Alveolar Epithelial Cell Line A549 Research Articles

The functional role of CST1 and CCL26 in asthma development.

Asthma is the most common chronic disease in children with an increasing prevalence. Its development is caused by genetic and environmental factors and allergic sensitization is a known trigger. Dog allergens affect up to 30% of all children and dog dander-sensitized children show increased expression of cystatin-1 (CST1) and eotaxin-3 (CCL26) in nasal epithelium. The aim of our study was to investigate the functional mechanism of CST1 and CCL26 in the alveolar basal epithelial cell line A549. A549 cells were transfected with individual overexpression vectors for CST1 and CCL26 and RNA sequencing was performed to examine the transcriptomics. edgeR was used to identify differentially expressed genes (= DEG, |log2 FC | ≥ 2, FDR < 0.01). The protein expression levels of A549 cells overexpressing CST1 and CCL26 were analyzed using the Target 96 inflammation panel from OLINK (antibody-mediated proximity extension-based assay; OLINK Proteomics). Differentially expressed proteins were considered with a |log2 FC| ≥ 1, p < .05. The overexpression of CST1 resulted in a total of 27 DEG (1 upregulated and 26 downregulated) and the overexpression of CCL26 in a total of 137 DEG (0 upregulated and 137 downregulated). The gene ontology enrichment analysis showed a significant downregulation of type I and III interferon signaling pathway genes as well as interferon-stimulated genes. At the protein level, overexpression of CST1 induced a significantly increased expression of CCL3, whereas CCL26 overexpression led to increased expression of HGF, and a decrease of CXCL11, CCL20, CCL3 and CXCL10. Our results indicate that an overexpression of CST1 and CCL26 cause a downregulation of interferon related genes and inflammatory proteins. It might cause a higher disease susceptibility, mainly for allergic asthma, as CCL26 is an agonist for CCR-3-carrying cells, such as eosinophils and Th2 lymphocytes, mostly active in allergic asthma.

Antiinflammatory effect of the ethanolic extract of Korean native herb Potentilla rugulosa Nakai in Bisphenol-a-stimulated A549 cells

ABSTRACT Potentilla rugulosa Nakai (P. rugulosa) is a perennial herb in the Rosaceae family and found in the Korean mountains. Previously, our findings demonstrated that P. rugulosa contains numerous polyphenols and flavonoids exhibiting important antioxidant and anti-obesity bioactivities. Bisphenol A (BPA) is a xenoestrogen that was shown to produce pulmonary inflammation in humans. However, the mechanisms underlying BPA-induced inflammation remain to be determined. The aim of this study was to examine whether ethanolic extract of P. rugulosa exerted an inhibitory effect on BPA-induced inflammation utilizing an adenocarcinoma human alveolar basal epithelial cell line A549. The P. rugulosa extract inhibited BPA-mediated cytotoxicity by reducing levels of reactive oxygen species (ROS). Further, P. rugulosa extract suppressed the upregulation of various pro-inflammatory mediators induced by activation of the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, inhibition of the NF-κB and MAPK signaling pathways by P. rugulosa extract was found to occur via decrease in the transcriptional activity of NF-κB. Further, blockade of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) was noted. Thus, our findings suggest that the ethanolic extract of P. rugulosa may act as a natural anti-inflammatory therapeutic agent.

Effects of particle-bound polycyclic aromatic hydrocarbons and plasticisers from different traffic sources on the human alveolar epithelial cell line A549

