DOI: 10.5061/dryad.qfttdz0t4
title: Culture media strongly influences primary human bronchial epithelial cells' transcriptomic response to ozone exposure

description: Exposure to the ambient air pollutant ozone induces acute and chronic
 respiratory health effects in part by causing inflammation of the airways.
 Several aspects of the inflammatory response to ozone can be modeled in
 vitro using primary human bronchial epithelial cells (HBECs) cultured at
 an air-liquid interface. We tested two commonly used HBEC culture media
 systems, one proprietary and one non-proprietary, to identify which system
 yielded the most in vivo-like pro-inflammatory response to acute ozone
 exposure as indicated by gene expression. Cells from six donors were grown
 in each culture system in parallel followed by examination of epithelial
 morphology and cell type proportions prior to ozone exposure. Cultures
 grown in the proprietary system were notably thicker and contained more
 ciliated and secretory cells, as well as internal cyst-like structures.
 The transcriptomic response to acute ozone exposure (0.5 parts per million
 ozone x 2 hours) was strongly affected by media. HBECs grown in the
 proprietary system exhibited minimal changes after ozone, with only 7
 differentially expressed genes (DEGs). In contrast, HBECs grown in the
 non-proprietary system exhibited a more dynamic response with 128 DEGs,
 including hallmark response genes indicative of inflammation (CXCL8) and
 oxidative stress (HMOX1). Gene set enrichment analysis using the 128 DEGs
 further corroborated upregulation of oxidative stress and inflammation
 pathways. In total, our results indicate that the choice of HBEC culture
 media should be carefully considered to best model the in vivo response to
 ozone.
description: # Culture media strongly influences primary human bronchial epithelial
 cells' transcriptomic response to ozone exposure Dataset DOI:
 [10.5061/dryad.qfttdz0t4](10.5061/dryad.qfttdz0t4) ## Description of the
 data and file structure Transcriptomic data for differential expression
 and gene set enrichment analysis to compare primary bronchial epithelial
 cell responses to ozone exposure when grown in different culture medias.
 ### Files and variables #### File: TableS1_UNC_O3vsFA.xlsx
 **Description:** Full DESeq2 results from ozone vs filtered air-exposed
 UNC-grown cultures. Missing values indicated with blank cells. #####
 Variables * "hgnc_symbol" = HGNC approved symbol for the
 accompanying Ensembl ID. * "baseMean" = Mean base expression
 level across all samples after DESeq2 library size normalization. *
 "log2FoldChange" = log2 fold change calculated by DESeq2 for the
 given comparison. * "lfcSE" = standard error of the log2 fold
 change values. * "pvalue" = nominal p value calculated by
 DESeq2. * "padj" = the Benjamini-Hochberg adjusted p value. *
 "geneID" = the Ensembl gene ID with the Ensembl version suffix.
 * "gene_biotype" = gene biotype as listed by Ensembl. *
 "DE_status" = one of "Up", "NS"
 (non-significant), or "Down". To be up or down, the gene must
 pass an adjusted p value threshold of 0.05 and an absolute log2 fold
 change threshold of 0.58 (equivalent to a 1.5 fold change). #### File:
 TableS2_PNEU_O3vsFA.xlsx **Description:** Full DESeq2 results from ozone
 vs filtered air-exposed PneumaCult-grown cultures. Missing values
 indicated with blank cells. ##### Variables * "hgnc_symbol" =
 HGNC approved symbol for the accompanying Ensembl ID. *
 "baseMean" = Mean base expression level across all samples after
 DESeq2 library size normalization. * "log2FoldChange" = log2
 fold change calculated by DESeq2 for the given comparison. *
 "lfcSE" = standard error of the log2 fold change values. *
 "pvalue" = nominal p value calculated by DESeq2. *
 "padj" = the Benjamini-Hochberg adjusted p value. *
 "geneID" = the Ensembl gene ID with the Ensembl version suffix.
