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What drives persistent
immune
activation/inflammation in
cART-treated HIV-1?
Giulia Marchetti, MD, PhD
Dept of Health Sciences, Clinic of Infectious Diseases - University of Milan,
San Paolo Hospital, Italy
The revolution of cART
CD4+
HIV-RNA
Hammer, S et al. NEJM 1997; Palella, F et al. NEJM 1998
Full restoration of
health upon cART?
Life expectancy in cART-treated
patients
ART-Cohort Collaboration
22.937 individuals (82,022 personyear)
At age 20
Samji et al. ART-Cohort Collaboration. PlosOne 2013
Serious non-AIDS events during long-term
virologically suppressive cART
Tenorio et al. JID 2014
HIV as an inflammatory
disease
• Acute HIV associated with rapid/intense release of pro-inflammatory cytokines
(IL-6, IP-10, TNF-a) and dramatic increase of activated innate immune cells T-,
B-cells
• Chronic HIV: T-cell activation steady state
High proportion of activated CD8+ T-cells in
HIV
Giorgi, J et al. J Immunol 1993
CD8 T-cell activation predicts CD4+ T-cell
count over time
Deeks et al. Blood 2004
Shorter survival is associated with Tlymphocyte activation
Giorgi, J et al. JID 1999
In untreated infection T-cell activation
is associated to HIV viral replication
Deeks S Blood 2004
What happens upon
cART?
CD8+ T cell activation fails to fully
normalize during effective cART
Hunt PW, et al. J Infect Dis. 2003;187:1534-1543.
Chronic inflammation is
a much more important
determinant of mortality
in treated HIV
Kuller L PLOS Medicine 2008; also Hunt et al. AIDS 2011; Lok et al, AIDS 2013; Hunt et al. JID 2014; Tenorio et al JID
2014; …..
What drives persistent
immune
activation/inflammation
in cART-treated
disease?
What drives persistent immune
activation/inflammation in cART-treated
disease?
• Gut epithelial barrier dysfunction, microbiome and
microbial translocation
• Co-infections (CMV et al….)
• Residual HIV replication
• Thymic dysfunction and residual defects in adaptive
immune responses
• Lack of immunoregulatory responses- Lymphoid fibrosis
• Co-morbid conditions (metabolic syndrome, central
adiposity)
What drives persistent immune
activation/inflammation in cART-treated
disease?
• Gut epithelial barrier dysfunction, microbiome and
microbial translocation
• Co-infections (CMV et al….)
• Residual HIV replication
• Thymic dysfunction and residual defects in adaptive
immune responses
• Lack of immunoregulatory responses- Lymphoid fibrosis
• Co-morbid conditions (metabolic syndrome, central
adiposity)
Gut epithelial barrier
dysfunction,
microbiome and
microbial translocation
The GI tract as a site of HIV pathogenesis
Brenchley et al. Nat Med 2006
Sandler & Douek, Nat Reviews 2012
Persistent Depletion of CD4+ T cells in the
GI Tract despite Normalization in the
Peripheral Blood
54 HIV+ patients (acute)
Mehandru S, Plos Med 2006
Persistent damage to the gut tight epithelial
barrier despite cART
Chung; Plos. Path. 2014; (see also Somsouk AIDS 2015)
HIV negative
HIV+ cART-treated
Tincati C et al. CROI 2014
Chung et al. Plos. Path. 2014; (see also Somsouk AIDS 2015)
Plasma LPS (pg/mL)
Altered gut tight junctions associate
with microbial translocation
P = 0.001
300
P = 0.002
200
100
0
HIV
Negative
HAART
VL < 75
Untreated
Brenchley J et al. Nat Med 2006; also Jiang et al. J Infect Dis 2009
(Altered) gut tight
epithelial barrier as
driver of inflammation?
Altered gut tight junctions associate with
immune activation
Chung et al Plos. Path. 2014; (see also Somsouk AIDS 2015)
Tincati C et al. CROI 2014
Altered gut microbioma in SIV/HIV
Brenchley Nat Med 2006; in humans: Gori et al. JCM 2008
Only partial recovery of gut
microbioma upon successful cART
50 HIV+ patients
before (T0) and
after 12 months
cART (T12)
(Altered) intestinal
microbioma as driver of
inflammation?
Greater representation of
proinflammatory/inflammationthriving class-level bacteria
Correlation between gut
microbioma and systemic immune
activation
Ellis et al. JAIDS 2011, also Dillon et al. Mucosal Immunol 2014
Plasma LPS (pg/mL)
Persistent microbial translocation
during cART
P = 0.001
300
P = 0.002
200
100
0
HIV
Negative
HAART
VL < 75
Untreated
Brenchley J et al. Nat Med 2006; also Jiang et al. J Infect Dis 2009
Microbial translocation hampers CD4+
T-cell recovery upon cART
Marchetti G et al. AIDS 2008; Brenchley J et al. Nat Med 2006; also Jiang et al. J Infect Dis 2009
Microbial translocation
and immune activation:
what is the cause what
is the effect?
