HCV and Host Innate Immunity · CTRL CTRLHCV HCV 0 20 40 60 80p=0.0050 % 100 % Phenotype skewed...

HCV and Host Innate Immunity

Mario U. Mondelli

Laboratori di Infettivologia, Dipartimento di Malattie Infettive, Fondazione IRCCS Policlinico San Matteo, e Università di Pavia.

First International Course on Translational Hepatology - Florence, 9-11 march 2011

First International Course of Translational Hepatology, Florence, 2011

Host Immunity Against Pathogens

• Innate immunity: – Evolutionarily ancient

– Universal, all multicellular organisms

– Constitutive, germ line configuration

– No memory (?)

– Rapid response, pattern recognition central

– Effector cells: NK, NKT, T, M, DC, B-1

• Adaptive immunity: – Delayed responses

– Rearranged TCR or IgR

– Memory

– Highly specific, responsible for pathogen clearance


Liver Gene Expression Profiles

HCV

HBV

0 6 9 14 18 23 0 5 8 11 14 27 32 0 8 14 18 24 2896A 1581 1590

0.8

1.8

2.8

0 6 9 14 18 23 0 5 8 11 14 27 32 0 8 14 18 24 2896A 1581 1590

0.8

1.8

2.8

0 4 6 12 16 20 27 0 2 4 6 14 18 22 26 0 2 4 6 10 14 18 28Ch 1627 Ch 5835 Ch 1615

0.8

1.8

2.8

0 4 6 12 16 20 27 0 2 4 6 14 18 22 26 0 2 4 6 10 14 18 28Ch 1627 Ch 5835 Ch 1615

0.8

1.8

2.8

0.1

1

10

100

0.01

0.1

1

10

100

No

rma

lize

d E

xp

res

sio

n

No

rma

lize

d E

xp

res

sio

n

Vire

mia

(% m

ax

) V

irem

ia (%

ma

x)

Chimp

138

131

0 4 6 12 16 20 27 0 2 4 6 14 18 22 26 0 2 4 6 10 14 18 281627 5835 1615

0.8

1.8

2.8

0 4 6 12 16 20 27 0 2 4 6 14 18 22 26 0 2 4 6 10 14 18 281627 5835 1615

0.8

1.8

2.8

0 6 9 14 18 23 0 5 8 11 14 27 32 0 8 14 18 24 2896A 1581 1590

0.8

1.8

2.8

0 6 9 14 18 23 0 5 8 11 14 27 32 0 8 14 18 24 2896A 1581 1590

0.8

1.8

2.8

0.1

1

10

100

0.01

0.1

1

10

100

No

rma

lize

d E

xp

res

sio

n

No

rma

lize

d E

xp

res

sio

n

Vire

mia

(% m

ax

) V

irem

ia (%

ma

x)

Chimp

Chimp

25

0

Clearance-related Virus-induced

Chimp

Wieland S, et al. Proc Natl Acad Sci USA. 2004;101:6669-74.

No activation of IFN- genes by gene chip analysis.


IFN-3 (IL28B): Mechanism of Action

Asselah et al. J Hepatol 2010

Gad et al. JBC 2009 First International Course of Translational Hepatology, Florence, 2011

IL28B Genotype and Spontaneous Clearance

rs12979860 SNP

Tillmann HL et al., Gastroenterology 2010;139:1586–1592


Innate Immunity in Chronic HCV

Saito T. and Gale M. Hepatology Research 2008; 38: 115–122

Immune evasion by hepatitis C virus NS3/4A protease-

mediated cleavage of the Toll-like receptor 3 adaptor protein

TRIF. Li, K. et al. Proc. Natl Acad. Sci. USA 102, 2992–2997 (2005).

IFN- antagonistic activity of HCV core protein involves

induction of suppressor of cytokine signaling-3. Bode, J. G. et al. FASEB J. 17, 488–490 (2003).

Inhibition of the interferon inducible protein kinase PKR by

HCV E2 protein. Taylor, D. R., et al. Science 285, 107–110 (1999).

Viral protein interference with host response: first host response blocked

Control of antiviral defenses through hepatitis C virus

disruption of retinoic acid-inducible gene-I signaling. Foy, E. et al. Proc. Natl Acad. Sci. USA 102, 2986–2991 (2005).


Cell-Associated HCV Inhibits IFN Production by NK Cells

through Engagement of CD81 in Chronic HCV Infection

Crotta et al., J Hepatol 2010;52:183-90.


