved and in accordance with the provision of the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Science. Male C57/BL6J mice were randomly divided into 4 groups: control group, model group, DYY group (800 mg.kg-1), and positive control sildenafil group (100 mg.kg-1). The animals were given control solvents or drugs by gavage three days in advance. On day 4, the animals in the model group, DYY group and sildenafil group were kept in a hypoxic chamber containing 10% +/- 0.5% oxygen, and the animals in the control group were kept in a normal environment, and the control solvent or drugs continued to be given continuously for 14 days. The right ventricular systolic pressure, right ventricular hypertrophy index, organ indices and other metrics were measured in the experimental endpoints. Meantime, the expression levels of the inflammatory factors in mice lung tissues were measured. The potential therapeutic targets of DYY on pulmonary hypertension were predicted using network pharmacology, the expression of nuclear factor kappa B (NF- kappaB) signaling pathway-related proteins were measured by Western blot assay. It was found that DYY significantly reduced the right ventricular systolic pressure, attenuated lung injury and decreased the expression of inflammatory factors in mice. It can also inhibit hypoxia-induced activation of NF- kappaB signaling pathway. DYY has a protective effect on lung function, as demonstrated by DYY has good efficacy in HPH, and preventive administration can slow down the disease progression, and its mechanism may be related to inhibit the activation of NF-kappaB and signal transducer and activator of transcription 3 (STAT3) by DYY.Copyright © 2023, Chinese Pharmaceutical Association. All rights reserved.

SARS-CoV-2 BA.5 CAUSES HIGHER REPLICATION AND LETHAL INFECTION IN K18-hACE2 MICE

Background: The continuous evolution of SARS-CoV-2 in the diverse immune landscape (natural, vaccine, hybrid) is giving rise to novel immune escape mutations. So far, the resulting new variants (BA.1, BA.2, BA.2.12.1) were observed to cause mild infections, however, BA.5 infections are associated with an increased risk of hospitalization.1 Therefore it is essential to investigate the pathogenesis of BA.5. Method(s): Here we compared the pathogenicity of Pre-Omicron (B.1.351) and Omicron (BA.1, BA.2.12.1, and BA.5) variants in wild-type C57BL/6J mice and K18-hACE2 mice. The virus replication kinetics was also studied in human Calu3, pulmonary alveolar type 2 (AT2) cells, and airway organoids (HAO). Cell-to-cell spread of virus was measured by syncytia formation assay and immunohistochemistry (IHC) of infected lungs. Result(s): In the results, infection in C57BL/6J mice showed severe weight loss ( >15%) for B.1.351 infected mice and moderate ( >5%) for BA.5 infected. C57BL/6J mice showed higher virus replication of B.1.351 followed by BA.5, BA.1, and BA.2.12.1. At the peak of virus replication (2 days) plaque-forming units from lung extract of BA.5 infected mice were two, and three logs higher compared to BA.1 and BA.2.12.1 respectively. BA.5 infection was lethal to 80% of infected K18-hACE2 mice, whereas the mice looked normal after infection with BA.1 and BA.2.12.1. BA.5 infected mice showed high virus replication in brain tissue. Surprisingly the syncytia formation assay and IHC for BA.5 was comparable to that of B.1.351, indicating the higher cell-to-cell spread of BA.5 and B.1.351 compared to BA.1 and BA.2.12.1, which is one of the measures of pathogenicity. Calu3 and HAO showed the same trend of virus replication as was observed in-vivo experiments however AT2 cells were found to be resistant to BA.5 replication. Conclusion(s): These results suggest that the BA.5 variant (lineage) of Omicron has the potential to regain the pathogenicity as it shows increased virulence compared to other Omicron sub-variants. Lethal infection of BA.5 in K18-hACE2 mice may be attributed to catastrophic encephalitis and increased cell-to-cell spread.

Mitigation of Post-Septic Microcirculation Dysfunction by Drag-Reducing Polymers

INTRODUCTION: Sepsis and septic shock in multiorgan dysfunction syndrome (MODS) are characterized by inflammation, coagulopathy, and vascular collapse with the endothelial and microvascular breakdown of endothelial function, the primary cause of mortality among hospitalized patients. We evaluated here whether drag-reducing polymers (DRP) can alleviate sepsis and MODS-associated panvascular dysregulation of microvascular blood flow using a mouse model of LPS-induced sepsis. Since the pathophysiology of MODS and Covid-19 share many characteristics in inflammation, coagulopathy, and low blood flow as a constellation of factors culminating in low microvascular shear rate and loss of endothelial function common to the pathophysiology of both diseases, the model utilized in this study also can be considered as the Covid-19 model. METHOD(S): To induce acute sepsis and MODS, lipopolysaccharide (LPS-Salmonella Thyphosa) was injected i.v. (10 mg/kg) in C57BL/6J mice. In-vivo 2-photon laser scanning microscopy was used to monitor systemic cerebral (parietal cortex) and peripheral (ear) microcirculation, NADH (hypoxia), and oxidative stress. Blood samples obtained at autopsy were analyzed for Inflammation, coagulopathy, and endothelial glycocalyx disintegration biomarkers. Brain, lungs, kidney, liver, muscle, and intestine (rectum) were histologically evaluated. Differences between groups were determined using two-way ANOVA for multiple comparisons and posthoc testing using the Mann-Whitney U-test. RESULT(S): LPS injection induced inflammatory reaction and microvascular dysfunction. DRP alleviated the inflammation, microthrombosis formation, and microvascular dysfunction in all organs evaluated (p< 0.05). Blood samples analysis by ELISA revealed reduced inflammation, coagulopathy, and endothelial glycocalyx disintegration in the DRP-treated group (p< 0.05). CONCLUSION(S): Hemorheological modulation of blood flow by DRP effectively improves systemic and peripheral circulation, reducing microthrombosis formation, inflammation and microvascular dysfunction, alleviating sepsis and MODS.

