Rat Lung Epithelial Cells Research Articles

Mesenchymal Stem Cells Reduce the Extracellular Mitochondrial DNA-Mediated TLR9 Activation in Neonatal Hyperoxia-Induced Lung Injury.

Mitochondrial DNA (mtDNA) released from dead or injured cells can activate inflammation, and mesenchymal stem cell (MSC) transplantation can reduce inflammation and injury. However, it has not been tested whether the release of mtDNA can be reduced by MSC transplantation. We hypothesized that the level of extracellular mtDNA would be increased after hyperoxia-induced lung injury but reduced after lung injury attenuation by MSC therapy in our newborn rat model. In an in vitro study using a rat lung epithelial L2 cell line, we found that the level of extracellular mtDNA was significantly increased with H2O2-induced cell death but reduced after MSC co-incubation. In an in vivo study, we confirmed that the levels of cell death, extracellular mtDNA, and inflammatory cytokines were significantly increased in hyperoxic newborn rat lungs but reduced after MSC transplantation. The levels of extracellular mtDNA were significantly and positively correlated with the levels of the inflammatory cytokines. The TLR9/MyD88/NF-κB pathway, which is activated by binding to mtDNA, was also significantly upregulated but downregulated after MSC transplantation. We found a significant positive correlation between inflammatory cytokines and extracellular mtDNA in intubated neonates. The levels of inflammatory cytokines and extracellular mtDNA changed over time in a similar pattern in transtracheal aspirate samples from intubated neonates. In conclusion, increased levels of extracellular mtDNA are associated with increased inflammation in hyperoxia-induced lung injury, and attenuation of lung inflammation by MSC therapy is associated with reduced levels of extracellular mtDNA.

Generation of rat-derived lung epithelial cells in Fgfr2b-deficient mice retains species-specific development.

Regenerative medicine is a tool to compensate for the shortage of lungs for transplantation, but it remains difficult to construct a lung in vitro due to the complex three-dimensional structures and multiple cell types required. A blastocyst complementation method using interspecies chimeric animals has been attracting attention as a way to create complex organs in animals, although successful lung formation using interspecies chimeric animals has not yet been achieved. Here, we applied a reverse-blastocyst complementation method to clarify the conditions required to form lungs in an Fgfr2b-deficient mouse model. We then successfully formed a rat-derived lung in the mouse model by applying a tetraploid-based organ-complementation method. Importantly, rat lung epithelial cells retained their developmental timing even in the mouse body. These findings provide useful insights to overcome the barrier of species-specific developmental timing to generate functional lungs in interspecies chimeras.

Discovery of a Druggable, Cryptic Pocket in SARS-CoV-2 nsp16 Using Allosteric Inhibitors.

A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a, a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S-adenosylmethionine binding cleft in a manner that prevents occupation by S-adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.

MALAT1 binds to miR-188–3p to regulate ALOX5 activity in the lung inflammatory response of neonatal bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) causes high morbidity and mortality in infants, but no effective preventive or therapeutic agents have been developed to combat BPD. In this study, we assessed the expression of MALAT1 and ALOX5 in peripheral blood mononuclear cells from BPD neonates, hyperoxia-induced rat models and lung epithelial cell lines. Interestingly, we found upregulated expression of MALAT1 and ALOX5 in the experimental groups, along with upregulated expression of proinflammatory cytokines. According to bioinformatics prediction, MALAT1 and ALOX5 simultaneously bind to miR-188–3p, which was downregulated in the experimental groups above. Silencing MALAT1 or ALOX5 and overexpressing miR-188–3p inhibited apoptosis and promoted the proliferation of hyperoxia-treated A549 cells. Suppressing MALAT1 or overexpressing miR-188–3p increased the expression levels of miR-188–3p but decreased the expression levels of ALOX5. Moreover, RNA immunoprecipitation (RIP) and luciferase assays showed that MALAT1 directly targeted miR-188–3p to regulate ALOX5 expression in BPD neonates. Collectively, our study demonstrates that MALAT1 regulates ALOX5 expression by binding to miR-188–3p, providing novel insights into potential therapeutics for BPD treatment.

4,7-Didehydro-neophysalin B Protects Rat Lung Epithelial Cells against Hydrogen Peroxide-Induced Oxidative Damage through Nrf2-Mediated Signaling Pathway.

