Isorce Nathalie, Testoni B., Luangsay S., Locatelli M., Zannetti C., Henry T., Hasan U., Zoulim F., Durantel D.

Transcription

1 Activité antivirale de différents interférons (IFNs) et cytokines proinflammatoires dans des modèles pertinents d hépatocytes infectés par le virus de l hépatite B (HBV) Isorce Nathalie, Testoni B., Luangsay S., Locatelli M., Zannetti C., Henry T., Hasan U., Zoulim F., Durantel D. Centre de Recherche en Cancérologie de Lyon CRCL (France) UMR Inserm 1052 CNRS 5286 CLB

2 I have no disclosure.

3 OBJECTIVES Interplay between HBV and innate immunity Circulating HBV DC LSEC KC Recognition by sensors NK/N KT SD Cytokines (e.g. IL-6, IL-1β, TNFα, IFNs, ) Stimulation of specialized s Recognition by host sensors Antiviral actions BC Prevention against infection Hepato-protection and survival Hepatocyte Infected Infected Protected Adapted from Ait-Goughoulte et al., Viruses 2010

4 OBJECTIVES Interplay between HBV and innate immunity TLR9 Circulating HBV DC LSEC Inflammasome KC Recognition by sensors NK/N KT SD Cytokines (e.g. IL-6, IL-1β, TNFα, IFNs, ) IFNs BC Hepatocyte Infected Infected Protected Gruffaz et al, EASL 2013 & HBV meeting 2013 ; Zannetti, Isorce, et al, EASL 2013 ; Vincent et al, PLoS One 2011

5 OBJECTIVES Interplay between HBV and innate immunity Circulating HBV DC LSEC KC Recognition by sensors NK/N KT SD Cytokines (e.g. IL-6, IL-1β, TNFα, IFNs, ) Hepatocyte Recognition by host sensors Infected Antiviral actions Infected 1. Anti-HBV effect of cytokines in relevant in vitro BC models? 2. Mechanisms and effectors Protected? Define new immunotherapeutic approaches against HBV

6 OBJECTIVES Interplay between HBV and innate immunity Circulating HBV DC LSEC KC Recognition by sensors NK/N KT SD Cytokines (e.g. IL-6, IL-1β, TNFα, IFNs, ) Recognition by host sensors Antiviral actions 1. Anti-HBV effect of cytokines? Hepatocyte Infected Infected Protected

7 EXPERIMENTAL APPROACH Differentiated HepaRG s or PHH D0 HBV infection 100 vge/ CYTOKINE SINGLE ADMINISTRATION D7 Established infection * D10 -IFN-I,II,III -Inflammatory cytokines days PI Analysis of viral parameters Viral DNA* Viral proteins* days PI HBV: 100 vge/ vge/ vge/ Hantz et al., J Gen Virol, 2009 ; Luangsay et al., submitted

8 HepaRG Effect on intraular total HBV DNA accumulation (qpcr) All cytokines dose-dependently decrease HBV DNA Units: pg/ml

9 HepaRG Effect on intraular total HBV DNA accumulation (qpcr) IL-1β still exerts inhibition of HBV DNA at very low doses Units: pg/ml

10 HepaRG IFNβ (IFNs) STAT1/2-dependent IL-1β (Inflammatory cytokines) NFκB-dependent HBV DNA HBV DNA HBV RNA HBV RNA HBV Ag HBV Ag

11 IFNβ IL-1β HepaRG STAT1/2-dependent NFκB-dependent HBV DNA HBV DNA HBV RNA HBV RNA HBV Ag HBV Ag Different mechanism of action between IFNs and inflammatory cytokines

12 IFNβ IL-1β HepaRG STAT1/2-dependent NFκB-dependent HBV DNA HBV DNA HBV RNA HBV RNA HBV Ag HBV Ag Confirmation in PHH in progress. Next step: Investigation of the effect on cccdna (silencing or degradation?) and pgrna?

13 OBJECTIVES Circulating HBV DC Inflammasome KC NK/N KT LSEC SD Cytokines IL-1β (e.g. IL-6, IL-1β, TNFα, IFNs, ) (very low doses) 2. Mechanisms and effectors? Antiviral actions Hepatocyte Infected Infected Protected Zannetti, Isorce, et al, EASL 2013

14 Comparison among IL-1 family members Adapted from Dinarello, 2009

15 Comparison among IL-1 family members Adapted from Dinarello, 2009 Downstream signaling of IL-1RI is involved in IL-1β antiviral effect against HBV

16 HYPOTHESIS? IL-1β IL-1RI IL-1RAcP NFκB Target transcripts IFNs TGFβ IL-6 TNFα IL-8 MIP1α IL-1α INDIRECT antiviral effect? (potentially induced secreted cytokines)

17 HYPOTHESIS? IL-1β IL-1RI IL-1RAcP NFκB Target transcripts IFNs IL-6 IL-8 TGFβ TNFα MIP1α IL-1α INDIRECT antiviral effect? (potentially induced secreted cytokines) DIRECT inhibitory effect? Putative binding sites of NFκB on HBV genome Kwon et al., 2002 Inhibitory transcriptional role of NFκB Hasan et al., 2013

18 Early and direct antiviral effect of IL-1β Antiviral effect of IL-1β already at 8h post-treatment and not related to IL-6

19 Early and direct antiviral effect of IL-1β Antiviral effect of IL-1β already at 8h post-treatment and not related to IL-6