Intratumoral Administration of Sars-Cov-2 Reduces Tumor Progression and Alters the Tumor Immune Microenvironment Similar to That of the Seasonal Influenza Vaccine

Background A significant challenge in using immunotherapies to treat solid tumors is that these treatments are largely ineffective due to lack of immune cell infiltration or are dominated by suppressive immune cell populations. To overcome this, we previously demonstrated that intratumoral administration of influenza converts an immune barren tumor to a tumor that is loaded with inflammatory factors, thus can be targeted by the immune system. With the onset of the COVID-19 pandemic, and there being several shared characteristics between the influenza and SARS-CoV-2 we sought to determine the cancer immunotherapeutic potential of SARSCoV- 2. Here, we have shown that inactivated SARS-CoV-2 can reduce tumor growth in murine tumor models and can shift composition of the tumor microenvironment paralleling that of treatment using the influenza vaccine. Methods To determine the anticancer response, 4T1 breast cancer and B16 melanoma tumors were induced in BALB/C and C57BL/6 mice, respectively. Tumors were treated with inactivated SARS-CoV-2, seasonal influenza vaccine, or PBS via intratumoral injection. Tumor growth was evaluated via caliper measurements. Determination of immune cell population changes within the tumor following each treatment was determined via flow cytometry analysis. Results Intratumoral injection of inactivated SARS-CoV-2 and the influenza vaccine showed significant reduction in tumor growth compared to a PBS control (p < 0.001) in both 4T1 and B16 tumor models. Within B16 tumors, both SARS-CoV- 2 and influenza vaccine increased CD45+ cell populations (p < 0.0001) compared to PBS. Notably, both B16 and 4T1 tumors treated with SARS-CoV-2 and influenza experienced a significant increase in CD8+ T-cell infiltration (p < 0.05, p < 0.01). Additionally, CD11b+ Ly6G/Gr-1+ myeloid derived suppressor cell populations were decreased in B16 melanoma tumors following inactivated SARS-CoV-2 or influenza vaccine treatment. Conclusions These findings indicate that introducing inactivated SARS-CoV-2 into the tumor microenvironment reduces tumor progression and is able to shift the immune profile of a tumor from an immune-suppressed to a more inflamed, immunologically targeted status. Further, the changes in immune cell populations within the tumor as well, paralleling those of influenza vaccine treated tumors.

Inducible nitric oxide synthase deficiency leads to early and severe demyelination in a murine coronavirus induced disease model of multiple sclerosis

Introduction: Inducible nitric oxide synthase (iNOS) catalyses production of nitric oxide during an inflammatory stimuli and is a signature marker of M1-like microglia/macrophages. iNOS mRNA and protein were found in brain lesions of MS patients however its role in demyelination remains unclear. We employed RSA59, a mild hepatoneurotropic strain of Mouse hepatitis virus (MHV) which in 4-weeks-old C57BL/6 mice causes biphasic CNS disease characterised by acute neuroinflammation (day5 p.i.) and chronic demyelination and axonal loss (day30 p.i.). Microglia/ macrophages are central to the disease pathology and require assistance from infiltrating CD4+ T cells to mount protective host immune response. The CNS immune interactions during the acute-adaptive transition stage thus determine disease trajectory. Objective(s): To understand the role of iNOS in microglia/macrophage and peripheral T cell communication and assess its effect on demyelination. Aim(s): To study the role of iNOS in demyelination. Method(s): 4-5-weeks-old MHV-free wildtype C57BL/6 (WT) and iNOS knockout (iNOS-/-) mice were infected intracranially with 20000 or 10000 pfus of RSA59 and assessed daily for weight loss and disease score. Mice were sacrificed at day9/10 and day30 p.i. CNS viral titers were detected by plaque assay. Transcript levels of anti-inflammatory and phagocytic M2-like phenotype markers were analysed by qRT PCR. Differential CNS immune cell infiltration was assessed by flow cytometry. LFB and Iba1 staining was used to study demyelination and microglia/macrophage activation in the CNS. Result(s): iNOS-/- mice infected with RSA59 at 20000 pfus exhibited aggravated disease and high mortality at the acute-adaptive transition stage i.e., day9/10 p.i. as compared to wildtype controls despite being no differences in virus clearance by the CNS. Histopathology at this stage showed early demyelination in the spinal cords accompanied by presence of amoeboid microglia macrophages;high CNS mRNA expression of M2-like phenotype markers, TGFbeta, Arg1, CD206 and TREM2;and more infiltration of T regulatory cells. iNOS-/- mice infected at low pfus of virus i.e., 10000 also showed significantly more chronic demyelination at day30 p.i. Conclusion(s): Our studies reveal a protective role of iNOS against RSA59 induced demyelination by regulating the CNS inflammatory phenotype specifically the phenotypic transition of microglia/macrophages and thereby their interaction with peripheral immune cells.

