1. The role of innate immunity in the pathogenesis of Serious Non-AIDS Events
Suzanne Crowe
Associate Director, Burnet Institute
Consultant Infectious Diseases Physician, The Alfred Hospital
Melbourne

2. Today’s presentation Serious non-AIDS Events
HIV, LPS trigger inflammation
How HIV &
inflammation
are linked
How breaches in gut mucosa trigger inflammation
Contribution of monocyte activation
Pro-inflammatory cytokines
How monocyte activation
Impacts these pathways
Involvement of innate immune pathways in
Fibrosis
Atherosclerotic plaque formation
Pro-coagulant activity
Metabolic dysregulation
Immune senescence
Links between monocyte
metabolism &
inflammation
Today I have been asked to talk about the contribution of one aspect of the immune system, innate immunity, to the development of serious non-AIDS events
I’ll cover how HIV and inflammation are linked
How HIV disrupts the gut mucosa and triggers the production of pro inflammatory cytokines that predict SNAEs
How monocyte activation contributes to these innate immune, pathways
Including links between altered metabolism in monocytes and inflammation
And lastly I’ll mention the contribution of innate immune senescence, to SNAEs. …….But note that these arrows are only dotted
Innate immune senescence
& risk of events---
Serious non-AIDS Events

3. Today’s presentation Serious non-AIDS Events
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
And there are huge gaps in what we know about how these pathways loop back and intersect
Serious non-AIDS Events

4. 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
Chronic immune activation and inflammation are central to HIV pathogenesis, both playing a pivotal role in both untreated and treated individuals.
This starts very early, in fact during seroconversion

5. Intense cytokine storm in acute HIV infection prior to peak viremia
Cytokine response in acute HIV Stacey 2009
Changes in plasma cytokine levels
When there is an intense cytokine storm,
with high plasma levels of a range of proinflammatory cytokines including IL12, IL18 and TNF
accompanied by intense T cell activation
Time since HIV infection (days)
Stacey et al J Virol :

6. T cell immune activation declines during suppressive ART
T cell activation, shown here on CD8+ T cells, declines dramatically in patients on suppressive ART
Hunt et al, JID, 2003; PLoS One, 2011

7. But T cell activation remains elevated during suppressive ART
But remains still significantly higher that in uninfected controls
Hunt et al, JID, 2003; PLoS One, 2011

8. What about
Innate immunity and
pro-inflammatory pathways?

9. What is meant by pro-inflammatory pathways?
Activation of cells eg monocytes & mϕ that produce pro-inflammatory cytokines
Pro-inflammatory cytokines include IL-6, TNF-alpha
Chronic inflammation leads to fibrosis and end-organ disease
When we talk about pro-inflammatory pathways we are referring to monocyte activation in particular that produces cytokines capable of promoting inflammation such as IL-6 and TNF.
Uncontrolled chronic inflammation leads to fibrosis and organ damage.
R Medzhitov Cell 2010; 140:771

10. 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
T cell activation is most relevant to HIV pathogenesis in untreated patients where it is tightly linked with T cell death and decline
Monocytes contribute more to the pathogenesis of longer term non-AIDS complications that are associated with chronic inflammation
The proportion of CD16+ monocytes increases in the blood with HIV and is only partly restored to normal levels with ART
CD11b and CX3CR1 independently predicted cIMT in HIV+, after controlling for Framingham risk score: imporrtant role for monocyte activaoin in CVD oathogenesis
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

11. CD16-positive monocytes
increase with inflammation [pro-inflammatory]
CD14+CD16++ non-classical
7-10%
CD14++CD16+ intermediate
5-8%
monocyte subsets are classified based on their expression of CD16 and CD14
CD16+ monocytes are a minor monocyte population, generally considered to be pro-inflammatory
They are further subdivided into non classical and intermediate subsets
classical CD14++CD16- monocytes
85%

