Biliary system and liver: new insights from immune system for

understanding the natural history, therapy and outcomes

AISF Annual Meeting 2016 Rome

Ana Lleo, MD PhDLiver Unit and

Center for Autoimmune Liver DiseasesHumanitas Clinical and Research Center

ana.lleo@humanitas.it

Il sottoscritto dichiara di non aver avuto negli ultimi 12 mesi conflitto d’interesse in relazione a questa presentazione

e

che la presentazione contiene discussione di farmaci in studio o ad uso off-label

An interdisciplinary branch of the biomedical field supported by three main pillars: benchside, bedside and community.

The goal of TM is to combine disciplines, resources, expertise, and techniques within these pillars to promote enhancements in prevention, diagnosis, and therapies.

Translational Medicine

European Society for Translational Medicine

An interdisciplinary branch of the biomedical field supported by three main pillars: benchside, bedside and community.

The goal of TM is to combine disciplines, resources, expertise, and techniques within these pillars to promote enhancements in prevention, diagnosis, and therapies.

Translational Medicine

European Society for Translational Medicine

TURNING BASIC RESEARCH INTO MEDICINES AND TREATMENTS

PBC

From Cirrhosis to Cholangitis

(1) the name ‘primary biliary cirrhosis’ should be changed [as advocated by Dame Sheila Sherlock in 1959] since the majority of patients are free of cirrhosis at the time of diagnosis and the the disease has a good prognosis

(2) the acronym ‘PBC’ should be kept if possible (3) a simple and short term should be used as long as the exact

pathogenesis of primary biliary cirrhosis remained undefined and, therefore, an ‘ideal’ replacement is not available

European Association for the Study of the Liver (EASL) American Association for the Study of Liver Disease (AASLD)

American Gastroenterological Association (AGA) Asian Pacific Association for the Study of the Liver (APASL)

1. Primary Biliary Cholangitis (PBC) is an autoimmune liver disease that predominantly affects adult women and leads to destruction of intrahepatic bile ducts and eventually biliary cirrhosis

2. multi-lineage loss of tolerance to the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2): presence of highly specific anti-mitochondrial antibodies (AMA) and circulating autoreactive T cells, both directed against components of the inner mitochondrial membrane, mainly PDC-E2.

3. genetic susceptibility and environmental factors that modify the auto-antigen motif contribute to the breakdown of tolerance

Primary Biliary Cholangitis

1. Primary Biliary Cholangitis (PBC) is an autoimmune liver disease that predominantly affects adult women and leads to destruction of intrahepatic bile ducts and eventually biliary cirrhosis

2. multi-lineage loss of tolerance to the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2): presence of highly specific anti-mitochondrial antibodies (AMA) and circulating autoreactive T cells, both directed against components of the inner mitochondrial membrane, mainly PDC-E2.

3. genetic susceptibility and environmental factors that modify the auto-antigen motif contribute to the breakdown of tolerance

Primary Biliary Cholangitis

1. Primary Biliary Cholangitis (PBC) is an autoimmune liver disease that predominantly affects adult women and leads to destruction of intrahepatic bile ducts and eventually biliary cirrhosis

2. multi-lineage loss of tolerance to the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2): presence of highly specific anti-mitochondrial antibodies (AMA) and circulating autoreactive T cells, both directed against components of the inner mitochondrial membrane, mainly PDC-E2.

3. genetic susceptibility and environmental factors that modify the auto-antigen motif contribute to the breakdown of tolerance

Primary Biliary Cholangitis

1. Primary Biliary Cholangitis (PBC) is an autoimmune liver disease that predominantly affects adult women and leads to destruction of intrahepatic bile ducts and eventually biliary cirrhosis

2. multi-lineage loss of tolerance to the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2): presence of highly specific anti-mitochondrial antibodies (AMA) and circulating autoreactive T cells, both directed against components of the inner mitochondrial membrane, mainly PDC-E2.