Proteome and phosphoproteome analyses of SARS-CoV-2-infected humanized K18-ACE2 mice reveal hyperactive phenotype of circulating platelets

Background: Platelets are effectors of hemostasis and play a major role in coordinating immune and inflammatory activities. Suitable animal models are needed to study COVID-19-associated coagulopathy and platelet effector functions in COVID-19, which are currently poorly understood. Aim(s): We aimed to characterize alterations of platelets isolated from K18-hACE2 transgenic mice infected with SARS-CoV-2. Method(s): Heterozygous K18-hACE2 (human ACE2) and C57BL/6J mice were used to study SARS-CoV-2 infectivity. Lung infection, infiltration, and platelet aggregation were characterized with histology and immunohistochemistry. Platelet response to SARS-CoV-2 infection was quantified by mass spectrometry analysis of proteomics and phosphoproteomics. Western blotting, ELISA, and multiplex plasma profiling were performed to validate the proteomics and phosphoproteomics data. Result(s): SARS-CoV-2 inoculated (10E6PFU, i.n.) K18-hACE2 mice started to lose weight at 4 days post-infection (dpi) and showed 90% lethality at 7-dpi in association with viral neuroinvasion. Histopathologic findings of infected K18-hACE2 mice included progressive lymphohistiocytic interstitial pneumonia with absence of diffuse alveolar damage. Lungs of infected K18-hACE2 mice (2-/ 4-dpi) showed mild increase in CD61+ aggregates compared to sham mice, but no overt tissue thrombosis. Gene ontology and pathway analyses of platelet proteomics and phosphoproteomics revealed that SARS-CoV-2 infection significantly upregulates the complement-coagulation cascades (F2/12/13, Tfpi, C1ra, Cd55, C4bp) and platelet activation-adhesion-degranulation proteins (Vwf, Itgb3/5, Selp, Pecam1) and chemokine (Pf4, Cxcl5/12) signaling at 2-dpi. However, interferon (Ddx58, Trim25, Mapk3) signaling was dominant at 4-dpi. Activation of proteomics and phosphoproteomics protein markers were highly correlated with platelet activation and interferon signaling at 2-/ 4-dpi, respectively. Plasma chemokine (e.g., Ccl8 and Pf4) and cytokines (e.g., IL6) were significantly elevated at 2-/ 4-dpi. SARS-CoV-2 spike protein was abundant at 2-/ 4-dpi in the lungs but not in platelets and kidneys, which correlated with no infectious virus in the serum. Conclusion(s): Platelet re-programming towards activation-degranulation-aggregation is likely attributable to a pneumonia-induced elevated circulatory factors (e.g., cytokines)-driven response rather than direct platelet infection.

Biphasic thrombus formation and resolution during a coronavirus infection in the liver

Background: The liver is the main producer of coagulation factors, which are activated in response to a variety of viruses, presumably to contain and limit virus spreading. Neutrophils and their extracellular traps (NETs) often build the molecular and structural basis for such thrombi that can cause adverse complications and organ damage in patients. Using the murine coronavirus m-CoV, we analysed the consequences of a coronavirus infection in the liver. Aim(s): Analysis of haemostatic alterations and immunothrombosis in a virus infected liver. Method(s): C57Bl/6 mice were nasally infected with m-CoV (MHV-A59). Livers were collected 2, 4 and 10 days after infection, and virus burden, tissue damage, thrombus formation, expression of coagulation factors and neutrophil activation were analysed via histology and qPCR. Result(s): NET formation occurred rapidly and U-shaped over the course of the infection: 1.55 thrombi/mm2 liver section were present 2 days after m-CoV infection, which decreased to 0.50 thrombi/ mm2 at day 4 and increased again to 1.04 thrombi/mm2 at day 10. Neutrophil counts significantly increased until day 4 and showed highest MPO activity on day 2. M-CoV virus is mainly present 4 days after infection and coincided with highest percentage of damaged tissue (4.58%). Although mRNAs of factors II, V, VII, VIII, IX, and X were not significantly altered, we could detect a significant peak in plasminogen mRNA on day 2 and PAI 1 mRNA on day 4. Plasma concentrations of MCP-1, IFN-gamma, IL-6 and TNF alpha were also highest at day 4. Conclusion(s): We describe a U-shaped liver thrombosis development during a coronavirus infection. Liver NETs are rapidly formed after a viral infection and are resolved due to increased plasminogen expression. Peak virus burden of the liver and increased systemic inflammation markers induce expression of liver PAI-1 and activation of recruited neutrophils, which favours NET formation anew.

