Knockout Lung Research Articles

Adam19 Deficiency Impacts Pulmonary Function: Human GWAS Follow-up in a Mouse Knockout Model

PurposeOver 550 loci have been associated with human pulmonary function in genome-wide association studies (GWAS); however, the causal role of most remains uncertain. Single nucleotide polymorphisms in a disintegrin and metalloprotease domain 19 (ADAM19) are consistently related to pulmonary function in GWAS. Thus, we used a mouse model to investigate the causal link between Adam19 and pulmonary function.MethodsWe created an Adam19 knockout (KO) mouse model and validated the gene targeting using RNA-Seq and RT-qPCR. Mouse body composition was assessed using dual-energy X-ray absorptiometry. Mouse lung function was measured using flexiVent.ResultsContrary to prior publications, the KO was not neonatal lethal. KO mice had lower body weight and shorter tibial length than wild-type (WT) mice. Their body composition revealed lower soft weight, fat weight, and bone mineral content. Adam19 KO had decreased baseline respiratory system elastance, minute work of breathing, tissue damping, tissue elastance, and forced expiratory flow at 50% forced vital capacity but higher FEV0.1 and FVC. Adam19 KO had attenuated tissue damping and tissue elastance in response to methacholine following LPS exposure. Adam19 KO also exhibited attenuated neutrophil extravasation into the airway after LPS administration compared to WT. RNA-Seq analysis of KO and WT lungs identified several differentially expressed genes (Cd300lg, Kpna2, and Pttg1) implicated in lung biology and pathogenesis. Gene set enrichment analysis identified negative enrichment for TNF pathways.ConclusionOur murine findings support a causal role of ADAM19, implicated in human GWAS, in regulating pulmonary function.

Targeting CEBPA to restore cellular identity and tissue homeostasis in pulmonary fibrosis.

Fibrosis in the lung is thought to be driven by epithelial cell dysfunction and aberrant cell-cell interactions. Unveiling the molecular mechanisms of cellular plasticity and cell-cell interactions is imperative to elucidating lung regenerative capacity and aberrant repair in pulmonary fibrosis. By mining publicly available RNA-Seq data sets, we identified loss of CCAAT enhancer-binding protein alpha (CEBPA) as a candidate contributor to idiopathic pulmonary fibrosis (IPF). We used conditional KO mice, scRNA-Seq, lung organoids, small-molecule inhibition, and potentially novel gene manipulation methods to investigate the role of CEBPA in lung fibrosis and repair. Long-term (6 months or more) of Cebpa loss in AT2 cells caused spontaneous fibrosis and increased susceptibility to bleomycin-induced fibrosis. Cebpa knockout (KO) in these mice significantly decreased AT2 cell numbers in the lung and reduced expression of surfactant homeostasis genes, while increasing inflammatory cell recruitment as well as upregulating S100a8/a9 in AT2 cells. In vivo treatment with an S100A8/A9 inhibitor alleviated experimental lung fibrosis. Restoring CEBPA expression in lung organoids ex vivo and during experimental lung fibrosis in vivo rescued CEBPA deficiency-mediated phenotypes. Our study establishes a direct mechanistic link between CEBPA repression, impaired AT2 cell identity, disrupted tissue homeostasis, and lung fibrosis.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts.

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study using genetic mouse models, we dissected the roles of bone morphogenetic protein (BMP) receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study using genetic mouse models, we dissected the roles of bone morphogenetic protein (BMP) receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

CAMSAP3-mediated regulation of HMGB1 acetylation and subcellular localization in lung cancer cells: Implications for cell death modulation

BackgroundDeregulation of cell death is a common characteristic of cancer, and resistance to this process often occurs in lung cancer. Understanding the molecular mechanisms underlying an aberrant cell death is important. Recent studies have emphasized the involvement of calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) in lung cancer aggressiveness, its influence on cell death regulation remains largely unexplored. MethodsCAMSAP3 was knockout in lung cancer cells using CRISPR-Cas9 system. Cell death and autophagy were evaluated using MTT and autophagic detection assays. Protein interactions were performed by proteomic analysis and immunoprecipitation. Protein expressions and their cytoplasmic localization were analyzed through immunoblotting and immunofluorescence techniques. ResultsThis study reveals a significant correlation between low CAMSAP3 expression and poor overall survival rates in lung cancer patients. Proteomic analysis identified high mobility group box 1 (HMGB1) as a candidate interacting protein involved in the regulation of cell death. Treatment with trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs) resulted in increased HMGB1 acetylation and its translocation to the cytoplasm and secretion, thereby inducing autophagic cell death. However, this process was diminished in CAMSAP3 knockout lung cancer cells. Mechanistically, immunoprecipitation indicated an interaction between CAMSAP3 and HMGB1, particularly with its acetylated form, in which this complex was elevated in the presence of TSA. ConclusionsCAMSAP3 is prerequisite for TSA-mediated autophagic cell death by interacting with cytoplasmic acetylated HMGB1 and enhancing its release. SignificantThis finding provides molecular insights into the role of CAMSAP3 in regulating cell death, highlighting its potential as a therapeutic target for lung cancer treatment.

