Suzanne Crowe
Associate Director, Burnet Institute
Consultant Infectious Diseases Physician, The Alfred Hospital
Melbourne
Today’s presentation
HIV, LPS trigger inflammation
How breaches
in gut mucosa
trigger
inflammation
How monocyte
activation
Impacts these
pathways
Innate immune
senescence
& risk of
events---
How HIV &
inflammation
are linked
Contribution of monocyte activation
Pro-inflammatory cytokines
Involvement of innate immune pathways in
Fibrosis
Atherosclerotic plaque formation
Pro-coagulant activity
Metabolic dysregulation
Immune senescence
Serious non-AIDS Events
Links
between
monocyte
metabolism
&
inflammation
Today’s presentation
HIV, LPS trigger inflammation
Contribution of monocyte activation
Pro-inflammatory cytokines
Involvement of innate immune pathways in
Fibrosis
Atherosclerotic plaque formation
Pro-coagulant activity
Metabolic dysregulation
Immune senescence
Serious non-AIDS Events
Chronic inflammation and immune activation
• Chronic immune activation & inflammation
is central to HIV pathogenesis
– In both treated and untreated individuals
– Starts during seroconversion
• Pro-inflammatory pathways triggered by
– HIV replication and viremia
– Co-infection eg with CMV
– Microbial translocation across the gut mucosa
Changes in plasma cytokine levels
Intense cytokine storm in acute HIV infection
prior to peak viremia
Cytokine response in acute HIV Stacey 2009
Time since HIV infection (days)
Stacey et al J Virol 2009 83:3719-3733
T cell immune activation declines during
suppressive ART
Hunt et al, JID, 2003; PLoS One, 2011
But T cell activation remains elevated
during suppressive ART
Hunt et al, JID, 2003; PLoS One, 2011
What about
Innate immunity
and
pro-inflammatory
pathways?
What is meant by pro-inflammatory pathways?
• Activation of cells eg monocytes & mϕ that produce proinflammatory cytokines
• Pro-inflammatory cytokines include IL-6, TNF-alpha
• Chronic inflammation leads to fibrosis and end-organ disease
R Medzhitov Cell 2010; 140:771
Differing pathogenetic roles of T cells &
monocytes in HIV infection
• T cell activation
– Main effect: Central role in T cell decline
• Monocyte activation
– Main effect: Central role in chronic inflammation
– Important in pathogenesis of longer-term, non-AIDS
morbidity
– CD16+ pro-inflammatory monocytes increased in HIV
& only partly normalized by cART
1. Westhorpe C et al Immunol & Cell Biol 2014;92:1338; 2.Tenorio AR et al J Infect Dis 2014;
3. Lederman MM et al Adv Immunol 2013; 119”51; 4. Sandler &Sereti Curr Opin HIV AIDS 2014;9:72
CD16-positive monocytes
• increase with inflammation [pro-inflammatory]
CD14+CD16++ non-classical
7-10%
CD14++CD16+ intermediate
5-8%
classical CD14++CD16- monocytes
85%
CD16+ monocyte subsets are increased in
uncontrolled viremia & may normalize with ART
HIVHIV+ VL<400
HIV+ VL>400
CD14++CD16Traditional
Classical
CD14++ CD16+
CD14+CD16++
Inflammatory
Intermediate
Non
classical
Patrolling

HIV infection increases the proportions of CD16+ monocytes in blood

cART restores the proportions of monocytes in blood1
o CD16+ monocytes remain elevated in elite controllers2
1. Funderburg NT et al Blood 2012 120: 4599
2. Krishnan S et al J Infect Dis 2014; 209:931
Pro-inflammatory CD14++CD16+ monocytes remain
expanded despite cART & virologic suppression
HIVHIV+
Hearps, A, Angelovich T et al unpublished data 2014; Lichtfuss G et al., J Immunol 2012;189:149 ;
Hearps A et al AIDS 2012;24:843; Martin G et al PLoSOne 2013;8:e55279l
Elevated levels of intracellular proinflammatory
cytokines IL-6 & TNF in monocytes from HIV+ pts
Monocyte subsets
Classical
Intermediate
Non-Classical
IL-6
HIV- HIV+ VL<20
HIV- HIV+ VL<20
HIV-
HIV+ VL<20
TNF
HIV- HIV+ VL<20
HIV-
HIV+ VL<20
HIV-
HIV+ VL<20
Angelovich T et al 2014 unpublished data 2014
Markers of inflammation and coagulation, but not