Road traffic represents a major source of particulate matter (PM), which is considered the most harmful pollutant for humans. This work aimed to evaluate the toxic effects of polycyclic aromatic hydrocarbons (PAHs) and plasticisers extracted from PM emitted by traffic, including exhaust samples, samples collected in a road tunnel and simultaneously in a nearby urban background atmosphere, and samples from the wear of distinct types of brakes. Different biological endpoints were investigated after exposing human alveolar epithelium A549 cells to the organic extracts: viability, cell cycle alterations and levels of intracellular reactive oxygen species (ROS). The particulate extracts from the wear of low steel brake pads induced greater decreases in cell viability. It was observed that cytotoxicity is more dependent on the individual composition of PAHs than on the global concentration. Exhaust samples from heavy-duty Euro V vehicles induced more toxic effects than Euro VI. Among Euro V vehicles, the exhaust of GTL was the most toxic. The composite weekly sample of the urban background atmosphere showed to be extremely toxic, decreasing cell viability to 2.66%, while the daily road tunnel samples caused a decrease of up to 83.6% at most. In the particulate extracts of the urban background atmosphere, retene and naphthalene were found, which were absent or present at trace levels in the tunnel. Irrespective of the sample, although some changes were observed in the A549 cell cycle after 24 h exposure in all the phases, the differences in the mean values were not great enough to exclude the possibility of random variability. When the results from the three phases (G0/G1, S and G2/M) of the cells exposed to all PM extracts were compared to the control, no statistically significant difference was detected. Regardless of the sample type, no significant increases in cellular ROS levels were observed either.

Rescue and in vitro characterization of a divergent TBEV-Eu strain from the Netherlands

Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland.

Coumarinolignoid and Indole Alkaloids from the Roots of the Hybrid Plant Citrus × paradisi Macfad (Rutaceae).

A phytochemical investigation of the roots of Citrus × paradisi Macfad. (Rutaceae) led to the isolation of two new compounds, namely 1-formyl-5-hydroxy-N-methylindolin-1-ium (1) and decyloxycleomiscosin D (2), along with ten known compounds: 1,1-dimethylpyrrolidin-1-ium-2-carboxylate (3), furan-2,3-diol (4), 5-methoxyseselin (5), umbelliferone (6), scopoletin (7), citracridone I (8), citracridone II (9), citracridone III (10), limonin (11) and lupeol (12). The structures were determined through the comprehensive spectroscopic analysis of 1D and 2D NMR and EI- and ESI-MS, as well as a comparison with the published data. Notably, compounds 3 and 4 from the genus Citrus are reported here for the first time. In addition, the MeOH extract of the roots and compounds 1-7 were screened against the human adenocarcinoma alveolar basal epithelial cell line A549 and the Caucasian prostate adenocarcinoma cell line PC3 using the MTT assay. While the extract showed significant activity, with IC50 values of 35.2 and 38.1 µg/mL, respectively, compounds 1-7 showed weak activity, with IC50 values of 99.2 to 250.2 µM and 99.5 to 192.7 µM, respectively.

Akt Inhibition Promotes Autophagy and Clearance of Group B Streptococcus from the Alveolar Epithelium.

Group B Streptococcus (GBS) is a gram-positive bacterium that is harmless for healthy individuals but may provoke invasive disease in young infants and immunocompromised hosts. GBS invades the epithelial barriers to enter the bloodstream, and thus strategies that enhance epithelial cell responses may hamper GBS invasion. In the present study, we sought to investigate whether the inhibition of Akt, a kinase that regulates host inflammatory responses and autophagy via suppression of mTOR, can enhance the response of non-phagocytic alveolar epithelial cells against GBS. Treatment of the alveolar epithelial cell line A549 with the Akt inhibitor MK-2206 resulted in the enhanced production of reactive oxygen species and inflammatory mediators in response to GBS. Additionally, Akt inhibition via MK-2206 resulted in elevated LC3II/I ratios and increased autophagic flux in alveolar epithelial cells. Importantly, the inhibition of Akt promoted GBS clearance both in alveolar epithelial cells in vitro and in lung tissue in vivo in a murine model of GBS pneumonia. The induction of autophagy was essential for GBS clearance in MK-2206 treated cells, as knockdown of ATG5, a critical component of autophagy, abrogated the effect of Akt inhibition on GBS clearance. Our findings highlight the role of Akt kinase inhibition in promoting autophagy and GBS clearance in the alveolar epithelium. The inhibition of Akt may serve as a promising measure to strengthen epithelial barriers and prevent GBS invasion in susceptible hosts.

Role of Nrf2 in Methotrexate-Induced Epithelial-Mesenchymal Transition in Alveolar A549 Cells.