Microbial translocation causes
immune activation: colocalization of
E.coli and IFN-a in colon
Estes J et al. PLoS Pathogens 2010
Microbial translocation is associated to
immune activation
Brenchley J et al. Nat Med 2006
Marchetti G et al. AIDS 2008; Jiang et al. J Infect Dis 2009
Exogenous LPS administration enhances
immune activation and HIV replication
Pandrea et al J Immunol 2008; Pandrea et al. Blood 2012
Bacterial products drive monocyte
expression of thrombosplastin
Funderburg N et al Blood 2010
In vitro LPS stimulation of monocyte-derived
macrophages: cytokine/chemokine expression of
genes involved in the TLR pathway
35 HIV+ cARTtreated
Merlini E et al ICI, International Congress of Immunology 2013
Stimulation of peripheral blood cells by TLR
ligands increases expression of CD38 on CD4+
and CD8+ T-lymphocytes
- HIV-negative
Funderburg N et al. PLoS One 2008
In vitro LPS stimulation on PBMC: CD4 and CD8 Tcell activation, proliferation and apoptosis
35 HIV+ cARTtreated
Merlini E et al ICI, International Congress of Immunology 2013
Sevelamer treatment reduces MT during early SIVsab
infection of PTMs
LNs stained for LPS core antigen
(brown)
Kristoff J, JCI, 2014: 124 (6)
Sevelamer treatment reduces immune
activation/inflammation during early SIV
infection in PTMs
Kristoff J, J Clin Invest 2014
Sevelamer does not reduce LPS and sCD14
in chronic early-stage untreated HIV
Sandler N, J Infect Dis 2014
Should we test
sevelamer in cARTtreated HIV?
Altered balance of gut
immunoregulatory cells
(e.g. Th17/Th22, guthoming T-cells) as
driver of inflammation?
Only partial recovery of gut-homing T-cells
upon cART
20 HIV+ before and at
12 months cART
20 HIV+ cARTtreated
Basilissi M ICAR 2015
Mavigner et al. JCI 2012
Only partial recovery of gut-homing and
Th17/Th22 T-cells upon cART
20 HIV+ before and at
12 months cART
Basilissi M ICAR 2015
Low Th17/Treg ratio despite
cART
20 HIV+ cARTtreated
Favre et al Science Transl Med 2010
Low Th17/Treg ratio is associated
to immune activation
20 HIV+ cARTtreated
Favre et al Science Transl Med 2010
What drives persistent immune
activation/inflammation in cART-treated
disease?
• Gut epithelial barrier dysfunction, microbiome and
microbial translocation
• Co-infections (CMV et al….)
• Residual HIV replication
• Thymic dysfunction and residual defects in adaptive
immune responses
• Lack of immunoregulatory responses- Lymphoid fibrosis
• Co-morbid conditions (metabolic syndrome, central
adiposity)
Higher non-AIDS morbidity/mortality in
HIV+/CMV-Ab+ patients
6111 HIV+ (5119 CMV-Ab+),
12% cART-treated
Lichtner M et al. J Infect Dis 2015
Naeger D et al. PlosOne 2010
cART-treated asymptomatic CMV
seminal shedders present higher Tcell activation/proliferation
53 HIV+ cARTtreated
Gianella S et al. J Virol 2015
cART-treated asymptomatic CMV
seminal shedders present higher
T-cell expression of PD-1
45 HIV+ cARTtreated
Dan J et al. CROI 2015
Higher innate immunity markers in HIV/CMV
co-infected patients on cART
p=0.018
sCD163 ng/ml
2500
2000
1500
1000
500
0
HIV/CMV -
Fig. 1A
HIV/CMV +
Serostatus
69 HIV+ cARTtreated
(46/69 CMV
Ab+)
Vita S et al. CROI 2015
Reduction of CD8 T-cell activation by
valganciclovir……
30 HIV+, 70%
cART-treated
Hunt et al., JID, 2011
……but not valacyclovir
40
HIV+/HSV2+
cART-treated
Yi TJ et al., CID, 2013
*ARR, 3.87
*ARR, 3.15
*ARR, 2.68
HCV coinfection was
associated with increased
risk of developing an ADI
(adjusted relative rate [ARR],
2.61; 95% confidence interval
[CI], 1.88–3.61)
127 HIV-infected hepatitis viruses co-infected patients (118 HCV, 9 HBV)
- ART naïve, CD4 cell count >200/μl
- known date of prior HIV neg/pos tests
→immune activation (IA): IL-6,TNFα
→microbial translocation (MT): LPS, sCD14
34 : 14
HCV+/HIV+
cART-treated;
11 HCV+; 9
HIV+ treated
Gonzalez et al et al. J Virol 2009
Hampered T-cell dynamics in
HIV/HCV co-infected patients 356 HIV+ cART-
treated : 130 HCV
co-infected
Zaegel-Fauchel O et al. AIDS 2015
HCV co-infection is associated to higher Tlymphocyte activation on cART
Hunt et al. JID 2003; also Greub G Lancet 2000
HCV treatment reduces
immune activation ?
