LRRC15 inhibits SARS-CoV-2 cellular entry in trans.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is mediated by the entry receptor angiotensin-converting enzyme 2 (ACE2). Although attachment factors and coreceptors facilitating entry are extensively studied, cellular entry factors inhibiting viral entry are largely unknown. Using a surfaceome CRISPR activation screen, we identified human LRRC15 as an inhibitory attachment factor for SARS-CoV-2 entry. LRRC15 directly binds to the receptor-binding domain (RBD) of spike protein with a moderate affinity and inhibits spike-mediated entry. Analysis of human lung single-cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not coexpressed in the same cell types in the lung. Strikingly, expression of LRRC15 in ACE2-negative cells blocks spike-mediated viral entry in ACE2+ cell in trans, suggesting a protective role of LRRC15 in a physiological context. Therefore, LRRC15 represents an inhibitory attachment factor for SARS-CoV-2 that regulates viral entry in trans.

Transcriptomics of acute myeloid leukaemia core bone marrow biopsies reveals distinct therapy response-specific osteo-mesenchymal profiles.

While the bone marrow (BM) microenvironment is significantly remodelled in acute myeloid leukaemia (AML), molecular insight into AML-specific alterations in the microenvironment has been historically limited by the analysis of liquid marrow aspirates rather than core biopsies that contain solid-phase BM stroma. We assessed the effect of anthracycline- and cytarabine-based induction chemotherapy on both haematopoietic and non-haematopoietic cells directly in core BM biopsies using RNA-seq and histological analysis. We compared matched human core BM biopsies at diagnosis and 2 weeks after cytarabine- and anthracycline-based induction therapy in responders (<5% blasts present after treatment) and non-responders (≥5% blasts present after treatment). Our data indicated enrichment in vimentin (VIM), platelet-derived growth factor receptor beta (PDGFRB) and Snail family transcriptional repressor 2 (SNAI2) transcripts in responders, consistent with the reactivation of the mesenchymal population in the BM stroma. Enrichment of osteoblast maturation-related transcripts of biglycan (BGN), osteopontin (SPP1) and osteonectin (SPARC) was observed in non-responders. To the best of our knowledge, this is the first report demonstrating distinct osteogenic and mesenchymal transcriptome profiles specific to AML response to induction chemotherapy assessed directly in core BM biopsies. Detailing treatment response-specific alterations in the BM stroma may inform optimised therapeutic strategies for AML.

Inhibition of lipid phosphatase SHIP1 expands myeloid-derived suppressor cells and attenuates rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of unknown cause, characterized by infiltration and accumulation of activated immune cells in the synovial joints where cartilage and bone destructions occur. Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) was shown to be involved in the regulation of MDSC differentiation. The purpose of the present study was to investigate the effect of inhibition of SHIP1 on the expansion of MDSCs in RA using a collagen-induced inflammatory arthritis (CIA) mouse model. In DBA/1 mice, treatment with a small molecule-specific SHIP1 inhibitor 3α-aminocholestane (3AC) induced a marked expansion of MDSCs in vivo. Both pretreatment with 3AC of DBA/1 mice prior to CIA induction and intervention with 3AC during CIA progression significantly reduced disease incidence and severity. Adoptive transfer of MDSCs isolated from 3AC-treated mice, but not naïve MDSCs from normal mice, into CIA mice significantly reduced disease incidence and severity, indicating that the 3AC-induced MDSCs were the cellular mediators of the observed amelioration of the disease. In conclusion, inhibition of SHIP1 expands MDSCs in vivo and attenuates development of CIA in mice. Small molecule-specific inhibition of SHIP1 may therefore offer therapeutic benefit to patients with RA and other autoimmune diseases.

Lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development.