Methotrexate (MTX) is known to induce serious lung diseases, such as pulmonary fibrosis. Although we demonstrated that MTX is associated with epithelial-mesenchymal transition (EMT), the underlying mechanism remains unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2), an oxidative stress response regulator, is related to EMT induction. In the present study, we examined the association of Nrf2 with the MTX-induced EMT in the alveolar epithelial cell line A549. MTX treatment decreased the mRNA expression of heme oxidase-1 (HO-1), a target of Nrf2, which was inhibited by co-treatment with diethyl maleate (DEM), an Nrf2 activator. Additionally, the MTX-induced increase in reactive oxygen species (ROS) production was significantly suppressed by DEM. Furthermore, DEM decreased mRNA/protein expression levels of α-smooth muscle actin (SMA), a representative EMT marker, which were upregulated by MTX. Nuclear expression and localization of Nrf2 were suppressed by MTX treatment, which led to a decrease in Nrf2 activity. Finally, in Nrf2 knockdown cells, the MTX-induced enhancement of α-SMA mRNA/protein expression was not observed, indicating that downregulation of Nrf2 may play a critical role in the MTX-induced EMT in A549 cells. These results suggest that Nrf2-regulated transcriptional activity would be associated with the MTX-induced EMT induction.

RUNX1 inhibits the antiviral immune response against influenza A virus through attenuating type I interferon signaling

BackgroundInfluenza A viruses (IAVs) are zoonotic, segmented negative-stranded RNA viruses. The rapid mutation of IAVs results in host immune response escape and antiviral drug and vaccine resistance. RUNX1 is a transcription factor that not only plays essential roles in hematopoiesis, but also functions as a regulator in inflammation. However, its role in the innate immunity to IAV infection has not been well studied.MethodsTo investigate the effects of RUNX1 on IAV infection and explore the mechanisms that RUNX1 uses during IAV infection. We infected the human alveolar epithelial cell line (A549) with influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) and examined RUNX1 expression by Western blot and qRT-PCR. We also knocked down or overexpressed RUNX1 in A549 cells, then evaluated viral replication by Western blot, qRT-PCR, and viral titration.ResultsWe found RUNX1 expression is induced by IAV H1N1 PR8 infection, but not by poly(I:C) treatment, in the human alveolar epithelial cell line A549. Knockdown of RUNX1 significantly inhibited IAV infection. Conversely, overexpression of RUNX1 efficiently promoted production of progeny viruses. Additionally, RUNX1 knockdown increased IFN-β and ISGs production while RUNX1 overexpression compromised IFN-β and ISGs production upon PR8 infection in A549 cells. We further showed that RUNX1 may attenuate the interferon signaling transduction by hampering the expression of IRF3 and STAT1 during IAV infection.ConclusionsTaken together, we found RUNX1 attenuates type I interferon signaling to facilitate IAV infection in A549 cells.

Are In Vitro Cytotoxicity Assessments of Environmental Samples Useful for Characterizing the Risk of Exposure to Multiple Contaminants at the Workplace? A Systematic Review.

In some occupational environments risk characterization is challenging or impossible to achieve due to the presence of multiple pollutants and contaminants. Thus, in vitro testing using the most relevant cell lines will provide information concerning health effects due to the co-exposure to multiple stressors. The aim of this review article is to identify studies where the cytotoxicity assessment was performed in environmental samples, as well as to describe the main outputs and challenges regarding risk characterization and management. This study is based on a study of the available information/data on cytotoxicity assessment performed on environmental samples following the PRISMA methodology. Different cell lines were used depending on the environment assessed and exposure routes implicated. The A549 alveolar epithelial cell line was applied in four studies for occupational exposure in the waste sorting industry and for outdoor environments; lymphocytes were used in two studies for occupational and outdoor environments; swine kidney cells were used in three studies performed in the waste industry and hepatocellular/Hep G2 in one study in the waste industry. Cytotoxicity assessments in environmental samples should have a more prominent role due to their contribution for identifying and better understanding the associations between co-exposure to environmental contaminants and adverse human health effects as a prioritization for risk management.

