1. Research background

Obesity represents a major public health problem these days. Given the current increase in life expectancy, the prevalence of obesity also raises among aged individuals. This is often accompanied with additional years of susceptibility to chronic diseases associated with obesity in elderly people. Some of these diseases develop in the presence of the low grade inflammation associated with obesity where white adipose tissue macrophages provide chronic levels of inflammatory cytokines systemically. While this systemic chronic inflammation results in numerous fatal disorders such as the development of insulin resistance, non alcoholic steatohepatitis (NASH) and type 2 diabetes mellitus, it has been proposed that it initially originates from the obesity-associated leaky gut where it spreads to the liver and ultimately to circulation. In the leaky gut, the mucosal immune system is in contact with the commensal microbiota that not only increases systemic endotoxemia but also inflammation. These increased obesity-induced inflammatory cytokines impair insulin action in metabolic organs and the intestine to ultimately result in the development of insulin resistance. Ultimately, the leaky gut in obesity directly fuels the liver with microbial products to result in NASH. Furthermore, NASH as well as the obesity-induced inflammation predispose individuals to certain types of cancer.

2. Research questions addressed by the group

The Wunderlich lab aims at dissecting the link between obesity-induced inflammation and associated disorders such as the development of insulin resistance, NASH and cancer. To this end, we use self generated sophisticated mouse models allowing for cell type specific manipulation of inflammatory and metabolic signaling pathways in disease models. Collectively, our research should shed light on the cellular and molecular mechanisms of NASH to ultimately develop novel therapeutic strategies for obese individuals.

3. Possible projects

Modulating hepatic sympathetic inputs in NASH and obesity to reveal effects on hepatic stellate cells
Despite its clinical importance, our knowledge of autonomic control of metabolic organs, such as the liver or white adipose tissue (WAT), via sympathetic and/or parasympathetic fibers is rather limited. Whereas sympathetic innervation of liver and WAT has been documented, a parasympathetic (vagal) input is discussed controversially (1, 2). Further, the ultimate target cells as well as the mode of neuronal transmission (synapses vs. en passant secretion of neurotransmitters) of autonomic fibers in WAT and liver are still under debate (3). Importantly, despite a lack of in-depth knowledge of the neuronal transmission and innervated target cells, the functional impact of autonomic control of metabolic organs is beyond doubt (3, 4). Despite the lack of vagal (cholinergic) innervation, there is clear evidence that acetylcholine (ACh) regulates metabolic inflammation upon nerve stimulation (5). Since sympathetic innervation releases norepinephrine (NE) (instead of ACh), there needs to be an unidentified cell that secretes ACh in a paracrine manner. An obesity-induced inflammatory liver disease is non-alcoholic steatohepatitis (NASH). In NASH, hepatic stellate cells (HSC) differentiate into myofibroblasts that provide collagen in fibrosis. Interestingly, HSCs are able to produce ACh and express choline acetyltransferase (ChAT), the enzyme responsible for ACh generation. Thus, we hypothesize that sympathetic input to stroma cells decreases metabolic inflammation and, thus, NASH as shown for spleen (6). We will use our transgenic dual recombinase system (7) (Lrat Cre in stellate cells and Dbh Dre in sympathetic inputs) to specifically label and modulate stellate cells and sympathetic inputs in diet-induced obese mouse models. Here, we follow the hypothesis that sympathetic input regulates HSCs to produce ACh that controls inflammation. We will use liver-specific retrograde AAVs expressing mCherry, DREADDs or Diphtheria toxin Receptor (DTR) that upon Dre mediated invertion label, activate or ablate hepatic neuronal inputs. We will use mice that besides DbhDre transgene will also carry Lrat Cre (specific for HSCs) and a Cre mediated reporter (EGFP-RPL10a R26). Thus, we will be able to stain red fluorescent neuronal inputs together with HSC specific green fluorescent EGFP-RPL10a in triple transgenic mice. We will determine synaptic connections and quantify those upon exposure of mice to diet-induced obesity. We will perform sectioned livers as well as whole mount clearance and staining. These experiments will reveal whether there is a connection between HSCs and sympathetic inputs and whether affected in obesity. Next, we aim at identifying the molecular make up of HSCs in NASH with or without activation of sympathetic inputs. To this end, we will inject a FREX hm3d AAV construct into triple transgenic mice that upon CNO injection activates hepatic sympathetic innervation. Since EGFP-RPL10a is expressed in HSCs of these mice, we can pull down the cell type specific translatome of HSCs with or without activation of sympathetic innervation. We will do this in control fed and diet-induced obese mice and compare gene expression between these conditions. These experiments will reveal whether hepatic sympathetic input activation regulates gene expression in HSCs. Similarly, we will ablate sympathetic hepatic inputs by using a FREX DTR AAV construct. All mice will also be phenotypically characterized and examined in hyperinsulinemic euglycemic clamps to determine organ and CNS insulin sensitivity. Collectively, these experiments will reveal whether sympathetic neuronal innervation to the liver regulates HSCs in NASH and obesity.