The administration of 4,7-didehydro-neophysalin B is expected to be a promising strategy for mitigating oxidative stress in respiratory diseases. This study was aimed at investigating the efficacy of 4,7-didehydro-neophysalin B for apoptosis resistance of rat lung epithelial cells (RLE-6TN) to oxidative stress and evaluating its underlying mechanism of action. The RLE-6TN cells treated with hydrogen peroxide (H2O2) were divided into five groups, and 4,7-didehydro-neophysalin B was administered into it. To evaluate its mechanism of action, the expression of oxidative stress and apoptotic proteins was investigated. 4,7-Didehydro-neophysalin B significantly inhibited H2O2-induced RLE-6TN cell damage. It also activated the Nrf2 signaling pathway which was evident from the increased transcription of antioxidant responsive of KLF9, NQO1, Keap-1, and HO-1. Nrf2 was found to be a potential target of 4,7-didehydro-neophysalin B. The protein levels of Bcl-2 and Bcl-xL were increased while Bax and p53 were decreased significantly. Flow cytometry showed that 4,7-didehydro-neophysalin B protected RLE-6TN cells from apoptosis and has improved the oxidative damage. This study provided a promising evidence that 4,7-didehydro-neophysalin B can be a therapeutic option for oxidative stress in respiratory diseases.

Silica exposure activates non-canonical inflammasome complex in intratracheal instilled rat model.

Inhalation of silica crystals in occupational settings is a main cause of silicosis, a chronic irreversible pulmonary disorder. Our prior studies demonstrated the activation of inflammasome sensors AIM2 and NLRP3, effector protein caspase-1, and significant increase in IL-1β in silica exposed rats, suggesting that the canonical inflammasome activation may be associated with silica-induced tissue damage and inflammation. In our current study using the same animal model system, we further evaluated the components of non-canonical inflammasome, including NEK7, caspase-11, and GSDMD following silica exposure. We demonstrated sustained NEK7 elevation in the rat lung epithelial cells and macrophages following 1- and 3-day exposure. Enhanced NEK7 expression was also detected in lung homogenate by western blot. Similarly, caspase-11 expression was induced by silica exposure in lung sections and homogenate. Elevated GSDMD was observed both in lung sections by immunohistochemical staining and in lung tissue homogenate by western blot. In summary, our current study demonstrated increase in NEK7, caspase-11, and GSDMD in silica exposed rats, indicating activation of non-canonical inflammasome complex, thereby providing a broad inflammasome activation pathway caused by silica exposure.

Derepression of the IL-10 Gene by CRISPR-Cas Genome Editing as a Therapeutic Strategy for Persistent Immunomodulation of Donor Lungs for Transplantation

<h3>Purpose</h3> Anti-rejection immunosuppression is a persistent challenge in lung transplantation. We envisioned creating less immunogenic donor organs to obviate or reduce the need for immunosuppression. To induce persistent expression of the immunomodulatory cytokine IL-10, we aimed to harness CRISPR-Cas-mediated genome editing. We hypothesized that mutating the regulatory region of the IL-10 could induce persistent IL-10 upregulation with practicality in organ modification. To prove our concept, we investigate the efficacy and persistence of this approach in vitro. <h3>Methods</h3> Plasmids encoding Streptococcus pyogenes Cas9 (SpCas9), a puromycin cassette, and a single guide RNA (gRNA), designed to bind the promoter of the rat IL-10 gene, were delivered into a rat lung epithelial cell line. Transfected cells were enriched using puromycin and assessed for mutations and gene expressions after 14 and 28 days. The treatment group (targeting gRNA group) was compared to a no gRNA group and a non-targeting gRNA group. <h3>Results</h3> Insertions/deletions at the target site were detected in 77±20 % of alleles at day 14 and 87±11% at day 28 in the treatment group (Fig. 1A). The treatment group showed enhanced rat IL-10 expression while other groups showed mostly undetectable IL-10 expression (457±338- and 865+573- fold increase compared to negative control at day 14 and 28 respectively, Fig.1B). Importantly, IL-10 upregulation lasted for 28 days despite a decline in SpCas9 expression (Fig. 1C) suggesting an effective and persistent expression after one round of genome editing. <h3>Conclusion</h3> We have demonstrated that CRISPR-Cas9-mediated genome editing at the promoter region induces stable IL-10 expression. Targeted editing outside of the coding region potentially leads to a safer application of whole organ gene editing. Our findings expand the potential of genome editing towards engineering optimized donor organs for transplantation.

