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SYSTEM BIOLOGY AND IMMUNE RESPONSES Les Pensières, Veyrier sur Lac - June 2010

SYSTEM BIOLOGY AND IMMUNE RESPONSES Les Pensières, Veyrier sur Lac - June 2010

Michaela Müller-Trutwin Unité de Régulation des Infections Rétrovirales

Primate Models for a system biology approach

to understanding AIDS pathogenesis

Non-human primate models for HIV/AIDS Non-human primate models for HIV/AIDS

Asian nonhuman primates

(macaques)

Macaque / SIVsm, SIVmac Pt Macaque / SIVagm.90 Pt Macaque / SIVlhoest

AIDSAIDS

HUMANS

HIV-1

AIDSAIDS

African nonhuman primates: natural hosts of SIV

(~40 species)

ChimpanzeeChimpanzee / / SIVcpzSIVcpz GorillaGorilla //SIVgorSIVgor

SootySooty Mangabey Mangabey / / SIVsmSIVsm AfricanAfrican Green Green MonkeyMonkey / / SIVagmSIVagm

LL’’HoestHoest monkeymonkey //SIVlhoestSIVlhoest Mandrill Mandrill / / SIVmndSIVmnd

ASYMPTOMATICASYMPTOMATIC

HIV-2

✔✔✔✔ ✔✔✔✔ Anti-viral T and B cell responses

✔✔✔✔ - Chronic T cell activation

✔✔✔✔ ✔✔✔✔ High viral genetic variability

✔✔✔✔ - Progressive loss of blood CD4 +T cells + AIDS

✔✔✔✔ ✔✔✔✔ Severe loss of CD4 + T cells in gut

✔✔✔✔ ✔✔✔✔ High viral load in blood and gut

HIV-1, SIVmac

SIVsm, SIVagm

Major common and distinct features between non pathogenic (AGM , SM)

and pathogenic (HUMAN, MAC) HIV/SIV infections

(Müller-Trutwin et al, Virology, 1996; Rey-Cuillé et al, 1998; Chakrabarti et al, 2000; Diop et al, 2000; Goldstein et al, 2000; Broussard et al, 2001; Silvestri et al, Immunity, 2003; Gueye A et al, 2004; Gordon et al 2007; Pandrea et al, Blood 2007; Lozano et al, JVI, 2009; Zahn et al, 2009.)

Natural host: excellent model for searching determi nants of protection against bystander immune activation

Mechanism of protection against bystander immune activation in natural hosts of SIV?

• Viral determinants (CD3 down-regulation by NEF) (Schindler et al, Cell, 2006)

Hypotheses from the litterature :

• Preservation of tissues: - lymph nodes (Beer et al, 1995; Diop et al, 2000; Cumont et al, 2008; Lederer et al, 2009) - intestinal barrier (Brenchley et al, Nat Med 2006; Li et al, JID 2008)

• Specific CD4 + T cell characteristics (low CCR5, CD4 down, Th17) (Murayama et al, 1997, Int Immunol; Pandrea et al, Blood, 2007; Brenchley et al, Blood, 2008, Favre et al, Plos Pathogens 2009; Beaumier et al, Nat Med 09)

CD

3+ C

D8+

DR

+ K

i67

+

0

400

800

1200

1600

0 10 20 30 40

0

400

800

1200

1600

0 10 20 30 40

Diop et al, JVI, 2008

Vire

mia

IFN

- αα αα

(pla

sma)

Kornfeld et al, JCI, 2005

weak or rapidly controlled?

MAC

AGM MAC

AGM

Diop et al, JVI, 2000 days p.i.

MAC

AGM

°°°°Rapid negative immunoregulation (Kornfeld et al, JCI 2005; Estes et al JI 2008; Favre et al 2009)

Gene expression profiles in CD4+ T cells (blood, lymph nodes) from

African green monkeys (AGMs) and Rhesus Macaques (RM) before and after infection (day 1 - day 600)

Functional assay to study the regulation of these genes ( in vitro )

OBJECTIVES

Identification of genes whose expression is distinct between SIV -infected AGM and RM

Analysis of gene expression profiles in AGMs and RM s

Ficoll PBMC Magnetic

cell sorting dilaceration

CDCD4+4+

RNA

Blood * Lymph Node

Gene Expression ABI Microarray

Jacquelin et al, FASEB J. 2007

1 6 14 28 41 65 days -90 -70 -40 -8 115

N = 6 AGM N = 6 Mac (RM)

~13.000 genes expressed in AGM and RM CD4 + cells

Experimental approach

600

* * * * * * * *

205 data sets

**

***

***

*** ***

***

**

** ***

*** *

AGM peripheral CD4+ cells RM peripheral CD4+ cells AGM LN CD4+ cells RM LN CD4+ cells * p < 0.01 ** p < 0.001 *** p < 0.0001

ONTOLOGYONTOLOGY: : Immunity and Defense subcategories (acute infection )