NK CELL FUNCTIONS

100

101

102

103

104

FL4-H: CD16 u APC

100

101

102

103

104

FL

3H

: C

D5

6 G

2b

PE

-Cy

5

10.3 80.2

2.656.78

CD16 (Fc III R) APC

CD

56 P

E-C

y5

CD56dim/CD16+/-

CD56bright/CD16-

Cytotoxicity vs virus infected or

tumour targets

Cytokine secretion

(TNF-, IFN-, TGF-, GM-CSF)


CD94/

NKG2C

Activating Receptors

Natural Cytotoxicity Receptors (NCRs)

MICA/MICB ULBP

HLA-E

Lectin-like Receptors

Induced by

cell stress

Co-receptors


Lectin-like Receptors

HLA-specific Receptors Non-HLA-specific Receptors

IRp60 (NK, Mø, Granulocytes, T-cell subsets)

p75/AIRM1

High degree of amino acid sequence

identity with CD33. Binds Sialic Acid.

Inhibitory Receptors

Killer Ig-like Receptors

(KIRs)

KIR2DL1

KIR2DL2

KIR3DL1

Leukocyte Immunoglobulin-like

Receptor

LIR1/ILT2

NKG2A

HLA-A, B, C, G


NK CELL-TARGET INTERACTION: THE ”MISSING-SELF” CONCEPT

NK cells test qualitative/quantitative expression of MHC I on target cell

NK Normal

cell

+

Inhibitory signal No action

MHC I

Target cell (infected or

transformed)

+

No inhibitory signal

+ activating signal

NK

Action killing, cytokine secretion


• Controversial findings:

– Phenotype:

• Activating receptors: NKG2D, NKp30, NKp46,

NKp44, NKG2C, DNAM-1.

• Inhibitory receptors: KIRs, NKG2A.

• Activation: CD69, …

– Cytolytic activity: CD107a, TRAIL, Perforin

– Cytokine production: IFN-, TNF-.

NK Cell Function in Chronic HCV

Infection


NK Cell Functional Dichotomy in Patients with

Chronic HCV Infection

Oliviero et al Gastroenterology 2009;137:1151-60.

Increased cytotoxic activity

p=0.003 p=0.001

%C

D1

07

a N

K c

ell

s

IL2+IL12 IL2+IL21 Media

0

10

20

30

40

50

60

p=0.010

CTRL HCV CTRL HCV CTRL HCV

Dysfunctional cytokine production

p=0.041 p=0.009

IFN TNF

CTRL HCV CTRL HCV 0

20

40

60

80

100 %

%

Phenotype skewed towards activation

NKG2D

CTRL HCV 0

25

50

75

100 p=0.012

KIR3DL1

CTRL 0

10

20

30 p=0.0050 %

HCV

Oliviero B, et al. Gastroenterology 2009;137:1151-60.


Ahlenstiel, et al. Gastroenterology 2010;138:325–335

In Vitro Exposure to IFN- Up-Regulates Markers of Cytotoxicity

on NK Cells


Chronic Exposure to HCV-Induced IFN Contributes to Liver Inflammation via Cytotoxic

Mechanisms but not to Viral Clearance Because of Insufficient IFN production.

NK Cells Are Polarized Towards Cytotoxicity

in Chronic HCV Infection: A Model for Virus-Induced Inflammation and Immune Escape

Ahlenstiel, et al. Gastroenterology 2010;138:325–335


Time (weeks)

0 2 4 12 ET

***

***

***

*

Time (weeks)

0 2 4 12 ET

*** ***

***

***

0 2 4 12 ET

Peg-IFN/RBV Treatment Induces Early NK Cell Activation %

CD

69

+ N

K c

ell

s

% N

Kp

30

+ N

K c

ell

s

Time (weeks)

% P

erf

ori

n+

NK

ce

lls

Time (weeks)

0 2 4 12 ET

% C

D1

07

a+

NK

ce

lls

Oliviero, EASL 2011 First International Course of Translational Hepatology, Florence, 2011

CD

69 F

old

In

cre

ase

CD

69 F

old

In

cre

ase

% P

erf

ori

n+

NK

cell

s

SVR Rel/NR

P< 0.05

Early NK Cell Activated Phenotype Predicts Treatment

Outcome in Chronic HCV Infection

Oliviero, EASL 2011

2 weeks on treatment


Early Effector NK Cell Functional Activation Predicts

Treatment Outcome in Chronic HCV Infection

BASELINE EARLY STAGE ON TREATMENT

Degranulation

4 w

ee

ks

p=0.004

IFN p=0.03

SVR Rel/NR

Oliviero, EASL 2011 First International Course of Translational Hepatology, Florence, 2011

Modified from O’Farrelly Immunol Rev 2000.

