Functional interpretation of transcript analysis

As before, we performed a gene ontology enrichment analysis with the 116 hours samples, using the ClusterProfiler¹⁶⁷ classification system. GO-terms were considered significantly enriched with a Bonferroni corrected p-value of ≤ 0.05 and p-adjust cutoff of 0.1. A dot plot visualization of overexpressed genes in the OA_ctrl_20h_LPS or PA_ctrl_20h_LPS stimulated samples compared to Ctrl_ctrl_20h_LPS or to each other is shown in Figure 5.7.5.1.

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Figure 5.7.5.1: GO-term-enrichment analysis of up- and down-regulated DE-genes of fatty acid trained monocyte-derived cells after re-stimulation with LPS. GO-terms were considered significantly enriched with a Bonferroni corrected p-value of ≤ 0.05 and p-adjust cutoff of 0.1.

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The GO-enrichment analysis is visualized in Figure 5.7.5.1. ‘Fatty acid metabolic process’ is the only GO-term appearing for up-regulated DE-genes in OA_Ctrl_20h_LPS samples compared to Ctrl_Ctrl_20h_LPS, with a gene ratio of 0.15.

When compared to PA_Ctrl_20h_LPS instead of to Ctrl_Ctrl_20h_LPS samples, much more GO-terms, taking only 26 up-regulated DE-genes into account, are predicted.

These terms describe processes involved in ‘small molecule biosynthetic processes’, the ‘regulation of triglyceride, lipid, sterol and cholesterol transport’, the ‘regulation of lipid localization’, as well as in ‘acylglycerol and triglyceride homeostasis’.

The term that describes the tendency of functional involvement of down-regulated DE-genes in OA_Ctrl_20h_LPS compared to PA_Ctrl_20h_LPS samples is termed

‘negative regulation of lipid transport’, which goes along with the up-regulated processes.

GO-terms describing product involvement of up-regulated DE-genes in PA_Ctrl_20h_LPS, compared to the Ctrl_Ctrl_20h_LPS comprise ‘complement activation, classical pathway’, ‘lipid biosynthetic process’ (large gene ratio) and

‘humoral immune response mediated by circulating immunoglobulin’. The gene ratio for ‘lipid biosynthetic process’ is with 0.20 comparably high and also significant with a p-value of 0.02. Conspicuously, the terms describing the gene product involvement of down-regulated DE-genes in PA_Ctrl_20h_LPS cultures compared to Ctrl_Ctrl_20h_LPS encompass several processes involved in the inflammatory response of myeloid-derived cells and in the initiation of adaptive immune responses.

Predicted GO-terms for this condition comprise ‘response to IFN-ɤ’, ‘antigen processing and presentation of peptide antigen via MHC class II’, ‘positive regulation of leukocyte activation’, ‘cytokine-mediated signaling pathway’, ‘positive regulation of cell adhesion’, and many more. Since genes whose products are involved in such processes are down-regulated in PA_Ctrl_20h_LPS cultures it is suggested that PA priming sets monocyte-derived cells into a state of tolerance to LPS.

In addition to GO-term analysis, KEGG enrichment analysis was performed using pre-defined DE-genes of the 116 hours samples. The KEGG-enrichment analysis was performed by Dr. Thomas Ulas (LIMES-Institute, University of Bonn). Settings described for 24 hours samples were overtaken. The cutoffs of included DE-genes were set to FC ≥ 2 and p-value ≤ 0.05.

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Figure 5.7.5.2: KEGG-enrichment analysis. Scatterplot of enriched KEGG pathways for DE-genes up-regulated in OA_Ctrl_20h_LPS and PA_Ctrl_20h_LPS monocyte-derived cells. The gene ratio indicates the ratio of the differentially expressed gene number to the total gene number in a certain pathway. The size and color of the dots represent the gene number and the range of p-values, respectively. A.) Represents pathways upregulated in OA_Ctrl_20h_LPS stimulated monocyte-derived cells compared to Ctrl_Ctrl_20h_LPS cells. B.) Shows pathways to which up-regulated DE-genes of PA_Ctrl_20h_LPS versus Ctrl_Ctrl_20h_LPS cultures contribute, while in C.) pathways are visualized that are upregulated in OA_Ctrl_20h_LPS monocyte-derived cells when compared to PA_Ctrl_20h_LPS cultures. DE-genes were defined by fold changes of ≥ 2 and p-values ≤ 0.05.

Figure 5.7.5.2 A.) shows KEGG pathways of up-regulated DE-genes established by comparison of OA_Ctrl_20h_LPS and Ctrl_Ctrl_20h_LPS cultures. The gene count is quite low although the number of up-regulated differentially expressed genes (60) set in for analysis was quite high. Likewise, as in 24 hours OA stimulated monocyte-derived cells, ‘PPAR signaling pathway’ and ‘Fatty acid metabolism’ are the terms with highest gene counts (4 genes), gene ratios and significance (p-adjust ≤ 0.005). In the remaining pathways described for this comparison, the gene counts and gene ratios are with two genes involved and a gene ratio of 0.06 relatively low. Pathway descriptions of genes with p-adjust lying between 0.015 and 0.02 are ‘fatty acid degradation’, ‘propanoate metabolism’, ‘chemical carcinogenesis’, ‘glycine, serine and threonine metabolism’ and ‘metabolism of xenobiotics by cytochrome P450’. The pathways assumed with higher significance (p-adjust between 0.01 and 0.005) include

‘porphyrin and chlorophyll metabolism’, ‘drug metabolism – cytochrome P450’ and

‘butanoate metabolism’.

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In Figure 5.7.5.2 B.) KEGG pathways determined for up-regulated DE-genes in PA_Ctrl_20h_LPS compared to Ctrl_Ctrl_20h_LPS monocyte-derived cells, are described. Here the gene counts of gene products involved in described pathways are with ≤ 5 slightly higher as visualized for OA_Ctrl_20h_LPS cultures, but the number of implicated up-regulated DE-genes was also much higher (105). The gene rations are with highest ≤ 0.1 quite low. The pathways with highest gene ratios and gene counts comprise ‘Focal adhesion’ and ‘RAP1 signaling pathway’. ‘Micro RNAs in cancer’ and

‘phosphatidyl inositol system’ both count 4 DE-genes involved and a gene ratio of 0.08.

Pathways described with gene ratios of 0.06 and three genes implicated are described as ‘inositol phosphate metabolism’, ‘aldosterone synthesis and secretion’, ‘circardian entrainment’ and ‘morphine addiction’. The pathways given with lower gene ratios (0.04) and involvement of 2 genes include ‘fructose and mannose metabolism’ and

‘glyoxylate and dicarboxylate metabolism’.

Figure 5.7.5.2 C.) shows pathways that are up-regulated in OA_Ctrl_20h_LPS compared to PA_Ctrl_20h_LPS cultures. In this comparison only 32 DE-genes were taken under investigation. Here, involvement of three pathways could be determined with gene ratios of 0.1 and two genes involved, respectively. Implied pathways are the

‘PPAR signaling pathway’, ‘cholesterol metabolism’ and ‘steroid biosynthesis’.