Interferon Mediated ImmunityInterferon Mediated Immunity

Jacquelin et al, JCI, Dec 2009

HIV-1/SIVmac non-viremic viremic ISG + +++

Bosinger et al, JI, 2004, Dandekar et al; Favre et al, 2009, and many others

Genes that were most upregulated: ISG

ISG (Interferon Stimulated Genes) PRR signaling

Immunomodulatory blood CD4 +

0

+ + 2

- - 2

AGM RM

0

+ + 2

- - 2

Typ

e I I

SG

s

AGM and RM

TRIMs Tetherine Mx1 OAS IRF7 RIG-I CXCL10 IDO …

Resolution after d28p.i. (p<0.001)

Induction of

AGM

days

Antiviral

Lymph Nodes Lymph Nodes : : strongstrong , , transient induction of ISG in AGMtransient induction of ISG in AGM AGM RM

LN CD4+

0

+ + 2

- - 2

only in RM RANTES TRAIL IRF8

chronic only in RM CCL2 CCL8 CXCL9 CXCL10 CXCL11

Species-specific up-regulations:

Resolution after d28p.i. (p<0.001)

AGM

days

Number of ISGs (type I)

SIVmac/RM AGM/SIVagm Blood CD4 + 4343 6767 Lymph node CD4 + 63 44

Typ

e I I

SG

s

IFN-αααα plasma levels

Plasma IFN- αααα : Associated with viral load (p< 0.0001) and ISG profiles (p<0.002) 3 times lower in AGM than RM (p<0.005) In some AGMs as high as in RM that progress to AIDS

tight sampling… RM AGM

JOURNAL OF VIROLOGY , 2010, in press

Down-Regulation of Robust Acute Type I IFN Response s Distinguishes Non-Pathogenic SIV Infection of Natural Hosts from Pathogenic SIV Infection of Rhesus Macaques

L.D. Harris, B.Tabb, D.Sodora, M.Paiardini, N.Klatt , D.Douek, G.Silvestri, M.Müller-Trutwin, I.Vasile-Pandrea, C.Apetrei, V.Hirsch, J.Lifson, J.M. Brench ley and J.D. Estes

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Mx1 RM AGM

0 10 2 10 3 10 4 10 5

Lineage( CD3 CD20) <APC-Cy7>

0

1000

2000

3000

4000

FS

C-A

21.7

0 10 2 10 3 10 4 10 5

CD123 <PE>

0

10 2

10 3

10 4

10 5

HLA

-DR

<P

E-C

y7>

0.44

0 10 2 10 3 10 4 10 5

CD14 <FITC>

0

1000

2000

3000

4000

FS

C-A

94

0 10 2 10 3 10 4 10 5

IFNa <APC>

0

10 2

10 3

10 4

10 5

CD

123

<P

E>

99.8 0.21

0 10 2 10 3 10 4 10 5

IFNa <APC>

0

10 2

10 3

10 4

10 5

CD

123

<P

E>

11.9 88.2

0 10 2 10 3 10 4 10 5

IFNa <APC>

0

10 2

10 3

10 4

10 5

CD

123

<PE

>

93.9 6.12

IFN-αααα

pDC NS pDC HSV 0,21% 88,2% 6,1 %

���� AGM pDC are responsible for SIVagm -stimulated IFN- αααα production in vitro

The cells were labelled after 6h stimulation.

Similar to human and Mac PBMC, among AGM PBMC, only pDC were positive for IFN-αααα after stimulation with SIV.

pDC SIVagm

SIVmac

251 SIVagm 92018

SIVmac 251 Stimulus

AGM RM Species

0

25

5050

125

200

ng/ml SIV Gag p27

Similar IFN-αααα production by AGM and MAC pDC after in vitro SIV stimulation

0 100 200 300 400

Medium HSV SIV

AGM N=3

IFN-αααα Production after 18h stimulation

500 1000 1500

IFN-αααα production by AGM pDC after in vitro stimulation with HSV and SIVagm

AGM (non-infected) N=12

10

100

1500

5000

10

100 1500

5000 0

2 4 6 8

10

Mx1

gen

e ex

pres

sion

(Lo

g 2 Q)

0 0

IFN-αααα sufficient to induce ISG in AGM cells? Why strong ISG in vivo irrespective of plasma IFN- αααα levels?

��Low threshold for ISG inductionLow threshold for ISG induction

IFNIFN--αααααααα Dose Dose --Response Response

RM AGM

rIFN (UI/ml)

(10 I

U/ml)

αααα r-I

FN- (100

IU/m

l)

r-IFN-αααα

(150

0 IU/m

l)

r-IFN-αααα

/ml)

50

TCID

5

SIVag

m (10

0 2 4 6 8

10 12

TR

AIL ge

ne e

xpre

ssio

n (L

og2

Q)

Moc

k

Why some ISGs are not seen induced in vivo ?

AGMAGM

----> > Efficient Efficient in vitroin vitro induction of TRAIL induction of TRAIL

*

* r-IFN-αααα no stimulus or r-IFN- αααα

0 18h 46h 64h

no stimulus

cell harvest washing

RM AGM

* p<0.01

Design of repeated in vitro stimulations of PBMC:

Are cells of AGM more refractory to repeated stimulation than RM cells ?

