Innate Immune Responses to Hepatitis C Virus Infection

3rd ACHA

10 May, 2014

Kui Li

Dept. of Microbiol., Immunol. & Biochem. University of Tennessee Health Science Center

HCV History Recognized as PT-NANBH in 1970s Cloned and sequenced: 1989 Antibody screening tests available: 1990 Infectious clone: 1997 HCV RNA replicon: 1999 Pseudovirus particles: 2003 Culture HCV in hepatoma cells: 2005

Therapy for Hepatitis C

Pegylated Interferon + Ribavirin + Direct-acting Antivirals (since 2011) NS3/4A Protease inhibitors (2011) NS5B polymerase inhibitor (2013)

Ultimate control of HCV will require a vaccine!

Hepatitis C Virus ~9.6 kb Single-stranded, positive-sense RNA genome 7 major genotypes, many subtypes 5’

3’ C

E1

E2

p7

NS2

NS3

NS4A

NS4B

NS5A

NS5B

3000-aa polyprotein IRES

5’ cis replication signal

3’ cis replication signal

The Hepatitis C Virus Life Cycle

Lindenbach et al, Nature 436:933

Challenges in Working with HCV

Sorry, I am about to retire!

 Lack of convenient small animal models chimpanzee: availability; $$$$$$; a higher rate of spontaneous resolution SCID mice engrafted with human hepatocytes (2001) Transgenic mice expressing human CD81 and occludin (2013)  Study closely related viruses: GB virus B  Can not grow HCV in cell culture until 2005 HCV RNA replicons (since 1999) only JFH-1 strain (genotype 2a) efficiently produces infectious HCV in cell culture until recently: J6 (2a); J8 (2b); H77S & TN (1a) Efficient propagation of HCV is confined to the Huh7 cell line

Factors Associated with HCV Clearance

 Humoral immune responses are not able to clear HCV infection.  Vigorous, broadly-directed, and sustained CD8+ and CD4+ T cell responses are critical in HCV clearance.  Innate immune responses offer early viral control and help orchestrate the development of adaptive immunity Host genetic variation in the IL28B (IFN-λ3) gene is critical for predicting response to IFN therapy as well as spontaneous clearance in patients Multiple HCV proteins disrupt innate immunity through distinct mechanisms

Cellular Control of Early Antiviral Responses - Induction of IFNs & inflammatory cytokines 5’ppp-RNA dsRNA

5’ppp

Endosome

Card Card Helicase CTD Helicase CTD

MAVS

RIG-I/MDA5 LGP2

TLR7/8

TLR3

Card TIR TIR

TIR

TRIF

PAMP

ssRNA

dsRNA

PRR

MyD88

U(n)

TIR

Adaptor IKKγ

TBK1/IKKε

IKKβ IKKα IKKα

p50

p65

IRF7

P P

P

P

IRF7

IFN-β, IFN-λ1 cytokines chemokines

IRF7

p50

p65

P

IRF3/7

P

Transcription Factors Cytoplasm

Nucleus IRF3/7

IRF3

IκBα

IRF7

P

P

Kinases

IFN-α IFN-λ2/3

IFNs, cytokines, chemokines Li & Lemon, Semin Immunopathol 2013

How do Liver cells Detect HCV Infection and Initiate Innate Immune Responses?  Microarray analysis has demonstrated that HCV infection is generally associated with induction of a strong interferonstimulated gene (ISG) response in the liver in vivo.  Intrahepatic chemokines such as the CCR5 ligands, RANTES, MIP-1β and MIP-1α, and the CXCR3 ligand, IP-10, are elevated in hepatitis C patients.  Yet, the cellular sources of IFN(s) and cytokines/chemokines in HCV-infected liver are not known, and the signaling pathways responsible for their induction are just being uncovered.