Transcriptomic analysis of glucosidase II beta subunit (GluIIß) knockout A549 cells reveals its roles in regulation of cell adhesion molecules (CAMs) and anti-tumor immunity

Glucosidase II beta subunit (GluIIß), encoded from PRKCSH, is a subunit of the glucosidase II enzyme responsible for quality control of N-linked glycoprotein folding and suppression of GluIIß led to inhibitory effect of the receptor tyrosine kinase (RTKs) activities known to be critical for survival and development of cancer. In this study, we investigated the effect of GluIIß knockout on the global gene expression of cancer cells and its impact on functions of immune cells. GluIIß knockout lung adenocarcinoma A549 cell line was generated using CRISPR/Cas9-based genome editing system and subjected to transcriptomic analysis. Among 23,502 expressed transcripts, 1068 genes were significantly up-regulated and 807 genes greatly down-regulated. The KEGG enrichment analysis showed significant down-regulation of genes related extracellular matrix (ECM), ECM-receptor interaction, cytokine-cytokine receptor interaction and cell adhesion molecules (CAMs) in GluIIß knockout cells. Of 9 CAMs encoded DEG identified by KEGG enrichment analysis, real time RT-PCR confirmed 8 genes to be significantly down-regulated in all 3 different GluIIß knockout clones, which includes cadherin 4 (CDH4), cadherin 2 (CDH2), versican (VCAN), integrin subunit alpha 4 (ITGA4), endothelial cell-selective adhesion molecule (ESAM), CD274 (program death ligand-1 (PD-L1)), Cell Adhesion Molecule 1 (CADM1), and Nectin Cell Adhesion Molecule 3 (NECTIN3). Whereas PTPRF (Protein Tyrosine Phosphatase Receptor Type F) was significantly decreased only in 1 out of 3 knockout clones. Microscopic analysis revealed distinctively different cell morphology of GluIIβ knockout cells with lesser cytoplasmic and cell surface area compared to parental A549 cells and non-targeted transfected cells.Further investigations revealed that Jurkat E6.1 T cells or human peripheral blood mononuclear cells (PBMCs) co-cultured with GluIIß knockout A549 exhibited significantly increased viability and tumor cell killing activity compared to those co-cultured with non-target transfected cells. Analysis of cytokine released from Jurkat E6.1 T cells co-cultured with GluIIß knockout A549 cells showed significant increased level of angiogenin and significant decreased level of ENA-78. In conclusion, knockout of GluIIß from cancer cells induced altered gene expression profile that improved anti-tumor activities of co-cultured T lymphocytes and PBMCs thus suppression of GluIIß may represent a novel approach of boosting anti-tumor immunity.

ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to spread around the world with serious cases and deaths. It has also been suggested that different genetic variants in the human genome affect both the susceptibility to infection and severity of disease in COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) has been identified as a cell surface receptor for SARS-CoV and SARS-CoV-2 entry into cells. The construction of an experimental model system using human iPS cells would enable further studies of the association between viral characteristics and genetic variants. Airway and alveolar epithelial cells are cell types of the lung that express high levels of ACE2 and are suitable for in vitro infection experiments. Here, we show that human iPS cell-derived airway and alveolar epithelial cells are highly susceptible to viral infection of SARS-CoV-2. Using gene knockout with CRISPR-Cas9 in human iPS cells we demonstrate that ACE2 plays an essential role in the airway and alveolar epithelial cell entry of SARS-CoV-2 in vitro. Replication of SARS-CoV-2 was strongly suppressed in ACE2 knockout (KO) lung cells. Our model system based on human iPS cell-derived lung cells may be applied to understand the molecular biology regulating viral respiratory infection leading to potential therapeutic developments for COVID-19 and the prevention of future pandemics.