SH2-containing inositol-5'-phosphatase-1 (SHIP-1) controls the phosphatidylinositol-3'-kinase (PI3K) initiated signaling pathway by limiting cell membrane recruitment and activation of Akt. Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the PI3K signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis. Here, we report that the lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development through its expression in osteochondroprogenitor cells. Global SHIP-1 knockout led to accelerated chondrocyte hypertrophy and premature formation of the secondary ossification center in the bones of postnatal mice. Drastically higher vascularization and greater number of c-kit + progenitors associated with sinusoids in the bone marrow also indicated more advanced chondrocyte hypertrophic differentiation in SHIP-1 knockout mice than in wild-type mice. In corroboration with the in vivo phenotype, SHIP-1 deficient PDGFRα + Sca-1 + osteochondroprogenitor cells exhibited rapid differentiation into hypertrophic chondrocytes under chondrogenic culture conditions in vitro. Furthermore, SHIP-1 deficiency inhibited hypoxia-induced cellular activation of Akt and extracellular-signal-regulated kinase (Erk) and suppressed hypoxia-induced cell proliferation. These results suggest that SHIP-1 is required for hypoxia-induced growth signaling under physiological hypoxia in the bone marrow. In conclusion, the lipid phosphatase SHIP-1 regulates skeletal development by modulating chondrogenesis and the hypoxia response of the osteochondroprogenitors during endochondral bone formation.

M1 Macrophage-Induced Endothelial-to-Mesenchymal Transition Promotes Infantile Hemangioma Regression.

Infantile hemangiomas are benign tumors of vascular endothelial cells (ECs), characterized by three distinct stages: proliferating phase, involuting phase, and involuted phase. The mechanisms that trigger involution of hemangioma into fibro-fatty tissue remain unknown. We report a novel mechanism by which M1-polarized macrophages induce endothelial-to-mesenchymal transition (EndMT) and promote hemangioma regression. M1- but not M2-polarized macrophages induced EndMT in ECs. Tumor necrosis factor-α and, to a lesser extent, IL-1ß and interferon-γ were the most potent cytokines produced by the M1 macrophages that induce in vitro EndMT. Western blot analysis and gene expression profiling showed that ECs treated with M1 macrophages, tumor necrosis factor-α, or IL-1ß decreased the expression of endothelial markers, whereas mesenchymal markers increased concomitantly. Immunohistochemical staining of patient samples revealed that a significant perivascular infiltration of M1, but not M2, macrophages coincides with endothelial expression of the critical EndMT transcription factors Snail/Slug in involuting hemangiomas. Most strikingly, M1 macrophage-treated ECs isolated from patient hemangiomas (HemECs) but not untreated HemECs readily differentiated into adipocytes on adipogenic induction. Thus, in vitro EndMT and adipogenesis of HemECs have, in part, recapitulated the natural history of hemangioma regression. In conclusion, our findings indicate that EndMT induced by M1 macrophages promotes infantile hemangioma regression and may lead to novel therapeutic treatments for this vascular tumor.

Transgenic expression of non-structural genes of Theiler's virus suppresses initial viral replication and pathogenesis of demyelination.