Toxicological assessment of nanocrystalline metal alloys with potential applications in the aeronautical field

The development of new candidate alloys with outstanding characteristics for their use in the aeronautical field is one of the main priorities for the sector. In this context, nanocrystaline (nc) alloys are considered relevant materials due to their special features, such as their exceptional physical and mechanical properties. However, another important point that needs to be considered with newly developed alloys is the potential toxicological impact that these materials may have in humans and other living organisms. The aim of this work was to perform a preliminary toxicological evaluation of three nc metal alloys (WCu, WAl and TiAl) in powder form produced by mechanical alloying, applying different in vitro assays, including a mix of W-Cu powders with standard grain size in the experiments to stablish comparisons. The effects of the direct exposure to powder suspensions and/or to their derived leachates were analysed in three model organisms representative of human and environmental exposures (the adenocarcinomic human alveolar basal epithelial cell line A549, the yeast Saccharomyces cerevisiae and the Gram negative bacterium Vibrio fischeri). Altogether, the results obtained provide new insights about the potential harmful effects of the selected nc alloys, showing that, from a toxicological perspective, nc TiAl is the safest candidate in the model organisms and conditions tested.

Increased LHX9 expression in alveolar epithelial type 2cells of patients with chronic obstructive pulmonary disease.

Alveolar epithelial type 2 (AT2) cells serve as stem cells in alveolar epithelium and are assumed to lose their stem cell function in the lungs of chronic obstructive pulmonary disease (COPD). Although we previously reported that LHX9 mRNA expression was up-regulated in AT2 cells of COPD lung tissues, it is yet to be elucidated how LHX9 is associated with the vulnerability of AT2 cells in COPD. AT2 cells were isolated from lung tissues of 10 non-COPD subjects and 11 COPD patients. LHX9 mRNA expression was determined by quantitative RT-PCR. To identify up-stream molecules, an alveolar epithelial cell line A549 was exposed to pro-inflammatory cytokines invitro. siRNA-mediated Lhx9 knockdown was performed to determine how Lhx9 affected the cellular viability and the cell-division cycle. LHX9 mRNA expression was increased in AT2 cells from COPD lung tissues, compared to those from non-COPD tissues. The airflow obstruction was independently correlated with the increase in LHX9 expression. Among several pro-inflammatory cytokines, interferon-γ was a strong inducer of LHX9 expression in A549cells. Lhx9 was involved in the increased susceptibility to serum starvation-induced death of A549cells. Our data suggest that IFN-γ predominantly increases the LHX9 expression which enhances the susceptibility to cell death. Considering the independent association of the increased LHX9 expression in AT2 cells with airflow obstruction, the IFN-γ-Lhx9 axis might contribute to the vulnerability of AT2 cells in the lungs of COPD patients.

Effect and Mechanism of Fuzheng Oral Liquid on Anlotinib Resistance in Non-Small Cell Lung Cancer Cells

To explore the reversal effect of Fuzheng oral liquid on anlotinib resistance in non-small cell lung cancer cells and its mechanism is the objective of the study. 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay and flow cytometric assay were performed to evaluate the effects of anlotinib on the proliferation and apoptotic cycles of adenocarcinomic human alveolar basal epithelial cell line A549 cells, respectively. Western blot was performed to detect the expression levels of unfolded protein response, endoplasmic reticulum-associated degradation, E-cadherin, vimentin, beta-catenin, transcription factor 4 and Axin in anlotinib-resistant cell line A549/AL3818 cells. Anlotinib-resistant A549 cells were successfully constructed and named as A549/AL3818. Fuzheng oral solution had proliferation inhibition, apoptosis induction and cycle blocking effects on A549/AL3818 cells. High dose of Fuzheng oral liquid was able to inhibit unfolded protein response and endoplasmic reticulum-associated degradation, expression in A549/ AL3818 cells and suppress wingless-related integration signaling pathway and epithelial-mesenchymal transition progression in A549/AL3818 cells. The inhibitory effect of Fuzheng oral liquid on winglessrelated integration signaling pathway and epithelial-mesenchymal transition progression was the same as that of paired box protein 6. Fuzheng oral liquid inhibited epithelial-mesenchymal transition progression through regulating unfolded protein response-endoplasmic reticulum-associated degradation signaling pathway, thereby reversing the resistance of non-small cell lung cancer cells to anlotinib.

