Session du Club pour l Etude de la Fibrose (CEF)

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1 Session du Club pour l Etude de la Fibrose (CEF) Modérateur : Chantal Housset est responsable à l UMR_S 938 (Inserm & UPMC), CdR Saint- Antoine, Paris, de l Equipe Maladies fibro-inflammatoires d'origine métabolique et biliaire du foie à laquelle est associée la compagnie de biotechnologie BioPredictive.

2 La réponse au stress du réticulum endoplasmique active l'inflammasome dans la NASH: bénéfice d'un traitement avec le TUDCA Dr. Béatrice Bailly-Maitre Lille, 2 Octobre 213 INSERM U165 Mediterranean Centre for Molecular Medicine (C3M), Nice, France Team 8 (Pr Tran & Dr Gual)

3 Aucun conflit d intérêt

4 The epidemic of obesity 5 million worldwide obese adults ( 72 million obese adults in the United States ( Premature death Insulin resistance Type 2 diabetes NAFLD Atherosclerosis Hypertension Stroke OBESITY Cancer Asthma Sleep apnea Osteoarthritis Neurodegeneration Gall bladder disease Metabolic diseases clusters NAFLD = Non-Alcoholic Fatty Liver Disease the leading cause of chronic liver diseases in Western countries Nonalcoholic fatty liver disease. P. Angulo. N Engl J Med,

5 Non Alcoholic Fatty Liver Diseases (NAFLD) Normal liver Steatosis Intrahepatic triglyceride accumulation Nonalcoholic steatohepatitis (NASH) Inflammation Hepatocyte death Hepatocellular ballooning Fibrosis Cirrhosis Hepatocellular carcinoma Calle EE, N Engl J Med. 23

6 Obesity, ER stress and NASH Normal liver Steatosis NASH Insulin Resistance Inflammation Lipolysis ER stress links obesity, insulin action and type 2 diabetes. Ozcan et al. Science (24). ER STRESS Fibrose Cirrhose HCC TG TG TG TG Insulin Resistance ER STRESS? TG TG TG TG TG TG TG TG TG TG Hepatocyte Death Apoptosis Inflammation ER STRESS?

7 ER stress and NASH OBESITY TUDCA and 4-PBA BI-1 Perk is essential for translational regulation and cell survival l during the UPR. Ron D. Mol Cell. (2). IRE1-mediated unconventional mna splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 UPR. Kaufman. Genes Dev (22). bi-1 is required for intrinsic protection from ER stress. Bailly-Maitre, B. PNAS. (26) and Cancer Research (27) Hepatic Bax inhibitor-1 inhibits IRE1alpha and protects from obesity-associated. Bailly-Maitre B et al. JBC (21).

8 Rational and Experimental procedure 1/ ER Stress Inhibition ob/ob PBS or TUDCA (5µg/g/j) 5 days PBS or LPS (2µg/g) ob/ob 6h Liver injury (Histology,Transaminases, Apoptosis) 2/ ER Stress Activation PBS or Tuni (2µg/g) 6h Hepatic ER stress markers Hepatic and systemic Inflammasome markers ob/ob

9 ER stress inhibition significantly reduces liver injury and apoptosis after LPS challenge Treatment with TUDCA before LPS injection reduced serum transaminases levels Increased apoptosis in livers of LPS injected obob mice is prevented by TUDCA UI/L ALT $$$ PBS TUCDA LPS LPS +TUCDA % of TUNEL positive hepatocytes PBS TUDCA LPS $$ TUDCA+LPS n=6 UI/L AST $$$ PBS TUCDA LPS LPS +TUCDA n = 7-9 each group PBS TUDCA LPS TUDCA+LPS Casp-3 p17 HSP9 Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Feldstein AE et al. Gastroenterology. 23. n=9

10 TUDCA inhibits LPS-induced PERK IREα-mediated XBP-1 processing and PERK activation LPS ATF6 IRE1 GRP78 IRE1α pathway PBS TUDCA LPS TUDCA+LPS sxbp1 ATF4 ATF6c sxbp1 PERK pathway HSP 9 CHOP p-eif2α t-eif2 Bcl-X L Apoptosis ATF4 PBS TUDCA LPS TUDCA+LPS GRP78 CHOP HSP 9 Bcl-2 family HSP9 PBS TUDCA LPS TUDCA+LPS Bax Bcl-XL PBS TUDCA LPS TUDCA+LPS Fold induction # GRP78/CHOP n= 9 Fold induction PBS TUDCA LPS TUDCA+LPS # ### ### Xbp-1s p-eif2 ATF4 Fold induction 2,5 2 1,5 1,5 ### Bax/Bcl-XL

11 TUDCA protects from inflammasome activation Hyperglycemia FFAs Endotoxinemia Bcl-2 Bcl-xL TUDCA decreases inflammation H&E (X2) LPS LPS+TUDCA ASC Martinon F et al. Moll Cell. 22. Inflammation Pyroptosis Bruey J.M et al. Cell. 27. TUDCA decreases inflammasome activation Number of inflammatory foci # PBS TUDCA LPS TUDCA+LPS n=9 PBS TUDCA LPS TUDCA+LPS Casp-11 p43 Casp-11 p26 Casp-1 p2 HSP9 NLRP3 HSP9 IL-1β (pg/ml) 25 2 ## PBS TUDCA LPS TUDCA +LPS IFNγ (pg/l) ### PBS TUDCA LPS TUDCA +LPS n=7-9 n=7-9