Fgf9-Nolz-1-Wnt2 Axis Regulates Morphogenesis of the Lung.

Morphological development of the lung requires complex signal crosstalk between the mesenchymal and epithelial progenitors. Elucidating the genetic cascades underlying the signal crosstalk is essential to understanding lung morphogenesis. Here, we identified Nolz-1/Znf503 as a mesenchymal lineage-specific transcriptional regulator that plays a key role in lung morphogenesis. Nolz-1 null mutation resulted in a severe hypoplasia phenotype, including a decreased proliferation of mesenchymal cells, aberrant differentiation of epithelial cells, and defective growth of epithelial branches. Nolz-1 deletion also downregulated Wnt2, Lef1, Fgf10, Gli3 and Bmp4 mRNAs. Mechanistically, Nolz-1 regulated lung morphogenesis primarily through Wnt2 signaling. Loss of function and overexpression studies demonstrated that Nolz-1 transcriptionally activated Wnt2 and downstream β-catenin signaling to control mesenchymal cell proliferation and epithelial branching. Exogenous Wnt2 could causally rescue defective proliferation and epithelial branching in Nolz-1 knockout lungs. Finally, we identified Fgf9 as an upstream regulator of Nolz-1. Collectively, Fgf9-Nolz-1-Wnt2 signaling represents a novel axis in the control of lung morphogenesis. These findings are relevant to lung tumorigenesis in which a pathological function of Nolz-1 is implicated.

ADAM9 drives immunosuppressive environment for lung tumor progression

Abstract Cancer cells develop the immune suppressive tumor environment, but the underlying molecular mechanism for it is still elusive. Here, we demonstrated that ADAM9 contributed to lung tumor progression and promoted IFN-related gene expression in lung cancer cells that participated in immune regulation in the tumor environment. ADAM9 knockout (KO) lung cancer cells reduced lung cancer metastasis with a decrease in neutrophil infiltration and an increase in CD8+ T cell infiltration. By performing the genome-wide approach to explore ADAM9-regulated genes in lung cancer cells, we revealed that ADAM9 regulated cytokine production and lymphocyte activation. It enhanced the master transcription factor of IFN signaling, STAT1 activation in lung cancer cells. Moreover, ADAM9 promoted IL6/IL6 receptor-STAT3 axis signaling that lead to suppression of IL12p40 secretion and MHC class II expression in dendritic cells and macrophages. Notably, we found that type I IFN related-proteins and steroid biosynthesis-related proteins were related to ADAM9-mediated STAT signaling activation by LC-MS/MS analysis. We demonstrated that ADAM9 created an immune suppressive environment by regulating IFN signaling for tumor progression. Supported by grants from NHRI (NHRI-EX112-11219BI)

Abstract 313: Mechanisms of oncogenic tyrosine kinase fusion formation and selection in human cancers

Abstract Introduction: Tyrosine kinase (TK) fusions are common events in the development of resistance to targeted therapy for non-small cell lung cancer (NSCLC) and others. The mechanisms by which recurrent breakpoints are generated in solid tumors and by which they are selected are unclear. APOBEC3 proteins are the main factors for mutations in solid cancers and the mutagenic activity of APOBEC3 can activate the DNA damage response by contributing to double-strand break (DSB) formation. However, the possibility that APOBEC3 proteins could induce chromosomal translocations in solid tumors has not been investigated. In addition, mechanistic explanations on how recurrent chromosomal translocation occur in lung cancer and solid tumors in precise genomic breakpoints and with specific partners are lacking. In this work, we present a newly developed functionally active chromosomal translocation sequencing (FACTS) approach to provide insights on the mechanisms that determine the selection of oncogenic fusions in human cancers. Methods: We applied FACTS to lung cells to map genome-wide translocations when DNA DSB is introduced in TK genes by the CRISPR/Cas9 system. We generated genomic maps of DNA breakpoints joining to anaplastic lymphoma kinase (ALK) before and after selection by high-throughput genome-wide translocation sequencing (HTGTS). We tested the effect of APOBEC3 proteins in an overexpression system or in an all seven APOBEC3 gene knockout lung cells by FACTS. Results: Overexpression of APOBEC3 proteins significantly increased the formation of functional fusions compared to control cells. In addition, deletion of APOBEC3 genes significantly decreased the formation of functional fusions compared to APOBEC3 wild-type cells. With the FACTS data and bioinformatics analyses, we found that gene expression rather than locus accessibility dictates the selection of TK fusions and that TK fusions are likely selected from a pool of translocations occurring at the right position in transcribe genes. In addition, we found that active transcription in silent genes was sufficient to introduce the spontaneous formation of functional fusions. By using HTGTS approach, we found that breakpoints before selection did not show a preferential strand bias, but after selection the breakpoints showed a strong bias. By comparing oncogenic potential of EML4-ALK fusion variants, we found that protein stability, but not subcellular localization, is key to determining the exon usage of EML-ALK fusions. In addition, we revealed that the selection of fusion partners for each TK is more likely dictated by protein stability rather than by mRNA expression. Conclusion: FACTS allows rapid and comprehensive mapping of functional chromosomal translocations and provides insights into the mechanisms of their formation and selection. The FACTS approach could be used to predict the oncogenic activity of fusion proteins to guide clinical decisions. Citation Format: Taek-Chin Cheong, Ahram Jang, Qi Wang, Giulia C. Leonardi, Maria K. Lehtinen, Marian H. Harris, Roberto Chiarle. Mechanisms of oncogenic tyrosine kinase fusion formation and selection in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 313.