Delivery of MiRNA-92a Inhibitor Using RP1-Linked Peptide Elicits Anti-Inflammatory Effects in an Acute Lung Injury Model.

Acute lung injury (ALI) is an inflammatory lung disease. miRNA-92a (miR92a) is induced in the lungs of ALI patients and mediates inflammatory reactions. In this study, a RP1-linked R3V6 (RP1R3V6) peptide was synthesized and evaluated as a carrier of anti-microRNA-92a oligonucleotide (AMO92a) into the lungs of an ALI animal model. In addition to the carrier function, the RP1-linked peptide can have anti-inflammatory effects in the lungs, since RP1 is an antagonist of the receptors for advanced glycation end-products (RAGEs). In a gel retardation assay, the RP1R3V6 peptide formed a spherical complex with AMO92a. In an in vitro delivery assay to L2 rat lung epithelial cells, RP1R3V6 had a lower AMO92a delivery efficiency than R3V6 and polyethyleneimine (PEI25k; 25 kDa). However, RP1R3V6 had an additional anti-inflammatory effect, reducing tumor necrosis factor-α (TNF-α) in lipopolysaccharide-activatedmacrophage cells. With the combined effects of AMO92a and RP1, the RP1R3V6/AMO92a complex reduced the miR92a level more efficiently than did the R3V6/AMO92a and PEI25k/AMO92a complexes. The RP1R3V6/AMO92a complex was administered into the lungs of ALI animals by intratracheal instillation. As a result, the expression of phosphatase and tensin homolog, a target of miR92a, was increased in the lungs. Furthermore, the RP1R3V6/AMO92a complex decreased the TNF-α and interleukin-1β (IL-1β) levels more efficiently than did the PEI25k/AMO92a and R3V6/AMO92a complexes, decreasing the damage in the lungs. These results suggest that RP1R3V6 is a useful carrier of AMO92a and has anti-inflammatory effects in an ALI animal model.

Association of microsomal epoxide hydrolase gene (fast genotype) with lung functions impairment in wood workers.

Exposure to wood dust may lead to impairment of the lung functions. Microsomal epoxide hydrolase enzyme (EPHX1) was shown to take part in protection against oxidative stress. An alteration in enzyme activity might be associated with its gene polymorphisms. In vitro polymorphisms in exons 3 (His113Tyr) and 4 (Arg139His) lead to reduced activity (slow allele) and increased activity (fast allele). Macrophage inflammatory protein 2 (MIP-2) is produced in rat lung epithelial cells after exposure to fine particles. We aimed to investigate the associations between mEPHX1 polymorphisms (in exon 3 and 4) and lung function in furniture workers and assessment of MIP-2 effect. Our study was performed on 70 wood dust exposed male workers and 70 matched normal controls subjects. Ventilatory function tests were measured by spirometer, MIP-2 was performed by ELISA methods and EPHX gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods for each participant. Significant reduction in forced vital capacity (FVC%) and forced expiratory volume in the first second (FEV1) levels in Tyr-Tyr and Tyr-Hist genotypes of EPHX (exon 3) was observed. Reduced peak expiratory flow (PEF) levels and significant rise in MIP-2 levels were detected in Tyr-Tyr genotype. While high significant reduction in FVC% and FEV1 levels were shown in different genotypes in exon 4. Significant rise was observed in MIP-2 levels in Hist-Hist genotype of exon 4. An increase in duration of exposure showed positive correlation with fall in ventilatory functions. It was concluded that in Hist139Arg of EPHX gene, fast genotype (Arg-Arg) was associated with impaired ventilatory functions.