T-cell activation, predict non-AIDS events
• Case-control study in virologically suppressed
patients
– Cases: non-AIDS events (AMI, CVA, non-AIDS
cancers, bacterial infection, death)
– Controls: well matched (age, sex, pre-ART CD4, ART
regimen)
• Blood taken for inflammatory markers
– at baseline, 1 yr post ART initiation, pre-event
• Conditional logistic regression analysis
Tenorio AR et al J Infect Dis 2014 May;
Markers of inflammation & coagulation, but not
T-cell activation, predict non-AIDS events
Pre-event marker
Odds ratio
1 IQR increase p value
OR
at baseline for
OR at baseline for:
Death
Ca MI/Stroke
AMI/stroke
Death CA
IL-6
IP-10
**
**
20.9** 3.1**
19.9** 2.9**
2.2**
2.1**
**
**
1.9
1.7*
1.7*
1.8
1.5
1.7*
**
3.3**
2.3**
2.1**
**
3.3*
2.2**
2.1**
**
**
2.6**
2.1**
1.9**
2.9**
1.9*
2*
**
**
2.7*
1.5
1.7
2.8*
1.4
1.7
**
**
8.4**
3.2**
2.6**
8.1**
3.1**
2.6**
1.4
1
1
0.8
0.9
0.9
sTNFR-I
sTNFR-II
sCD14
D-dimer
CD38+DR+CD8
Tenorio AR et al J Infect Dis 2014 May ahead of print
• Increased innate immune inflammatory
markers are strongly associated with nonAIDS morbidity
• T cell activation is less related to non-AIDS
morbidity
Tenorio AR et al J Infect Dis 2014 May
So
where
does this
inflammation
come from?
(HIV,co-pathogens, and…)
Early loss of CD4+ cells in gut-associated lymphoid
tissue results in systemic inflammation
HIV-
HIV+
• Loss of CD4+ T cells in GALT
o Not in elite controllers
 Starts in acute HIV infection
 Permanently damages gut
mucosa
 Microbial translocation
o Systemic inflammation
 Incompletely restored by ART
• Mϕ accumulate in gut mucosa * in untreated HIV
 Further drive pro-inflammatory cytokine production
Guadalupe M et al J Virol 2003 77:11708;
Ciccone E et al J Virol 2011;85:5880
Brenchley et al Nat Med 2006 12:1365;.
Allers K et al JID 2014 209:739-48
Gut-derived bacterial products translocate to the
systemic circulation
Increased gut
permeability & microbial +
translocation
Gutderived
bacterial
products
LPS
peptido
glycan
lipotechoic
acid
unmethyl
ated CpG
DNA
Altered gut
microbiome
ribosomal
DNA
Portal vein
Liver
Innate immune activation &
systemic inflammation
(monocytes, mϕ)
End organ
disease
Data from multiple sources &reviews including Deeks SG et al; Immunity; 2013: 39 :633
Activation of Kynurenine/IDO pathway may be
involved in the pathogenesis of non-AIDS events
Increased gut
permeability & microbial +
translocation
Altered gut
microbiome
(dysbiosis)
LPS
activation of
IDO pathway
Tryptophan
Quinolinic acid
Data from multi[le sources including
Heyes MP et al J Neuroimmunol 1992;40:71;
Pro-inflammatory cytokine production (TNF, IL-6)
Innate immune activation &
inflammation
(monocytes, mϕ)
Vujkovic-Cvijin et al Aci Transl Med 2013;5:193;
End organ
disease
Activation of the IDO pathway is also linked
to premature mortality
Byakwaga H, et al J infect Dis 2014 August
Multi-pronged liver attack by HIV, copathogens and gut-derived bacterial products
Increased gut
permeability & microbial
translocation
Gutderived
bacterial
products
LPS
Portal vein
Kupffer mϕ
& hepatic
stellate cell
activation
peptido
glycan
lipotechoic
acid
unmethyl
ated CpG
DNA
Liver damage
Activation of innate
pro-inflammatory &
pro-fibrotic pathways
ribosomal
DNA
 clearance
LPS
End organ disease
Including
liver fibrosis
Tacke F& Zimmerman H J Hepatol 2014;60:1090; Balagopal A et al Gastroenterology 2008 135:226
Multi-pronged liver attack via HIV, co-pathogens,
gut-derived bacterial products, etc
Increased gut
permeability & microbial
translocation
Gutderived
bacterial
products
LPS
Portal vein
Kupffer mϕ
Hepatic
stellate cell
activation
peptido
glycan
lipotechoic
acid
Coinfection
Eg HCV
unmethyl
ated CpG
DNA
Impaired
hepatic function
Activation of innate
pro-inflammatory &
pro-fibrotic pathways
ribosomal
DNA
 clearance
LPS, etc