SODD Promotes Lung Cancer Tumorigenesis by Activating the PDK1/AKT and RAF/MEK/ERK Signaling.

The Bcl2-associated athanogene4 (BAG4/SODD) protein could be identified as a tumor marker for several malignancies and plays a major role in the occurrence, development, and drug resistance of tumors. However, the role of Silencer of death domains (SODD) in lung carcinogenesis is still elusive. To illuminate the effect of SODD on the proliferation, migration, invasion, and apoptosis of lung cancer cells and tumor growth in vivo and explore the corresponding mechanism. The expression of SODD in tumor and normal tissues was determined and compared via western blot. SODD gene knockout lung cancer cells (H1299 cells) were established through a CRISPR/Cas9 gene deleting system, and a transient SODD overexpression of H1299 cells was also constructed. Then, cell proliferation and invasion were assessed through colony formation and cell counting kit-8 assays, transwell migration assays, and wound healing assays. Cell drug sensitivity is also analyzed by Cell Counting Kit-8 assay. The flow cytometer was used to perform cell circle and apoptosis analysis. The interaction of SODD and RAF-1 was confirmed by co-immunoprecipitation, and the phosphorylated level of Phosphatidylinositol 3-kinase (PI3K), Serine/threonine-protein kinase (AKT), Rapidly accelerated fibrosarcoma (RAF)-1,and extracellular signal regulated kinase (ERK) in cells was examined by western blot to evaluate the activation of PI3K/PDK1/AKT and RAF/MEK/ERK pathways. In vivo, Xenograft tumor assay of SODD knockout H1299 cells was used to evaluate further the role of SODD on the proliferation of H1299 cells. SODD binds to RAF-1 and is over-expressed in lung tissues, and promotes the proliferation, migration, invasion, and drug sensitivity of H1299 cells. The reduced cells in the S phase and increased cells arrested in the G2/M phase were found in SODD knockout H1299 cells, and more cells got apoptosis. The expression of 3-phosphoinositide-dependent protein kinase 1(PDK1) protein in SODD knockout H1299 cells decreases distinctively, and the phosphorylated level of AKT, RAF-1, and ERK-1 kinase in SODD knockout H1299 cells is also less than that in normal H1299 cells. In contrast, SODD overexpression significantly increases the phosphorylation of AKT. In vivo, SODD promotes the tumorigenicity of H1299 cells in nude mice. SODD is overexpressed in lung tissues and plays a considerable role in the development and progression of lung cancer by regulating the PI3K/PDK1/AKT and RAF/MEK/ERK pathways.

Dipeptidyl peptidase 4 expression is not associated with an activated fibroblast phenotype in idiopathic pulmonary fibrosis.

