RESEARCH PROJECT-1 Mid-semester Report On Role of Bile acids on Macrophage function Submitted by Kotwal Shifa Bushra 2018PHXP0004H Under the Supervision of Dr

Mid-semester Report
Role of Bile acids on Macrophage function
Submitted by
Kotwal Shifa Bushra
Under the Supervision of
Dr. Trinath Jamma

Department of Biological Sciences
BITS-Pilani Hyderabad Campus

Role of Bile acids in the regulation of metabolic pathways
Bile acids (BA), components of human bile have an integral role in health, through the absorption of lipophilic nutrients and vitamins. Bile acids are produced by liver cells as 1? BA but undergo further conjugation to form 2? BA by intestinal bacteria , prior to their absorption and return to the liver. The pathways that governs BA recycling between the gut and the liver and the identi?cation of BA-speci?c receptors in different cell-types and tissues have marked their role in health and disease mainly in relation to metabolic and liver diseases.BA synthesis occurs mainly by two different pathways i.e., classic pathway and alternative pathway. In the classic pathway, hydroxylation at C7? position which is the first step is catalyzed by the enzyme cholesterol­7?-hydroxylase (CYP7A1). In the alternate pathway, first hydroxylation at C27 position is carried out by the enzyme sterol-27?-hydroxylase (CYP27A1). Generally the classic pathway is mainly more important pathway than the alternative pathway since it is responsible for maintaining cholesterol homeostasis.

1047750251714000 The BAs activates intestinal FXR which causes an increased expression of fibroblast growth factor (FGF)-15 in rodents and FGF-19 in humans. Bile acids are absorbed in the ileum activates FXR so as to induce production of FGF-15/19. FGF-15/19 passes through the portal vein to the hepatocytes and couples with FGF receptor 4 (FGFR4) which inturn induces receptor dimerization, autophosphorylation, and c-Jun N-terminal kinase pathway activation resulting in the repression of CYP7A1 transcription. TGR5/M-BAR, . a 2nd BA receptor also contributes for the regulation of Bile acid homeostasis. Phosphoenolpyruvate carboxykinase(PEPCK),which is the rate limiting enzyme of gluconeogenesis is suppressed by Bile acids. BAs repress enzymes, glucose 6-Phosphatase and fructose 1,6-bisphosphatase . BAs stimulates glucagon like peptide-1(GLP-1) which inturn promotes secretion of insulin by binding to the GLP-1 receptor in ? cells of pancreas. The target of FXR,SHP, suppressed up regulation of sterol regulatory element binding protein-1c(SREBP-1c) to mainly to reduce TG synthesis(fig 1).

Figure 1A. Farnesoid X receptor-dependent metabolic regulation in the liver. B. TGR5/M-BAR-dependent metabolic regulation (Ref-1)
Self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress is referred to as Autophagy. Stimulation of FXR supresses autophagy in the liver. At the promoter regions of autophagic genes FXR and PPAR? competitively bind and show effects on transcription mainly based on nutrional conditions. Basically in liver, under fasted conditions PPAR? activation promotes autophagic lipolysis. Stimulation of FXR caused by feeding disrupts the functional CREB-CRTC2 complex and eventually downregulates autophagy(Fig 2).


Figure 2. Farnesoid X receptor role in Autophagy regulation (Ref-1)
Figure 3. Intestinal FXR activity on conflicting mechanisms of metabolic regulation (Ref-1)

Induction of FGF-15/19 expression by intestinal FXR activation affects glucose and energy homeostasis. Increased ?-oxidation in the liver by overexpression of FGF-19. FXR activation by administration of fexaramine, an FXR agonist, improved insulin resistance and obesity by inducing FGF-15 which inturn changes BA composition in serum readily stimulates systemic TGR5. An FXR antagonist, T-?-MCA and microbiota affect mainly bile acid homeostasis by inhibition of intestinal FXR signalling and change in BA composition inturn improves lipid and glucose metabolism. Increased levels of T-?-MCA leads to reduced FXR activation in intestine and very low serum ceramide levels due to repression of ceramide synthesis which downregulates hepatic SREBP-1c leading to improvement in NAFLD and obesity. FXR deactivation in intestine also improves glucose and lipid metabolism. BABR(Bile acid binding resin) can induces GLP-1 secretion via activation of TGR5 and can decrease blood glucose levels only in high glucose situations (Fig 3).