End organ disease
eg
liver fibrosis
Tacke F& Zimmerman H J Hepatol 2014;60:1090; Balagopal A et al Gastroenterology 2008 135:226
Monocyte activation is associated with liver fibrosis
in HIV+ patients in Uganda
• Retrospective Case/Control study
– in Rakai Uganda
• HIV+ & HIV- subjects with liver fibrosis
(transient elastography >9.3kPa)
– Cases n = 133
– Controls n =133
• matched for age, gender, HIV, ART
– Less than 5% infected with HBV
Redd AD et al AIDS Res Hum Retroviruses 2013:29:1026
Patients with monocyte activation had higher
odds of liver fibrosis
Overall population
Cases (>9.3kPa)
Soluble CD14 (ng/ml)
1,770 (+/- 1163)
2,029 (+/- 1320)
Controls (<9.3 kPa)
HIV+
113 (+/-77.8)
1,840 (+/- 1369)
HIV-
1,682 (+/- 793)
• Higher sCD14 levels were associated with
– 19% increased odds of having liver fibrosis (adj OR 1.19 p 0.002)
• In HIV+ higher sCD14 was associated with
– 54% increased odds of having liver fibrosis (adj OR 1.54, p <0.001)
Redd AD et al AIDS Res Hum Retroviruses 2013:29:1026
How do
monocytes contribute
to the
development
of
cardiovascular disease
in
HIV+?
Contributing factors to CVD in HIV+ patients
Traditional risk
factors
cART toxicity
monocyte & mϕ
activation
other pro-inflammatory
& pro-coagulant pathways
chronic inflammation
Cardiovascular disease
Co-infection
with eg CMV
Role of monocytes in
atheromatous plaque development
Adhesion
m’cules
Slide produced by G Martin
HIV activates monocytes & endothelial cells (in conjunction with proatherogenic lipids),
 Increase monocyte transmigration
 Increase uptake of oxLDL
 Promote differentiation into foam cells
 And contribute to atherosclerotic plaque formation
Campbell J et al AIDS 2014 in press
HIV promotes monocyte foam cell formation
in vitro
monocyte
macrophage
Foam cell
• Sub-endothelial monocytes take up ox LDL via CD36 and
develop into foam cells after migrating across TNF-activated
endothelial cells1
– Endothelial activation is critical for this process1
– Foam cell formation by activated monocytes from HIV+2
1. Westhorpe C et al Exp Mol Pathol 2012;93:220
2. Maisa A et al submitted 2014
How does
monocyte metabolism
augment
inflammation
in
HIV+ patients?
Glucose metabolism in monocytes
• Glucose is important
substrate for ATP
production
• Glut1 is major glucose
transporter
• Activated monocytes
dramatically increase
Glut1 expression &
glucose uptake
• Change from oxidative to
glycolytic metabolism
Palmer CS & Crowe SM AIDS Res Human Retro 2014 30:335
Fold expression
Glucose uptake is linked to pro-inflammatory
responses of mΦ
Glut1
Glut2
Glut3
Glut4
• Murine MΦ with high
Glut1 expression
express high levels of
pro-inflammatory
cytokines
IL-6
TNF
p = .018
p = .006
• Glut1 is rate-limiting
transporter in mΦ
• (murine & human)
Glut1
LOW
Glut1
HIGH
Glut1
LOW
Glut1
HIGH
Freemerman AJ et al J Biol Chem 2014 289:7884
HIV increases glucose metabolic activity in
monocytes, linked to inflammation
Not restored by ART
p = 0 .0 0 8
p = 0 .0 0 5
p = 0 .4 8
%% Glut1+
G lu t1 +
C D 1 4 + monocytes
60
50
40
30
20
15
p = 0 .0 2 1
p = 0 .0 1 8
100
p = 0 .0 0 2
80
60
40
20
0
N C
I
C
N C
I
C
N C
I
C
NC I C NC I C NC I C
H IV H IV + /n a ïv e
H IV + /c A R T
HIVHIV+
naïve
HIV+
cART+
n =
15
14
8
10
• The increased Glut1
5
T
A
ïv
R
e
HIV+ naïve HIV+cART+
n= 16
17
+
11
IV
17
H
IV
H
N = 18
+
/n
/c
a
H
IV
HIV-
-
0
•
% Glut1+ monocytes
•
p = 0 .0 4 7
m o n o c y te s u b p o p u la tio n s
Increase in proportion of Glut1+
monocytes in HIV+
% G lu t1 + c e lls o n
•
expression is on intermediate
and non-classical (NC)
monocytes in HIV+
• Not restored by ART
11
Correlates with monocyte activation
& D-dimer expression
Palmer C , Anzinger J et al submitted, 2014
See POSTER MOPE-006 & TODAY WEPE-009
Does premature
Innate immune
senescence
contribute
to
premature risk
of
non-AIDS morbidities ?