BACKGROUND: Chronic infection with Theiler's murine encephalomyelitis virus (TMEV) in susceptible SJL/J mice induces an immune-mediated demyelinating disease and has extensively been used as a relevant infectious model for multiple sclerosis (MS). Infection of the host with many other viruses also leads to acute or chronic inflammatory diseases in the central nervous system (CNS). Levels of viral load in the host often play a critical role in the pathogenesis of virus-induced diseases. Thus, the inhibition of viral replication in the host against a broad spectrum of similar viruses is critically important for preventing the viral pathogenicity. METHODS: P2/P3-expressing transgenic (B6 X SJL)F1 founders were generated and bred onto the C57BL/6 and SJL/J backgrounds. Differences in the development of demyelinating disease were compared. Viral persistence, cytokine production, and immune responses in the CNS of infected control and P2/P3-Tg mice were analyzed after infection using quantitative PCR, ELISA, and flow cytometry. Various cell types from the control and P2/P3-Tg mice, as well as cells transfected in vitro with the P2 and/or P3 regions, were also analyzed for viral replication and innate cytokine production. RESULTS: P2/P3-transgenic (P2/P3-Tg) mice carrying the viral non-structural protein genes displayed significantly reduced virus-specific T cell responses in the CNS against both the structural and non-structural proteins. Consequently, viral loads in the CNS were greater in the Tg mice during the chronic infection. However, P2/P3-Tg SJL mice exhibited reduced disease incidence and less severe clinical symptoms than did their non-transgenic littermates. Interestingly, P2/P3-Tg mice showed low viral loads in the CNS at a very early period after infection (1-3 days) with TMEV and related EMCV but not unrelated VSV. Cells from P2/P3-Tg mice and cells transfected with the P2 and/or P3 regions in vitro yielded also lower viral replication but higher IFN-α/ß production. CONCLUSIONS: This study demonstrates that the expression of viral non-structural genes in mice inhibits initial viral replication and suppresses sustaining pathogenic anti-viral immune responses to broad viral determinants. It appears that the elevation of innate immune cytokines produced in the cells expressing the non-structural viral genes upon viral infection is responsible for the inhibitions. The inhibition is partially virus-specific as it is more efficient for a related virus compared to an unrelated virus, suggesting a role for the similarity in the viral genome structures. Therefore, the expression of viral non-structural genes may serve as a useful new method to prevent a broadly virus-specific pathogenesis in the hosts.

BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction.

BACKGROUND: BRAT1 (BRCA1-associated ATM activator 1) interacts with both BRCA1, ATM and DNA-PKcs, and has been implicated in DNA damage responses. However, based on our previous results, it has been shown that BRAT1 may be involved in cell growth and apoptosis, besides DNA damage responses, implying that there are undiscovered functions for BRAT1. METHODS: Using RNA interference against human BRAT1, we generated stable BRAT1 knockdown cancer cell lines of U2OS, Hela, and MDA-MA-231. We tested cell growth properties and in vitro/in vivo tumorigenic potentials of BRAT1 knockdown cells compared to control cells. To test if loss of BRAT1 induces metabolic abnormalities, we examined the rate of glycolysis, ATP production, and PDH activity in both BRAT1 knockdown and control cells. The role of BRAT1 in growth signaling was determined by the activation of Akt/Erk, and SC79, Akt activator was used for validation. RESULTS: By taking advantage of BRAT1 knockdown cancer cell lines, we found that loss of BRAT1 expression significantly decreases cell proliferation and tumorigenecity both in vitro and in vivo. Cell migration was also remarkably lowered when BRAT1 was depleted. Interestingly, glucose uptake and production of mitochondrial ROS (reactive oxygen species) are highly increased in BRAT1 knockdown HeLa cells. Furthermore, both basal and induced activity of Akt and Erk kinases were suppressed in these cells, implicating abnormality in signaling cascades for cellular growth. Consequently, treatment of BRAT1 knockdown cells with Akt activator can improve their proliferation and reduces mitochondrial ROS concentration. CONCLUSIONS: These findings suggest novel roles of BRAT1 in cell proliferation and mitochondrial functions.

Deregulation of DNA damage response pathway by intercellular contact.

Deregulation of the DNA damage response (DDR) pathway could compromise genomic integrity in normal cells and reduce cancer cell sensitivity to anticancer treatments. We found that intercellular contact stabilizes histone H2AX and γH2AX (H2AX phosphorylated on Ser-139) by up-regulating N/E-cadherin and γ-catenin. γ-catenin and its DNA-binding partner LEF-1 indirectly increase levels of H2AX by suppressing the promoter of the RNF8 ubiquitin ligase, which decreases levels of H2AX protein under conditions of low intercellular contact. Hyperphosphorylation of DDR proteins is induced by up-regulated H2AX. Constitutive apoptosis is caused in confluent cells but is not further induced by DNA damage. This is conceivably due to insufficient p53 activation because ChIP assay shows that its DNA binding ability is not induced in those cells. Together, our results illustrate a novel mechanism of the regulation of DDR proteins by the cadherin-catenin pathway.