12. CD16+ monocyte subsets are increased in uncontrolled viremia & may normalize with ART
HIV-
HIV+ VL<400
HIV+ VL>400
CD14+CD16++
Non classical
CD14++CD16-
Classical
CD14++ CD16+
Intermediate
Traditional Inflammatory Patrolling
HIV infection increases the proportions of CD16+ monocytes in blood
cART restores the proportions of monocytes in blood1
CD16+ monocytes remain elevated in elite controllers2
HIV increases the proportion of CD16+ monocytes in the blood (shown in green)
In this study from the Lederman lab ART restored the proportions of monocytes
However… patient populations differ
For example CD16+ monocytes remain elevated in elite controllers.
1. Funderburg NT et al Blood : 4599
2. Krishnan S et al J Infect Dis 2014; 209:931

13. Pro-inflammatory CD14++CD16+ monocytes remain
expanded despite cART & virologic suppression
And further, Data from our lab at Burnet suggest that the proinflammatory monocytes can remain expanded despite ART and virologic suppression
HIV-
HIV+
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

14. Elevated levels of intracellular proinflammatory cytokines IL-6 & TNF in monocytes from HIV+ pts
Monocyte subsets
Intermediate
Non-Classical
Classical
IL-6
HIV- HIV+ VL<20
HIV- HIV+ VL<20
HIV- HIV+ VL<20
TNF
Monocytes from HIV infected patients (shown in blue and red) produce high levels of Intracellular pro-inflammatory cytokines such as IL6 and TNF compared with uninfected controls (shown in black),
regardless of whether there is virologic suppression (red) or not
HIV- HIV+ VL<20
HIV- HIV+ VL<20
HIV HIV+ VL<20
Angelovich T et al 2014 unpublished data 2014

15. 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
And a study from Landay and colleagues provides Strong evidence that non-AIDS events are associated with inflammation and immune activation in virologically suppressed pts
This was a case control study
Blood was taken at study entry, during ART and pre-event
Tenorio AR et al J Infect Dis 2014 May;

16. Markers of inflammation & coagulation, but not T-cell activation, predict non-AIDS events
Pre-event marker Odds ratio IQR increase p value OR at baseline for
OR at baseline for:
Death CA
MI/Stroke
20.9**
3.1**
2.2**
19.9**
2.9**
2.1**
1.9
1.7*
1.8
1.5
3.3**
2.3**
3.3*
2.6**
1.9**
1.9* 2*
2.7*
1.7
2.8*
1.4
8.4**
3.2**
8.1**
 1.4 1 
 0.8
 0.9
Death Ca AMI/stroke
IL-6 ** **
IP-10 ** **
sTNFR-I ** **
sTNFR-II ** **
sCD14 ** **
D-dimer
Soluble innate markers of inflammation, activation & coagulation (shown in red) predicted a non AIDS event
Whereas markers of T cell activation (green) were not associated with morbidity
……………………………………………………………………………………..
Most of these markers (except sCD14) decreased after starting ART,
And Most, except IP10 (CXCL10 iFN gamma induced protein, remained elevated at year 1 and pre-event in all subjects, ** **
CD38+DR+CD8
Tenorio AR et al J Infect Dis 2014 May ahead of print

17. T cell activation is less related to non-AIDS morbidity
Increased innate immune inflammatory markers are strongly associated with non-AIDS morbidity
T cell activation is less related to non-AIDS morbidity
Thus Inflammatory markers are strongly associated with morbidity, even after controlling for both baseline and time-updated CD4 count
And T cell activation appears to be not significantly related to outcomes.
Tenorio AR et al J Infect Dis 2014 May

18. does this inflammation come from?So
where
does this inflammation
come from?
(HIV,co-pathogens, and…)
So where does this inflammation come from?