Lung Transplant Recipients with SARS-CoV2 Infection and Bnt162b2 Vaccination Induce Circulating Exosomes with Spike Protein S2 Capable of Inducing Immune Responses to SARS0-CoV2 in Mice

Purpose: Exosomes are small vesicles which are released by cells into body fluids. We have demonstrated the presence of circulating exosomes with viral antigens in lung transplant recipients (LTxRs) diagnosed with respiratory viral infections. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection results Covid-19 disease and SARS-CoV2 infection of LTxRs can be severe with poor clinical outcomes. The goal of this single center study is to determine the development of antibody responses specific to SARS-CoV2 in LTxRs, characterize the immune and molecular markers in the circulating exosomes induced and its role in eliciting immunity. Method(s): To determine that antibody responses and induction of circulating exosomes we enrolled LTxRs with SARS-CoV2 infection (n=50), following 2 doses of vaccination (n=100). Exosomes were isolated from plasma by exosome precipitation kit followed by 0.2 micron filtration and size determination by NanoSight300. Exosomes were subjected to transmission electron microscopy for spike (CSP) and nucleocapsid (CNP) antigens. Exosomes were also characterized by western blot for immune and molecular markers (NFkB, CIITA, 20S proteasome, beta catenin and VWF). C57BL/6 mice were immunized with circulating exosomes isolated from LTxRs with infection. Result(s): 78% of SARS-CoV2 infected LTxRs developed antibodies to CSP and CNP as opposed to normal infected individuals. In contrast, only 55% vaccinated LTxRs developed antibodies to SARS-CoV2 spike. Exosomes from SARS-CoV2 infected and vaccinated individuals contained CSP S2, CNP and immune and molecular markers. Transmission electron microscopy also revealed the presence of CSP and CNP on exosomes. C57BL/6 mice immunized with exosomes carrying CSP developed antibodies to SARS-CoV2 spike antigens. Severe inflammation and lung lesions were also demonstrated in the lungs of mice immunized with exosomes carrying CSP. Conclusion(s): In conclusion, we demonstrated that SARS-CoV2 infected and vaccinated LTxRs induced circulating exosomes with SARS-CoV2 CSP. In addition, exosomes contained important immune activating molecules suggesting that the exosomes induced by SARS-CoV2 may have a physiological role in inducing immune responses. Immunization of mice with exosomes from SARS-CoV2 infected and vaccinated LTxRs not only induced SARS-CoV2 spike specific antibody but also resulted in inflammation and lung lesions in the immunized animals.

Lipid Nanoparticles With Low Immunogenicity For Nucleic Acid Delivery

Purpose/Objectives: The delivery of nucleic acids to cells has revolutionized medicine and enabled new technologies such as mRNA vaccines and stem cell therapies. These recent advances rely on delivery vehicles to stabilize the genetic payload and increase cellular transfection. While engineered viruses are efficient vectors for ex vivo cellular reprogramming, they are not ideal for in vivo gene therapies as repeated dosing leads to anti-vector immunity. Lipid nanoparticles have thus emerged as the best alternative to viral vectors for in vivo nucleic acid delivery. However, all FDA-approved lipid nanoparticles have been linked to inflammatory responses, undesirable for regenerative medicine applications that require precise immunomodulation. Thus, non-immunogenic delivery materials must be developed to fulfill the immense potential of gene therapy in regenerative medicine. Lipid nanoparticles typically comprise 4 different lipids, with the ionizable amino lipid being the main driver of potency and immunogenicity. A way to reduce immunogenicity is to develop lipid nanoparticles that minimize the amount of lipids per gram of nucleic acids. To do so, we developed a novel class of ionizable amino lipids with high charge density. Our primary objective is to design a lipid nanoparticle that maximizes RNA delivery and minimizes immunogenicity. Methodology: We designed a library of proprietary ionizable lipids based on the structure of a poly(amido amine) dendron. The structure is modular, which allowed us to systematically vary molecular motifs to optimize important physiochemical parameters: Lipid-to-RNA ratio;apparent pKa;surface zeta potential;size distribution;and RNA encapsulation These structures are also designed to include a higher number of amines compared to current ionizable lipids. This improves ionization charge density of the lipid and lowers the amount of lipid required to encapsulate RNA. In this study, lipid nanoparticles contain an ionizable lipid selected from our library, cholesterol, a phospholipid, and a PEG-lipid. The lipids and formulation conditions were selected to mimic Moderna's COVID-19 vaccine (SpikeVax), albeit with different lipid-to-RNA ratios. C57BL/6 mice were injected intramuscularly with nanoparticles co-formulated with a firefly luciferase mRNA and ovalbumin mRNA to simultaneously study transfection efficiency and antigen-specific immune responses. Nanoparticles that comprise SM-102, the ionizable lipid used in SpikeVax, were used as a comparative control due to their high potency and immunogenicity. Luciferase activity was detected using an IVIS Spectrum, and key organs were harvested for immune phenotyping. Results: We have so far determined the effect of hydrophobic motifs on apparent pKa and RNA encapsulation. Our best lipids with optimized tails did not induce IFN-I responses in vitro and demonstrated comparable in vivo efficacy to SM-102. We are currently in the process of collecting immunogenicity data which we expect to complete prior to the conference. Conclusion/Significance: We have produced a novel set of lipid nanoparticles that efficiently transfect cells in vivo. These new particles deliver RNA with half of the lipid mass used in SpikeVax, which can reduce the amount of material-induced immunogenicity. This result opens the door to developing mRNA vaccines with fewer side effects and equitable gene therapies for untreatable diseases such as inflammatory and autoimmune disorders.