 * "gene_biotype" = gene biotype as listed by Ensembl. *
 "DE_status" = one of "Up", "NS"
 (non-significant), or "Down". To be up or down, the gene must
 pass an adjusted p value threshold of 0.05 and an absolute log2 fold
 change threshold of 0.58 (equivalent to a 1.5 fold change). #### File:
 TableS3_FA_PNEUvsUNC.xlsx **Description:** Full DESeq2 results from
 filtered air exposed cultures, PneumaCult-grown vs UNC-grown. Missing
 values indicated with blank cells. ##### Variables *
 "hgnc_symbol" = HGNC approved symbol for the accompanying
 Ensembl ID. * "baseMean" = Mean base expression level across all
 samples after DESeq2 library size normalization. *
 "log2FoldChange" = log2 fold change calculated by DESeq2 for the
 given comparison. * "lfcSE" = standard error of the log2 fold
 change values. * "pvalue" = nominal p value calculated by
 DESeq2. * "padj" = the Benjamini-Hochberg adjusted p value. *
 "geneID" = the Ensembl gene ID with the Ensembl version suffix.
 * "gene_biotype" = gene biotype as listed by Ensembl. *
 "DE_status" = one of "Up", "NS"
 (non-significant), or "Down". To be up or down, the gene must
 pass an adjusted p value threshold of 0.05 and an absolute log2 fold
 change threshold of 0.58 (equivalent to a 1.5 fold change). #### File:
 TableS4_UNC_enrichGO_Full.xlsx **Description:** Full enrichGO results from
 UNC DEGs. ##### Variables * "ID" = Gene Ontology ID for the gene
 set. * "Description" = Full name of the gene set. *
 "GeneRatio" = How many genes in the differentially expressed
 list that are observed in the gene set versus not observed. *
 "BgRatio" = How many genes in the background gene list that are
 observed in the gene set versus not observed. * "pvalue" =
 nominal p value for genes in that gene set being enriched in the
 differentially expressed list compared to the background list. *
 "p.adjust" = Benjamini-Hochberg adjusted p value. *
 "qvalue" = enrichGO calculated q value. * "geneID" =
 HGNC gene name of genes in differentially expressed list found in the gene
 set. * "Count" = count of genes in differentially expressed list
 found in the gene set. #### File: TableS5_UNC_enrichGO_Selected.xlsx
 **Description:** Selected gene sets from table 5 used to select genes for
 main figure 3C. ##### Variables * "ID" = Gene Ontology ID for
 the gene set. * "Description" = Full name of the gene set. *
 "GeneRatio" = How many genes in the differentially expressed
 list that are observed in the gene set versus not observed. *
 "BgRatio" = How many genes in the background gene list that are
 observed in the gene set versus not observed. * "pvalue" =
 nominal p value for genes in that gene set being enriched in the
 differentially expressed list compared to the background list. *
 "p.adjust" = Benjamini-Hochberg adjusted p value. *
 "qvalue" = enrichGO calculated q value. * "geneID" =
 HGNC gene name of genes in differentially expressed list found in the gene
 set. * "Count" = count of genes in differentially expressed list
 found in the gene set. #### File: TableS6_PNEUvsUNC_gseGO.xlsx
 **Description:** Full gseGO results from filtered air-exposed PneumaCult
 vs UNC DEGs. ##### Variables * "ID" = Gene Ontology ID for the
 gene set. * "Description" = Full name of the gene set. *
 "setSize" = Size of the gene set. * "enrichmentScore"
 = enrichment score as calculated by gseGO, positive values denote
 activated gene sets (more genes have positive log2 fold changes and are in
 the top part of the ranked list), negative values denote suppressed gene
 sets (more genes have negative log2 fold changes and are in the bottom
 part of the ranked list). * "NES" = normalized enrichment score.