Multiplex Targeted Epigenome Editing Utilizing CRSPR/Cas9 for Potent Anti-Inflammatory Gene Therapy in Lung Transplant

<h3>Purpose</h3> Flexible modulation of gene expressions in the donor lung holds great promise in lung transplant. We hypothesized that activation of multiple anti-inflammatory genes would synergistically induce more potent effects. CRISPR/Cas9 system has strength in multiplexing due to its simple mechanism of the programmable RNA-guided DNA targeting. This study investigated the efficacy of simultaneous activation of the IL-10 and IL-1 receptor antagonist (IL1RN) genes, both of which elicit anti-inflammatory effects, using CRISPR/Cas9-mediated epigenome editing <i>in vitro</i>. <h3>Methods</h3> gRNAs were designed to target the promoter regions of the IL-10 or IL1RN genes and expressed along with the Cas9 activator (Fig. A) in the rat lung epithelial cell line (L2). The concentrations of IL-10 and IL1RN in the culture supernatant were measured 48 hours after plasmid transfection (n=3). Cells were assessed in four groups; No gRNA (expressing Cas9 activator alone), IL-10 activating (expressing Cas9 activator and two gRNAs for the IL-10 gene), IL-10+IL1RN activating (expressing Cas9 activator and two gRNAs for the IL-10 gene and two gRNAs for the IL1RN gene), and negative control (no plasmid). <h3>Results</h3> In L2 cells (Fig. B), the IL-10 activating group showed increased IL-10 (2353 ± 551 pg/ml) compared to nearly undetectable in the no gRNA and negative control groups. The IL-10+IL1RN activating group increased IL1RN (769 ± 104 pg/ml vs 8 ± 9.5 pg/ml, p=0.0023) compared to the IL-10 activating group, accompanied by a retained increase in IL-10 (2047 ± 493 pg/ml). Similar observations were found in primary lung fibroblasts cells (data not shown) supporting the efficacy of this strategy in diverse cell types. <h3>Conclusion</h3> We have achieved simultaneous activation of the IL-10 and IL1RN genes through epigenome editing using CRISPR/Cas9 <i>in vitro</i>. This study demonstrates the potential of manipulating the expression of multiple genes using DNA targeting via CRISPR/Cas9 for engineering optimized organs for transplant.

Transcriptional Downregulation of a Type III Secretion System under Reducing Conditions in Bordetella pertussis.

Bordetella pertussis uses a type III secretion system (T3SS) to inject virulence proteins into host cells. Although the B. pertussis T3SS was presumed to be involved in host colonization, efficient secretion of type III secreted proteins from B. pertussis has not been observed. To investigate the roles of type III secreted proteins during infection, we attempted to optimize culture conditions for the production and secretion of a type III secreted protein, BteA, in B. pertussis We observed that B. pertussis efficiently secretes BteA in ascorbic acid-depleted (AsA-) medium. When L2 cells, a rat lung epithelial cell line, were infected with B. pertussis cultured in the AsA- medium, BteA-dependent cytotoxicity was observed. We also performed an immunofluorescence assay of L2 cells infected with B. pertussis Clear fluorescence signals of Bsp22, a needle structure of T3SS, were detected on the bacterial surface of B. pertussis cultured in the AsA- medium. Since ascorbic acid is known as a reducing agent, we cultured B. pertussis in liquid medium containing other reducing agents such as 2-mercaptoethanol and dithioerythritol. Under these reducing conditions, the production of type III secreted proteins was repressed. These results suggest that in B. pertussis, the production and secretion of type III secreted proteins are downregulated under reducing conditions.IMPORTANCE The type III secretion system (T3SS) of Bordetella pertussis forms a needlelike structure that protrudes from the bacterial cell surface. B. pertussis uses a T3SS to translocate virulence proteins called effectors into host cells. The culture conditions for effector production in B. pertussis have not been investigated. We attempted to optimize culture medium compositions for producing and secreting type III secreted proteins. We found that B. pertussis secretes type III secreted proteins in reducing agent-deprived liquid medium and that BteA-secreting B. pertussis provokes cytotoxicity against cultured mammalian cells. These results suggest that redox signaling is involved in the regulation of B. pertussis T3SS.

Long non-coding RNA MALAT1 targeting STING transcription promotes bronchopulmonary dysplasia through regulation of CREB.