19. Early loss of CD4+ cells in gut-associated lymphoid tissue results in systemic inflammation
HIV-
Loss of CD4+ T cells in GALT
Not in elite controllers
Starts in acute HIV infection
Permanently damages gut mucosa
Microbial translocation
Systemic inflammation
Incompletely restored by ART
HIV+
Mϕ accumulate in gut mucosa * in untreated HIV
Further drive pro-inflammatory cytokine production
A profound Loss of +CD4+ T cells from gut-associated lymphoid tissue during acute HIV infection permanently damages the gut mucosa,
This allows microbial products including LPS to enter the circulation.
Meanwhile macrophages accumulate in the gut especially in untreated individuals and can contribute to pro inflammatory cytokine production.
Guadalupe M et al J Virol :11708;
Ciccone E et al J Virol 2011;85:5880
Brenchley et al Nat Med :1365;.
Allers K et al JID :739-48

20. Gut-derived bacterial products translocate to the systemic circulation
Increased gut permeability & microbial translocation
Altered gut microbiome +
Gut-
derived
bacterial
products
LPS
peptidoglycan
lipo-techoic acid
unmethylated CpG DNA
ribosomal DNA
Portal vein
As a result of increased gut permeability,
Gut-derived bacterial products translocate from the gut to the systemic circulation
via the portal vein,
leading to systemic inflammation, innate immune activation, end organ damage and Non-AIDS events.
But it isnt just this simple… other pathways interact
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

21. 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
Pro-inflammatory cytokine production (TNF, IL-6)
For example Translocated LPS can also activate the KYNURENINE IDO pathway that converts tryptophan to the neurotoxic quinolinic acid
LPS activation of IDO also contributes to innate immune activation with synthesis of TNF and IL-61, 2
Activation of the IDO pathway is also linked with dysbiosis and inflammatory cytokine production
Tryptophan
Innate immune activation & inflammation
(monocytes, mϕ)
End organ disease
Quinolinic acid
Data from multi[le sources including
Heyes MP et al J Neuroimmunol 1992;40:71; Vujkovic-Cvijin et al Aci Transl Med 2013;5:193;

22. Activation of the IDO pathway is also linked to premature mortality
In a paper from Peter Hunt’s gp just published showed that in Ugandan patients initiating ART,
activation of the IDO pathway predicted poor T cell recovery and premature mortality
Byakwaga H, et al J infect Dis 2014 August

23. Multi-pronged liver attack by HIV, co-pathogens and gut-derived bacterial products
Increased gut permeability & microbial translocation
Gut-
derived
bacterial
products
LPS
peptidoglycan
lipo-techoic acid
unmethylated CpG DNA
ribosomal DNA
Portal vein
Liver damage
 clearance
LPS
Gut derived bacterial products also stimulate Kuppfer cells and hepatic stellate cells
These in turn activate proinflammatory and pro-fibrotic pathways .
Which damage the liver, further exacerbating the clearance of LPS and accelerate end organ disease
Kupffer mϕ
& hepatic stellate cell activation
Activation of innate pro-inflammatory & pro-fibrotic pathways
End organ disease
Including
liver fibrosis
Tacke F& Zimmerman H J Hepatol 2014;60:1090; Balagopal A et al Gastroenterology :226

24. Multi-pronged liver attack via HIV, co-pathogens, gut-derived bacterial products, etc
Increased gut permeability & microbial translocation
Gut-
derived
bacterial
products
LPS
peptidoglycan
lipo-techoic acid
unmethylated CpG DNA
ribosomal DNA
Portal vein
Impaired hepatic function
 clearance
LPS, etc
Coinfection
Eg HCV
All of this is potentially worsened by coinfection with viruses like HCV
Kupffer mϕ
Hepatic stellate cell
activation
Activation of innate pro-inflammatory & pro-fibrotic pathways
End organ disease eg
liver fibrosis
Tacke F& Zimmerman H J Hepatol 2014;60:1090; Balagopal A et al Gastroenterology :226

