Disturbances in the gut-brain axis resulting in hypothalamic inflammation are important in the pathogenesis of obesity. Recently we found in humans that a diet rich in saturated fatty acids induces a specific change in the gut microbiome (decrease in Parasutterella spec.) associated with hypothalamic inflammation. Of interest, this effect was most prominent in human subjects carrying common SNP in the c-Jun N-terminal kinase (JNK) gene, implicating that gene-environment interactions are important in the development of hypothalamic inflammation in human obesity. These results were recently published: Kreutzer et al., Diabetes (2017), 66:2407-2415.
The main goal is identify novel pro-inflammatory mediators in the communication of the gut with the brain in the context of hypothalamic inflammation in obesity. To this end, the analysis will combine bioinformatics studies using data from our FoCus cohort and also experimental studies using our well established infrastructure in cell and molecular biology.
Work package (WP) 1: Screening for inflammatory mediators of the gut-brain axis in the FoCus cohort:
From 2011 to 2016 our research group has recruited the FoCus cohort in the North of Germany including n=1500 cross-sectional controls and n=500 subjects with severe obesity. All study participants have been pheno- and genotyped in detail including 16S rRNA gene sequencing of stool samples and 12-month nutritional analysis. In addition, magnetic resonance images (MRI) were performed to detect hypothalamic inflammation and serum samples were stored in our PopGen Biobank for future analyses. In the first set of experiments it is assumed that the RTG student will measure several cytokines in an appropriate number of FoCus serum samples including also the novel pro-inflammatory wnt5a. The reason to use wnt5a as a potential candidate is that wnt5a has been shown to be a pro-inflammatory factor secreted by macrophages and that the wnt-signalling pathway has been shown to be important in the function of the gut mucosa. Afterwards, the cytokine and wnt5a levels will be related to nutritional factors, the microbiome (esp. Parasutterella) and to the hypothalamic MRI data in a systems medicine approach in order to detect potential candidates mediating hypothalamic inflammation in response to environmental factors (nutrition and microbiome). In a second analysis, the levels of the so identified cytokines/mediators will be related to the above mentioned common JNK SNP in order to identify, if these common polymorphisms influence the cytokine levels which would indicate a specific genetic predisposition.
WP2: Functional analysis of the identified pro-inflammatory factors:
In a second set of experiments it will be examined on cellular and molecular level (1.) how the identified pro-inflammatory factors (cytokines, wnt5a …) are getting released in the colonic mucosa [gut site] and (2) how these factors affect neuronal cell and/or microglia [brain site]. On the gut site colonic cell lines will be incubated in vitro with certain nutritional factors and/or certain gut bacteria (Parasutterella) and the release of the identified cytokines into the medium will be analyzed by ELISA. On the brain site, a cell culture system will be implemented by the RTG student for both, neurons and microglia. Afterwards, these cells will be characterized in respect to the response to the incubation with the identified pro-inflammatory molecules. In these analyses standard technics in molecular biology (RT-PCR, western blotting etc.) will be used. Also, it is assumed to specifically knock-down potential intracellular cytokine pathways in the cell system by siRNA to prove specificity.