NK

13%

T

72%

NKT

B T

10%

BLOOD

T

72%

T 3%

B

10% NK

NKT

13% 2%

NKT

LIVER

T

T

37%

T 6%

B

6% NK

NKT

31%

20%

Relative Distribution of Lymphocyte

Subsets in PB and Liver


CTRL CTRL HCV HCV

%C

D56+

CD

3-

ce

lls

PBMC LIVER

0

5

10

15

20

25

30

35

p<0.0001 p=0.0006

%C

D56+

CD

3+

cells

0

5

10

15

20

25

CTRL CTRL HCV HCV

PBMC LIVER

p<0.0002

The Proportions of Intrahepatic Innate Immune

Cells Are Reduced in Chronic HCV Infection

NK CELLS NKT CELLS

Varchetta et al., AASLD 2009. Abstract 175


IH NK Cells Expressing NCRs Are Enriched

in the HCV-Infected Liver

0

25

50

75

100

NKp30

%N

Kp

30+

NK

cells

CTRL CTRL HCV HCV

PBMC LIVER

p=0.02 p=0.04

NKp46

0

50

100

150

CTRL CTRL HCV HCV

PBMC LIVER

p=0.03

%N

Kp

30+

NK

cells



CTRL

NKG2D+ NK Cells Are Enriched in

the HCV-Infected Liver

HCV

PBMC LIVER 25

50

75

100

PBMC LIVER %

NK

G2D

+ N

K c

ells

25

50

75

100

% N

KG

2D

+ N

K c

ells


PBMC LIVER PBMC LIVER0

25

50

75

100p=0.03

p=0.001 p=0.01

PBMC LIVER PBMC LIVER 0

5

10

15

20

25 p=0.03

PBMC LIVER PBMC LIVER 0

10

20

30

40

50

p=0.0005

p=0.034

PBMC LIVER PBMC LIVER0

20

40

60

80

100

p=0.002

p=0.014

p=0.027

K562 P815

P815+anti-NKG2D P815+anti-NKp30

HCV

HCV

HCV

HCV

CTRL CTRL

CTRL CTRL

%C

D107a+

NK

cells

%

CD

107a+

NK

cells

%C

D107a+

NK

cells

%

CD

107a+

NK

cells

IH NK Cells from HCV+ Patients Show Lower Cytotoxic

Potential than Control IHNK Cells

TRAIL MFI

0.0

2.5

5.0

7.5

10.0

p=0.02

CTRL CTRL HCV HCV

PBMC LIVER

MF

I T

RA

IL+

NK

cells

Expression of TRAIL on IH NK Cells Is

Lower in HCV-Infected Patients


CHRONIC HCV

INFECTION

Unresponsiveness ?

NK

Degranulation

Exhaustion ?

↓ TRAIL

NKR ligands

Continuous NK receptor

engagement and modulation

NK


Accapezzato et al., J Clin Invest 2004;113:963-72

CD8+ IL-10 Producing Treg Cells Are Enriched in the Liver of

Patients with Chronic HCV Infection


Intrahepatic, IL-10-Producing CD8+ T cells

Perform Regulatory Function

Accapezzato et al., J Clin Invest 2004;113:963-72


CHRONIC HCV

INFECTION

Unresponsiveness ?

NK

Degranulation

Exhaustion ?

↓ TRAIL

Inhibitory

Cytokine(s),

e.g. IL-10 ?

TGF ?

NKR ligands

Continuous NK receptor

engagement and modulation

NK


Conclusions

• Impaired intrahepatic NK cytolytic function in

HCV infection may represent an additional

mechanism contributing to viral escape and

persistence.


Acknowledgements

Lab Team: • Stefania Varchetta • Barbara Oliviero • Stefania Mantovani • Eleonora Cremonesi • Dalila Mele

Clinical Team: • Giuseppe Michelone • Marco Zaramella • Serena Ludovisi

S.C. Laboratori di Infettivologia, Dipartimento di Malattie Infettive, Fondazione IRCCS Policlinico San Matteo e Università di Pavia

Collaborators: • Savino Bruno, Liver Unit, Dept. of Medicine, Ospedale Fatebenefratelli, Milan • Paolo Dionigi, Andrea Pietrabissa, 1st and 2nd Divisions of Surgery, San Matteo • Marco Montorsi, Riccardo Rosati, Division of Surgery, ICH, Rozzano. • Elisabetta Degasperi, Massimo Colombo, IRCCS Ospedale Maggiore…, Milano


HCV and Host Innate Immunity · CTRL CTRLHCV HCV 0 20 40 60 80p=0.0050 % 100 % Phenotype skewed...

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Transcript of HCV and Host Innate Immunity · CTRL CTRLHCV HCV 0 20 40 60 80p=0.0050 % 100 % Phenotype skewed...