25. Retrospective Case/Control study
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
In a recent study in Uganda monocyte activation was shown to be linked with liver fibrosis.
This was a retrospective case control study in Uganda
of HIV+ and HIV- pts with liver fibrosis and matched controls.
Less than 5% had coinfection with HBV; none with HCV
Redd AD et al AIDS Res Hum Retroviruses 2013:29:1026

26. Patients with monocyte activation had higher odds of liver fibrosis
Soluble CD14 (ng/ml)
Overall population
1,770 (+/- 1163)
Cases (>9.3kPa)
2,029 (+/- 1320)
Controls (<9.3 kPa)
113 (+/-77.8)
HIV+
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)
Overall Patients with monocyte activation shown by high plasma levels of sCD14 had increased odds of liver fibrosis. (1.19)
In pts with HIV infection and high sol CD14 the odds of having liver fibrosis were 54% increased
Redd AD et al AIDS Res Hum Retroviruses 2013:29:1026

27. How do
monocytes contribute
to the
development of
cardiovascular disease in
HIV+?

28. Contributing factors to CVD in HIV+ patients
Traditional risk factors
cART toxicity
Co-infection with eg CMV
monocyte & mϕ
activation
other pro-inflammatory
& pro-coagulant pathways
The pathogenesis of CVD in HIV-infected patients is multifactorial.
Monocytes and chronic inflammation
Are central to innate immune pathways that are linked to the pathogenesis of CVD
But a number of other pathways including the coagulation pathway also critically important and beyond what I can cover today
chronic inflammation
Cardiovascular disease

29. atheromatous plaque development
Role of monocytes in
atheromatous plaque development
Adhesion
m’cules
HIV in conjunction with pro-atherogenic lipids up-regulates adhesion molecules on endothelia
HIV also activates monocytes increasing their expression of adhesion molecule expression, increasing their migration across activated endothelia into the intima of the blood vessel.
HIV can also increase their capacity to scavenge ox LDL via CD36 and become foam cells in the developing atherosclerotic plaque.
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

30. HIV promotes monocyte foam cell formation in vitro
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
Our in vitro data show that HIV promotes foam cell from monocytes that have transmigrated across activated endothelium where they take up cholesterol and modified lipoproteins
And foam cell is increased using monocytes from HIV-infected patients.
1. Westhorpe C et al Exp Mol Pathol 2012;93:220
2. Maisa A et al submitted 2014

31. How does
monocyte metabolism
augment
inflammation in
HIV+ patients?

32. 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
Glucose is needed in the cell for energy
When monocytes are activated there is an increase in Glut1 expression,
with a concomitant increase in glucose uptake
and a switch from oxidative to glycoytic metabolism
Palmer CS & Crowe SM AIDS Res Human Retro :335

33. Glucose uptake is linked to pro-inflammatory responses of mΦ
Murine MΦ with high Glut1 expression express high levels of pro-inflammatory cytokines
Fold expression
Glut1 Glut2 Glut3 Glut4
IL TNF
p = .018
p = .006
Glut1 is rate-limiting transporter in mΦ
(murine & human)
While there are a number of possible glucose transporters Glut1 is the rate limiting transporter in macrophages
Macrophages with high glut1 expression have high intracellular levels of proinflammatory cytokines IL-6 and TNF
……………………………………………………..
Classically activated M1 macrophages activated by TH1 cytokines eg TNF, IFN-g secrete IL-6 and TNF
Secrete pro-inflammatory cytokines
Preferentially metabolize glucose for energy
Glut1 mRNA upregulated 10-fold by M1 polarization and is the main transporter of gluc in humans as well as mice
Murine macrophages that express high levels of Glut1 contain sig greater IL-6 and TNF
Resident macrophages with a M2 anti-inflammatory phenotype present in some tissues: change to a M1 pro-inflammatory phenotype when adversely stressed
Glut1
LOW
Glut1
HIGH
Glut1
LOW
Glut1
HIGH
Freemerman AJ et al J Biol Chem :7884