Bronchopulmonary dysplasia (BPD) is a severe complication of preterm infants characterized by increased alveolarization and inflammation. Premature exposure to hyperoxia is believed to be a key contributor to the pathogenesis of BPD. No effective preventive or therapeutic agents have been created. Stimulator of interferon gene (STING) is associated with inflammation and apoptosis in various lung diseases. Long non‐coding RNA MALAT1 has been reported to be involved in BPD. However, how MALAT1 regulates STING expression remains unknown. In this study, we assessed that STING and MALAT1 were up‐regulated in the lung tissue from BPD neonates, hyperoxia‐based rat models and lung epithelial cell lines. Then, using the flow cytometry and cell proliferation assay, we found that down‐regulating of STING or MALAT1 inhibited the apoptosis and promoted the proliferation of hyperoxia‐treated cells. Subsequently, qRT‐PCR, Western blotting and dual‐luciferase reporter assays showed that suppressing MALAT1 decreased the expression and promoter activity of STING. Moreover, transcription factor CREB showed its regulatory role in the transcription of STING via a chromatin immunoprecipitation. In conclusion, MALAT1 interacts with CREB to regulate STING transcription in BPD neonates. STING, CREB and MALAT1 may be promising therapeutic targets in the prevention and treatment of BPD.

Metabolomics profiling to investigate nanomaterial toxicity in vitro and in vivo

Nanomaterials (NMs) can be produced in plenty of variants posing several challenges for NM hazard and risk assessment. Metabolomic profiling of NM-treated cells and tissues allows for insights into underlying Mode-of-Action (MoA) and offers several advantages in this context. It supports the description of Adverse Outcome Pathways (AOPs) and, therefore, tailored AOP-based hazard testing strategies. Moreover, it bears great potential for biomarker discovery supporting toxicity prediction. Here, we applied metabolomics profiling to cells treated with four well-selected SiO2 variants, differing in structure, size and surface charge. TiO2 NM-105 served as a benchmark. Responses were studied in vitro in rat lung epithelial cells (RLE-6TN) and alveolar macrophages (NR8383) and compared to in vivo responses in rat lung tissues obtained from in vivo instillation and short-term inhalation studies (STIS). Time- and concentration-dependent changes were observed in both in vitro models but with cell-type specific responses. Overall, the levels of lipids and biogenic amines (BAs) tended to increase in epithelial cells but decreased in macrophages. Many identified metabolites like Met-SO, hydroxy-Pro and spermidine were related to oxidative stress, indicating that oxidative stress contributes to the MoA for the selected NMs. Several biomarker candidates such as Asp, Asn, Ser, Pro, spermidine, putrescine and LysoPCaC16:1 were identified in vitro and verified in vivo. In this study, we successfully applied a metabolomics workflow for in vitro and in vivo samples, which proved to be well suited to identify potential biomarkers, to gain insights into NM structure–activity relationship and into the underlying MoA.

LiCoO2 particles used in Li-ion batteries induce primary mutagenicity in lung cells via their capacity to generate hydroxyl radicals

BackgroundLi-ion batteries (LIB) are used in most portable electronics. Among a wide variety of materials, LiCoO2 (LCO) is one of the most used for the cathode of LIB. LCO particles induce oxidative stress in mouse lungs due to their Co content, and have a strong inflammatory potential. In this study, we assessed the mutagenic potential of LCO particles in lung cells in comparison to another particulate material used in LIB, LTO (Li4Ti5O12), which has a low inflammatory potential compared to LCO particles.ResultsWe assessed the mutagenic potential of LCO and LTO particles in vitro by performing a cytokinesis-block micronucleus (MN) assay with rat lung epithelial cells (RLE), as well as in vivo in alveolar type II epithelial (AT-II) cells. LCO particles induced MN in vitro at non-cytotoxic concentrations and in vivo at non-inflammatory doses, indicating a primary genotoxic mechanism. LTO particles did not induce MN. Electron paramagnetic resonance and terephthalate assays showed that LCO particles produce hydroxyl radicals (•OH). Catalase inhibits this •OH production. In an alkaline comet assay with the oxidative DNA damage repair enzyme human 8-oxoguanine DNA glycosylase 1, LCO particles induced DNA strand breaks and oxidative lesions. The addition of catalase reduced the frequency of MN induced by LCO particles in vitro.ConclusionsWe report the mutagenic activity of LCO particles used in LIB in vitro and in vivo. Our data support the role of Co(II) ions released from these particles in their primary genotoxic activity which includes the formation of •OH by a Fenton-like reaction, oxidative DNA lesions and strand breaks, thus leading to chromosomal breaks and the formation of MN. Documenting the genotoxic potential of the other LIB particles, especially those containing Co and/or Ni, is therefore needed to guarantee a safe and sustainable development of LIB.