3. genetic susceptibility and environmental factors that modify the auto-antigen motif contribute to the breakdown of tolerance

Primary Biliary Cholangitis

Primary Biliary Cholangitis

1. Primary Biliary Cholangitis (PBC) is an autoimmune liver disease that predominantly affects adult women and leads to destruction of intrahepatic bile ducts and eventually biliary cirrhosis

2. multi-lineage loss of tolerance to the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2): presence of highly specific anti-mitochondrial antibodies (AMA) and circulating autoreactive T cells, both directed against components of the inner mitochondrial membrane, mainly PDC-E2.

3. genetic susceptibility and environmental factors that modify the auto-antigen motif contribute to the breakdown of tolerance

PBC

Area Year Patients(No.)

Prevalence(per mln)

Incidence(per mln/yr)

Age(years)

Sex(M:F)

Europe 1984 569 23 54 54 1:10Sweden 1985 111 151 13.3 55 1:6Newcastle, UK 1989 347 154 19 58 1:9Ontario, Canada 1990 225 22 3.3 59 1:13Victoria, Australia 1995 84 19 - - 1:11Estonia 1995 69 27 2.3 - 1:22Newcastle, UK 1997 160 240 22 66 1:10Norway 1998 21 146 16 - 1:9Minnesota, USA 2000 46 402 27 - 1:8Newcastle, UK 2001 770 251 31 - 1:10Victoria, Australia 2004 249 51 - 61 1:9Japan 2005 9761 78 - - 1:9

Canada 2009 137 227 30 53 1:5

Geoepidemiology of PBC

Zhang, Lleo, et al, Dig Dis 2015

Administrative databases

LOMBARDIA:Inhabitants at 1-Jan-2010: 9.742.676

DENMARK: Inhabitants at 1-Jan-2010: 5.534.738

(2000-2010)

Sex ratio

LOMBARDIA:Inhabitants at 1-Jan-2010: 9.742.676

DENMARK: Inhabitants at 1-Jan-2010: 5.534.738

M:F RATIO1:2

M:F RATIO1:4

Lleo A, et al., submitted

0.2

5.5

.75

1

0 12 24 36 48 60 72 84 96 1 08 120months

2970 2373 2077 1851 1634 1390 1185 983 781 5 52 297 N umber at risk

95% CI Survival function

Survival from diagnosis

86% (CI 85%-87%) at 1 year70% (CI 69%-72%) at 5 years61% (CI 59%-64%) at 10 years

LOMBARDIA

0.2

5.5

.75

1

0 12 24 36 48 60 72 84 96 108 120months

722 575 473 396 321 259 198 135 88 42 3 Number at risk

95% C I Survival function

Survival from diagnosis

87% (CI 84%-89%) at 1 year 67% (IC 63%-71%) at 5 years 54% (IC 48%-60%) at 10 years

DENMARK

0.2

5.5

.75

1

0 12 24 36 48 60 72 84 96 1 08 120months

897 627 516 442 382 3 24 273 230 173 1 20 64Maschi2073 1746 1561 1409 1252 1 066 912 753 608 4 32 233Femmine

Number at risk

95% CI 95% CIFemale Male

Survival from diagnosis

0.2

5.5

.75

1

0 12 24 36 48 60 72 84 96 1 08 120months

897 627 516 442 382 3 24 273 230 173 1 20 64Maschi2073 1746 1561 1409 1252 1 066 912 753 608 4 32 233Femmine

Number at risk

95% CI 95% CIFemale Male

Survival from diagnosis

Lleo A, et al., submitted

Survival rate

0.2

5.5

.75

1

0 12 24 36 48 60 72 84 96 1 08 120months

2970 2373 2077 1851 1634 1390 1185 983 781 5 52 297 N umber at risk

95% CI Survival function

Survival from diagnosis

86% (CI 85%-87%) at 1 year70% (CI 69%-72%) at 5 years61% (CI 59%-64%) at 10 years

