1. Ci sono presupposti biologici per una diversa risposta al trattamento in relazione al genere? Giulia Marchetti, MD, PhD Dept of Health Sciences- Clinic of Infectious Dis- University of Milan, San Paolo Hospital, Milano HCV e Donne

2. Today’s outline  Response to Interferon-based anti HCV therapy: any role for hormonal-mediated inflammatory milieu?  Any reason to think of gender-based differences in DAA therapy?

3. Higher rates of SVR to interferon- based therapy according to gender? Manns MP et al. Lancet 2001; McHutchinson JG et al. NEJM 2009…….

4. Hayashi J et al. Arch Intern Med 1998; also: Floreani A et al. Eur J Gastroenterol Hepatol 2011 311 HCV+ patients (199 men; 112 women)

5. Men Women Hayashi J et al. Arch Intern Med 1998; also: Floreani A et al. Eur J Gastroenterol Hepatol 2011 Higher rates of SVR to IFN-a in women <40 yo

6. Effect of sex on SVR related to reproductive status/hormonal asset?

7. Risk factors of severe fibrosis Villa E et al. Gastroenterology 2011 442 women CHC

8. Villa E et al. Gastroenterology 2011 Risk factors of severe necroinflammation

9. Villa E et al. Gastroenterology 2011 Risk factors of SVR failure (442 women CHC)

10. Effect of sex on SVR is related to different (gender-related) immunologic asset?

11. Pro-inflammatory milieu in HCV+ patients according to sex and menopause Only in menopausal women baseline IL-6 correlated with higher necroinflammation (OR 3.571; 95%CI 1.494-8.536, p=.004) Villa E et al. Gastroenterology 2011

12. Increased pro-inflammatory milieu after the onset of menopause Villa E et al. Gastroenterology 2011 TNF-a, 3 yrs pre- menopause TNF-a, 2 yrs post-menopause

13. Post-menopausal G1 non-responder TNF-a

14. Intrahepatic TNF-a substantially affects response to IFN-based anti-HCV therapy Predictors of SVR – Multivariable analysis Taliani G et al. Gastroenterology 2011 23 CHC patients

15. And TNF-a in the periphery….. Neuman MG et al. Clin Biochem 1999 18 CHC patients

16. HCV signalling Gale et al. Nature 2005 ss-RNA and ds-RNA PAMP: Pathogen- associated molecular patterns

17. HCV attenuates IFN signalling Gale et al. Nature 2005 SOCS: suppressor of cytokine signalling Liver toxins Cytokines (TNF-a, IL-6…)

18. Higher SCOCS-3 protein expression in HCV non non-responder vs responder Non responderResponder HCV-negative Walsh MJ et al. Gut 2006

19. SOCS-3 gene expression is associated with non- response to IFN-a therapy Persico M Hepatology 2007 74 G1b CHC patients

20. Gene expression of SOCS3 in non-transformed PBMC from HCV+ NR vs SVR Persico M Hepatology 2007

21. Walsh MJ et al. Gut 2006

22. Post-menopausal G1 non-responder TNF-a SOCS3

23. Menopausal women (might) have resistance to IFN-based therapies through hepatic pro- inflammatory milieu

24. Increased pro-inflammatory cytokines following surgical menopause is reversed by estrogen therapy Pfeilschifter J et al. Endocrinol Rev 2002

25. Bone marrow cultures: cytokine production in SN Pfeilschifter J et al. Endocrinol Rev 2002

26. Adjuvant therapy with raloxifene (selective estrogen receptor modulator) improves response to antiviral therapy Furusyo N et al. J Hepatol 2012 123 CHC post- menopausal women

27. Is the effect of sex less relevant with direct-acting antivirals? i.e. a less-relevant question from a clinical standpoint; still an interesting question

28. NK cells constitute the main innate immune cell population in the liver Spengler – Clin Science 2007

29. The interaction between HCV and the host weakens innate immunity in the liver: Inhibition of TLR pathways Counteraction of type1 IFNs Inhibition of NK cells Impaired trafficking of DC Acute HCV infection- innate immunity

30. NK cells are a crucial component of HCV-specific response

31. NK Cells Constrict HCV Replication Adapted from Balagopal, CROI 2015

32. NK Cells Constrict HCV Replication via NKG2D Adapted from Balagopal, CROI 2015

33. In chronic HCV NK cells are activated, polarized toward cytotoxicity and deficient IFN-g production (functional dychotomy)

34. Mondelli M Gastroenterology 2015

35. Viral cure ≠ immunologic cure

36. NK cells upon DAA?

37. NK phenotype “normalization” 13 G1b NR to Peg- IFN/Riba NR, DCV/ASV Serti E et al. Gastroenterology 2015

38. NK function “normalization” Serti E et al. Gastroenterology 2015 13 G1b NR to Peg- IFN/Riba NR, DCV/ASV

39. Mondelli M Gastroenterology 2015 Viral cure = immunologic cure

41. Racial- and gender-related differences in Natural Killer (NK) cell populations may explain altered natural history and treatment responses NKp46 receptor: - Major human NCR involved in NK cell cytotoxicity - Significant role in anti-tumor immunity - Important for antiviral immunity (influenza virus, CMV, HPV- 16) - Up-regulation in response to IFN-α treatment is predictive of SVR

42. Higher NKp46 in females and Caucasians Healthy uninfected controls: 29 African-American (AA; 55% male); 29 Caucasian- American (CA; 48% male) Golden-Mason L Hepatology 2012

43. NKp46 expression is associated with increased expression of killing genes/proteins and higher induction of degranulation

44. NKp46 expression is associated with increased antiviral (HCV) NK activity

45. HCV-infected hepatocytes demostrate a cell surface pattern of staining for NKp46 ligand Immunological findings corroborate epidemiological data that both race and gender are associates with spontaneous recovery from HCV infection

46. Differences in NK function and phenotype in HCV+ women versus men? NK cells in DAA non-responders?

47. Thanks  Esther Merlini  Camilla Tincati  Giusi M Bellistrì  Javier Sanchez-Martinez  Matteo Basilissi  Antonella d ’ Arminio Monforte ICONA Foundation Laboratory at the Clinic of Infectious Dis- Dept of Health Sciences, Univ of Milan (S Paolo Hospital) Clinica di Malattie Infettive e Tropicali, Università di Brescia- Paola Nasta, Francesca Gatti, Giampiero Carosi Malattie Infettive, Ospedale Niguarda Massimo Puoti Pathology Unit, San Paolo Hospital Paola Braidotti Delfina Tosi Federica Savi Solange Romagnoli Gastroenterology Unit, San Paolo Hospital Benedetto Mangiavillano