Shared immunologic features between
HIV infection & ageing
Ageing
HIV
Immunologic
changes
Chronic immune
activation/inflamm
ation
Immune
senescence
:
CVD, cancers, kidney, bone
& liver disease,
neurocognitive impairment
• Telomeres shorten during
each cell division
• As telomeres shorten,
cells age
• Telomere length is a
classical marker of
immune ageing
M o n o c y t e t e lo m e r e le n g th (% o f c o n t r o l)
Shortened telomeres in young HIV+ and in
healthy elderly
Telomere length is shorter in
healthy elderly and
young HIV+ on cART with
T e lo m e r e l e n gVL<50
t h in m o n o c y t e s
15
p = 0 .0 3
p = 0 .0 2
p = 0 .0 0 1
p = 0 .0 0 8
10
5
+
+
Classical
CD16+
C D 1 4 m omonocytes
n o c y te s
C D 1 6 m monocytes
o n o c y te s
0
Y o u n g E ld e r ly Y o u n g
Y o u n g E ld e r ly
Young
Young
Aged Young
Young
Aged
Young
H IV +
H IV +
HIV+
HIV+
Median age: 28
72
29
Range (yrs): 20-32 70-82 27-45
28
72
Hearps A et al AIDS 2012; 26: 843
29 yrs
HIV accelerates innate immune changes
• HIV induces age-related changes in
monocytes
– In HIV+ men1
– In HIV+ women2
– Changes appear approx 10-14 years earlier in HIV+
compared with HIV- women2.
• Does the clinical phenotype of ageing
emerge earlier in HIV+ patients with innate
senescence?
– No clear evidence
Hearps A et al AIDS. 2012;24:843-53
Martin G et al 2013;8(1):e55279;
Summary
• Pathogenesis of SNAEs is
complex & multifactorial
• Chronic endotoxemia
activates innate immune
cells
• Pro-inflammatory
environment with
associated coagulation,
fibrotic & immune
metabolic changes
 Heightened risk of serious
non-AIDS events
Palmer CS & Crowe SM AIDS Res Human Retro 2014 30:335
Conclusions:
Strong role of innate immune pathways in
pathogenesis of Serious Non-AIDS Events
• Most innate immune changes in HIV+ only partly
reversed by ART
– Residual immune dysregulation syndrome
– Persistent immune activation, inflammation &
coagulation
• Circulating biomarkers of inflammation are
strongest predictors of non-AIDS events
• Less evidence for T cell involvement
• Innate immune senescence occurs earlier in HIV+
– Not clear whether this translates into premature risk of
SNAEs
Acknowledgements
Dmitri Sviridov
Anthony Jaworowski
Anna Hearps
Genevieve Martin
Clovis Palmer
Tom Angelovich
Anna Maisa
Gregor Lichtfuss
Wan-Jung Cheng
Jingling Zhou
Tim Spelman
M Gouillou
Infectious Diseases
Sharon Lewin
Jenny Hoy
Julian Elliott
Kate Cherry
Janine Trevillyon
Alan Landay
Cardiovascular Medicine
William Muller
Anthony Dart
Liz Dewar
Sofie Karapanagiotidis
The HaCH Study
volunteers
Univ West indies,
Jamaica
Josh Anzinger
Funding
Clare Westhorpe
Mike McCune
Also with grateful thanks to colleagues who contributed slides for this presentation, some
“The Ageing Team”
not included because of time limitations