Liver Microanatomy Hepatocyte

KC

Sinusoidal lumen HSC

LSEC

pDC & mDC2

Sinusoidal lumen NK/NKT Hepatocyte

Hepatocyte

Productively infected

LSEC HSC Kupffer cell (KC) NK/NKT

Bystanders responding to virus exposure

pDC, mDC2 Modified from Jenne & Kubes, Nat Immunol 2013

TLR3 and RIG-I Constitute Two Distinct Viral RNA Sensing Pathways in Non-neoplastic Hepatocytes

Relative Luciferase Activity

Relative Luciferase Activity

IFN-β Mock

Luciferase M-pIC

10

120

8

90

6

PH5CH8 cells

60

4

30

2 0

SeV Se

Ctrl TLR3 siRNA

0

5

50

4

40

3

30

2

20

1

10

0

0

Ctrl RIG-I siRNA

Ctrl TLR3 siRNA

Ctrl RIG-I siRNA

Li et al, JBC 2005

Huh7.5 Cells Highly Permissive for HCV Replication Are Defective for RIG-I Signaling 5’ NTR C Neo

EMCV IRES

3’ NTR NS3

4A NS4B

NS5A

NS5B

SenV RIG-I ISG56 Actin

Blight et al, J. Virol. 2002

Sumpter et al, J. Virol. 2005

Reconstitution of RIG-I Signaling Restricts HCV RNA Replication in Huh7.5 cells RIG-I (lethal point mutation)

C Luc

EMCV IRES

Fold increase

5’ NTR

3’ NTR NS3

4A NS4B

NS5A

NS5B

poly-U/UC track in 3’NTR + 5’-ppp 5’ppp

U(n)

Sumpter et al, J. Virol. 2005 Saito et al, Nature 2008

 Although the RIG-I defect likely contributes to the highly permissiveness phenotype of Huh7.5 cells, it does not explain why parental Huh7 cells, which contain an active RIG-I pathway, are already unique among the hepatoma cell lines for the ability to efficiently support HCV replication.  Is there a role for TLR3 in hepatocellular innate responses to HCV?

Hepatoma Huh7 Cells are Defective in TLR3 Signaling due to Lack of TLR3

Fold Stimulation by M-pIC

IFN-β

Luciferase

10 8

TLR3

6 4

TRIF

2

actin

0

Huh7

PH5CH8

Li et al, JBC 2005

Ectopic Expression of TLR3 Reconstitutes the Signaling Defect to Extracellular dsRNA in Huh7 cells − −

− +

+ −

+ TLR3 + M-pIC ISG15 TLR3 Actin

1

2

3

4

Wang et al, J Virol 2009

Primary Human Hepatocytes Express TLR3 in situ

Merge

α TLR3

Ctrl IgG

Wang et al, J Virol 2009

Primary Human Hepatocytes Contain a Robust TLR3 Signaling Pathway (Induction of ISGs) ISG56

5

M-pIC SeV R-848

0

PHH

30 20 10 0

PHH

40 30 20 10 0

M-pIC SeV R-848

10

Fold Change

15

M-pIC SeV R-848

20

50

70 60 50 40

mock

Fold change

25

Huh7 mock

Fold Difference

30

MxA

mock

TLR3

PHH

Wang et al, J Virol 2009

Primary Human Hepatocytes Contain a Robust TLR3 Signaling Pathway (Induction of cytokines)

10000

10

SeV

M-pIC

Mock

R-848

SeV

1 0.1

1

M-pIC

10

R-848

0

100

100

SeV

50

1000

Mock M-pIC

100

IP-10 (ng/ml)

150

1000

R-848

IL-6 (pg/ml)

200

Mock

TNFα (pg/ml)

250

Li et al, Hepatology 2012

Reconstitution of TLR3 Signaling in Huh7.5 Cells Inhibits HCV Infection 6

Vect

Virus Titers (Log10TCID50/ml

WT ∆TIR

5

H539E N541A

4

3

2

Wang et al, J Virol 2009

1 1

2

3

4

Days Post Infection

H539E & N541A: TLR3 mutants defective for dsRNA binding

5

6

TLR3 mediates Production of Chemokines and Proinflammatory Cytokines in HCV-infected Hepatoma cells

3000 2500 2000 1500 1000

300 250 200 150 100

500

50

0

0

N541A

3500

TLR3 H539E

H539E

Vect

Vect TLR3

0

0

350

Vect

300

4000

IP-10 (µg/ml)