20 HYPOTHESIS? IL-1β IL-1RI IL-1RAcP NFκB Target transcripts IFNs IL-6 IL-8 TGFβ TNFα MIP1α IL-1α INDIRECT antiviral effect? (potentially induced secreted cytokines) DIRECT inhibitory effect? Putative binding sites of NFκB on HBV genome Kwon et al., 2002 Inhibitory transcriptional role of NFκB Hasan et al., 2013

21 Does IL-1β have an inhibitory effect on HBV transcription? Plating of proliferating HepaRG s D0 D1 Transfection SINGLE ADMINISTRATION IL-1β D2 2h Medium change D3 time Luciferase assay

22 Does IL-1β have an inhibitory effect on HBV transcription? Plating of proliferating HepaRG s D0 D1 Transfection SINGLE ADMINISTRATION IL-1β D2 2h Medium change D3 time Luciferase assay IL-1β inhibits both Core and X activity promoters within 2 hours, suggesting that IL-1β may target HBV transcription directly

23 Does IL-1β activate the NF-κB promoter in our model? IL-1β can activate NF-κB promoter, suggesting that IL-1β may target HBV transcription through this pathway. Next step: Confirmation in ChIP with NF-kB

24 CONCLUSION Panel of cytokines: type I, II, III IFNs IL-6, TNFα, IL-1α/β BC Antiviral actions BC Infected Hepatocyte Infected Protected

25 CONCLUSION Panel of cytokines: type I, II, III IFNs IL-6, TNFα, IL-1α/β BC Antiviral actions BC Infected Hepatocyte Infected Protected HBV DNA (Watashi et al., 2013) HBV RNA (+++) HBeAg (not HBsAg) IL-1β (low doses) HBx/Core promoters NF-κB promoter

26 CONCLUSION Panel of cytokines: type I, II, III IFNs IL-6, TNFα, IL-1α/β Mechanisms? BC Antiviral actions BC Infected Hepatocyte Infected Protected HBV DNA (Watashi et al., 2013) HBV RNA (+++) HBeAg (not HBsAg) IL-1β (low doses) HBx/Core promoters NF-κB promoter cccdna? pgrna and others?

27 Acknowledgments Teams 15&16 of INSERM U1052 (CRCL, Lyon, France) And particularly Fabien Zoulim David Durantel Barbara Testoni Souphalone Luangsay Marion Gruffaz Dulce Alfaiate Judith Fresquet Maud Michelet Lydie Lefrançois Marie-Laure Plissonnier Maëlle Locatelli Collaborations: Uzma Hasan, Thomas Henry, Claudia Zannetti INSERM U1111 (CIRI), Lyon Nathalie Bendriss-Vermare, Christophe Caux INSERM U1052 (CRCL) Travel Grant:

28 Viability Assays No cytotoxicity Units: pg/ml, excepted for positive control Puromycin(antibiotic) in µg/ml

29 No impairement of extracted RNA rate by IL-1β treatments

30 HBV infected Primary Human Hepatocytes Southern Blot (HBV) on intraular total DNA Batch #1 IFNβ IFNγ TNFα IL-28B IL-29 IL-6 IL-1β (ng/ml) IL-18 Ribavirin IFNα 60-80% inhibition

31 HBV infected Primary Human Hepatocytes

32 IL-1α results in HepaRG s: Intraular HBV DNA (qpcr) Secreted HBeAg and HBsAg (ELISA)

33 What is the effect of IL-1β/ IFNα or Tenofovir co-treatment on HBV? Chronically infected HBV patients could be treated with IFNα or Tenofovir 30% 75% 85% No antagonism between IL-1β and IFNα or Tenofovir (Potentiation of the combination (with IFN) demonstrated in PLC/PRF/5, Hamasaki et al, 1992)

34 1. Indirect antiviral effect via other potential cytokines? SUMMARY 50% inhibition of HBeAg secretion by 100pg/ml of IL-1β Cytokines secreted Upon 100pg/ml IL-1β treatment during 72h IL-6 ~3000pg/ml <50% TNFα <10 pg/ml No IL-1α <20 pg/ml No IL-29 <50 pg/ml No Individual antiviral activity (HBeAg) IP10 ~1000 pg/ml Not determined IL-4 Not detected Not determined Other cytokines? Luminex analyses? Hypothesis: Antiviral activity of IL-1β could not be totally due to IL-6 Confirm this hypothesis using (excess of) anti-il-6 neutralizing antibody (>10ng/ml) and anti-tnfα neutralizing antibody (>100pg/ml), and IL-1Ra (150µg/ml) to confirm specificity of action of IL-1 Identify other cytokines?

35 Cellular factors associated with IL-1β anti-hbv activity? Analysis of some hepatocyte markers: mrna quantification by qrt-pcr Secreted protein quantification by ELISA Next step: Confirmation at the protein level (FACS HNF-4α) has to be done

36 Are HepaRG expressing receptors of inflammatory cytokines? What about IL-1 family? IL-1 Receptor (IL-1RI) IL18 Receptor (IL-18Ra) Receptors from IL-1 family are present, but we still have to check the expression of ST2 (IL-33R) and IL1RAcP (associated receptor of IL-1RI and ST2)

37 Are HepaRG expressing receptors of inflammatory cytokines? What about other receptors of inflammatory cytokines? IL-6 Receptor (gp80) TNFα Receptor (TNFRI) Receptors of IL-6 and TNFα are expressed

38 IL-1β signaling Dinarello, Ann Rev Immunol 2009