The role of PPARβ/δ in human macrophages

Macrophages represent the most diverse cell type in biology. They adapt selectively to many stimuli allowing for precise functionality in any environment without harming the organism. Consequently, they monitor their surroundings carefully and react to a plethora of signals. Fatty acids and their derivatives are important signaling mediators in this context, which besides other signals impinge on the lipid-regulated nuclear receptor peroxisome proliferator-activated receptor (PPARβ/δ). Studies conducted in mice have shown that ablation of PPARβ/δ results in the inability of adipose and liver macrophages to adopt an alternative anti-inflammatory activation state, demonstrating a prominent role of PPARβ/δ in macrophage function with implications for immune regulation. To date, however, systematic studies focusing on PPARβ/δ's role in human macrophages have not been reported. The first part of this thesis addresses the role of PPARβ/δ in human macrophages including its transcriptional network affecting a multitude of cellular processes. A major part of this network involves cell type independent canonical regulation, which is characterized by the binding of PPARβ/δ with its obligatory dimerization partner retinoid X receptor (RXR) to specific sites in the regulatory region of established and previously unreported target genes, their induction by agonists and repression by inverse agonists. Additionally, a new set of non-canonical regulated target genes is described. These genes lack chromatin-bound PPARβ/δ complexes, are repressed by agonists (inverse regulation) and are macrophage-selective. Consistent with the prevailing opinion and the induction of an IL4-like morphological phenotype by agonists, this mode of regulation inhibits pro-inflammatory signaling. Surprisingly, anti-inflammatory genes, such as CD32B, IDO1 and CD274 (PD-L1) were also repressed. Consistent with these results, immune functions such as CD8+ T cell activation were stimulated by these ligands. In combination, these findings point to a unique macrophage activation state induced by PPARβ/δ agonists with context dependent functions in immune regulation. The second part describes the PPARβ/δ-regulated transcriptome for tumor-associated macrophages (TAMs) from human serous ovarian carcinoma ascites. Interestingly, most canonical PPARβ/δ target genes were found to be upregulated and refractory to synthetic agonists as compared to monocyte-derived macrophages. This was not due to a TAM specific increase in PPARβ/δ protein level or recruitment to target genes. However, the unaffected response of these genes to inverse agonists hinted at the presence of endogenous activating ligands. Lipidomic analysis of malignancy-associated ascites indeed revealed very high concentrations of dietary polyunsaturated fatty acids (PUFAs), mainly linoleic and arachidonic acid. These PUFAs induced lipid droplet formation in macrophages which provide a potential reservoir for PPARβ/δ agonists and may serve as the causal nexus for target gene deregulation. Among the deregulated genes, ANGPTL4 is associated with shorter relapse-free survival, illustrating the potential clinical implications of these finding

Comparison of Various Reducing Agents for Methane Production by Methanothermobacter marburgensis.

peer reviewedBiological methanation is driven by anaerobic methanogenic archaea, cultivated in different media, which consist of multiple macro and micro nutrients. In addition, a reducing agent is needed to lower the oxidation-reduction potential (ORP) and enable the growth of oxygen-sensitive organisms. Until now, sodium sulfide (Na2S) has been used mainly for this purpose based on earlier published articles at the beginning of anaerobic microbiology research. In a continuation of earlier investigations, in this study, the usage of alternative reducing agents like sodium dithionite (Na2S2O4) and L-Cysteine-HCl shows that similar results can be obtained with fewer environmental and hazardous impacts. Therefore, a newly developed comparison method was used for the cultivation of Methanothermobacter marburgensis. The median methane evolution rate (MER) for the alternatives was similar compared to Na2S at different concentrations (0.5, 0.25 and 0.1 g/L). However, the use of 0.25 g/L Na2S2O4 or 0.1 g/L L-Cys-HCl led to stable MER values over consecutive batches compared to Na2S. It was also shown that a lower concentration of reducing agent leads to a higher MER. In conclusion, Na2S2O4 or L-Cys-HCl can be used as a non-corrosive and non-toxic reducing agent for ex situ biological methanation. Economically, Na2S2O4 is cheaper, which is particularly interesting for scale-up purposes