Melanoma differentiation-associated gene 5 is critical for protection against Theiler's virus-induced demyelinating disease.

Infection of dendritic and glial cells with Theiler's murine encephalomyelitis virus (TMEV) induces various cytokines via Toll-like receptor- and melanoma differentiation-associated gene 5 (MDA5)-dependent pathways. However, the involvement and role of MDA5 in cytokine gene activation and the pathogenesis of TMEV-induced demyelinating disease are largely unknown. In this study, we demonstrate that MDA5 plays a critical role in the production of TMEV-induced alpha interferon (IFN-α) during early viral infection and in protection against the development of virus-induced demyelinating disease. Our results indicate that MDA5-deficient 129SvJ mice display significantly higher viral loads and apparent demyelinating lesions in the central nerve system (CNS) accompanied by clinical symptoms compared with wild-type 129SvJ mice. During acute viral infection, MDA5-deficient mice produced elevated levels of chemokines, consistent with increased cellular infiltration, but reduced levels of IFN-α, known to control T cell responses and cellular infiltration. Additional studies with isolated CNS glial cells from these mice suggest that cells from MDA5-deficient mice are severely compromised in the production of IFN-α upon viral infection, which results in increased cellular infiltration and viral loads in the CNS. Despite inadequate stimulation, the overall T cell responses to the viral determinants were significantly elevated in MDA5-deficient mice, reflecting the increased cellular infiltration. Therefore, the lack of MDA5-mediated IFN-α production may facilitate a massive viral load and elevated cellular infiltration in the CNS during early viral infection, leading to the pathogenesis of demyelinating disease.

Integrin/TGF-ß1 inhibitor GLPG-0187 blocks SARS-CoV-2 Delta and Omicron pseudovirus infection of airway epithelial cells which could attenuate disease severity.

As COVID-19 continues to pose major risk for vulnerable populations including the elderly, immunocompromised, patients with cancer, and those with contraindications to vaccination, novel treatment strategies are urgently needed. SARS-CoV-2 infects target cells via RGD-binding integrins either independently or as a co-receptor with surface receptor angiotensin-converting enzyme 2 (ACE2). We used pan-integrin inhibitor GLPG-0187 to demonstrate blockade of SARS-CoV-2 pseudovirus infection of target cells. Omicron pseudovirus infected normal human small airway epithelial (HSAE) cells significantly less than D614G or Delta variant pseudovirus, and GLPG-0187 effectively blocked SARS-CoV-2 pseudovirus infection in a dose-dependent manner across multiple viral variants. GLPG-0187 inhibited Omicron and Delta pseudovirus infection of HSAE cells more significantly than other variants. Pre-treatment of HSAE cells with MEK inhibitor (MEKi) VS-6766 enhanced inhibition of pseudovirus infection by GLPG-0187. Because integrins activate TGF-ß signaling, we compared plasma levels of active and total TGF-ß in COVID-19+ patients. Plasma TGF-ß1 levels correlated with age, race, and number of medications upon presentation with COVID-19, but not with sex. Total plasma TGF-ß1 levels correlated with activated TGF-ß1 levels. In our preclinical studies, Omicron infects lower airway lung cells less efficiently than other COVID-19 variants. Moreover, inhibition of integrin signaling prevents SARS-CoV-2 Delta and Omicron pseudovirus infectivity, and may mitigate COVID-19 severity through decreased TGF-ß1 activation. This therapeutic strategy may be further explored through clinical testing in vulnerable and unvaccinated populations.

Integrin/TGF-ß1 Inhibitor GLPG-0187 Blocks SARS-CoV-2 Delta and Omicron Pseudovirus Infection of Airway Epithelial Cells In Vitro, Which Could Attenuate Disease Severity.