4. Applied Methods and model organisms

All experiments will be exclusively performed in transgenic mice and tissue culture. Methods include mouse experiments, metabolic experiments (ITT, GTT, Hyperinsulinemic euglycemic CLAMPS, HOMA IR, adiposity etc.), FACS, Western blot, qPCR, ELISA/Multiplex, Immunohistochemistry.

5. Desirable skills and qualifications

The student should be aware that this project includes in vivo mouse experimentation. A FELASA B course and mouse handling experience would be helpful but can also be acquired/learned during the thesis. Basic immunology, metabolic and molecular biology skills are of advantage but not essentially necessary.

6. References

1. Kreier F, Kap YS, Mettenleiter TC, van Heijningen C, van der Vliet J, Kalsbeek A, Sauerwein HP, Fliers E, Romijn JA, Buijs RM. Tracing from fat tissue, liver, and pancreas: a neuroanatomical framework for the role of the brain in type 2 diabetes. Endocrinology. 2006; 147(3):1140-7. doi:10.1210/en.2005-0667.
2. Giordano A, Song CK, Bowers RR, Ehlen JC, Frontini A, Cinti S, Bartness TJ. White adipose tissue lacks significant vagal innervation and immunohistochemical evidence of parasympathetic innerva-tion. Am J Physiol Regul Integr Comp Physiol. 2006; 291(5):R1243-55. doi: 10.1152/ajpregu.00679.2005.
3. Ruud J, Brüning JC. Metabolism: Light on leptin link to lipolysis. Nature. 2015; 527(7576):43-4. doi: 10.1038/527043a.
4. Zeng W, Pirzgalska RM, Pereira MM, Kubasova N, Barateiro A, Seixas E, Lu YH, Kozlova A, Voss H, Martins GG, Friedman JM, Domingos AI. Sympathetic neuro-adipose connections mediate leptin-driven lipolysis. Cell. 2015; 163(1):84-94. doi: 10.1016/j.cell.2015.08.055.
5. Kimura K, Inaba Y, Watanabe H, Matsukawa T, Matsumoto M, Inoue H. Nicotinic alpha-7 acetylcholine receptor deficiency exacerbates hepatic inflammation and fibrosis in a mouse model of non-alcoholic steatohepatitis. J Diabetes Investig. 2019 May;10(3):659-666. doi: 10.1111/jdi.12964
6. Rosas-Ballina M, Olofsson PS, Ochani M, Valdés-Ferrer SI, Levine YA, Reardon C, Tusche MW, Pavlov VA, Andersson U, Chavan S, Mak TW, Tracey KJ. Acetylcholine-synthesizing T cells relay neural sig-nals in a vagus nerve circuit. Science. 2011; 334(6052):98-101. doi: 10.1126/science.1209985.
7. Biglari N, Gaziano I, Schumacher J, Radermacher J, Paeger L, Klemm P, Chen W, Corneliussen S, Wunderlich CM, Sue M, Vollmar S, Klöckener T, Sotelo-Hitschfeld T, Abbasloo A, Edenhofer F, Reimann F, Gribble FM, Fenselau H, Kloppenburg P, Wunderlich FT, Brüning JC. Functionally distinct POMC-expressing neuron subpopulations in hypothalamus revealed by intersectional targeting. Nat Neurosci. 2021; 24(7):913-929. doi: 10.1038/s41593-021-00854-0.