LOMBARDIA

0.2

5.5

.75

1

0 12 24 36 48 60 72 84 96 108 120months

722 575 473 396 321 259 198 135 88 42 3 Number at risk

95% C I Survival function

Survival from diagnosis

87% (CI 84%-89%) at 1 year 67% (IC 63%-71%) at 5 years 54% (IC 48%-60%) at 10 years

DENMARK

0.2

5.5

.75

1

0 12 24 36 48 60 72 84 96 1 08 120months

897 627 516 442 382 3 24 273 230 173 1 20 64Maschi2073 1746 1561 1409 1252 1 066 912 753 608 4 32 233Femmine

Number at risk

95% CI 95% CIFemale Male

Survival from diagnosis

0.2

5.5

.75

1

0 12 24 36 48 60 72 84 96 1 08 120months

897 627 516 442 382 3 24 273 230 173 1 20 64Maschi2073 1746 1561 1409 1252 1 066 912 753 608 4 32 233Femmine

Number at risk

95% CI 95% CIFemale Male

Survival from diagnosis

Lleo A, et al., submitted

Survival rate

Factor Hazard Ratio 95% Confidence Interval

Male sex 2,36 2,08 2,69

Age at diagnosis 1,06 1,06 1,07

Year of diagnosis 1,29 1,24 1,34

International data base

Since 20101379 patients

Since 20124845 patients

www.uk-pbc.com www.globalpbc.com

To assess the proportion of patients achieving:

– ALP <1.67x ULN (& ≥15% reduction)

– bilirubin ≤ULN

ResponderNon-Responder

Lammers W, Lleo A et al. Gastroenterology. 2014

The Global PBC Study Group (N=4845) confirmed ALP <1.67 xULN and normal bilirubin after 1 year of UDCA highly predictive of outcome1

Prognosis: surrogate end-points

Response to UDCA and symptoms are related to sex and age at presentation.

Men were significantly less likely to have responded to UDCA than women; male sex was an independent predictor of nonresponse on multivariate analysis.

Lowest response rates to UDCA in those younger than age 30

Carbone M et al. Gastroenterology. 2013

Prognosis: sex and age

Gender differences in the age-related likelihood of achieving UDCA response criteria.

Male

Female

Long-term prediction of end-stage liver disease in PBC

Scoring system

Carbone M et al. Hepatology. 2015 Lammers W et al. Gastroenterology. 2015

1. Males with PBC have worse prognosis and survival rates and seem to be more frequent than previously expected

2. ALP is a good surrogate marker of outcome.

Epidemiology

PBC

PBC

- Geo-epidemiology of PBC prevalence

- Local clustering

- Risk factors

- Experimental evidence for a role of xenobiotics, infectious agents

ENVIRONMENTALFACTORS

GENETICFACTORS

Etiopathogenesis of PBC

- Familial clustering

- High concordance rate of PBC in monozygotictwins

- Polymorphisms associated with susceptibility and progression

Genetics of PBC

Risk variantsHLA

IL12A IL12RB2

IRF5IKZF3/ORMDL3

SPIB

536 Canadian & US PBC vs. 1536 controls (300K SNPs)PLUS 457 Italian PBC vs. 947 controls (1Mb SNPs)

Invernizzi, Italian PBC Genetics Study Group, et al. Nat Genetics, 2010

Genetics of PBC: list of genes

Webb, et al. J Autoimmun, 2016

Genetics of PBC: pathways

Cordell, Italian PBC Genetics Study Group, et al. Nat Commun, 2015

PBC

Apoptotic cholangiocyte

Apoptotic NON-cholangiocyte

Odin JA, J Clin Invest 2001

Intact PDC-E2

Excess glutathione

Reduced environment

glutathione

Oxidated PDC-E2

PDC-E2 remains immunologically active during apoptosis in BECs

Lleo A, Hepatology 2009

Non apoptotic HIBEC

*

*

Lleo A, Hepatology 2009

Non apoptotic HIBEC

*

*

HIBEC HeLa CACO-2 Human keratinocytes Human bronchial cells

0

1000

2000

3000

4000 ***TN

F (p

g/m

l)

Figure 3. Macrophages from PBC cultured withapoptotic bodies from HIBEC secrete pro-inflammatory cytokines in the presence of AMA.MDMΦ from patients with PBC or healthy controls(HC) were cultured with apoptotic bodies and antibody(AMA IgG or IgG control). The triad of MDMΦ frompatients with PBC, but not from controls, apoptoticbodies from HIBEC cells, and AMA leads tosignificantly increased secretion of TNF-α comparedto IgG control. ***p<0.001, based on a two tailed Mann-Whitney test with 95% CI.