CD177 and PR3 in the pathogenesis of P. aeruginosa-induced keratitis

Purpose : Bacterial keratitis is a prevalent eye infection that causes corneal opacification and purulent discharge, especially among contact lens wearers. Such infections recruit innate immune cells into the cornea, predominately neutrophils (PMN). CD177 is a GPIanchored protein expressed on ∼50% of circulating PMN. Proteinase-3 (PR3) is a serine protease that binds CD177 and is shown to be released from PMN granules upon activation or expressed on the plasma membrane (mPR3). CD177 PMN can be protective or pathogenic in various diseases ranging from IBD to COVID-19. On the other hand, elevated PR3 is found in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated systemic vasculitis. With little known regarding the eye, this study investigates the expression and role(s) of CD177 and PR3 in the cornea following bacterial keratitis. Methods : This work uses an experimental model of bacterial keratitis carried out in 8-week-old, female susceptible C57BL/6 (B6) and resistant BALB/c mice. The left eye of each mouse was scarified then infected with P. aeruginosa ATCC strain 19660 (5 μL of 1 x 106 CFU). Corneas from naïve, uninfected mice from both strains served as controls. Corneas were harvested at 1, 3, and 5 days post-infection (p.i.). Levels of CD177 and PR3 were determined at the protein level by Western blot and by phenotypic profiling using flow cytometry. In vitro assessment was carried out using HL-60, a human promyelocytic cell line, and siCD177 knockdown. Results : Results from the in vivo model showed no differences in protein levels at 1 day p.i., but significantly higher levels of both CD177 and PR3 in B6 vs. BALB/c at 3 and 5 days p.i. Flow cytometry data revealed CD177+ and PR3+ expression on both PMN and macrophages from B6 and BALB/c infected corneas with differential mean fluorescence intensities detected between the strains under normal conditions and following infection. In vitro results indicated that cell activation was altered following CD177 knockdown with differences in downstream signaling. Conclusions : As one of the first studies to explore the role of CD177 and PR3 in the pathogenesis of bacterial keratitis, our findings reveal strain-specific expression profiles for PMN that may contribute to resistance vs. susceptibility. In addition, we show the presence of CD177 PR3 macrophages. Overall, these findings may uncover novel therapeutic targets to treat bacterial keratitis.

Bunazosin inhibited axial elongation with an increase of the choroidal blood perfusion in a murine model of myopia

Purpose : The prevalence of myopia is accelerating worldwide possibly because of the decrease in outdoor activity including COVID-19 home confinement. We have reported the effective treatments of suppressing myopia progression, including oral administration of crocetin (Mori K et al. Sci Rep. 2019) and violet light exposure (Jiang X et al. Proc Natl Acad Sci USA. 2021). In this study, we examined the therapeutic effects of bunazosin, known as one of the α1-adrenergic receptor antagonists, in a lens-induced myopia mouse model. Methods : C57BL/6J mice were induced myopia at 3-week-old by a method established in our research group (Jiang X et al. Sci Rep. 2018). For 3 weeks, mice were equipped with lenses in both eyes, a left for 0 D lens as internal control and a right for -30 D lens as myopia induction. During this period, we administered 0.01% bunazosin hydrochloride solution by intraperitoneal injection (IP group) and eye drop (E group) once a day, and PBS as control. Ocular components including refraction error, axial length, and choroidal thickness before and after myopia induction were measured by an infrared photorefractor and an SD-OCT. The choroidal blood flow was evaluated by an SS-OCT angiography. Results : In the eye with -30D lens of control group, significant changes in a myopic shift of refraction (p < 0.01), axial elongation (p < 0.05), and choroidal thinning (p < 0.01) compared to 0D lens were observed. In contrast, IP or E groups showed no significant difference between both eyes, suggesting myopia progression was suppressed by bunazosin treatment. The choroidal blood flow of the eye with -30D in E group (58.9±8.9%) was higher than that of the control group -30D (44.0±6.4%)(p < 0.05). Conclusions : Bunazosin has a preventive effect on myopia progression by suppressing axial elongation and choroidal thinning together with an increase of choroidal blood flow.