Reduction of T-cell activation
by anti-HCV treatment
356 HIV+
cART-treated :
130 HCV coinfected
Gonzalez et al et al. J Virol 2009; also Massanella M
et al. Antiviral Therapy 2010
What drives persistent immune
activation/inflammation in cART-treated
disease?
• Gut epithelial barrier dysfunction, microbiome and
microbial translocation
• Co-infections (CMV et al….)
• Residual HIV replication
• Thymic dysfunction and residual defects in adaptive
immune responses
• Lack of immunoregulatory responses- Lymphoid fibrosis
• Co-morbid conditions (metabolic syndrome, central
adiposity)
Despite cART, HIV viremia persists
indefinitely at very low level
Adapted from Deeks S – International Congress on Drug Therapy in HIV
Infection, Glasgow UK 2-6 Nov 2014
Immune activation does not
correlate with residual plasma
viremia……
Case: 123 HIV+
cART-treated
with transient
low level viremia
(>50 <400 cp/ml)
Control: HIV+
cART-treated
RNA<50cp/ml
Taiwo B et al. JAIDS 2013; also Chun TW et al. JID 2011 (including C-reactive
protein, D-dimer, IL-6, soluble TNF receptor I); Steel A et al. Antiviral Therapy
2007…..
Steel A et al. Antiviral Therapy 200.
…but may associate with residual plasma
viremia in the setting of poor immune
recovery on cART….
Mauvigner M et al. PlosOne 2009
Marchetti G et al. AIDS 2006
….Immune activation (and senescence) does
associate with cell-associated HIV-DNA/RNA in
peripheral blood ….
190 HIV+ cARTtreated
Hatano H et al. JID 2012; also
Stone SF HIV Med 2005
….and in tissues
23 HIV+ cARTtreated
Sheth PM et al. Mucosal Immuno 2008; also Yukl SA JID 2010;
d’Ettorre G et al. Curr HIV Res 2011
Hypothesis: if residual
HIV replication sustains
immune activation upon
cART, then cART
intensification should
lower immune
activation
Any benefit by maraviroc intensification?
Wilkin et al., JID 2012
Any benefit by maraviroc intensification?
45 HIV+ cART-treated with
low CD4+ immune recovery
Hunt et al., Blood, 2013
97 HIV+ cARTtreated with low
CD4+ immune
recovery
Rusconi et al., PLOSOne, 2013
Any benefit by integrase
inhibitors intensification?
30 HIV+ cART-treated with
low CD4+ immune recovery
Peripheral blood
Gut
Hatano H et al., JID, 2011; also Hatano H et al JAIDS 2012
Raltegravir induced a specific reduction of
CD38 expression in CD8 T cells
69 HIV+ cART-treated
Buzon MJ et al. Nat Med 2010;
Massanella et al., AIDS, 2012; also Vallejo A et al. AIDS 2012
Any differences in the
effect on immune
activation by diverse
cART class?