24-Dehydrocholesterol Reductase alleviates oxidative damage-induced apoptosis in alveolar epithelial cells via regulating Phosphatidylinositol-3-Kinase/Protein Kinase B activation

ABSTRACT Apoptosis of alveolar epithelial cells during acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a critical pathological event that seriously endangers the health of patients. Suppressing apoptosis of alveolar epithelial cells was shown to alleviate functional damage of lung, and modulator of the reactive oxygen species (ROS)-induced apoptosis becomes a promising approach to the ALI therapy. Previous little studies showed that DHCR24 possessed anti-oxidative and anti-apoptotic property in ALI. Thus, H2O2 was utilized to mimic oxidative damage in vitro in alveolar epithelial cell line A549 in the present study. Our results exhibited that H2O2 treatment of A549 cells reduced the level of SOD and increased the level of ROS. Moreover, H2O2 inhibited Bcl-2 expression in A549 cells, but increased Bax and the activity of Caspase-3. In addition, the apoptosis rate in the H2O2 treatment group also increased significantly. And the expression of 24-dehydrocholesterol reductase (DHCR24) was markedly reduced in the H2O2 treatment group. Overexpression of DHCR24 can remarkably inhibit H2O2-induced oxidative stress and apoptosis. We found that overexpression of DHCR24 increased the phosphorylation level of PI3K and AKT, however, the PI3K inhibitor LY294002 (LY) eliminated the protective effect of DHCR24 in ALI. DHCR24 was down-regulated in H2O2-induced ALI, and overexpression of DHCR24 could inhibit H2O2-induced oxidative stress and apoptosis of A549 cells by activating the Phosphatidylinositol-3-Kinase/Protein Kinase B (PI3K/AKT) signaling pathway. The above exhibited a protective effect of DHCR24 on alveolar epithelial cells exposed to oxidative stress-mediated apoptosis and provided a novel therapeutic method for ALI.

Baicalin attenuates LPS-induced alveolar type II epithelial cell A549 injury by attenuation of the FSTL1 signaling pathway via increasing miR-200b-3p expression.

In China, baicalin is the main active component of Scutellaria baicalensis, which has been used in the treatment of inflammation-related diseases, such as inflammation-induced acute lung injury. However, its specific mechanism remains unclear. This study examined the protective effect of baicalin on LPS-induced inflammation injury of alveolar epithelial cell line A549 and explored its protective mechanism. Compared with the LPS-induced group, the proliferation inhibition rates of alveolar type II epithelial cell line A549 intervened by different concentrations of baicalin decreased significantly, as did the levels of inflammatory factors IL-6, IL-1β, prostaglandin 2 and TNF-α in the supernatant. The expression levels of inflammatory proteins inducible NO synthase (iNOS), NF-κB65, phosphorylated ERK (p-ERK1/2), and phosphorylated c-Jun N-terminal kinase (p-JNK1) significantly decreased, as did the protein expression of follistatin-like protein 1 (FSTL1). In contrast, expression of miR-200b-3p significantly increased in a dose-dependent manner. These results suggested that baicalin could significantly inhibit the expression of inflammation-related proteins and improve LPS-induced inflammatory injury in alveolar type II epithelial cells. The mechanism may be related to the inhibition of ERK/JNK inflammatory pathway activation by increasing the expression of miR-200b-3p. Thus, FSTL1 is the regulatory target of miR-200b-3p.

