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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