As COVID-19 continues to pose major risk for vulnerable populations, including the elderly, immunocompromised, patients with cancer, and those with contraindications to vaccination, novel treatment strategies are urgently needed. SARS-CoV-2 infects target cells via RGD-binding integrins, either independently or as a co-receptor with surface receptor angiotensin-converting enzyme 2 (ACE2). We used pan-integrin inhibitor GLPG-0187 to demonstrate the blockade of SARS-CoV-2 pseudovirus infection of target cells. Omicron pseudovirus infected normal human small airway epithelial (HSAE) cells significantly less than D614G or Delta variant pseudovirus, and GLPG-0187 effectively blocked SARS-CoV-2 pseudovirus infection in a dose-dependent manner across multiple viral variants. GLPG-0187 inhibited Omicron and Delta pseudovirus infection of HSAE cells more significantly than other variants. Pre-treatment of HSAE cells with MEK inhibitor (MEKi) VS-6766 enhanced the inhibition of pseudovirus infection by GLPG-0187. Because integrins activate transforming growth factor beta (TGF-ß) signaling, we compared the plasma levels of active and total TGF-ß in COVID-19+ patients. The plasma TGF-ß1 levels correlated with age, race, and number of medications upon presentation with COVID-19, but not with sex. Total plasma TGF-ß1 levels correlated with activated TGF-ß1 levels. Moreover, the inhibition of integrin signaling prevents SARS-CoV-2 Delta and Omicron pseudovirus infectivity, and it may mitigate COVID-19 severity through decreased TGF-ß1 activation. This therapeutic strategy may be further explored through clinical testing in vulnerable and unvaccinated populations.

Targeting RUNX1 as a novel treatment modality for pulmonary arterial hypertension.

AIMS: Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. Previously, we found that transcription factor RUNX1-dependent haematopoietic transformation of endothelial progenitor cells may contribute to the pathogenesis of PAH. However, the therapeutic potential of RUNX1 inhibition to reverse established PAH remains unknown. In the current study, we aimed to determine whether RUNX1 inhibition was sufficient to reverse Sugen/hypoxia (SuHx)-induced pulmonary hypertension (PH) in rats. We also aimed to demonstrate possible mechanisms involved. METHODS AND RESULTS: We administered a small molecule specific RUNX1 inhibitor Ro5-3335 before, during, and after the development of SuHx-PH in rats to investigate its therapeutic potential. We quantified lung macrophage recruitment and activation in vivo and in vitro in the presence or absence of the RUNX1 inhibitor. We generated conditional VE-cadherin-CreERT2; ZsGreen mice for labelling adult endothelium and lineage tracing in the SuHx-PH model. We also generated conditional Cdh5-CreERT2; Runx1(flox/flox) mice to delete Runx1 gene in adult endothelium and LysM-Cre; Runx1(flox/flox) mice to delete Runx1 gene in cells of myeloid lineage, and then subjected these mice to SuHx-PH induction. RUNX1 inhibition in vivo effectively prevented the development, blocked the progression, and reversed established SuHx-induced PH in rats. RUNX1 inhibition significantly dampened lung macrophage recruitment and activation. Furthermore, lineage tracing with the inducible VE-cadherin-CreERT2; ZsGreen mice demonstrated that a RUNX1-dependent endothelial to haematopoietic transformation occurred during the development of SuHx-PH. Finally, tissue-specific deletion of Runx1 gene either in adult endothelium or in cells of myeloid lineage prevented the mice from developing SuHx-PH, suggesting that RUNX1 is required for the development of PH. CONCLUSION: By blocking RUNX1-dependent endothelial to haematopoietic transformation and pulmonary macrophage recruitment and activation, targeting RUNX1 may be as a novel treatment modality for pulmonary arterial hypertension.

LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans.