Carbon black nanoparticles induce pulmonary fibrosis through NLRP3 inflammasome pathway modulated by miR-96 targeted FOXO3a.

Carbon black nanoparticle (CBNP) is a core constituent of air pollutants like fine particulate matter (PM2.5) as well as a common manufactural material. It was proved to pose adverse effects on lung function and even provoke pulmonary fibrosis. However, the underlying mechanisms of CBNPs-induced pulmonary fibrosis remain unclear. The present study aimed to investigate the mechanism of fibrotic effects caused by CBNPs in rat lung and human bronchial epithelial (16HBE) cells. Forty-nine male rats were randomly subjected to 7 groups, means the 14-day exposure group (30 mg/m3), the 28-day exposure groups (5 mg/m3 and 30 mg/m3), the 90-day exposure group (30 mg/m3) and their respective controls. Rats were nose-only-inhaled CBNPs. 16HBE cells were treated with 0, 50, 100 and 200 μg/mL CBNPs respectively for 24 h. Besides, Forkhead transcription factor class O (FOXO)3a and miR-96 overexpression or suppression 16HBE cells were established to reveal relative mechanisms. Our results suggested CBNPs induced pulmonary fibrosis in time- and dose-dependent manners. CBNPs induced persisting inflammation in rat lung as observed by histopathology and cytology analyses in whole lung lavage fluid (WLL). Both invivo and invitro, CBNPs exposure significantly increased the expression of NLRP3 inflammasome, accompanied by the increased reactive oxygen species (ROS), decreased miR-96 and increased FOXO3a expressions dose -and time-dependently. MiR-96 overexpression or FOXO3a suppression could partially rescue the fibrotic effects through inhibiting NLRP3 inflammasome. Conclusively, our research show that CBNPs-induced pulmonary fibrosis was at least partially depended on activation of NLRP3 inflammasome which modulated by miR-96 targeting FOXO3a.

Acid Sphingomyelinase Inhibition Attenuates Cell Death in Mechanically Ventilated Newborn Rat Lung.

Premature infants subjected to mechanical ventilation (MV) are prone to lung injury that may result in bronchopulmonary dysplasia. MV causes epithelial cell death and halts alveolar development. The exact mechanism of MV-induced epithelial cell death is unknown. To determine the contribution of autophagy to MV-induced epithelial cell death in newborn rat lungs. Newborn rat lungs and fetal rat lung epithelial (FRLE) cells were exposed to MV and cyclic stretch, respectively, and were then analyzed by immunoblotting and mass spectrometry for autophagy, apoptosis, and bioactive sphingolipids. Both MV and stretch first induce autophagy (ATG 5-12 [autophagy related 5-12] and LC3B-II [microtubule-associated proteins 1A/1B light chain 3B-II] formation) followed by extrinsic apoptosis (cleaved CASP8/3 [caspase-8/3] and PARP [poly(ADP-ribose) polymerase] formation). Stretch-induced apoptosis was attenuated by inhibiting autophagy. Coimmunoprecipitation revealed that stretch promoted an interaction between LC3B and the FAS (first apoptosis signal) cell death receptor in FRLE cells. Ceramide levels, in particular C16 ceramide, were rapidly elevated in response to ventilation and stretch, and C16 ceramide treatment of FRLE cells induced autophagy and apoptosis in a temporal pattern similar to that seen with MV and stretch. SMPD1 (sphingomyelin phosphodiesterase 1) was activated by ventilation and stretch, and its inhibition prevented ceramide production, LC3B-II formation, LC3B/first apoptosis signal interaction, caspase-3 activation, and, ultimately, FLRE cell death. SMPD1 inhibition also attenuated ventilation-induced autophagy and apoptosis in newborn rats. Ventilation-induced ceramides promote autophagy-mediated cell death, and identifies SMPD1 as a potential therapeutic target for the treatment of ventilation-induced lung injury in newborns.

