Transcript analysis of monocytes after 24 hours training

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effect on the transcriptome of tested cells which separate in clearly defined clusters.

The Hierarchical Clustering of the 1000 most variable genes as well as DE-gene analysis demonstrate however, that the transcriptomic differences between 24h_PA treated and control cells is much higher as for 24h_OA treated and control cells. In fact, transcriptional analysis of 24h_OA stimulated monocytes and 24h_Ctrl cells just gives rise to 61 DE-genes. Up-regulated DE-genes in 24h_OA samples play a role in long-chain fatty acid transport and the activation of lipid metabolic processes. An anti-inflammatory effect of OA on monocyte-derived cells was ascertained from KEGG enrichment analysis. Here, ‘PPAR signaling pathway’ was the term appearing with most gene counts and highest significance. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by fatty acids and their derivatives. PPARs bind fatty acids and eicosanoids. There are three subtypes of PPARs, namely PPAR-α, -β/δ, and -ɤ, whereby PPAR-α plays a role in the clearance of circulating or cellular lipids via the regulation of gene expression involved in lipid metabolism in liver and skeletal muscle. PPAR-β/δ is involved in lipid oxidation and cell proliferation and PPAR-ɤ promotes adipocyte differentiation to enhance blood glucose uptake²⁰⁷. As described in the introduction, the PPAR-signaling pathway is associated with a M-IL4-like anti-inflammatory phenotype¹⁴⁸. In contrast to 24h_OA stimulated monocytes, GOEA and KEGG pathway analysis of 24h_PA stimulated monocytes document that these cells highly express

pro-inflammatory mediators and pro-inflammatory response genes. These findings go in hand with prior literature, for instance explained by Palomer et. al., 2018, who review

mechanistic insights into the beneficial effects of oleic acid compared with palmitic acid on insulin resistance and T2DM, including its anti-inflammatory actions, and its capacity to inhibit endoplasmic reticulum (ER) stress²⁰⁸.

In our experimental set up, a higher activation state of 24h_PA treated monocyte-derived cells is specified by GO-terms like ‘regulation of MAPK cascade’ and ‘G-protein coupled receptor signaling cascade’ as these processes are highly involved in myeloid cell differentiation and polarization, microbial elimination, inflammation and resolution, as well as in macrophage-mediated pathologies.^193,194

The high number of overlapping DE-genes (192 genes) between 24h_PA stimulated and Ctrl_Ctrl_20h_LPS stimulated monocyte-derived cells as well as the GOEA of the

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24h_PA samples, exhibiting the term ‘response to lipopolysaccharide’, code for the high inflammatory potential of PA. Preceding studies described already that PA has a similar effect on macrophage polarization as LPS. Thereby, it has been described that the signaling mechanism induced by SFAs is mediated by TLR4 activation.^60,209 TLR4 is the best characterized of all PRRs and the earliest evidence linking TLR4 to lipid-induced inflammation can be backdated to the time it was identified as receptor for LPS²¹⁰. Osborn and Olefsky, 2012²¹¹ hypothesized that long-chain SFAs are TLR4 agonists as the major constituent of LPS responsible for its immunostimulatory activity is the lipid A (LPA) region, which contains numerous saturated fatty acyl-chains that are required for binding to and activating TLR4. Lancaster et al., 2018, however demonstrated that the inflammatory fatty acid palmitate is actually not a direct TLR4 agonist but that long-chain SFA-induced inflammatory signaling is completely TLR4-dependent and that TLR4-TLR4-dependent priming is necessary for long-chain SFAs to activate inflammatory signaling in macrophages. Though, TLR4 is required for palmitate-induced inflammation through TLR4-dependent priming and altered cellular metabolism.²¹²

Underlining the hypothesis that SFAs do force the activation of TLR4, we could show that only 24h_PA stimulated monocytes activated almost all of the MyD88-dependent downstream genes (see Figure 5.8.1.1 A.)) while in 24h_OA and 24h_Ctrl cultures downstream genes were comparably down-regulated. These results were strengthened by corresponding MyD88, TIRAP and final IL1B and TNF expression data and by the evaluation of TNF ELISA data, which showed increased values in 24h_PA stimulated monocytes compared to 24h_OA and 24h_Ctrl monocyte-derived cells. In line with former publications, we can conclude that even if PA is not a direct TLR4 agonist, it forces the activation of MyD88-dependent TLR4 activation in monocyte-derived cells, which is not indicated by OA stimulations. TRIF-dependent TLR4 activation was in neither case exhibited since CCL5 expression was down-regulated and as low as in the control situation.