12 ER stress significantly increases liver apoptosis, IRE1 & PERK activities and inflammasome activation Tuni increased hepatocyte death Tuni induces apoptosis IU/L PBS Tuni p =,4 Asat Alat % of TUNEL positive hepatocytes PBS Tuni PBS TUNI Casp-3 P19 p17 HSP9 Tuni overwhelms IRE1 and PERK activities Tuni leads to inflammasome activation IRE1/XBP1 Axis PBS TUNI PBS TUNI PBS TUNI CHOP GRP78 HSP9 PERK/ATF4 Axis sxbp1 HSP 9 Casp-11 p26 Casp-1 p2 Fold induction CHOP/GRP78 PBS Tuni PBS TUNI P-PERK t-perk HSP 9 IL-1β p17 IL-18 p14 HSP9

13 TUDCA protects from LPS-induced-CHOP dependent inflammasome activation in mouse primary hepatocytes TUDCA protects hepatocytes from LPS+Tuni induced apoptosis VIABILITY (%) C TUDCA LPS Tu # # Tu TUDCA +LPS +Tu+LPS TUNEL (% pos cells) C TUDCA # LPS Tu Tu +LPS # TUDCA +Tu+LPS chop Genetic Inhibition and TUDCA treatment abolished inflammasome activation Cont si chop Cont TUDCA C LPS Tu Tu+ LPS C LPS Tu Tu + LPS C LPS Tu Tu+ LPS C LPS Tu Tu + LPS CHOP Casp-11 p43 Casp-11 p26 Casp-1 p2 Pro- IL-1β IL-1β p17 HSP9

14 ER stress and inflammasome gene expression in Human NASH Increased ER stress and inflammasome gene expression in NASH patients Fold induction chop/grp78 Without NAFLD ## Steatosis NASH Caspase-1 Without NAFLD ## Steatosis NASH Caspase-4 Without NAFLD ### Steatosis NASH IL-1β Without NAFLD Steatosis ### NASH Correlation with biological parameters NAS Inflammation STEATOSIS ALT AST Fold r s P N r s P N r s P N r s P N r s P N CHOP/,445,16 29,633 <,1 29,181,347 29,442,19 29,37,48 29 GRP78 Casp1,52,3 3,517,3 3,493,6 3,434,17 3,421,2 3 Casp4,629 <,1 3,69 <,1 3,572,1 3,565,1 3,522,3 3 NLRP3,449,13 3,416,22 3,475,8 3,29,119 3,352,57 3 IL1b,636 <,1 29,642 <,1 29,434,19 29,474,9 29,499,6 29

15 Take home message TUDCA Hepatocyte ER Stress Inflammasome LPS/Tuni PERK IRE1 ATF4 sxbp1 NLRP3 Pro-IL-1β Pro-IL-18 CHOP ASC Caspase-11 Caspase-1 IL-1β IL-18 APOPTOSIS INFLAMMATION (IL-1β, IL-18, IFNγ)

16 AKNOWLEDGMENTS Collaborators Sanford-Burnham Medical Research Institute La Jolla, CA John C Reed IPMC, Sophia Antipolis Carole Rovere INSERM U165, C3M,Equipe 8, Nice CH. Desnoyer de Bieville E. Proics D. Rousseau, IR S. Patouraux, IE S. Bonnafous E. Gouze, CR1 S Garcia D. Crenesse, PUPH R. Anty, PH Ianelli A, PUPH A. Tran, PUPH P. Gual, DR2 B. Bailly-Maitre-Re, CR1. Institut Benjamin Delessert Société Française Diabétologie 212 AFEF 213

17 Does hepatic BI-1 protect from inflammation associated obesity? 1/ Hypothesis : ER Stress inhibition = NAFLD db/db Ad BI-1 vs Ad GFP 5 days db/db? Insulino Resistance Blood glucose Hepatic ER stress markers Inflammasome markers 2/ Hypothesis : ER Stress activation = NAFLD bi- 1 +/+ bi- 1 -/- ND (8 months) HFD 16 weeks? Insulino Resistance Blood glucose Hepatic ER stress markers Inflammasome markers

18 Hepatic BI-1 inhibits IRE1α and Inflammasome activation Ad BI-1 reversed hyperglycemia in db/db mice. Ad BI-1 inhibits Caspase-1 activation and IL-1β processing in db/db mice Ad GFP Ad BI-1 Procaspase-1 p35 p2 Pro-IL1β P17 IL1β Ad BI-1 decreases NRLP3 expression Hsp9 Nrlp3 Hsp9 Students : Licence Biotechnologie: E. Proics

19 Is bi- 1 deficiency predisposing for steatosis and type 2 diabetes? Hypothesis : ER Stress activation = NAFLD bi- 1 +/+ bi- 1 -/- ND (8 months) HFD (16 weeks)? Insulino Resistance Blood glucose Hepatic ER stress markers Inflammasome markers bi- 1 -/- have impaired glucose metabolism (8 months old) bi- 1 -/- are developping steatosis (8 months old) bi- 1 + /+ bi- 1 - /-

20 Open questions/perspectives 1/ Will diet-induced-obesity increase inflammasome activation in bi- 1 -/- mice correlating with overwhelming ER stress? Which are the mechanisms? Consequences on NAFLD? 2/ Might the breeding DKO caspase-1/caspase-11 X bi-1-/- rescue the bi-1 phenotype? 3/ Might BI-1 directly or indirectly be able to modulate inflammasome regulation? 4/ Which are the consequences of inflammasome invalidation on ER stress signaling pathways?