600

400

H539E N541A

300

900

4500

TLR3

600

MIP-1β (pg/ml)

900

1200

N541A

MIP-1α (pg/ml)

1200

TLR3 H539E N541A

RANTES (pg/ml)

Mock HCV

Vect

1500

1500

H539E & N541A: TLR3 mutants defective for dsRNA binding Li et al, Hepatology 2012

TLR3-mediated Chemokine Induction by HCV infection in Hepatocytes  depends on viral replication and first occurs at 36-48 h post-infection  depends on activation of NF-κB  HCV dsRNA intermediates generated during viral replication is the ligand for TLR3  HCV dsRNAs need to be ≥ ~80-100 bp for TLR3 activation, while are independent of the genome position or nucleotide composition (as opposed to the RIG-I ligand)

Li et al, Hepatology 2012

Short-range Exosomal Transfer of HCV RNA from Infected Cells to pDCs Triggers TLR7-dependent Type I IFN Production Huh7.5 HCV RNA+ HCV RNA

Co-culture

Exosome

Exosome release inhibitor

pDC

TLR7

IFN-α

Takahashi et al, PNAS 2010 Dreux et al, Cell Host Microbe 2012

HCV dsRNAs Released from Infected Cells Triggers TLR3-dependent Type III IFN Production in co-cultured BDCA3+ mDC2 Cells

Huh7.5

Co-culture mDC2

TLR3

HCV dsRNA

HCV dsRNA+

Inhibitors of endocytosis (cytochalasin D or chlorpromazine)

Inhibitors of endosome acidification (chloroquine or bafilomycin)

IFN-λs

Zhang et al, Gastroenterology 2013

Phagocytosis of HCV virions containing viral RNAs Activates the NLRP3 Inflammasome in Kupffer cells Leading to IL-1β Secretion

Signal 1

Signal 2

Negash et al, PLoS Pathogens 2013 Horner, JMB 2014

Summary 1. RIG-I and TLR3 constitute two parallel innate antiviral pathways in human hepatocytes that act to fend off HCV infection. 2. In addition to its antiviral role in sensing and restricting HCV replication, TLR3 also mediates inflammatory cytokine induction in HCV-infected hepatocytes, which may contribute to immune responses to the virus. 3. HCV ssRNAs and dsRNAs released from infected hepatocytes can be sensed by infiltrating pDCs and mDC2, respectively, leading to production of differing types of IFNs via distinct TLR pathways. 4. Intrahepatic IL-1β production by liver resident macrophages (Kupffer cells) after phagocytosis of HCV virions may contribute to liver inflammation.

Questions Remain Open 1. How do multiple arms (pathways) of the innate immune system interact to coordinately respond to HCV infection? (depending on novel culture systems) 2. How do various liver cell types act in concert to drive immunity against HCV? (depending on immuno-competent, permissive mouse models) 3. How do the innate immune responses to clinical isolates (especially the ones difficult to treat) differ from those to JFH-1 virus? 4. How do genetic variations (e.g., IL28B genotype) alter spontaneous viral clearance and treatment responses?

Acknowledgement Li Lab @ Univ of TN HSC Current N. Wang B. Liu S. N. Lester X. Li K. Kumthip

Alumni Z. Chen D. Wei S. Devaraj Q. Dong Z. Zhou Y. Shen J. Yi

Lawrence Pfeffer (Univ. of TN HSC) Stanley Lemon (UNC-Chapel Hill) Michael Gale, Jr (Univ. of Washington) Gyongyi Szabo (UMass Med. School) Francis Chisari (The Scripps Res. Inst.) Takaji Wakita (National Instit. of Infect. Dis., Japan) Charles Rice (Rockefeller Univ.) NIH: $$$

Hepatitis C is a Global Health Problem 170-200 million infected worldwide

~70-85% people infected progress to chronic liver diseases

Dependence on miR-122 for HCV Replication

Protect uncapped HCV RNA from exonuclease attack Sheild 5’-end triphosphate from innate immune recognition by RIG-I Stimulate translation of HCV RNA

Scheel & Rice, Nature Med 2013