Dipeptidyl peptidase 4 (DPP4) has been proposed as a marker for activated fibroblasts in fibrotic disease. We aimed to investigate whether a profibrotic DPP4 phenotype is present in lung tissue from patients with idiopathic pulmonary fibrosis (IPF). The presence of DPP4+ fibroblasts in normal and IPF lung tissue was investigated using flow cytometry and immunohistology. In addition, the involvement of DPP4 in fibroblast activation was examined in vitro, using CRISPR/Cas9 mediated genetic inactivation to generate primary DPP4 knockout lung fibroblasts. We observed a reduced frequency of primary DPP4+ fibroblasts in IPF tissue using flow cytometry, and an absence of DPP4+ fibroblasts in pathohistological features of IPF. The in vivo observations were supported by results in vitro showing a decreased expression of DPP4 on normal and IPF fibroblasts after profibrotic stimuli (transforming growth factor β) and no effect on the expression of activation markers (α-smooth muscle actin, collagen I and connective tissue growth factor) upon knockout of DPP4 in lung fibroblasts with or without activation with profibrotic stimuli.

Kub3 Deficiency Causes Aberrant Late Embryonic Lung Development in Mice by the FGF Signaling Pathway

As a Ku70-binding protein of the KUB family, Kub3 has previously been reported to play a role in DNA double-strand break repair in human glioblastoma cells in glioblastoma patients. However, the physiological roles of Kub3 in normal mammalian cells remain unknown. In the present study, we generated Kub3 gene knockout mice and revealed that knockout (KO) mice died as embryos after E18.5 or as newborns immediately after birth. Compared with the lungs of wild-type (WT) mice, Kub3 KO lungs displayed abnormal lung morphogenesis and pulmonary atelectasis at E18.5. No difference in cell proliferation or cell apoptosis was detected between KO lungs and WT lungs. However, the differentiation of alveolar epithelial cells and the maturation of type II epithelial cells were impaired in KO lungs at E18.5. Further characterization displayed that Kub3 deficiency caused an abnormal FGF signaling pathway at E18.5. Taking all the data together, we revealed that Kub3 deletion leads to abnormal late lung development in mice, resulting from the aberrant differentiation of alveolar epithelial cells and the immaturation of type II epithelial cells due to the disturbed FGF signaling pathway. Therefore, this study has uncovered an essential role of Kub3 in the prenatal lung development of mice which advances our knowledge of regulatory factors in embryonic lung development and provides new concepts for exploring the mechanisms of disease related to perinatal lung development.

Deletion of the miR-144/451 cluster aggravates lethal sepsis-induced lung epithelial oxidative stress and apoptosis.

BackgroundSepsis is associated with a high mortality rate. A major cause of death in sepsis patients is respiratory failure, which is characterized by oxidative injury, epithelial apoptosis, and increased lung permeability. MicroRNAs (miRs) are important regulators of sepsis progression.MethodsThis study aimed to explore the role of miR-144/451 in sepsis in mice. Experimental sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP).ResultsCLP significantly induced systemic inflammation, lung permeability, and lung epithelial apoptosis with downregulated messenger RNA (mRNA) levels of antioxidant enzymes. The miR-144/451 knockout mice had a lower 48-hour survival rate, higher plasma tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) levels, and greater pulmonary permeability compared with wild-type mice after CLP. CLP also markedly increased interstitial hemorrhage, collapsed more alveolar sacs, and increased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive and Bcl-2-associated X (Bax)-positive cells in miR-144/451 knockout lung tissues, with elevated mRNA levels of Bax and reduced activities of catalase (Cat), glutathione peroxidase 1(Gpx1). MiR-451 negatively regulated 14-3-3ζ expression evidenced in miR-144/451 knockout lungs and the A549 cell line. In lipopolysaccharide (LPS)-induced A549 cells, miR-451 overexpression remarkably suppressed the production of reactive oxygen species, inhibited cell apoptosis, and enhanced levels of FoxO3 protein and related enzymes.ConclusionsDeletion of the miR-144/451 cluster aggravated sepsis-induced oxidative injury of lung epithelial cells.

Pig-to-baboon lung xenotransplantation: Extended survival with targeted genetic modifications and pharmacologic treatments

Galactosyl transferase knock-out pig lungs fail rapidly in baboons. Based on previously identified lung xenograft injury mechanisms, additional expression of human complement and coagulation pathway regulatory proteins, anti-inflammatory enzymes and self-recognition receptors, and knock-down of the β4Gal xenoantigen were tested in various combinations. Transient life-supporting GalTKO.hCD46 lung function was consistently observed in association with either hEPCR (n=15), hTBM (n=4), or hEPCR.hTFPI (n=11), but the loss of vascular barrier function in the xenograft and systemic inflammation in the recipient typically occurred within 24h. Co-expression of hEPCR and hTBM (n=11) and additionally blocking multiple pro-inflammatory innate and adaptive immune mechanisms was more consistently associated with survival >1day, with one recipient surviving for 31days. Combining targeted genetic modifications to the lung xenograft with selective innate and adaptive immune suppression enables prolonged initial life-supporting lung function and extends lung xenograft recipient survival, and illustrates residual barriers and candidate treatment strategies that may enable the clinical application of other organ xenografts.