Modified mRNA delivery to the heart using Lipid Nanoparticles

Myocardial infarction is a global health burden for which there is no treatment available that aims to recover the damaged tissue after the ischemic event. Lipid nanoparticles (LNPs) represent a well characterized class of mRNA delivery systems, which were recently approved for clinical usage in their application for mRNA-based covid-19 vaccines. After myocardial infarction, endogenous mechanisms that enable repair of the functional damaged tissue can be triggered by modified mRNA (modRNA) delivery, locally in the infarcted area. As a first step, in order to optimize the LNP formulation for effective myocardial delivery and study cellular tropism of the LNPs in the heart, different LNPs formulations will be evaluated as delivery systems in a murine healthy heart model. Different LNP formulations varying in type and amount of helper lipid were used as delivery systems for modRNA encoding the reporter genes luciferase or eGFP. In vitro, LNPs were evaluated for modRNA delivery in a human endothelial cell line (HMEC-1), induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) and induced pluripotent stem cell -derived fibroblasts (iPS-FBs). In vivo, modRNA delivery was evaluated in C57BL-6 mice, undergoing open chest heart surgery under general anaesthesia in order to infuse LNPs into the left ventricular wall. For determination of luciferase expression levels, animals were infused with luciferin substrate intraperitoneally 24 hrs after injection. Heart, liver, lungs, spleen and kidneys were extracted for imaging in a bioluminescence imaging system. The organs were then stored in liquid nitrogen for further ex-vivo modRNA delivery analysis. For determining cellular tropism, histology was performed on mice treated with eGFP modRNA. Both bioluminescence imaging and luminescence analysis in tissue lysates showed that mRNA transfection is achieved in the myocardium 24 hours after LNP intramyocardial administration. However, all LNP formulations also resulted in high expression levels in other organs, including liver and spleen. Changes in type or amount of helper lipid in LNPs strongly affected transfection levels. Histology of the treated hearts revealed a distinct transfection pattern. The targeted, interstitial cells were negative for CD31 (marker for endothelial cells and monocytes) and Troponin I3 (marker for cardiomyocytes) (Figure 1). We show that, using an optimized LNP formulation, a significant degree of modRNA local transfection of the heart can be achieved. However, despite the local route of administration (into the left ventricular wall), the highest LNP transfection is shown in remote organs such as liver and spleen. More improvements of the LNP formulations must be done to increase their tropism towards the heart tissue for their optimization as cardiac delivery systems. Determining which cell types are being targeted is also important in order to establish a therapeutic target when applying the LNPs for cardiac therapy. (Figure Presented).

Effect of Subacute Angiotensin II Elevations on Sphingolipid Levels and Lung Injury in Mice

Background: There is a paucity of therapies for acute lung injury (ALI) induced by respiratory viruses. A previously demonstrated key mechanism of ALI, particularly in the setting of severe acute respiratory syndrome coronavirus infections, has been ascribed to decreased cell surface angiotensin converting enzyme 2 (ACE2) leading to increased circulating levels of angiotensin II (Ang2). In turn, supraphysiological Ang2 levels trigger a cascade of events that culminates with endothelial injury in the systemic circulation via acid sphingomyelinase (ASMase) activation. ASMase has been implicated in several models of ALI, but its specific involvement in Ang2-induced ALI is unknown. ASMase hydrolyzes sphingomyelin to pro-apoptotic, edemagenic ceramide, which can be metabolized to endothelial-protective sphingosine-1-phosphate (S1P). Therefore, the ratio of ceramide/S1P can determine endothelial cell fate and lung vascular permeability. We hypothesized that ceramide levels are increased relative to S1P in mice with Ang2-induced ALI. Methods: Following a published protocol of Ang2-induced ALI (Wu et al, 2017), we delivered Ang2 via osmotic pumps (1 ug/kg/min, 7 days;Ang2-mice), using saline (sham) or untreated C57BL/6 mice as controls. We evaluated pulmonary function (FlexiVent);albumin, IgM (ELISA), and inflammatory cell abundance in bronchoalveolar lavage fluid (BALF);and lung parenchyma inflammation and fibrosis (Ashcroft score) on H/E-stained lungs. Sphingolipid levels in lungs and plasma were measured by tandem liquid chromatography/mass spectrometry. Results: Inspiratory capacity, lung compliance, and body weight all decreased in Ang2-mice (by 13-14%, p<0.05 each) compared to sham. Lung pressure-volume loops exhibited a right-shift in Ang2- vs. sham or untreated mice. There was no significant change in BALF albumin, IgM, or inflammatory cells, or in lung histology inflammation or fibrosis scores in Ang2-mice. Compared to sham, S1P levels were significantly increased in plasma and unlavaged lung in Ang2-mice, decreasing ceramide/S1P ratios (from 3.1 to 2.0, and 26 to 20, respectively, p<0.05 each). Conclusions: Sustained subacute systemic elevations of Ang2 increased lung stiffness, but did not cause severe ALI in mice. Lung and circulatory elevations of S1P but not ceramide may have protected against lung edema and inflammatory injury. Although the cause of increased lung stiffness in this model remains to be elucidated, it is notable that chronic (months) supraphysiological elevations of either Ang2 or S1P have been associated with lung fibrosis. In conclusion, a second-hit injury may be necessary to augment the susceptibility of murine lung to Ang2-induced endothelial damage and inflammation relevant to coronavirus.

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.

TREK-1 Potassium Channel Activation Protects Against Influenza-A Induced Lung Injury