Regulation of ACE-2 enzyme by hyperoxia in lung epithelial cells by post-translational modification

Bronchopulmonary Dysplasia (BPD) occurs in premature neonates with respiratory distress who require supplemental oxygen in the first days after birth. BPD involves uniform arrest of alveolar development and variable interstitial cellularity and/or fibroproliferation. Previous studies by our lab showed that the enzyme, angiotensin converting enzyme-2 (ACE-2) and its product Ang1-7 exerting action on the receptor Mas oncogene in what is known as ACE-2/Mas axis is protective to lung cells. We also showed that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas lung injury, an effect caused by ACE-2 enzyme shedding mediated by TNF-alpha-converting enzyme (TACE/ADAM17). However, no reports yet exist about the regulation of ACE-2 in the alveolar epithelia in hyperoxic lung injury. In this study we aim to define the effects of hyperoxic lung injury on the protective ACE-2 enzyme in the human lung alveolar epithelial cell line A549. Cultured A549 cells were exposed to hyperoxia (95% O2) or normoxia (21% O2) for 3 or 7 days in serum-free nutrient media. Cells were lysed and culture media were collected to test for cellular ACE-2 enzymatic activity and for ACE-2, Mas receptor, TACE/ADAM17, and ubiquitin proteins abundance by immunoblotting. Cells were harvested in Trizol for RNA extraction and ACE-2 qRT-PCR. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Total ubiquitinated proteins were increased by hyperoxia treatment, while ACE-2 and Mas receptor proteins abundance and ACE-2 enzymatic activity were decreased significantly in A549 cells exposed to hyperoxia relative to the normoxia controls. The percent decrease in ACE-2 activity corresponded with increased time of hyperoxic gas exposure. However, in contrast to our data from lung fibroblasts, no significant change was noted in ACE-2 protein released into the media or in ACE-2 mRNA levels by the hyperoxic treatment. Ubiquitin immunoreactive bands were detectable in the ACE-2 immunoprecipitate. These data suggest that hyperoxic exposure of the lung epithelial cells decreases the protective enzyme ACE-2 by cell type specific mechanisms independent of shedding by TACE/ADAM17. The data also suggest a regulatory level of ACE-2 downstream of transcription may involve ACE-2 ubiquitination and targeting for degradation.

Vincamine, a safe natural alkaloid, represents a novel anticancer agent

Vincamine, a well-known plant alkaloid, has been used as a dietary supplement and as a peripheral vasodilator to combat aging in humans. In this study, for the very first time, we demonstrated that vincamine can function as an anticancer agent in a human alveolar basal epithelial cell line A549 (IC50 = 309.7 μM). The anticancer potential of vincamine in A549 cells was assessed by molecular assays to determine cell viability, generation of intracellular ROS, nuclear condensation, caspase-3 activity and inhibition, and change in mitochondrial membrane potential (ΔΨm). In silico studies predicted that the anti-proliferative potential of vincamine is enhanced by its interaction with the apoptotic protein caspase-3, and that this interaction is driven by two hydrogen bonds and has a high free energy of binding (−5.64 kcal/mol) with an estimated association constant (Ka) of 73.67 μM. We found that vincamine stimulated caspase-3-dependent apoptosis and lowered mitochondrial membrane potential, which ultimately led to cytochrome C release. Vincamine was also found to quench hydroxyl free radicals and deplete iron ions in cancer cells. As a dietary supplement, vincamine is almost non-toxic in BEAS-2B and 3T3-L1 cells. Therefore, we propose that vincamine represents a safe anticancer agent in lung cancer cells. Its role in other cancers has yet to be explored.

Human Placental Hematopoietic Stem Cell Derived Natural Killer Cells (CYNK-001) Mediate Protection Against Influenza A Viral Infection