The role of PPARβ/δ in human macrophages

Macrophages represent the most diverse cell type in biology. They adapt selectively to many stimuli allowing for precise functionality in any environment without harming the organism. Consequently, they monitor their surroundings carefully and react to a plethora of signals. Fatty acids and their derivatives are important signaling mediators in this context, which besides other signals impinge on the lipid-regulated nuclear receptor peroxisome proliferator-activated receptor (PPARβ/δ). Studies conducted in mice have shown that ablation of PPARβ/δ results in the inability of adipose and liver macrophages to adopt an alternative anti-inflammatory activation state, demonstrating a prominent role of PPARβ/δ in macrophage function with implications for immune regulation. To date, however, systematic studies focusing on PPARβ/δ's role in human macrophages have not been reported. The first part of this thesis addresses the role of PPARβ/δ in human macrophages including its transcriptional network affecting a multitude of cellular processes. A major part of this network involves cell type independent canonical regulation, which is characterized by the binding of PPARβ/δ with its obligatory dimerization partner retinoid X receptor (RXR) to specific sites in the regulatory region of established and previously unreported target genes, their induction by agonists and repression by inverse agonists. Additionally, a new set of non-canonical regulated target genes is described. These genes lack chromatin-bound PPARβ/δ complexes, are repressed by agonists (inverse regulation) and are macrophage-selective. Consistent with the prevailing opinion and the induction of an IL4-like morphological phenotype by agonists, this mode of regulation inhibits pro-inflammatory signaling. Surprisingly, anti-inflammatory genes, such as CD32B, IDO1 and CD274 (PD-L1) were also repressed. Consistent with these results, immune functions such as CD8+ T cell activation were stimulated by these ligands. In combination, these findings point to a unique macrophage activation state induced by PPARβ/δ agonists with context dependent functions in immune regulation. The second part describes the PPARβ/δ-regulated transcriptome for tumor-associated macrophages (TAMs) from human serous ovarian carcinoma ascites. Interestingly, most canonical PPARβ/δ target genes were found to be upregulated and refractory to synthetic agonists as compared to monocyte-derived macrophages. This was not due to a TAM specific increase in PPARβ/δ protein level or recruitment to target genes. However, the unaffected response of these genes to inverse agonists hinted at the presence of endogenous activating ligands. Lipidomic analysis of malignancy-associated ascites indeed revealed very high concentrations of dietary polyunsaturated fatty acids (PUFAs), mainly linoleic and arachidonic acid. These PUFAs induced lipid droplet formation in macrophages which provide a potential reservoir for PPARβ/δ agonists and may serve as the causal nexus for target gene deregulation. Among the deregulated genes, ANGPTL4 is associated with shorter relapse-free survival, illustrating the potential clinical implications of these finding

Antigen receptor-mediated depletion of FOXP3 in induced regulatory T-lymphocytes via PTPN2 and FOXO1

Regulatory T-cells induced via IL-2 and TGFβ in vitro (iTreg) suppress immune cells and are potential therapeutics during autoimmunity. However, several reports described their re-differentiation into pathogenic cells in vivo and loss of their key functional transcription factor (TF) FOXP3 after T-cell antigen receptor (TCR)-signalling in vitro. Here, we show that TCR-activation antagonizes two necessary TFs for foxp3 gene transcription, which are themselves regulated by phosphorylation. Although the tyrosine phosphatase PTPN2 is induced to restrain IL-2-mediated phosphorylation of the TF STAT5, expression of the TF FOXO1 is downregulated and miR-182, a suppressor of FOXO1 expression, is upregulated. TGFβ counteracts the FOXP3-depleting TCR-signal by reassuring FOXO1 expression and by re-licensing STAT5 phosphorylation. Overexpressed phosphorylation-independent active versions of FOXO1 and STAT5 or knockdown of PTPN2 restores FOXP3 expression despite TCR-signal and absence of TGFβ. This study suggests novel targets for stabilisation and less dangerous application of iTreg during devastating inflammation

Quantitative Long-Term Monitoring of the Circulating Gases in the KATRIN Experiment Using Raman Spectroscopy.

The Karlsruhe Tritium Neutrino (KATRIN) experiment aims at measuring the effective electron neutrino mass with a sensitivity of 0.2 eV/c2, i.e., improving on previous measurements by an order of magnitude. Neutrino mass data taking with KATRIN commenced in early 2019, and after only a few weeks of data recording, analysis of these data showed the success of KATRIN, improving on the known neutrino mass limit by a factor of about two. This success very much could be ascribed to the fact that most of the system components met, or even surpassed, the required specifications during long-term operation. Here, we report on the performance of the laser Raman (LARA) monitoring system which provides continuous high-precision information on the gas composition injected into the experiment's windowless gaseous tritium source (WGTS), specifically on its isotopic purity of tritium-one of the key parameters required in the derivation of the electron neutrino mass. The concentrations cx for all six hydrogen isotopologues were monitored simultaneously, with a measurement precision for individual components of the order 10-3 or better throughout the complete KATRIN data taking campaigns to date. From these, the tritium purity, εT, is derived with precision of <10-3 and trueness of <3 × 10-3, being within and surpassing the actual requirements for KATRIN, respectively

Quantitative Long-Term Monitoring of the Circulating Gases in the KATRIN Experiment Using Raman Spectroscopy.

The Karlsruhe Tritium Neutrino (KATRIN) experiment aims at measuring the effective electron neutrino mass with a sensitivity of 0.2 eV/c2, i.e., improving on previous measurements by an order of magnitude. Neutrino mass data taking with KATRIN commenced in early 2019, and after only a few weeks of data recording, analysis of these data showed the success of KATRIN, improving on the known neutrino mass limit by a factor of about two. This success very much could be ascribed to the fact that most of the system components met, or even surpassed, the required specifications during long-term operation. Here, we report on the performance of the laser Raman (LARA) monitoring system which provides continuous high-precision information on the gas composition injected into the experiment's windowless gaseous tritium source (WGTS), specifically on its isotopic purity of tritium-one of the key parameters required in the derivation of the electron neutrino mass. The concentrations cx for all six hydrogen isotopologues were monitored simultaneously, with a measurement precision for individual components of the order 10-3 or better throughout the complete KATRIN data taking campaigns to date. From these, the tritium purity, εT, is derived with precision of <10-3 and trueness of <3 × 10-3, being within and surpassing the actual requirements for KATRIN, respectively