Introduction and Rationale: No targeted therapies exist that improve the outcomes of patients with Acute Respiratory Distress Syndrome (ARDS), in part to the multifactorial etiology of this devastating disease. Infectious agents remain the most common initiating insults, and besides SARS-CoV-2, Influenza-A virus (IAV) is responsible for more ARDS cases and deaths than any other agent. In fact, IAV increases the risk of mortality in ARDS patients three-fold, and accounts for almost half of all ARDS deaths. We recently identified TREK-1 potassium channels on epithelial cells as important regulators of alveolar inflammation and barrier function, two hallmarks of ARDS, and found that pharmacological activation of TREK-1 protects against hyperoxia-induced lung injury. However, whether TREK-1 channels convey similar protection in a clinically more relevant IAVinduced lung injury model, remains unknown. Methods: We infected adult C57BL/6 wildtype mice intra-tracheally (i.t.) with IAV (PR8 strain;TCID50 400), followed by once-daily i.t. injections (days 5, 6 and 7 post-IAV) with the novel TREK-1 activating compounds ML335 (60mcg/kg), BL1249 (100mcg/kg), or a vehicle control, to create a clinically-relevant treatment model. To evaluate the role of epithelial cells in this model, we infected primary human alveolar epithelial cells (HAEC) with IAV (0.01 pfu) for 24 hours. Endpoint analysis consistent in quantification of quasi-static lung compliance;BAL fluid total protein, cell counts, and ROS concentrations;cytokine levels in BAL fluid and cell supernatants;and HAEC viability (XTT assay). In addition, we measured alterations in epithelial potassium currents (fluorometric FLIPR assays) and in IAV-induced signaling cascades (real-time PCR) following IAV infection and treatment with our TREK-1 activators. Results: Oncedaily treatment of mice with the TREK-1 activating compounds ML335 or BL1249 following IAV infection improved lung compliance, and BAL fluid total protein levels, cell counts, IL-6, CXCL-10, MIP-1alpha, and TNF-alpha concentrations, but not ROS, CCL-2 or IL-10 levels. In HAEC, TREK-1 activation improved IAV-induced IL-6, CXCL-10, and CCL-2 levels, while MIP-1alpha, TNF-alpha and IL-10 levels remained unchanged. XTT assays confirmed that in our model IAV infection did not cause significant cell death. Interestingly, IAV infection decreased HAEC potassium currents, which could be counteracted by TREK-1 activation and cell hyperpolarization. Finally, TREK-1 activationmediated cell hyperpolarization inhibited TLR4- and TNFSF13-mediated downstream signaling in IAV-infected HAEC, whereas NFkB, RIG1, TLR3, and TLR7 signaling was not affected. Conclusions: TREK-1 potassium channel activation may represent a novel approach to protect against IAV-induced acute lung injury.

A Novel Ogremorphin Group of First-in-Class GPR68 Inhibitors Upregulate Endothelial Barrier Function and Rescue Bacterial Pathogens or Acidosis-Induced Inflammation in Lung Endothelium

Rationale: An increase in endothelial permeability resulting from the disruption of endothelial barrier and aggravated inflammatory responses are two major pathological hallmarks of various lung disorders including the current global pandemic COVID-19. Drugs that enable the preservation and restoration of endothelial function represent attractive therapeutic targets to treat endothelial dysfunction-derived cardiopulmonary diseases. A role of G protein-coupled receptors (GPCRs), especially a sub-family of proton-sensing GPCRs including GPR4 and GPR68, has been suggested in modulation of endothelial function. In this study, we analyzed the barrier protective and anti-inflammatory effects of two recently developed novel class of GPR68 inhibitors: ogremorphins OGM8345 and OGM-1.Methods: Transendothelial electrical resistance (TER) was monitored in human pulmonary arterial endothelial cells (HPAECs) to evaluate endothelial barrier function. Quantitative real time PCR and western blot analyses were performed to determine mRNA and protein expression of endothelial inflammation markers, respectively. Acidic pH (6.5) medium was used to induce acidosis, and luciferase-based Tango assay was employed to evaluate GPR68 activation. C57BL/6 mice were exposed to lipopolysaccharide (LPS from Escherichia coli) or heatkilled Staphylococcus aureus (HKSA), and vascular leak/inflammation was assessed by determining the extravasation of intravenously injected Evans blue tracer into lungs and total cells/protein count in bronchoalveolar lavage samples. Results: A robust dose-dependent increase in basal EC barrier function was observed with OGM8345 (1-5 μM) and OGM-1 (0.3-1.5 μM) evident by an 150-200% increase in TER values. Both inhibitors also effectively rescued LPS- and HKSA-induced EC hyperpermeability. RT-PCR analysis demonstrated that LPS or HKSA-induced upregulation of inflammatory cytokines/chemokines genes TNF-α, ICAM-1, VCAM-1, IL-6, IL-8, IL- 1β, and CXCL5 was significantly attenuated by OGMs. Consistently, both OGMs suppressed LPSand HKSA-induced protein expression of VCAM-1 and ICAM-1. In contrast, pharmacologic inhibition of GPR4 by NE 52-QQ57 failed to alleviate LPS or HKSA-induced EC barrier dysfunction and inflammation. Importantly, LPS, HKSA or acidosis stimulation resulted in increased GPR68 mRNA expression and GPR68 activity that was inhibited by OGMs. Intratracheal injection of LPS or HKSA in C57BL/6 mice caused vascular leak and lung inflammation that was attenuated by both OGMs as illustrated by reduced Evans blue accumulation in the lungs and significant inhibition of accumulation of inflammatory cells and protein content in bronchoalveolar lavage samples. Conclusion: These results establish a critical role of GPR68 in endothelial dysfunction and strongly suggest a therapeutic potential of GPR68-selective inhibitors in improving endothelial dysfunction caused by bacterial infections and acidosis associated with acute and chronic lung injury.