MDMФ PBC PBC PBC HC PBCAb AMA IgG ctr AMA AMA AMABlebs HIBEC HIBEC Kerat HIBEC HIBEC

Other - - - - anti-CD16

PBC HC M1 HC0

1000

2000

3000

4000***

*

ns

TNF

(pg/

ml)

Figure 4. Stimulation of PBC macrophages in thepresence of AMA and HIBEC apoptotic bodies is aconsequence of M1 polarization. Comparison of TNFαsecretion from MDM from patients with PBC (n = 8)and healthy controls (n = 8) after M1 polarization (HCM1), and untreated (HC) cultured with HIBECapoptotic bodies in the presence of AMA. (***p<0.001,*p<0.05). For full M1 polarization, MDM from healthycontrols were stimulated with 100 U/ml IFN-γ(Boehringer Ingelheim) and 1 ng/ml LPS (Cambrex)during the last 24 h of culture.

Lleo A, Hepatology 2010

1. Following apoptosis human BECs translocate PDC-E2 immunologically intact into the apoptotic bodies

2. Monocyte derived macrophages (MDMf), from patients with PBC to produce pro-inflammatory cytokines in response to biliary apotopes in the presence of AMAs.

Biliary epithelial cells and macrophages

Trypsin digestionIE fractionation

LC-MS/MSHiBEC n=4

BrEPC n=6

HRPTEpiC n=6

ISOLATIONOF APOPTOTIC

BODIES

STATISTICAL COMPARISON OF

PROTEIN INVENTORIES DIFFERENCIAL

PROTEINLIST

Shotgun proteomic analysis of apoptotic bodies

Apoptotic bodies

HiBEC HRPTepiC BrEPC

Lleo A, Hepatology 2014

LRP1

RAB11A ANXA6

Lleo A, Hepatology 2014

PBC

NORMAL APOPTOTIC

CHOLAGIOCYTE

PDC-E2 GLUTATHIOLATION

Intact PDC-E2

NORMAL APOPTOTIC

CHOLAGIOCYTE

PDC-E2 containing apoptotic

bodies

PDC-E2 GLUTATHIOLATION

Intact PDC-E2

NORMAL APOPTOTIC

CHOLAGIOCYTE

PDC-E2 containing apoptotic

bodies

AMAs

Immune complex

formation

PDC-E2 GLUTATHIOLATION

Intact PDC-E2

NORMAL APOPTOTIC

CHOLAGIOCYTE

PDC-E2 containing apoptotic

bodies

AMAs

Immune complex

formation

PDC-E2 GLUTATHIOLATION

Intact PDC-E2

NORMAL APOPTOTIC

CHOLAGIOCYTE

PDC-E2 containing apoptotic

bodies

AMAs

Immune complex

formation

Phagocytocis ↓

Proinflammatory cytokine secretion

PDC-E2 GLUTATHIOLATION

Intact PDC-E2

Lleo A, et at Hepatology 2009Lleo A, et at Hepatology 2010

NORMAL APOPTOTIC

CHOLAGIOCYTE

PDC-E2 containing apoptotic

bodies

AMAs

Immune complex

formation

Phagocytocis

Proinflammatory cytokine secretion

PDC-E2 GLUTATHIOLATION

Intact PDC-E2

1. BECs apoptotic bodies2. Macrophages from PBC patients3. AMA

Lleo A, J Hepatol 2012

PBC: multi-faceted pathophysiology

PBC

UDCA in a dose of 13-15 mg/kg/day orally is recommended for patients with PBC who have abnormal liver enzyme values regardless of

histologic stage (Class I, Level A).