SARS-CoV-2 infection is mediated by the entry receptor ACE2. Although attachment factors and co-receptors facilitating entry are extensively studied, cellular entry factors inhibiting viral entry are largely unknown. Using a surfaceome CRISPR activation screen, we identified human LRRC15 as an inhibitory receptor for SARS-CoV-2 entry. LRRC15 directly binds to the receptor-binding domain (RBD) of spike protein with a moderate affinity and inhibits spike-mediated entry. Analysis of human lung single cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not co-expressed in the same cell types in the lung. Strikingly, expression of LRRC15 in ACE2-negative cells blocks spike-mediated viral entry in ACE2+ cell in trans, suggesting a protective role of LRRC15 in a physiological context. Therefore, LRRC15 represents an inhibitory receptor for SARS-CoV-2 regulating viral entry in trans.

Sexual dimorphism in aging hematopoiesis: an earlier decline of hematopoietic stem and progenitor cells in male than female mice.

Adult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) ensuring homeostasis of blood production and immune response throughout life. Sex differences in immunocompetence and mortality are well-documented in humans. However, whether HSPCs behave dimorphically between sexes during aging remains unknown. Here, we show that a significant expansion of BM-derived HSPCs occurs in the middle age of female but in the old age of male mice. We then show that a decline of HSPCs in male mice, as indicated by the expression levels of select hematopoietic genes, occurs much earlier in the aging process than that in female mice. Sex-mismatched heterochronic BM transplantations indicate that the middle-aged female BM microenvironment plays a pivotal role in sustaining hematopoietic gene expression during aging. Furthermore, a higher concentration of the pituitary sex hormone follicle-stimulating hormone (FSH) in the serum and a concomitant higher expression of its receptor on HSPCs in the middle-aged and old female mice than age-matched male mice, suggests that FSH may contribute to the sexual dimorphism in aging hematopoiesis. Our study reveals that HSPCs in the BM niches are possibly regulated in a sex-specific manner and influenced differently by sex hormones during aging hematopoiesis.

BST204, a Rg3 and Rh2 Enriched Ginseng Extract, Upregulates Myotube Formation and Mitochondrial Function in TNF-α-Induced Atrophic Myotubes.

The loss of skeletal muscle mass and function is a serious consequence of chronic diseases and aging. BST204 is a purified ginseng (the root of Panax ginseng) extract that has been processed using ginsenoside-ß-glucosidase and acid hydrolysis to enrich ginsenosides Rg3 and Rh2 from the crude ginseng. BST204 has a broad range of health benefits, but its effects and mechanism on muscle atrophy are currently unknown. In this study, we have examined the effects and underlying mechanisms of BST204 on myotube formation and myotube atrophy induced by tumor necrosis factor-α (TNF-α). BST204 promotes myogenic differentiation and multinucleated myotube formation through Akt activation. BST204 prevents myotube atrophy induced by TNF-α through the activation of Akt/mTOR signaling and down-regulation of muscle-specific ubiquitin ligases, MuRF1, and Atrogin-1. Furthermore, BST204 treatment in atrophic myotubes suppresses mitochondrial reactive oxygen species (ROS) production and regulates mitochondrial transcription factors such as NRF1 and Tfam, through enhancing the activity and expression of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Collectively, our findings indicate that BST204 improves myotube formation and PGC1α-mediated mitochondrial function, suggesting that BST204 is a potential therapeutic or neutraceutical remedy to intervene muscle weakness and atrophy.

Ginsenoside Rb1 and Rb2 upregulate Akt/mTOR signaling-mediated muscular hypertrophy and myoblast differentiation.