The NLRP3 inflammasome is an important innate immune pathway that regulates at least two host responses to protect against infections. One is the secretion of the pro-inflammatory cytokines IL-1β and IL-18 and the other one the induction of pyroptosis of infected cells.²⁰¹ The NLRP3 inflammasome is involved in many obesity-associated diseases, such as T2DM, atherosclerosis, and gouty arthritis, through its ability to

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induce IL-1β release²¹³. In this study we also could show that PA stimulation leads to over-expression of NLRP3 and IL1B in monocyte-derived cells. These findings fit very well to data from A. Christ et. al., 2018 who demonstrated that Western diet, high in SFAs, induced systemic inflammation, myeloid progenitor proliferation and re-programming in myeloid cells which again depended on NLRP3 inflammasome activation. They interpreted that NLRP3 mediates trained immunity following Western diet in myeloid cells of tested mice.²¹⁴ As previously stated, with our 24h_PA experiment we also could show that monocyte-derived cells stimulated with PA for 24 hours highly express NLRP3 and the inactive form of IL1β, but intense activation of the NLRP3 inflammasome was not indicated since the gene CASP1 coding for the IL1β activating enzyme caspase 1 was not notably upregulated.

Laurent L’homme et. al., 2013 reported that UFAs like oleate and linoleate do not activate the NLRP3 inflammasome and instead rather reveal an anti-inflammatory property through preventing inflammasome activation by SFAs.¹⁸⁴ Unexpectedly, the NLRP3 network data of 24h_OA stimulated monocytes show also high expression of NLRP3 and IL1B, however, in line with published results, NLRP3-inflammasome activation was again not indicated since CASP1 expression was equally down-regulated as described for 24h_PA cultures. These results give space for speculations and indicate no necessary involvement of NLRP3 inflammasome activation in PA induced inflammatory signaling in monocyte-derived cells. The inclusion of secreted IL-1β values would now give the full picture and clear information about actual NLRP3-inflammasome activation.

The monocyte isolation procedure and transfer to culturing medium which induced stress signals to the cells may be an explanation for increased expression of NLRP3 components in all 24h conditions. TNF, which is secreted in high amounts in all 24h samples compared to 96h samples (see Figure 5.8.2.1 A.) & C.)) is described to be an important transcriptional regulator of NLRP3 inflammasome components in murine inflammasomopathies²¹⁵ and might also be the reason for over-expression of these components in all 24h cultured monocyte-derived cells.

In line with former publications, the complete expression data profile of 24h_OA cultures indicates that OA highly induces fatty acid metabolic processes in monocyte-derived cells, most pronounced in fatty acid oxidation, which drive the cells towards an anti-inflammatory M-IL4 like phenotype²⁰⁸. 24h_PA cultured monocyte-derived

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cells in contrast developed into pro-inflammatory phenotypes, initiating innate immune responses similar to those described to be induced by LPS activated macrophages, while fatty acid metabolic processes seem to play a minor role.

Surprisingly, the overall picture of the 24h_Ctrl cultures exhibited expression profiles of activated monocyte-derived cells with typical moDC characteristics (see Figure 5.5.6.1 A.) (CD207, CLEC4F, NDRG2)) while 24 hours fatty acid stimulations either indicated monocyte-derived cells with typical macrophage characteristics, however with divergent polarizations.

Interestingly, TREM2, a gene coding for a receptor protein which was found to be highly expressed on one of two atherosclerosis-specific macrophage populations described by Cochain et. al., 2018, was up-regulated in 24h_OA stimulated monocytes⁴⁶. Cochain et. al., 2018 described that Trem2^high macrophages appear to be endowed with specialized functions in lipid metabolism and catabolism and presented a gene expression signature reminiscent of osteoclasts, suggesting a lesion calcification. Moreover, Trem2 expression was detected in human lesional macrophage populations which were also present in advanced stages of atherosclerosis.⁴⁶ These typical characteristics go in line with our findings of 24h_OA stimulated monocyte-derived cells.

That 24h_PA stimulated monocyte-derived cells evolved not only pro-inflammatory, but also pro-atherosclerotic characteristics is not surprising and either proposed by the overexpression of the ABC1A gene (Figure 5.5.6.1). The gene product of ABC1A has been described to promote cholesterol efflux to extracellular acceptors in vitro and in vivo (reverse cholesterol transport) and to influence atherosclerosis in mice²¹⁶.

A probable conclusion from these data would be that in atherosclerotic lesions either inflammatory and anti-inflammatory acting myeloid-derived cells do play a likewise important role, facilitating the simultaneous progression and resolution of atherosclerotic plaques. Atherosclerosis and Diabetes are diseases which stand in close proximity to overweight and excess saturated fatty acid consumption, thus it is not surprising that monocytes stimulated with high amounts of palmitic acid produce gene products that are crucial for such complications.

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6.2 Transcript analysis of monocyte-derived cells after wash-out of the