Binding of Pneumocystis carinii to the lung epithelial cell receptor HSPA5 (GRP78).

The importance of lung macrophages in Pneumocystis-host interaction is well known, but little is known about the initial binding/colonization of the airway epithelium. Our prior studies have documented cell-signalling events that occur following binding of the organisms to lung epithelial cells; however, the receptors that mediate Pneumocystis attachment to lung surfaces have not yet been fully defined. Using affinity chromatography, we identified heat shock protein 5 (HSPA5), also known as GRP78, as a potential host receptor that may have relevance in Pneumocystis lung colonization. Pneumocystis carinii (Pc) organisms not only bound HSPA5 on a rat lung epithelial cell line, but also on primary rat airway epithelial cells (AECs). Furthermore, Pc bound CHO1 cells overexpressing HSPA5 more than the CHO1 parent line alone, supporting a role for Pc-HSPA5 protein interaction in mediating organism attachment. These results provide new insights into the interactions of Pneumocystis with host lung epithelium.

Abstract A35: Vesicular secretion of suppressor of cytokine signaling 3 by alveolar macrophages is dysregulated in NSCLC and its provision inhibits tumor cell function

Abstract Rationale: Inadequate expression of suppressor of cytokine signaling 3 (SOCS3) with subsequent activation of its target, the transcription factor STAT3, has been implicated in tumorigenesis and cancer progression in the lung and other organs. Our lab has recently reported the novel capability of alveolar macrophages (AMs) to secrete SOCS3 within microvesicles (MVs). While AM delivery of MV-encapsulated SOCS3 was shown to suppress inflammatory signaling in recipient lung epithelial cells, the potential significance of this process in restraining the development of lung cancer has not been studied. Methods: A KRAS G12D mutant mouse model was utilized to determine dysfunction of AM SOCS3 secretion in lung cancer. Mice were administered adenoviral Cre recombinase via intratracheal instillation, resulting in formation of lung tumors after 16 weeks. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from the lungs of KRAS and wild-type (WT) mice, and analysis of SOCS3 secretion in BAL or AM cell culture medium was done via ELISA after sonication to disrupt vesicles. In vitro experiments utilized human adenocarcinoma cells (A549) or KRAS mutant rat lung epithelial cells (RLE-G12V). Proliferation, apoptosis and transformation were assessed by Cyquant assay, Annexin V staining, and soft agar assay, respectively. For SOCS3 provision studies, natural AM-derived MVs (isolated by ultracentrifugation) or synthetic liposomes containing recombinant SOCS3 were utilized. Results: Levels of secreted SOCS3 were ~50% lower in KRAS mice BAL than in WT BAL fluid. Additionally, although AMs isolated from KRAS mice contained similar amounts of intracellular SOCS3 and released similar numbers of MVs as those from WT mice, their ex vivo capacity for SOCS3 secretion was significantly lower than that of WT AMs. To determine whether provision of exogenous SOCS3 could inhibit tumorigenesis, synthetic SOCS3 liposomes were administered to RLE-G12V cells prior to chemical transformation with N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG). Addition of exogenous SOCS3 had the capacity to significantly inhibit colony formation in soft agar. To investigate effects of exogenous SOCS3 on established tumor cell function, A549 cells were exposed to both natural AM-derived MVs and SOCS3-containing liposomes and effects on proliferation and apoptosis were measured. Both liposomes and natural MVs significantly induced apoptosis and inhibited proliferation. Finally, the reduction in secreted SOCS3 observed in the mouse model was confirmed in BAL samples of a cohort of NSCLC lung cancer patients compared to healthy volunteers. Conclusion: We report a novel dysregulation of immune surveillance in the form of decreased SOCS3 secretion by AMs that is elicited by the tumor microenvironment, and that may promote tumorigenesis via sustained STAT3 activation. Future studies will focus on the mechanism underlying this defect and whether rescuing SOCS3 secretion can inhibit cancer progression in vivo. Citation Format: Jennifer M. Speth, Loka R. Penke, Joseph Bazzill, Daniel J. Schneider, Douglas A. Arenberg, James J. Moon, Venkateshwar G. Keshamouni, Marc Peters-Golden. Vesicular secretion of suppressor of cytokine signaling 3 by alveolar macrophages is dysregulated in NSCLC and its provision inhibits tumor cell function [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A35.