BACKGROUND: As a process of aging, skeletal muscle mass and function gradually decrease. It is reported that ginsenoside Rb1 and Rb2 play a role as AMP-activated protein kinase activator, resulting in regulating glucose homeostasis, and Rb1 reduces oxidative stress in aged skeletal muscles through activating the phosphatidylinositol 3-kinase/Akt/Nrf2 pathway. We examined the effects of Rb1 and Rb2 on differentiation of the muscle stem cells and myotube formation. METHODS: C2C12 myoblasts treated with Rb1 and/or Rb2 were differentiated and induced to myotube formation, followed by immunoblotting for myogenic marker proteins, such as myosin heavy chain, MyoD, and myogenin, or immunostaining for myosin heavy chain or immunoprecipitation analysis for heterodimerization of MyoD/E-proteins. RESULTS: Rb1 and Rb2 enhanced myoblast differentiation through accelerating MyoD/E-protein heterodimerization and increased myotube hypertrophy, accompanied by activation of Akt/mammalian target of rapamycin signaling. In addition, Rb1 and Rb2 induced the MyoD-mediated transdifferentiation of the rhabdomyosarcoma cells into myoblasts. Furthermore, co-treatment with Rb1 and Rb2 had synergistically enhanced myoblast differentiation through Akt activation. CONCLUSION: Rb1 and Rb2 upregulate myotube growth and myogenic differentiation through activating Akt/mammalian target of rapamycin signaling and inducing myogenic conversion of fibroblasts. Thus, our first finding indicates that Rb1 and Rb2 have strong potential as a helpful remedy to prevent and treat muscle atrophy, such as age-related muscular dystrophy.

MEK inhibitors reduce cellular expression of ACE2, pERK, pRb while stimulating NK-mediated cytotoxicity and attenuating inflammatory cytokines relevant to SARS-CoV-2 infection.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N = 9) versus control (N = 11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1α, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression.

Natural Killer cell activation, reduced ACE2, TMPRSS2, cytokines G-CSF, M-CSF and SARS-CoV-2-S pseudovirus infectivity by MEK inhibitor treatment of human cells.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1α, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression.

Theiler's virus infection induces TLR3-dependent upregulation of TLR2 critical for proinflammatory cytokine production.

Theiler's murine encephalomyelitis virus (TMEV) infection directly induces many proinflammatory genes, including type I interferon (IFN) and a variety of cytokine genes. These virus-induced cytokines are a critical factor in developing TMEV-induced demyelinating disease. We have previously reported that the major activation signal for the cytokine genes is mediated via TLR3. In this study, we describe that TLR2 is upregulated via TLR3 signal and cooperatively participates in the expression of IL-6, IL-1beta, CCL2, and CCL5 genes following TMEV infection. The expression of these genes was significantly impaired in both TLR2-deficient and TLR3-deficient primary astrocytes. However, the induction of type I IFNs was not affected by TLR2 deficiency in the primary cells. TMEV infection led to TLR2-mediated NF-kappaB activation, but not IRF3 or IRF7 activation, critical for type I IFN production. More importantly, TLR3 was required for TMEV-induced early TLR2 upregulation in primary astrocytes leading to the production of TLR2-dependent cytokines such as IL-6. Interestingly, soluble factor(s) produced via TLR2/3-dependent signals appears to be partially associated with the downstream cytokine production. These results indicate that TMEV utilizes TLR3-induced TLR2 to induce inflammatory cytokines, which are critical to the development of immune-mediated demyelinating disease.

Role of dendritic cells in differential susceptibility to viral demyelinating disease.

Although persistent viral diseases are a global health concern, the mechanisms of differential susceptibility to such infections among individuals are unknown. Here, we report that differential interactions between dendritic cells (DCs) and virus are critical in determining resistance versus susceptibility in the Theiler murine encephalomyelitis virus-induced demyelinating disease model of multiple sclerosis. This virus induces a chronic demyelinating disease in susceptible mice, whereas the virus is completely cleared in resistant strains of mice. DCs from susceptible mice are more permissive to viral infection, resulting in severe deficiencies in development, expansion, and function, in contrast to DCs from resistant mice. Although protective prior to viral infection, higher levels of type I interferons (IFNs) and IFN-gamma produced by virus-infected DCs from susceptible mice further contribute to the differential inhibition of DC development and function. An increased DC number and/or acquired resistance of DCs to viral infection render susceptible mice resistant to viral persistence and disease progression. Thus, the differential permissiveness of DCs to infectious agents and its subsequent functional and developmental deficiencies determine the outcome of infection- associated diseases. Therefore, arming DCs against viral infection-induced functional decline may provide a useful intervention for chronic infection-associated diseases.