Role of NOX4 in Modulating Alveolar Type II (AT2) Epithelial Cell Phenotype and Mitochondrial Dynamics in Hyperoxic Acute Lung Injury

Introduction: Management of acute respiratory distress including COVID-19 pneumonia involves O2 supplementation, which is lifesaving, but causes severe hyperoxic acute lung injury (HALI). AT2 cells are the most affected cell type in hyperoxia (HO). NADPH oxidase (NOX) is a major source of reactive oxygen species (ROS) in HO. NOX4, the only functionally active NOX present in mitochondria, and primarily produces H2O2 as well as mtROS has been shown to be involved in several human pathologies. Not much is known about NOX4-induced mitochondrial injury in HALI. The current study aims to determine the role of AT2 epithelial cell NOX4 in HALI and the impact of HO on the modulation of mtROS and mitochondrial dynamics in HALI. Methods: Nox4-/-Spc-Cre animals were generated using tamoxifen induction and the knockdown was validated. The Nox4- /-Spc-Cre knockout (KO) and wild type (WT) mice were exposed to room air (NO) or 95% O2 (HO) for 66h to study the structural and functional changes in the lung. Transmission Electron Microscopy (TEM) was used to study the HO-induced changes in mitochondria. Isolated primary AT2 and/ mouse lung epithelial (MLE) cell line was investigated for mtROS, mt dynamics and apoptosis. Mitochondrial injury was assessed in Nox4 WT and Nox4 silenced cells. Results: C57BL/6J WT animals subjected to HO for 66h showed increased expression of NOX4, determining the role of NOX4 in HALI. The H&E staining demonstrated significant HALI in Nox4 WT animals exposed to HO compared to Nox4 KO as determined by increased infiltration of neutrophils, alveolar wall thickening and presence of proteinaceous debris in the alveolar space. Further, increased BAL cell count and protein levels, increased AT2 cell death and elevation of the proinflammatory cytokine IL- 6 and the chemokine KC was seen in WT animals compared to Nox4 KO. Analysis of lung tissues by TEM showed mitochondrial swelling, cristae damage and mitophagy in AT2 cells due to HO. Changes in mt injury markers were also observed. HO-induced NOX4 increase in primary AT2/ MLE-12 cells resulted in increased mtROS production and apoptosis, which was reduced with Nox4 siRNA silencing. Conclusion: This study suggests that the HO induced NOX4 expression in mouse lung, and deletion of Nox4 gene in AT2 cells reduced mtROS production and apoptosis and protected the lungs from severe hyperoxic lung injury. These results suggest NOX4 as a potential target for the treatment of HALI.

DIFFERENTIAL TRANSCRIPTOMIC CHANGES IN THE CENTRAL NERVOUS SYSTEM AND NEUROGENIC BLADDERS OF MICE INFECTED WITH A CORONAVIRUS

INTRODUCTION AND OBJECTIVE: Neurodegenerative diseases, such as multiple sclerosis (MS), often lead to the development of neurogenic lower urinary tract symptoms (LUTS). We previously characterized neurogenic bladder dysfunction in a mouse model of MS induced by a coronavirus, mouse hepatitis virus (MHV). The objective of this study was to identify genes and pathways linking neuroinflammation in the central nervous system with urinary bladder dysfunction to enhance our understanding of the mechanisms underlying LUTS in demyelinating diseases. METHODS: Adult C57BL/6 male mice (N=12) received either an intracranial injection of MHV (6,000 PFU) or sterile saline (control). The lumbosacral (L6-S2) spinal cord (SC) segments and urinary bladders were collected during acute infection stage (week 1) and at the first peak of demyelination (week 4) after inoculation with the virus. Total RNA was isolated and analyzed using Nanostring nCounter Neuroinflammation panel. The expression levels of 770 genes associated with neuroinflammation were assessed and compared between the specimens. RESULTS: Transcriptome analysis of SC specimens confirmed a significantly increased expression of 132 genes in MHV mice (tens to hundreds fold change) involved in the regulation of astrocyte, microglia and oligodendrocyte functions, neuroinflammation and immune responses. Out of 132 genes up-regulated in the SC, only 2 genes (siglec1, 46-fold in the SC, 2.6-fold at 1 week and 1.8-fold at 4 weeks in the bladder;and zbp1, 568-fold in the SC, 2.8-fold at 1 week and 2.2-fold at 4 weeks in the bladder) were up-regulated in the urinary bladders of MHV-infected mice. Additionally, two genes were significantly up-regulated (ttr, 2.2-fold at 1week and 1.7-fold at 4 weeks;and ms4a4a, 2.3-fold at 1week and 1.6-fold at 4 weeks), and two were down-regulated (asb2, -1.8-fold at 1 week and -1.6-fold at 4 weeks, and myct1, -1.7-fold at 1week and -1.6-fold at 4 weeks) exclusively in the urinary bladders of MHV mice. CONCLUSIONS: Two genes, siglec1 (encodes type 1 transmembrane protein, expressed in microglia and macrophages, promotes neuroinflammation) and zbp1 (encodes a Z-DNA binding protein, plays role in the innate immune response) link the development of neuroinflammation in the central nervous system with neurogenic changes in the urinary bladders of MHV-infected mice. Further research is needed to establish a functional relationship between expression of these genes and neurogenic LUTS.