 * "pvalue" = nominal p value for genes in that gene set being
 enriched in the differentially expressed list compared to the background
 list. * "p.adjust" = Benjamini-Hochberg adjusted p value. *
 "qvalue" = gseGO calculated q value. * "rank" = the
 position in the ranked gene list where the maximum enrichment score was
 found. * "leading_edge" = three fields: tags - the percentage of
 genes in the gene set found in the ranked list before (if positive
 enrichment score) or after (if negative enrichment score) the peak rank
 score. list - the percentage of genes included or not included in the gene
 set found before (if positive enrichment score) or after (if negative
 enrichment score) the peak rank score. signal - enrichment signal
 strength, combines the previous two metrics. * "core_enrichment"
 = the subset of genes contributing most to the enrichment score. ####
 File: TableS7_PNEUvsUNC_gseGO_PNEUonly.xlsx **Description:** Filtered
 version of table S6 showing gene sets enriched in PneumaCult-grown
 cultures. ##### Variables * "ID" = Gene Ontology ID for the gene
 set. * "Description" = Full name of the gene set. *
 "setSize" = Size of the gene set. * "enrichmentScore"
 = enrichment score as calculated by gseGO, positive values denote
 activated gene sets (more genes have positive log2 fold changes and are in
 the top part of the ranked list), negative values denote suppressed gene
 sets (more genes have negative log2 fold changes and are in the bottom
 part of the ranked list). * "NES" = normalized enrichment score.
 * "pvalue" = nominal p value for genes in that gene set being
 enriched in the differentially expressed list compared to the background
 list. * "p.adjust" = Benjamini-Hochberg adjusted p value. *
 "qvalue" = gseGO calculated q value. * "rank" = the
 position in the ranked gene list where the maximum enrichment score was
 found. * "leading_edge" = three fields: tags - the percentage of
 genes in the gene set found in the ranked list before (if positive
 enrichment score) or after (if negative enrichment score) the peak rank
 score. list - the percentage of genes included or not included in the gene
 set found before (if positive enrichment score) or after (if negative
 enrichment score) the peak rank score. signal - enrichment signal
 strength, combines the previous two metrics. * "core_enrichment"
 = the subset of genes contributing most to the enrichment score. ####
 File: TableS8_PNEUvsUNC_gseGO_UNConly.xlsx **Description:** Filtered
 version of table 6 showing gene sets enriched in UNC-grown cultures. #####
 Variables * "ID" = Gene Ontology ID for the gene set. *
 "Description" = Full name of the gene set. * "setSize"
 = Size of the gene set. * "enrichmentScore" = enrichment score
 as calculated by gseGO, positive values denote activated gene sets (more
 genes have positive log2 fold changes and are in the top part of the
 ranked list), negative values denote suppressed gene sets (more genes have
 negative log2 fold changes and are in the bottom part of the ranked list).
 * "NES" = normalized enrichment score. * "pvalue" =
 nominal p value for genes in that gene set being enriched in the
 differentially expressed list compared to the background list. *
 "p.adjust" = Benjamini-Hochberg adjusted p value. *
 "qvalue" = gseGO calculated q value. * "rank" = the
 position in the ranked gene list where the maximum enrichment score was
 found. * "leading_edge" = three fields: tags - the percentage of
 genes in the gene set found in the ranked list before (if positive
 enrichment score) or after (if negative enrichment score) the peak rank
 score. list - the percentage of genes included or not included in the gene
 set found before (if positive enrichment score) or after (if negative
 enrichment score) the peak rank score. signal - enrichment signal
 strength, combines the previous two metrics. * "core_enrichment"
 = the subset of genes contributing most to the enrichment score. ##
 Code/software Raw read quality was checked using FastQC (v0.12.1).
 Residual adapter sequences were trimmed using cutadapt (v4.1). Reads were
 aligned to the human transcriptome (GRch38.p14, release 44) using Star
 (v2.7.11a) and quantified using Salmon (v1.10.2). All downstream analyses
 were performed using R (v4.3.1). Salmon counts were imported using
 tximport. Genes without at least 10 counts across 6 samples were filtered
 out. Differential expression analysis was performed using DESeq2; genes
 were considered differentially expressed with an adj.p.val &lt; 0.05 and
 an absolute log2FC &gt; 0.58. All processing and analysis commands can be
 found at
 [https://github.com/Kelada-Lab/HBEC-Media-Comparison](https://github.com/Kelada-Lab/HBEC-Media-Comparison).