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Inflammatory transcriptome profiling of human monocytes exposed acutely to cigarette smoke - PubMed

Inflammatory transcriptome profiling of human monocytes exposed acutely to cigarette smoke

William R Wright et al. PLoS One. 2012.

Abstract

Background: Cigarette smoking is responsible for 5 million deaths worldwide each year, and is a major risk factor for cardiovascular and lung diseases. Cigarette smoke contains a complex mixture of over 4000 chemicals containing 10(15) free radicals. Studies show smoke is perceived by cells as an inflammatory and xenobiotic stimulus, which activates an immune response. The specific cellular mechanisms driving cigarette smoke-induced inflammation and disease are not fully understood, although the innate immune system is involved in the pathology of smoking related diseases.

Methodology/principle findings: To address the impact of smoke as an inflammagen on the innate immune system, THP-1 cells and Human PBMCs were stimulated with 3 and 10% (v/v) cigarette smoke extract (CSE) for 8 and 24 hours. Total RNA was extracted and the transcriptome analysed using Illumina BeadChip arrays. In THP-1 cells, 10% CSE resulted in 80 genes being upregulated and 37 downregulated by ≥1.5 fold after 8 hours. In PBMCs stimulated with 10% CSE for 8 hours, 199 genes were upregulated and 206 genes downregulated by ≥1.5 fold. After 24 hours, the number of genes activated and repressed by ≥1.5 fold had risen to 311 and 306 respectively. The major pathways that were altered are associated with cell survival, such as inducible antioxidants, protein chaperone and folding proteins, and the ubiquitin/proteosome pathway.

Conclusions: Our results suggest that cigarette smoke causes inflammation and has detrimental effects on the metabolism and function of innate immune cells. In addition, THP-1 cells provide a genetically stable alternative to primary cells for the study of the effects of cigarette smoke on human monocytes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. THP-1 samples treated for 8 h with control media or 10% CSE group according to treatment conditions.

THP-1 cells were treated for 8 h with control media or 10% CSE, RNA was extracted and gene expression values measured using HumanRef-8v3 Expression BeadChip arrays. Principal component analysis (PCA) by conditions was performed on GeneSpring GX 11.0.2 and represented as a 3D scatterplot of THP-1 monocytes treated with control media or 10% cigarette smoke extract (CSE) for 8 hours from 6 individual experiments.. The PCA plot showed that samples clustered based on their treatment with medium (blue) or 10% CSE (red). Data represent n = 6. Component % variance; PC1 = 81.7%, PC2 = 14.1%, PC3 = 4.2%. RNA was extracted from each sample and gene expression values measured using 2 HumanRef-8v3 Expression BeadChip arrays. This represents stratification according to treatment with 10% CSE (red) and media treated controls (blue) over 6 individual experiments.

Figure 2
Figure 2. Expression of genes that were increased above a 1.5-fold cut-off in THP-1 monocytes treated with control medium or cigarette smoke extract (CSE) for 8 hours.

THP-1 monocytes were treated for 8 hours with RPMI-1640 medium (control 1–6) or CSE (1–6). RNA was extracted from each sample and gene expression values measured using the Illumina HumanRef-8v3 BeadChip array. (A) Heat map representation of normalized signal intensity values for genes altered by ≥1.5-fold. Red denotes high expression and turquoise denotes low expression. Order of samples was dictated by hierarchical clustering. (B) Table showing gene symbol, fold change and p-value for all genes upregulated by ≥1.5-fold. Statistical significance (p<0.05) was calculated using student's t-test followed by Benjamini-Hochberg false discovery rate correction on GeneSpring GX11.0.2 software. Fold change represents a comparison between mean normalised signal intensity for control (n = 6) versus smoke (n = 6) treated THP-1 monocytes. Refer to Data S1 for full data sets and Entrez Gene IDs.

Figure 3
Figure 3. Expression of genes that were decreased below a 1.5-fold cut-off in THP-1 monocytes treated with control medium or cigarette smoke extract for 8 hours.