Endogenous regulation of the Akt pathway by the aryl hydrocarbon receptor (AhR) in lung fibroblasts

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to mediate toxic responses to dioxin. However, the role of the AhR in the regulation of cellular physiology has only recently been appreciated, including its ability to control cell cycle progression and apoptosis by unknown mechanisms. We hypothesized that the AhR enhances the activation of the AKT serine/threonine kinase (Akt) pathway to promote cell survival. Utilizing AhR knock-out (Ahr−/−) and wild-type (Ahr+/+) mouse lung fibroblasts (MLFs), we found that Ahr−/− MLFs have significantly higher basal Akt phosphorylation but that AhR did not affect Akt phosphorylation in MLFs exposed to growth factors or AhR ligands. Basal Akt phosphorylation was dependent on PI3K but was unaffected by changes in intracellular glutathione (GSH) or p85α. There was no significant decrease in cell viability in Ahr−/− MLFs treated with LY294002—a PI3K inhibitor—although LY294002 did attenuate MTT reduction, indicating an affect on mitochondrial function. Using a mass spectrometry (MS)-based approach, we identified several proteins that were differentially phosphorylated in the Ahr−/− MLFs compared to control cells, including proteins involved in the regulation of extracellular matrix (ECM), focal adhesion, cytoskeleton remodeling and mitochondrial function. In conclusion, Ahr ablation increased basal Akt phosphorylation in MLFs. Our results indicate that AhR may modulate the phosphorylation of a variety of novel proteins not previously identified as AhR targets, findings that help advance our understanding of the endogenous functions of AhR.

Stress-Inducible Gene Atf3 Dictates a Dichotomous Macrophage Activity in Chemotherapy-Enhanced Lung Colonization.

Previously, we showed that chemotherapy paradoxically exacerbated cancer cell colonization at the secondary site in a manner dependent on Atf3, a stress-inducible gene, in the non-cancer host cells. Here, we present evidence that this phenotype is established at an early stage of colonization within days of cancer cell arrival. Using mouse breast cancer models, we showed that, in the wild-type (WT) lung, cyclophosphamide (CTX) increased the ability of the lung to retain cancer cells in the vascular bed. Although CTX did not change the WT lung to affect cancer cell extravasation or proliferation, it changed the lung macrophage to be pro-cancer, protecting cancer cells from death. This, combined with the initial increase in cell retention, resulted in higher lung colonization in CTX-treated than control-treated mice. In the Atf3 knockout (KO) lung, CTX also increased the ability of lung to retain cancer cells. However, the CTX-treated KO macrophage was highly cytotoxic to cancer cells, resulting in no increase in lung colonization—despite the initial increase in cell retention. In summary, the status of Atf3 dictates the dichotomous activity of macrophage: pro-cancer for CTX-treated WT macrophage but anti-cancer for the KO counterpart. This dichotomy provides a mechanistic explanation for CTX to exacerbate lung colonization in the WT but not Atf3 KO lung.

Knockout of FosB gene changes drug sensitivity and invasion activity via the regulation of Bcl-2, E-cadherin, β-catenin, and vimentin expression

Our previous research suggested the presence of a novel SETDB1-mediated FosB pathway that could be responsible for the regulation of cell proliferation and invasiveness during anticancer treatments. In this study, we prepared FosB knock-out (FosB-KO) A549 human lung cancer cells using the CRISPR/Cas9 system and examined the physiological and molecular changes that caused. Annexin V and TUNEL assays showed that FosB-KO clones were less sensitive to doxorubicin treatment compared to the control A549 cells. Bcl2 expression and mitochondrial membrane potential were also both markedly increased in FosB-KO clones, which suggests the involvement of Bcl2 in the doxorubicin mediated increase in cell viability demonstrated the FosB-KO clones. Moreover, we identified changes in the migration and transforming activities of the FosB-KO clones that coincided with changes in the expression levels of E-cadherin, β-catenin, and Vimentin. RT-PCR and qPCR analysis showed that the expressions of Bcl2, E-cadherin, β-catenin, and Vimentin were regulated at the transcriptional level. Importantly, FosB overexpression in FosB-KO clones restored the expression of Bcl2, Akt, E-cad, β-catenin, and Vimentin, suggesting that those proteins were tightly regulated by FosB. These data suggest that the FosB gene critically regulates both drug sensitivity and invasion related genes, and does so in a manner coordinated with the function of SETDB1. Therefore, we propose that the FosB gene regulates both drug sensitivity and invasion activity related genes, and also shows coordinated function with SETDB1 for the regulation of target proteins.

