Epithelial MAPK signaling directs endothelial NRF2 signaling and IL-8 secretion in a tri-culture model of the alveolar-microvascular interface following diesel exhaust particulate (DEP) exposure.

BACKGROUND: Particulate matter 2.5 (PM2.5) deposition in the lung's alveolar capillary region (ACR) is significantly associated with respiratory disease development, yet the molecular mechanisms are not completely understood. Adverse responses that promote respiratory disease development involve orchestrated, intercellular signaling between multiple cell types within the ACR. We investigated the molecular mechanisms elicited in response to PM2.5 deposition in the ACR, in an in vitro model that enables intercellular communication between multiple resident cell types of the ACR. METHODS: An in vitro, tri-culture model of the ACR, incorporating alveolar-like epithelial cells (NCI-H441), pulmonary fibroblasts (IMR90), and pulmonary microvascular endothelial cells (HULEC) was developed to investigate cell type-specific molecular responses to a PM2.5 exposure in an in-vivo-like model. This tri-culture in vitro model was termed the alveolar capillary region exposure (ACRE) model. Alveolar epithelial cells in the ACRE model were exposed to a suspension of diesel exhaust particulates (DEP) (20 µg/cm2) with an average diameter of 2.5 µm. Alveolar epithelial barrier formation, and transcriptional and protein expression alterations in the directly exposed alveolar epithelial and the underlying endothelial cells were investigated over a 24 h DEP exposure. RESULTS: Alveolar epithelial barrier formation was not perturbed by the 24 h DEP exposure. Despite no alteration in barrier formation, we demonstrate that alveolar epithelial DEP exposure induces transcriptional and protein changes in both the alveolar epithelial cells and the underlying microvascular endothelial cells. Specifically, we show that the underlying microvascular endothelial cells develop redox dysfunction and increase proinflammatory cytokine secretion. Furthermore, we demonstrate that alveolar epithelial MAPK signaling modulates the activation of NRF2 and IL-8 secretion in the underlying microvascular endothelial cells. CONCLUSIONS: Endothelial redox dysfunction and increased proinflammatory cytokine secretion are two common events in respiratory disease development. These findings highlight new, cell-type specific roles of the alveolar epithelium and microvascular endothelium in the ACR in respiratory disease development following PM2.5 exposure. Ultimately, these data expand our current understanding of respiratory disease development following particle exposures and illustrate the utility of multicellular in vitro systems for investigating respiratory tract health.

Characterizing the extracellular vesicle proteomic landscape of the human airway using in vitro organotypic multi-cellular models.

Extracellular vesicle (EV)-mediated intercellular communication significantly influences pulmonary cell health and disease, yet in vitro methods to investigate these mechanisms are limited. We hypothesize that organotypic models of the airway can be leveraged to investigate EV-mediated intercellular signaling, focusing on EV proteomic content as a case study. Two in vitro airway culture models were evaluated by mass spectrometry-based proteomics analysis: a tri-culture model consisting of alveolar epithelial, fibroblast, and lung microvascular endothelial cells and a co-culture model of alveolar epithelial and fibroblasts. EVs isolated from the tri-culture model were enriched with EV proteins regulating RNA-to-protein translation. EVs isolated from the co-culture model were enriched with EV biogenesis and extracellular matrix signaling proteins. These model-specific differences suggest that different pulmonary cell types uniquely affect EV composition and the biological pathways influenced by the EV proteome in recipient cells. These findings can inform future studies surrounding EV-related pulmonary disease pathogenesis and therapeutics.

A piggyBac-based toolkit for inducible genome editing in mammalian cells.

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is cotransfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. We show that CRISPR-Bac can be used to knock down proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.

Comprehensive analysis of The Cancer Genome Atlas reveals a unique gene and non-coding RNA signature of fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a unique liver cancer primarily affecting young adults and characterized by a fusion event between DNAJB1 and PRKACA. By analyzing RNA-sequencing data from The Cancer Genome Atlas (TCGA) for >9,100 tumors across ~30 cancer types, we show that the DNAJB1-PRKACA fusion is specific to FLCs. We demonstrate that FLC tumors (n = 6) exhibit distinct messenger RNA (mRNA) and long intergenic non-coding RNA (lincRNA) profiles compared to hepatocellular carcinoma (n = 263) and cholangiocarcinoma (n = 36), the two most common liver cancers. We also identify a set of mRNAs (n = 16) and lincRNAs (n = 4), including LINC00473, that distinguish FLC from ~25 other liver and non-liver cancer types. We confirm this unique FLC signature by analysis of two independent FLC cohorts (n = 20 and 34). Lastly, we validate the overexpression of one specific gene in the FLC signature, carbonic anhydrase XII (CA12), at the protein level by western blot and immunohistochemistry. Both the mRNA and lincRNA signatures support a major role for protein kinase A (PKA) signaling in shaping the FLC gene expression landscape, and present novel candidate FLC oncogenes that merit further investigation.