Background: Influenza A virus (IAV) infections are associated with a high healthcare burden around the world and there is an urgent need to develop more effective therapies. Natural killer (NK) cells provide the first line of innate defense against IAV by killing infected epithelial cells, by producing antiviral cytokines and affecting adaptive immunity. Preclinical studies have demonstrated that NK cells play a pivotal role in reducing IAV-induced pulmonary infection; however, little is known about the therapeutic potential of adoptively transferred NK cells for IAV infections. Celularity Inc. is developing human placental hematopoietic stem cell-derived allogeneic, off-the-shelf NK cell therapy (CYNK-001) for the treatment of viral infections, including coronavirus disease of 2019. Here, we report the evaluation of antiviral activities of CYNK-001 against IAV infection.Methods:In vitro antiviral activities of CYNK-001 were evaluated using human alveolar epithelial cell line A549, infected with IAV strain A/PR/8/34 (H1N1) at variable multiplicity of infection (MOI). The expression of ligands for NK cell receptors was analyzed on infected A549 cells using Fc-coupled recombinant proteins. CYNK-001 was added to A549 cells 16 hours post infection. CYNK-001 degranulation was measured after 4 hours of coculture, and CYNK-001 cytotoxicity against IAV-infected A549 was measured real-time using impedance-based xCELLigence platform. In vivo antiviral and immunomodulatory activities of CYNK-001 were assessed in A/PR/8/34 (H1N1)-induced severe acute lung injury mouse model. Mice were intranasally infected with 2500 PFU IAV. PBS or 1 x 107 CYNK-001 cells were intravenously administered twice at 1 and 3 days post infection (dpi). At 6 dpi, lungs were collected for the evaluation of viral load by qPCR, lung injury and immune cell profiling by histology. Bronchoalveolar lavage fluid (BALF) was collected at 6 dpi for cytokine analysis by multiplex assays, total protein concentration by ELISA and immune cell profiling by flow cytometry.Results:In vitro, IAV infection corresponded with dose-dependent expression of ligands to NK cell-activating receptors, including NKp44, NKp46 and NKG2D. CYNK-001 cells exhibited increased IFNγ, TNFα and GM-CSF production, and elevated level of degranulation upon coculture with IAV-infected A549 cells. Cytokines in culture supernatant and CD107a expression in CYNK-001 cells were upregulated in a virus dose-dependent manner. Consistent with this finding, CYNK-001 cytotoxicity against IAV-infected A549 cells increased from 35% at 0 MOI to 50%, 60% and 75% at 0.001, 0.01 and 0.1 MOI, respectively. These data indicate that CYNK-001 cells recognize virally infected cells, resulting in specific cytotoxic elimination of the source of infection. In vivo, treatment of IAV-infected mice with CYNK-001 reduced weight loss and increased their likelihood of survival. PBS control group developed a severe disease and 37.5% mortality was observed as early as day 4. In the group treated with CYNK-001, disease onset was delayed by 2 days. qPCR analysis of viral RNA showed that CYNK-001-treated mice had lower viral load in the lung than vehicle-treated mice, demonstrating antiviral function of CYNK-001 in vivo. CYNK-001-treated mice had reduced lung injury as assessed by lower total protein concentration in BALF. Moreover, CYNK-001 reduced BALF murine cytokines and chemokines, including IFNγ (p<0.001), IL-6, TNFα, MCP-1 (p<0.05), CXCL2 and CXCL9. Lastly, immunohistochemical analysis of the lung showed that CYNK-001-treated mice had an altered immune response to IAV with higher number of CD68+ macrophages and CD8+ T cells at 6 dpi.Conclusions: Our in vitro and in vivo data show the promising antiviral activities of CYNK-001 against IAV infection. In a severe IAV infection mouse model, CYNK-001 treatment demonstrates lower mortality rate, lower weight loss, lower lung viral load and reduced lung injury along with reduced inflammation. These results support our hypothesis that the adoptive transfer of CYNK-001 could reduce the burden of viral infection through the elimination of infected epithelial cells, coordinate a more effective immune response, and result in a clinical benefit in patients with severe viral infection.DisclosuresHe:Celularity Inc.: Current Employment. Mahlakõiv:Celularity Inc.: Current Employment. Gleason:Celularity Inc.: Current Employment, Current equity holder in private company. Van Der Touw:Celularity Inc.: Current Employment. Kang:Celularity Inc.: Current Employment. Hariri:Celularity Inc.: Current Employment, Current equity holder in private company. Zhang:Celularity Inc.: Current Employment, Current equity holder in private company.