Chenodeoxycholic acid activates NLRP3 inflammasome and contributes to cholestatic liver fibrosis.

Long-term interruption in the excretion of bile causes cholestasis. Increasing evidence marks the levels of multiple pro-inflammatory cytokines that are strongly increased in the cholestasis, including IL-1?, IL-6 and TNF-?.Being a key inflammatory mediator upstream of IL-6 and TNF-? signaling cascades, IL-1? is produced by activated macrophages and is involved in diverse acute and chronic liver injury, including liver fibrosis.

In the present study, the authors for the first time proved that CDCA, which is the major toxic bile acid involved in cholestasis, could dose dependently induce NLRP3 inflammasome activation and secretion of pro-inflammatory cytokine-IL-1? in murine J774A.1 macrophages, bone marrow derived macrophages as well as kupffer cells. An enormously increase in mature IL-1? levels in liver of BDL mouse model was observed . IL-1? promoted fibrogenesis and being an important inflammatory mediator, it also recruits other inflammatory cells, mainly neutrophils, which are implicated actively in tissue inflammation and worse liver damage. Consistently, the in vivo studies showed that using caspase-1 inhibitor lead to inhibition of IL-1? level with drastic low MPO activity in the liver and ameliorated liver fibrosis in BDL mouse model, which inturn suggests an important role of IL-1? in triggering tissue inflammation and fibrosis during cholestasis.
The data provided by authors demonstrates a new mechanism that excessive toxic bile acids initiate liver inflammation at least in part through NLRP3 inflammasome activation. The in vitro studies demonstrates that phagocytic uptake was not required for CDCA-induced IL-1? secretion in macrophages, and that is different from the mode of NLRP3 activation by various crystals. The following experiments confirms that through TGR5,CDCA activated NLRP3 inflammasome and induced IL-1? release partially.
TGR5 abundant expression on monocytes and macrophages was also observed to exhibit immunomodulatory effects. In contrast to results provided by authors, some studies have shown that CDCA exerts an inhibitory effect on inflammatory cytokines production in LPS-stimulated macrophages mainly through TGR5 AC-cAMP dependent pathways. Based on reports provided, bioactive IL-1? production requires priming signal (1st signal), which increases pro-IL-1? and NLRP3 level, and then followed by activation signal (2nd signal) which induces NLRP3 inflammasome assembly and activation.
In response to CDCA, the authors observed mature IL-1? release from LPS primed macrophages, therefore, one possible explanation that first macrophages were primed with LPS and then they used CDCA as second signal which activaes the inflammasome. The results showed that transactivation of EGFR, an alternative pathway independent of AC-cAMP lead to inflammasome activation and IL-1? release, which suggests that TGR5 may regulate inflammatory response through different mechanisms. The up-regulation of EGFR-ERK/AKT/JNK signaling involves activation of NLRP3 inflammasome by CDCA. Activation of NLRP3 inflammasome by CDCA requires ROS formation, which is evidenced by reduced IL-1? production after NAC treatment. Many studies have described that EGFR activation induces ROS formation. The present study shows the role of CDCA on inflammasome activation which is attributed to the induction of ATP release from macrophages, which inturn triggers K+ efflux through purinergic signalling. P2X7 receptor (purinergic receptor) is involved in induction of K+ efflux, which shows that ATP can bind to P2X7 receptor and then open the P2X7-associated ion channel pore, which increases K+ conductance(Fig 1).

Figure 1: Proposed model of CDCA-induced NLRP3 inflammasome activation and liver injury (Ref-2)
Hiroki Taoka, Yoko Yokoyama, Kohkichi Morimoto, Naho Kitamura, Tatsuya Tanigaki, Yoko Takashina, Kazuo Tsubota, Mitsuhiro Watanabe. Role of bile acids in the regulation of the metabolic pathways. World J Diabetes. 2016 ; 7(13): 260-270.

Zizhen Gong, Jiefei Zhou, Shengnan Zhao, Chunyan Tian, Panliang Wang, Congfeng Xu, Yingwei Chen, Wei Cai and Jin Wu. Chenodeoxycholic acid activates NLRP3 inflammasome and contributes to cholestatic liver fibrosis. Oncotarget.2016;7(51): 83951-83963.