TGR5 Activation Inhibits Atherosclerosis by Reducing Macrophage Inflammation and Lipid Loading

The G protein-coupled receptor TGR5 has been identified as an important component of the bile acid signaling network, and its activation has been linked to enhanced energy expenditure and improved glycemic control. Here, we demonstrate that activation of TGR5 in macrophages by 6 alpha-ethyl-23(S)-methylcholic acid (6-EMCA, INT-777), a semisynthetic BA, inhibits proinflammatory cytokine production, an effect mediated by TGR5-induced cAMP signaling and subsequent NF-kappa B inhibition. TGR5 activation attenuated atherosclerosis in Ld/r(-/-)Tgr5(+/+) mice but not in Ld/r(-/-)Tgr5(-/-) double-knockout mice. The inhibition of lesion formation was associated with decreased intraplaque inflammation and less plaque macrophage content. Furthermore, Ld/r(-/-)animals transplanted with Tgr5(-/-) bone marrow did not show an inhibition of atherosclerosis by INT-777, further establishing an important role of leukocytes in INT-777-mediated inhibition of vascular lesion formation. Taken together, these data attribute a significant immune modulating function to TGR5 activation in the prevention of atherosclerosis, an important facet of the metabolic syndrome.

Role of the bile acid membrane receptor TGR5 in macrophages and metabolism

Mitsunori Nomura

Activation of the bile acid responsive G protein-coupled receptor TGR5 improves metabolic homeostasis via induction of energy expenditure and release of glucagon-like peptide-1 (GLP-1). The goal of my PhD work was to explore the role of macrophage TGR5 with respect to inflammation and to evaluate its impact on chronic low-grade inflammatory and metabolic disorders, such as atherosclerosis and insulin resistance. In the first part of this work, we have focused on the role of TGR5 in the progression of atherosclerotic lesions. I showed that activation of TGR5 in macrophages by 6"-ethyl-23(S)-methyl-cholic acid (S-EMCA, INT-777), a semi-synthetic bile acid, inhibits pro-inflammatory responses via a cAMP-NF-!# pathway. Furthermore, INT-777 treatment of chimeric Ldlr knockout mice that were transplanted with bone marrow from either Tgr5 wild-type or Tgr5 knockout mice, revealed that TGR5 in leukocytes largely contribute to the protective effects on atherosclerotic lesion formation. In the second part of this work, I have determined the function of TGR5 in macrophage polarization and evaluated the relative contribution of macrophage TGR5 in the onset of diet-induced insulin resistance. I demonstrated that deficiency of TGR5 results in exacerbated insulin resistance in diet-induced obesity (DIO) mice with severe impairment of insulin responsiveness in adipose tissue. This was associated with an increase in M1 macrophages and a reduction of M2 macrophages in adipose tissue of Tgr5 knockout mice. In line with this, TGR5 activation by INT-777 in DIO mice increased M2 macrophages, attenuated adipose tissue inflammation and improved insulin resistance. Taken together, the research that is described in this thesis reveals a significant immune modulatory function of TGR5, which has a major impact on the prevention of atherosclerosis and insulin resistance in mouse models. Future studies in humans will be required to underscore the importance of TGR5 as a potential target to counteract the chronic low-grade inflammation that goes hand-in hand with many disorders of the metabolic syndrome.

EPFL2013

Establishment of polarized microglial cells derived from mouse embryonic stem cells

Céline Ruegsegger

Microglia are the resident immune cells of the central nervous system (CNS). Microglia undergo rapid activation in response to even minor pathological changes in the CNS. The activation state of microglia and tissue macrophages is characterized by two extremes that are known as M1 and M2. M1 is triggered upon pro-inflammatory stimuli and lead to the secretion of molecules involved in brain defense but also in neurotoxicity. M2 arises upon anti-inflammatory stimuli and is hallmarked by the secretion of molecules that mediate tolerance and neuroprotection. The aim of this study was to investigate the in vitro polarization capacity of microglia into M1 and M2 subtypes. Primary microglia are hard to obtain in sufficient number and therefore a recently developed protocol was applied to differentiate microglial precursors from mouse embryonic stem (ES) cells. The obtained ES cell derived microglial precursors (ESdM) were shown to be an appropriate substitute to primary microglia. Here we showed that ESdM stimulated with interferon-γ (IFN-gamma) and lipopolysaccharide (LPS) display typical M1 characteristics like higher transcription levels of pro-inflammatory cytokines as well as an increase in the expression level of M1 surface markers. They also secreted significantly higher levels of the chemokine CXCL10 and nitric oxide than untreated or interleukin-4 (IL-4) treated cells. These results indicate that ESdM can be polarized into a cytotoxic subtype in vitro using IFN-gamma and LPS. However, no evidence indicating that ESdM were polarized into a neuroprotective M2 subtype by IL-4 treatment has been found. Even though an increase in the transcription level of anti-inflammatory cytokines was observed upon IL-4 treatment, this increase was not significant. In addition, no increase in the expression levels of M2 markers or in the secretion of the anti-inflammatory cytokine IL-10 were found upon IL-4 treatment indicating that IL-4 might be insufficient to polarize ESdM into M2 subtype in vitro. Further experiments are required to better understand the mechanisms of microglial sub-differentiation and to determine the stability of the acquired M1 and M2 phenotypes over time

2010

Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells

Howard Riezman

Sphingolipids are not only important components of membranes but also have functions in protein trafficking and intracellular signaling. The LCB1 gene encodes a subunit of the serine palmitoyltransferase, which is responsible for the first step of sphingolipid synthesis. Here, we show that activation of the unfolded protein response (UPR) can restore normal ceramide levels and viability in yeast cells with a conditional defect in LCB1. Dependence on UPR was demonstrated by showing the HAC1-dependence of the suppression. A similar induction of ceramides by UPR seems to take place in mammalian cells. In rat pancreatic INS-1E cells, UPR activation induces the transcription of the CerS6 gene, which encodes a ceramide synthase. This correlates with the specific accumulation of ceramide with a C16 fatty acyl chain upon UPR activation. Therefore, our study reveals a novel connection between UPR induction and ceramide synthesis that seems to be conserved between yeast and mammalian cells.-Epstein, S., C. L. Kirkpatrick, G. A. Castillon, M. Mu iz, I. Riezman, F. P. A. David, C. B. Wollheim, and H. Riezman. Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells. J. Lipid Res. 2012. 53: 412-420.