Synthesis and Identification of a Novel Peptide, Ac-SDK (Biotin) Proline, That Can Elicit Anti-Fibrosis Effects in Rats Suffering from Silicosis.

BackgroundN-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a short peptide with an anti-silicosis effect. However, the short biological half-life and low plasma concentration of Ac-SDKP hamper discovery of specific targets in organisms and reduce the anti-silicosis effect. A novel peptide, Ac-SDK (biotin) proline, termed “Ac-B”, with anti-fibrotic properties was synthesized.MethodsAc-B was detected quantitatively by high-performance liquid chromatography. Phagocytosis of Ac-B by the alveolar epithelial cell line A549 was investigated by confocal laser scanning microscopy and flow cytometry. To further elucidate the cellular-uptake mechanism of Ac-B, chemical inhibitors of specific uptake pathways were used. After stimulation with transforming growth factor-β1, the effects of Ac-B on expression of the myofibroblast marker vimentin and accumulation of collagen type I in A549 cells were analyzed by Western blotting. Sirius Red staining and immunohistochemical analyses of the effect of Ac-B on expression of α-smooth muscle actin (SMA) in a rat model of silicosis were undertaken.ResultsAc-B had good traceability during the uptake, entry, and distribution in cells. Ac-B treatment prevented an increase in α-SMA expression in vivo and in vitro and was superior to that of Ac-SDKP. Caveolae-mediated uptake of Ac-B by A549 cells led to achieving anti-epithelial–mesenchymal transformation (EMT) effects.ConclusionAc-B had an anti-fibrotic effect and could be a promising agent for the fibrosis observed in silicosis in the future.

Plant growth regulators and Axl and immune checkpoint inhibitors from the edible mushroom Leucopaxillus giganteus.

A novel compound, (R)-4-ethoxy-2-hydroxy-4-oxobutanoic acid (1), and six known compounds (2-7) were isolated from the fruiting bodies of the wild edible mushroom Leucopaxillus giganteus. The planar structure of 1 was determined by the interpretation of spectroscopic data analysis. The absolute configuration of 1 was determined by comparing specific rotation of the synthetic compounds. In the plant regulatory assay, the isolated compounds (1-7) and the chemically prepared compounds (8-10) were evaluated their biological activity against the lettuce (Lactuca sativa) growth. Compounds 1 and 3-10 showed the significant regulatory activity of lettuce growth. 1 showed the strongest inhibition activity among the all the compounds tested. In the lung cancer assay, all the compounds were assessed the mRNA expression of Axl and immune checkpoints (PD-L1, PD-L2) in the human A549 alveolar epithelial cell line by RT-PCR. Compounds 1-10 showed significant inhibition activity against Axl and/or immune checkpoint.

Analysis of influenza virus-induced perturbation in autophagic flux and its modulation during Vitamin D3 mediated anti-apoptotic signaling

Vitamin D3/Calcitriol supplementation in humans is associated with reduced incidence and severity during influenza A virus (IAV) infection. Apoptosis in response to IAV infection is a major contributor to host cell death and tissue damage; however, its modulation by Vitamin D3 remains unclear. In this study, we demonstrate the efficacy of Vitamin D3 in preventing apoptosis induction by pandemic influenza A (H1N1)pdm09 virus in human alveolar cells (A549). Human alveolar epithelial cell line A549 was used to assess the cytotoxic effects of IAV infection. Immunoblotting and fluorescence microscopy were used to study apoptosis and autophagy. The results of the present study demonstrate that IAV induces apoptosis by subversion of host autophagy via down-regulating components of autophagic machinery involved in autophagosome-lysosome fusion and lysosomal activity. Vitamin D3 restores the autophagic flux inhibited by IAV by upregulating the expression of Syntaxin-17 (STX17) and V-type proton ATPase subunit (ATP6V0A2) thereby causing a concomitant decrease in cellular apoptosis via a Vitamin D3 receptor (VDR) dependent mechanism. The present study suggests that Vitamin D3 is a potentially useful agent for limiting IAV-induced cellular injury via its pro-autophagic action.