Enzymatic Synthesis, Characterization and Biocompatibility Studies of Cellulose Nanoparticles from Cotton Fibers

Interest in cellulose nanoparticles has been increasing exponentially in the past few decades due to its unique characteristics such as reinforcement properties, high tensile strength, and excellent thermal and electrical properties. Cellulose nanoparticles were produced by an enzymatic method including hydrolysis of cotton fibers by cellulase enzyme and sonication process. Further, cellulose nanoparticles were characterized to determine the morphology and purity of the material. Characterization of cellulose nanoparticles was performed by Scanning Electron Microscope (SEM) with Energy Dispersive X-Ray Spectroscopy (EDX) and Fourier Transform Infrared Spectroscopy (FT-IR). Biocompatibility studies of cellulose nanoparticles were carried out by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and Live/Dead viability assay. Using SEM, the average size of cellulose nanoparticles was found to be around 100-125nm and the particles were spherical in shape. FTIR spectrum showed the formation of cellulose nanoparticles from cotton fibers without any presence of impurities. MTT assay and Live/Dead viability assay showed no significant induction of cell death even at higher concentrations (100 μg) upon exposure to Rat Lung Epithelial cells. The results revealed that the synthesized cellulose nanoparticles could be used in wide range of emerging applications in the development of new energy storage devices, enzyme immobilization, antimicrobial and medical materials, green catalysis, bio-sensing and controlled drug delivery.

2165 Vesicular secretion of suppressor of cytokine signaling 3 by alveolar macrophages is dysregulated in NSCLC patients and its provision inhibits epithelial cell transformation and tumor cell function

OBJECTIVES/SPECIFIC AIMS: Insufficient endogenous expression of suppressor of cytokine signaling 3 (SOCS3) with subsequent over-activation of its target, the transcription factor STAT3, has been associated with tumorigenesis and cancer development in the lung and other organs. We have observed that a “backup” source of SOCS3 in the lung, namely that secreted in microvesicles (MVs) by alveolar macrophages, is reduced in the bronchoalveolar lavage fluid (BALF) of KRAS mutant mice harboring lung tumors. Here we sought to evaluate levels of SOCS3 in BALF of a cohort of non-small cell lung cancer (NSCLC) patients and to test the effects of vesicular SOCS3 administration on tumor cell transformation and function as potential therapeutic strategy. METHODS/STUDY POPULATION: In total, 22 BALF samples were obtained from healthy volunteers (n=11) as well as patients undergoing diagnostic bronchoscopies for suspected lung cancer (n=11). SOCS3 levels in the BALF were determined by ELISA after brief sonication to disrupt vesicles. In vitro experiments utilized the human adenocarcinoma cell line (A549) or human G12V mutant KRAS-expressing rat lung epithelial cells (RLE-G12V). Proliferation, Fas ligand (FasL)-induced apoptosis, and chemical transformation with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or cigarette smoke extract (CSE) were assessed by CyQuant assay, annexin V staining, and soft agar assay, respectively. For SOCS3 rescue, epithelial cells were treated with natural alveolar macrophages-derived MVs (isolated via ultracentrifugation) or synthetic unilamellar liposomes containing human recombinant SOCS3 for at least 1 hour before assay. RESULTS/ANTICIPATED RESULTS: SOCS3 levels were significantly reduced in BALF samples of patients determined to have NSCLC as compared with healthy volunteers (186.6±26.74 vs. 395.6±74.31 pg/mL, p=0.015, n=11). Addition of exogenous SOCS3-containing liposomes had the capacity to significantly inhibit MNNG and cigarette smoke extract-induced transformation and colony formation in soft agar. Exogenous SOCS3 provided in liposomes or in natural MVs significantly induced apoptosis (both in the presence and absence of FasL) and inhibited basal proliferation of A549 cells. DISCUSSION/SIGNIFICANCE OF IMPACT: These data identified a novel dysregulation of immune surveillance in the form of decreased SOCS3 secretion in the tumor-bearing lung that may contribute to tumorigenesis via sustained STAT3 activation. Future studies will focus on the mechanism underlying this defect and whether rescuing SOCS3 secretion can inhibit cancer progression in vivo.