Normal IFN- αααα production and ISG induction in PBMC from chronically infected AGMs in vitro --> in vivo regulation ….

Do chronically infected AGMs respond less?

AGM SIV- (N=5) AGM SIV+ (N=6)

ns ns: non significant

Mx1: after 18h of stimulation in vitro

IFNIFNIFNIFN----I I I I signalingsignalingsignalingsignaling

PRR PRR PRR PRR signalingsignalingsignalingsignaling

Positive feedbackPositive feedbackPositive feedbackPositive feedback

NegativeNegativeNegativeNegative feedback feedback feedback feedback

-90

-8

1 6 14

28

65

592

-8

1 14

28

65

592

AGM (LN) RM (LN) INDO (IDO) GPNMB ILT4 galectin-3 LAG3 BCMA CTLA4 LIF FGL2 TGF-beta IL10 CD160

Immunosuppressive genes ?

-90 -70 -40 -8 1 6 14 28 41 65 -8 1 14 28 65 592

* * * * * *

Negative feed back loop genes ?

before acute chronic infection

AGM

MAC

loss of «on» signals

signals «off»

persistent «on»

«off» signals lacking

�� Which genes are associated with the control of the innate response in vivo ?

still too many genes…

�� Meta-analysis • Lederer S, Favre D, Walters KA, Proll S, Kanwar B, Kasakow Z, Baskin CR, Palermo R, McCune JM, Katze MG. . Transcriptional profiling in pathogenic and non-pat hogenic SIV infections reveals significant distinct ions in kinetics and tissue compartmentalization. 2009. PLoS Pathog. 5(2):e1000296. (90 arrays, Rhesus, Agilent) (AGM, 3 tissues, 2 time points) • Jacquelin B, Mayau V, Targat B, Liovat AS, Kunkel D, Petitjean G, Dillies MA, Roques P, Butor C, Silvestri G, Giavedoni LD, Lebon P, Barré-Sinoussi F, Benecke A, Müller-Trutwin . Strong but Rapidly Controlled Interferon Type I Res ponse in Nonpathogenic SIV Infection of African Green Monkey s. 2009. J Clin Invest. 119(12):3544-55. (201 arrays, Human, Applied) (AGM, 2 tissues, 8 time points) • Bosinger SE, Li Q, Gordon SN, Klatt NR, Duan L, Xu L, Francella N, Sidahmed A, Smith AJ, Cramer EM, Zeng M, Masopust D, Carlis JV,Ran L, Vanderford TH, Paiardini M, Isett RB, Baldwin DA, Else JG, Staprans SI, Silvestri G, Haase AT, Kelvin D. Global genomic analysis reveals rapid control of a robust innate response in SIV-infected sooty mangab eys. 2009. J Clin Invest. 119(12):3556-72. (120 arrays, Rhesus, Affymetrix) (SM, 2 tissues, 6 time points)

NON

PROGRES

SORS

PROGRES

SORS

AG

M

LN

PB

MAC PB

LN

MA

NG

PB

LN

MAC

SIVm

ac2

39

PB

SIVSm

m PB

MM

T G

S

A. Benecke AFFYMETRIX

APPLIED (HS) AGILENT (HS)

H

S R

M

AP(h)2AG(h) AG(h)2AP(h)

AF(h)2AF(rh) AF(rh)2AF(h)

AB(h)2

AF(h)

AF(h)2

AB(h)

AF(h

)2AG

(h)

AG

(h)2A

F(h)

AP(h)2AF(rh)

AF(rh)2AP(h

)

AF(rh

)2A

G(h

)

AG

(h)2

AF(rh

)

Non-pathogenic SIVagm infection in AGM

Conclusion

Immediate, strong and broad systemic IFN-I response Immunoregulatory control at the transition from acu te to chronic stage

A little inflammation in acute phase is essential for virus and host

�Establishment of persistent infection �Partial control of viral replication

-- > Resolution of immune activation! Beneficial for the host, but HOW is it achieved ?

«Off» signals? Lack of second line «on» signals? local environment…

Primary infection

Chronic phase

INSTITUT PASTEUR, PARIS Régulations des Infections Rétrovirales F. Barré-Sinoussi TeamTeam »»Early determinants Early determinants of protection of protection against against AIDS AIDS »»

Beatrice JacquelinBeatrice Jacquelin GaGaëël l PetitjeanPetitjean AnneAnne --Sophie Sophie LiovatLiovat VVééronique ronique MayauMayau

IHES, France B.Targat, C.Bécavin, JF

GolibDzib, G. Brisbaert, A. Benecke

CEA, Fontenay-aux-Roses ANRS NHP core facility

N. Bousquet, P. Roques, B. Vaslin, R. Le Grand

U Paris 7, Cochin C. Butor, A. Hosmalin

INSTITUT PASTEUR, Animalerie Centrale Celine Gommet, Xavier Montagutelli

San Antonio, Texas L. Giavedoni

Meta-analysis S. Proll, M.Katze R. Dunham, JM McCune S. Bosinger, G. Silvestri A. Haase D. Kelvin

Hôpital St Vincent de Paul Pierre Lebon

NCI, Frederick J. Estes