THP-1 monocytes were treated for 8 hours with RPMI-1640 medium (control 1–6) or CSE (1–6). RNA was extracted from each sample and gene expression values measured using the Illumina HumanRef-8v3 BeadChip array. (A) Heat map representation of normalized signal intensity values for genes altered by ≥1.5-fold. Red denotes high expression and turquoise denotes low expression. Order of samples was dictated by hierarchical clustering. (B) Table showing gene symbol, fold change and p-value for all genes downregulated by ≥1.5-fold. Statistical significance (p<0.05) was calculated using student's t-test followed by Benjamini-Hochberg false discovery rate correction on GeneSpring GX11.0.2 software. Fold change represents a comparison between mean normalised signal intensity for control (n = 6) versus smoke (n = 6) treated THP-1 monocytes. Refer to Data S1 for full data sets and Entrez Gene IDs.

Figure 4
Figure 4. Expression of genes that were changed by 2.0-fold or above in THP-1 monocytes treated with control medium or cigarette smoke extract for 8 hours.

THP-1 monocytes were treated for 8 hours with RPMI-1640 medium (control 1–6) or CSE (1–6). RNA was extracted from each sample and gene expression values measured using the Illumina HumanRef-8v3 BeadChip array. (A) Heat map representation of normalized signal intensity values for genes altered by ≥2.0-fold. Red denotes high expression and turquoise denotes low expression. Order of samples was dictated by hierarchical clustering. (B) Table showing gene name, fold change and p-value for all genes upregulated by ≥2.0-fold. (C) Table showing gene name, fold change and p-value for all genes downregulated by ≥2.0-fold. Statistical significance (p<0.05) was calculated using student's t-test followed by Benjamini-Hochberg FDR correction on GeneSpring GX11.0.2 software. Fold change represents a comparison between mean normalised signal intensity for control (n = 6) versus smoke (n = 6) treated THP-1 monocytes. Refer to Data S1 full data sets and Entrez Gene IDs.

Figure 5
Figure 5. 3D Scatterplot from the principle conponent analysis of PBMCs treated with control media or cigarette smoke extract (CSE) for 8 and 24 hours.

PBMCs were treated with control media, 3% CSE or 10% CSE for 8 and 24 hours. RNA was extracted from each sample and gene expression values measured using HumanRef-8v3 Expression BeadChip arrays. Samples stratify according to treatment with 3% CSE (maroon), 10% CSE (red) and media treated controls (blue) from 3 individuals for (A) 8 hours and (B) 24 hours. Component % variance for 8 hours was; PC1 = 35.8%, PC2 = 27.6%, PC3 = 19.2%, PC4 = 17.4%. Component % variance for 24 hours was; PC1 = 44.1%, PC2 = 22.4%, PC3 = 18.8%, PC4 = 14.7%.

Figure 6
Figure 6. Expression of genes that were changed by 3.0-fold or above in PBMCs treated with control medium or cigarette smoke extract (CSE) for 8 hours.

PBMCs were treated for 8 hours with RPMI-1640 medium (control 1–3), 3% CSE (1–3) or 10% CSE (1–3). RNA was extracted from each sample and gene expression values measured using the Illumina HumanRef-8v3 BeadChip array. (A) Heat map representation of normalized signal intensity values for genes altered by ≥3.0-fold. Red denotes high expression and turquoise denotes low expression. Order of samples was dictated by hierarchical clustering. (B) Table showing gene name, fold change and p-value for all genes upregulated by ≥3.0-fold. (C) Table showing gene name, fold change and p-value for all genes downregulated by ≥3.0-fold. Statistical significance (p<0.05) was calculated using student's t-test followed by Benjamini-Hochberg FDR correction on GeneSpring GX11.0.2 software. Fold change represents a comparison between mean normalised signal intensity for control (n = 3) versus smoke (n = 3) treated PBMCs. Refer to Data S2 for full data sets and Entrez Gene IDs.

Figure 7
Figure 7. Expression of genes that were changed by 3.0-fold or above in PBMCs treated with control medium or cigarette smoke extract (CSE) for 24 hours.

PBMCs were treated for 24 hours with RPMI-1640 medium (control 1–3) or 3% CSE (1–3) or 10% CSE (1–3). RNA was extracted from each sample and gene expression values measured using the Illumina HumanRef-8v3 BeadChip array. (A) Heat map representation of normalized signal intensity values for genes altered by ≥3.0-fold. Red denotes high expression and turquoise denotes low expression. Order of samples was dictated by hierarchical clustering. (B) Table showing gene name, fold change and p-value for all genes upregulated by ≥3.0-fold. (C) Table showing gene name, fold change and p-value for all genes downregulated by ≥3.0-fold. Statistical significance (p<0.05) was calculated using student's t-test followed by Benjamini-Hochberg FDR correction on GeneSpring GX11.0.2 software. Fold change represents a comparison between mean normalised signal intensity for control (n = 3) versus smoke (n = 3) treated PBMCs. Refer to Data S2 for full data sets and Entrez Gene IDs.