34. HIV increases glucose metabolic activity in monocytes, linked to inflammation
Increase in proportion of Glut1+ monocytes in HIV+
Not restored by ART
Correlates with monocyte activation & D-dimer expression
% Glut1+ monocytes
The increased Glut1 expression is on intermediate and non-classical (NC) monocytes in HIV+
Not restored by ART
% Glut1+ monocytes
NC I C NC I C NC I C
HIV- HIV+ naïve HIV+cART+
Clovis Palmer at Burnet and his collaborator Josh Anzinger from Univ West indies have unpublished data showing that the proportion of monocytes expressing Glut1 is increased in HIV+
and this is not restored by ART.
The increase I Glut 1 expression is confined to CD16+ intermediate and non classical monocytes
and Glut 1 expression correlates with monocyte activation and d dimer expression
HIV- HIV+ naïve HIV+cART+
N =
Palmer C , Anzinger J et al submitted, 2014
See POSTER MOPE-006 & TODAY WEPE-009

35. Innate immune senescence contribute to premature risk of
Does premature
Innate immune senescence
contribute to
premature risk of
non-AIDS morbidities ?
IS contributes to the pathogenesis of non AIDS morbidities

36. Shared immunologic features between
HIV infection & ageing
Ageing
HIV
Immunologic changes
Chronic immune activation/inflamm ation :
Immune senescence
Ageing and HIV share a number of immunologic features including, chronic immune activation and inflammation
And in both there is evidence of immune senescence and a heightened risk of diseases normally seen in elderly people.
CVD, cancers, kidney, bone & liver disease, neurocognitive impairment

37. Shortened telomeres in young HIV+ and in healthy elderly
Telomere length is shorter in healthy elderly and
young HIV+ on cART with VL<50
Telomeres shorten during each cell division
As telomeres shorten, cells age
Telomere length is a classical marker of immune ageing
The best evidence of immune senescence is of shortened telomere length
Short telomeres mean the cell has aged
The TL in monocytes from young HIV+ (shown in red), median age 29 yrs with virologic suppression
is shorter than in uninfected controls, same age (black)
And similar to the TL in uninfected elderly with a mean age of 72 years (blue)
Showing that innate immune senescence occurs at a younger age in association with HIV infection
Classical monocytes
CD16+ monocytes
Young Aged Young
HIV+
Young Aged Young
HIV+
Median age: yrs
Range (yrs):
Hearps A et al AIDS 2012; 26: 843

38. HIV accelerates innate immune changes
HIV induces age-related changes in monocytes
In HIV+ men1
In HIV+ women2
Changes appear approx 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
Two studies from our group in collaboration with Alan Landay,
one in men and one in women,
using Age as a continuous variable show that innate immune senescence changes can emerge approx years earlier in HIV+ women
There have been similar reports regarding T cell immune senescence.
But this innate immunosenescent phenotype has not yet been supported by evidence of premature development of serious non AIDS events.
Hearps A et al AIDS. 2012;24:843-53
Martin G et al 2013;8(1):e55279;

39. 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 :335

40. 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
Particularly those with incomplete immune restoration
Currently provide the strongest predictors of

41. Acknowledgements
Dmitri Sviridov
Infectious Diseases
Sharon Lewin
Jenny Hoy
Julian Elliott
Kate Cherry
Janine Trevillyon
Anthony Jaworowski
Anna Hearps
Genevieve Martin
Clovis Palmer
Tom Angelovich
Anna Maisa
Gregor Lichtfuss
Wan-Jung Cheng
Jingling Zhou
Tim Spelman
M Gouillou
Alan Landay
Cardiovascular Medicine
Anthony Dart
Liz Dewar
Sofie Karapanagiotidis
William Muller
Univ West indies,
Jamaica
The HaCH Study volunteers
Josh Anzinger
Funding
Clare Westhorpe
Mike McCune
Also with grateful thanks to colleagues who contributed slides for this presentation, some not included because of time limitations
“The Ageing Team”