Is there need for new therapies in PBC?

Pares A, Gastroenterology 2006

Author Endpoint criteria Non-response

Barcelona ALP 40% decrease or normalization 39%

Paris IALP < 3 x ULNAST< 2 x ULNBil < 1 x ULN

39%

Paris IIALP < 1.5 x ULNAST< 21.5x ULN

Bil < 1 x ULN52%

Toronto ALP < 1.67 x ULN 43%

Primary Biliary Cholangitis: the future?

Obeticholic acid (Waiting for registration)

Bezafibrate in combination with UDCA in PBC. Phase 3.

Ustekinumab in patients with PBC who had an inadequate response to UDCA

Apical Sodium-dependent Bile Acid Transporter Inhibitor (ASBTi), open label.

UDCA plus budesonide versus UDCA alone in PBC

High-protein high-fiber diet in patients with PBC

Abatacept to treat PBC (CTLA-4)

Umbilical cord mesenchymal stem cells for patients with PBC

IBAT Inhibitor A4250 for Cholestatic Pruritus

ClinicalTrials.gov

IL-12

IL12 Genetic defect

Anti-IL12 Clinical trial

Ongoing trials

Nuclear Receptors (FXR, PPARs):at the crossroad of metabolism, inflammation and regeneration

Karpen, Trauner J Hepatol 2010

Farnesoid Receptor (FXR) protects against toxic effects of hydrophobic bile acids

Courtesy of U. Beuers

POISE: Significant Reductions in ALP Within 2 Weeks

* p-values from an ANCOVA model with baseline value as a covariate and fixed effects for treatment and randomization strata factor.Data are least squares mean(SE)

*p<0.0001 vs. placebo

% Drop inALP

Obeticholic Acid

Fibrates

Bile duct protection

Nakai et al, Am J Gastroenterol 2000Honda et al, Hepatology 2013

UDCA

UDCA+BF

UDCA

UDCA+BF

ALP (range 35-118)

Bil tot (mg/dl) PT INR MELD OCA

Apr 2012 932 1.77 0.89 4.2 -Dec 2013 713 1.82 1.36 8.2 10 mgMar 2014 654 1.78 1.55 10.3 10 mgJun 2014 300 3.15 1.30 10.5 10 mgSep 2014 264 3.00 1.50 12.0 10 mg

Female, DOB 22-Dec-1967PBC Stage IV - portal hypertension – UDCA 14 mg/kg

Clinical Case

June 2015: OLT

PBC

Conclusions

PBC

Conclusions- UDCA non-responders have a

poor prognosis

- Male and very young women have lower response to UDCA

- Male have lower survival rates than women

- ALP can be used as surrogate marker of outcome

PBC

Conclusions

- Genetics of PBC, the mechanisms of injury of BECs, and the immune response may offer important therapy opportunities

- Cholestasis is an important modifier of the disease process and may drive ongoing bile duct destruction after an initial immune response

- LOTS OF DATA AND TIME

- UDCA non-responders have a poor prognosis

- Male and very young women have lower response to UDCA

- Male have lower survival rates than women

- ALP can be used as surrogate marker of outcome

PBC- There is need for new therapies in PBC

- OCA and Fibrates seem to offer realistic alternatives to UDCA

Conclusions

- Genetics of PBC, the mechanisms of injury of BECs, and the immune response may offer important therapy opportunities

- Cholestasis is an important modifier of the disease process and may drive ongoing bile duct destruction after an initial immune response

- LOTS OF DATA AND TIME

- UDCA non-responders have a poor prognosis

- Male and very young women have lower response to UDCA

- Male have lower survival rates than women

- ALP can be used as surrogate marker of outcome

Acknowledgments

Humanitas Clinical & Research CenterSavino Bruno

Università degli Studi di Milano-BicoccaPietro Invernizzi

University of California - DavisM. Eric Gershwin

Università degli Studi di Milano - Ospedale San PaoloMassimo Zuin