Figure 8
Figure 8. Expression of genes that were changed in both 3 and 10% cigarette smoke extract by 1.5 fold or above compared to control in PBMCs.

PBMCs were treated for 8 hours or 24 hours with RPMI-1640 medium (control 1–3), 3% CSE (1–3) or 10% CSE (1–3). RNA was extracted from each sample and gene expression values measured using the Illumina HumanRef-8v3 BeadChip array. (A) Heat map representing normalized signal intensity values and list of genes altered by ≥1.5 fold in PBMCs treated with both 3 and 10% CSE after 8 hours. Red denotes high expression and turquoise denotes low expression. Order of samples was dictated by hierarchical clustering. (B) Heat map representing normalized signal intensity values and list of genes altered by ≥1.5 fold in PBMCs treated with both 3 and 10% CSE after 24 hours. Red denotes high expression and turquoise denotes low expression. Order of samples was dictated by hierarchical clustering. Statistical significance (p<0.05) was calculated using one-way analysis of variants followed by a Tukey's post-hoc test and Benjamini-Hochberg FDR correction on GeneSpring GX11.0.2 software. Fold change represents a comparison between mean normalised signal intensity for control (n = 3) versus smoke (n = 3) treated PBMCs. Refer to Data S2 for full data sets and Entrez Gene IDs.

Figure 9
Figure 9. Comparison of target gene expression in THP-1 cells and PBMCs treated with cigarette smoke extract (CSE).

We have previously shown that CSE increases the expression of HMOX1, TNF and IL8 in THP-1 cells and PBMCs. RNA was extracted from each sample and gene expression values measured using the Illumina HumanRef-8v3 BeadChip array. (A) Heat map representing normalized signal intensities of HMOX1, TNF and IL8 genes from THP-1 monocytes treated for 8 hours with RPMI-1640 (control 1–6) and 10% CSE (1–6). (B) Heat map representing normalized signal intensities of HMOX1, TNF and IL8 genes from PBMCs treated for 8 hours with RPMI-1640 (control 1–3), 3% and 10% CSE (1–3). (C) Heat map representing normalized signal intensities of HMOX1, TNF and IL8 genes from PBMCs treated for 24 hours with RPMI-1640 (control 1–3), 3% and 10% CSE (1–3). Heat maps were generated using GeneSpring GX11.0.2 software where, red denotes high expression and turquoise denotes low expression.

Figure 10
Figure 10. Top 5 canonical pathway gene interactions in THP-1 cells treated cigarettes smoke extract for 8 hours.

The top 5 canonical pathways identified from genes differentially expressed by ≥1.5-fold in THP-1 cells treated with 10% CSE. RNA was extracted from each sample and gene expression levels were measured using the Illumina HumanRef8-v3 BeadChip Array.Genes that were significantly modified by ≥1.5-fold according to the student's t-test and Benjamini-Hochberg FDR correction were imported into Ingenuity Pathway Analysis software. Interactions of these genes are represented in this schematic.

Figure 11
Figure 11. Top 5 canonical pathway gene interactions in PBMCstreated with 10% CSE for 8 hours.

The top 5 canonical pathways identified from genes differentially expressed by ≥1.5-fold in PBMCs treated with 10% CSE. RNA was extracted from each sample and gene expression levels were measured using the Illumina HumanRef8-v3 BeadChip Array.Genes that were significantly modified by ≥1.5-fold as assessed using student's t-test followed by Benjamini-Hochberg FDR correction were imported into Ingenuity Pathway Analysis software. Interactions of these genes are represented in the schematic as determined by experimentation in human cells.

Figure 12
Figure 12. Top 5 canonical pathway gene interactions in PBMCs treated with 10% CSE for 24 hours.

The top 5 canonical pathways identified from genes differentially expressed by ≥1.5-fold in PBMCs treated with 10% CSE. RNA was extracted from each sample and gene expression levels were measured using the Illumina HumanRef8-v3 BeadChip Array and genes that were modified by ≥1.5-fold and significantly changed according to student's t-test followed by Benjamini-Hochberg FDR correction were imported into Ingenuity Pathway Analysis software. Interactions of these genes are represented in the figure.

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