Anti-Non Gal Antibody Binding in Pig-to-Baboon and Pig-to-Human Lung Transplantation Models is Reduced by β4GalKO

Purpose Antibody-mediated xenograft rejection is inhibited by knock-out (KO) of porcine carbohydrates (CHO), including Galα1,3Gal. The β4Gal epitope was discovered using serum from xenograft-sensitized baboons. However, it remains controversial if humans and baboons have detectable titers of preformed anti-β4Gal antibody (as humans do for Neu5Gc), and whether anti-β4Gal antibody modulates lung xenograft injury. Methods In serum from 14 baboons, pre-lung transplant and post-op day1 anti-pig Ab binding against GalTKO versus GalTKO.β4GalKO (double KO (DKO)) porcine aortic endothelial cells (PAECs) was measured in animals that had received a lung from a GalTKO pig and compared to those in which the donor animal had β4GalKO (or knock down), with (TKO) or without (DKO) additional Neu5GcKO. To evaluate Ab binding to pig tissue, lung biopsies from wildtype, GalTKO, DKO and TKO pigs after 30min of ex vivo xeno-perfusion with human blood were stained for IgG and IgM binding. In one case Ab binding to GalTKO and DKO cells was also measured at 1 and 31d in a long-surviving xenolung recipient. Results IgM Ab binding in naive baboons was consistently lower on GalTKO.β4GalKO relative to GalTKO PAECs. Decline in anti-non-Gal Ab levels on post-op d1 in vivo was consistently less using GalTKO.β4GalKO lungs (31%±16%, n=3) when compared to single KO lungs (58%±16%, n=7). In a long-surviving LTX recipient animal, sensitized anti-non Gal antibody binding was almost completely abrogated when using TKO cells. Lungs with multiple CHO KOs appear to have decreased IgM tissue deposition relative to GalTKO lungs after perfusion with human blood when analyzed by Immunohistochemistry. Conclusion β4Gal is a dominant antigen after lung xenotransplantation. Binding of preformed antibody (IgM>IgG) in humans and baboons is reduced by β4GalKO. Since the β4Gal epitope presumably contributes to pig lung xenograft injury, including the β4GalKO modification in donor pigs will likely help to better control Ab-mediated xenorejection pathways and advance xenotransplantation of the lung and other organs towards the clinic.

Genetic Deletion of Polo-Like Kinase 2 Induces a Pro-Fibrotic Pulmonary Phenotype.

Pulmonary fibrosis is the chronic-progressive replacement of healthy lung tissue by extracellular matrix, leading to the destruction of the alveolar architecture and ultimately death. Due to limited pathophysiological knowledge, causal therapies are still missing and consequently the prognosis is poor. Thus, there is an urgent clinical need for models to derive effective therapies. Polo-like kinase 2 (PLK2) is an emerging regulator of fibroblast function and fibrosis. We found a significant downregulation of PLK2 in four different entities of human pulmonary fibrosis. Therefore, we characterized the pulmonary phenotype of PLK2 knockout (KO) mice. Isolated pulmonary PLK2 KO fibroblasts displayed a pronounced myofibroblast phenotype reflected by increased expression of αSMA, reduced proliferation rates and enhanced ERK1/2 and SMAD2/3 phosphorylation. In PLK2 KO, the expression of the fibrotic cytokines osteopontin and IL18 was elevated compared to controls. Histological analysis of PLK2 KO lungs revealed early stage remodeling in terms of alveolar wall thickening, increased alveolar collagen deposition and myofibroblast foci. Our results prompt further investigation of PLK2 function in pulmonary fibrosis and suggest that the PLK2 KO model displays a genetic predisposition towards pulmonary fibrosis, which could be leveraged in future research on this topic.