1
*
1.0
0.5
0.0
60
40
20
10
80
60
40
20
60
ID
Vr
EF
V
Vr
*
*
*
40
*
20
EF
V
LP
V/
r
40
*
*
30
T0
20
T12
10
0
D
R
V/
r
EF
V
A
TZ
/r
50
LP
V/
r
0
EF
V
Tincati et al. under review
B
20
ID
*
% CD8+CD38+DR+
*
%CD8+CD38+HLADR+
Immuno Study
CD4<50>250/uL; n=35
TDF+FTC+ EFV or DRV 800mg/r
A
30
0
Mirò et al. JAIDS, 2015
*
0
EF
V
100
C
r
ID
V/
EF
V
C
B
80
0
% CD8+CD38+
Advanz 3 Study
CD4<100/uL; n=89
TDF+FTC+ EFV or ATZ/r
or LPV/r
5
AT
Z/
r
Mirò et al. AIDS Res and Human Retrov 2010
10
40
CD8+CD38+DR+, %
%CD8+CD38+
Advanz Study
CD4<100/uL; n=65
AZT+3TC+EFV or LPV/r
A
*
100
*
15
0
r
ID
V/
B
A
EF
V
0
1.5
*
*
20
ID
V/
r
2
25
EF
V
3
2.0
%CD8+CD38+R0+
4
C
Rizzardini et al., HIV Clin Trials 2006
*
%CD8+CD25+
5
% CD8+DRII+
CD4 231/uL; n=76
AZT+ddI or AZT+3TC+
ABC or EFV or IDV/r
Data are presented as
median values
hs RCP
D-Dimer
0.3
400
mg/dL
200
100
pg/mL
2
LP
V/
r
2
Data are presented as
median values
D
R
V/
r
0
D
R
V/
r
T12
3
1
EF
V
A
TZ
/r
LP
V/
r
T0
*
4
4
10
0
EF
V
LP
V/
r
A
TZ
/r
*
*
*
5
sCD14
ug/mL
pg/mL
/r
1
5
0
See also: McComsey, AIDS, 2012
2
IL-6
8
*
15
EF
V
Tincati et al. under review
20
0
EF
V
0
6
LP
V
EF
V
5
Immuno Study
CD4<50>250/uL, n=35
TDF+FTC+
EFV or DRV 800mg/r
*
3
*
TNF-a
sCD14
*
EF
V
IL-6
ug/mL
10
LP
V/
r
EF
V
A
TZ
/r
0.0
160
120
80
40
pg/mL
Mirò et al. JAIDS, 2015
0
0.1
A
TZ
/r
Advanz 3 Study
CD4<100/uL; n=89
TDF+FTC+ EFV or ATZ/r
or LPV/r
0.2
A
TZ
/r
ng/mL
300
Similar reduction of T-cell activation by
different cART class
318 HIV+ starting first cART
(170 PI; 128 NNRTI; 20 INI)
What drives persistent immune
activation/inflammation in cART-treated
disease?
• Gut epithelial barrier dysfunction, microbiome and
microbial translocation
• Co-infections (CMV et al….)
• Residual HIV replication
• Thymic dysfunction and residual defects in adaptive
immune responses
• Lack of immunoregulatory responses- Lymphoid fibrosis
• Co-morbid conditions (metabolic syndrome, central
adiposity)
Bone marrow alterations upon cART
cART
cART
23 HIV+ cARTtreated
Isgro’ et al. CID 2008
Bellistrì et al. PlosOne 2010
Douek et al. Nature 1998
What drives persistent immune
activation/inflammation in cART-treated
disease?
• Gut epithelial barrier dysfunction, microbiome and
microbial translocation
• Co-infections (CMV et al….)
• Residual HIV replication
• Thymic dysfunction and residual defects in adaptive
immune responses
• Lack of immunoregulatory responses- Lymphoid fibrosis
• Co-morbid conditions (metabolic syndrome, central
adiposity)
In untreated HIV:
hyper-inflamed
cytokine milieu → Treg
response → TGF-β →
collagen deposition →
Fibrosis → Reduced IL7 → Reduced T cell
regeneration →
inflammation
Recovery of collagen deposition
according to the stage of cART start
Zeng et al. PlosPathogens 2012
Collagen deposition in lymphoid tissues before
cART substantially impacts the dynamics of Tlymphocyte reconstitution
LN
Schaker et al. JID 2002
GUT
Asmuth et al. AIDS 2015
Zeng et al. PlosPathogens 2012
Tissue fibrosis as driver
of immune activation?
Increased CD90+TLR4+ activated
myofibroblast in HIV+ duodenal mucosa (aSMA+FAP+)
Asmuth et al. AIDS 2015
Pinchuck IV et al. Curr Gatroenterol Rep 2010
LPS stimulation of cultured intestinal
myofibroblast from HIV+ patients
upregulates pro-fibrotic mediators
+ LPS
+ LPS
Asmuth et al. AIDS 2015
Klatt et al. Immunol Rev 2013
Thanks
*Dept of Health SciencesClinic of Infectious DiseasesUniv of Milan, San Paolo H
Esther Merlini
Camilla Tincati
Elvira S Cannizzo
Giuseppe Ancona
Giusi M Bellistrì
Francesca Bai
Matteo Basilissi
Antonella d’Arminio Monforte
***all the patients and staff
*Dept of Health SciencesPathology DeptUniv of Milan, S Paolo Ho
Delfina Tosi, Solange Romagnoli (now
Roche Diagnostics, Germany)
*Clinic of Infect Dis
Univ of Milan, L Sacco H
Stefano Rusconi, Massimo Galli
Alessandro Cozzi-Lepri, Miriam Lichtner,
Antonella d’Arminio Monforte