Suzanne Crowe Associate Director, Burnet Institute Consultant Infectious Diseases Physician, The Alfred Hospital Melbourne.

The role of innate immunity in the pathogenesis of Serious Non-AIDS Events

Suzanne CroweAssociate Director, Burnet Institute

Consultant Infectious Diseases Physician, The Alfred Hospital

Melbourne

Today’s presentation

HIV, LPS trigger inflammation

Contribution of monocyte activationPro-inflammatory cytokines

Involvement of innate immune pathways inFibrosis

Atherosclerotic plaque formationPro-coagulant activity

Metabolic dysregulationImmune senescence

Serious non-AIDS Events

How HIV & inflammation are linked

How breaches in gut mucosa trigger inflammation

Links between monocytemetabolism &inflammation

How monocyte activationImpacts these pathways

Innate immune senescence& risk of events---

Today’s presentation

HIV, LPS trigger inflammation

Contribution of monocyte activationPro-inflammatory cytokines

Involvement of innate immune pathways inFibrosis

Atherosclerotic plaque formationPro-coagulant activity

Metabolic dysregulationImmune 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

Stacey et al J Virol 2009 83:3719-3733

Intense cytokine storm in acute HIV infection prior to peak viremia

Cytokine response in acute HIV Stacey 2009

Ch

ang

es

in p

lasm

a c

ytok

ine

leve

ls

Time since HIV infection (days)

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 pro-inflammatory 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

• increase with inflammation [pro-inflammatory] CD16-positive monocytes

classical CD14++CD16- monocytes

CD14+CD16++ non-classical

CD14++CD16+ intermediate

7-10%

5-8%

85%

Traditional Inflammatory Patrolling

1. Funderburg NT et al Blood 2012 120: 45992. Krishnan S et al J Infect Dis 2014; 209:931

CD16+ monocyte subsets are increased in uncontrolled viremia & may normalize with ART

CD14++CD16-

Classical

CD14++ CD16+

Intermediate

CD14+CD16++

Non classical

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

HIV- HIV+ VL<400HIV+ VL>400

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

Pro-inflammatory CD14++CD16+ monocytes remain expanded despite cART & virologic suppression

HIV-

HIV+

Intermediate Non-ClassicalClassical

Angelovich T et al 2014 unpublished data 2014

Elevated levels of intracellular proinflammatory cytokines IL-6 & TNF in monocytes from HIV+ pts

IL-6

TNF

HIV- HIV+ VL<20 HIV- HIV+ VL<20 HIV- HIV+ VL<20

HIV- HIV+ VL<20 HIV- HIV+ VL<20 HIV- HIV+ VL<20

Monocyte subsets

• 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 and coagulation, but not T-cell activation, predict non-AIDS events

Markers of inflammation & coagulation, but not T-cell activation, predict non-AIDS events

IL6

Unadjusted <.0012.58 (1.91-3.48)

Adjusted* <.0012.48 (1.83-3.35)

IP-10

Unadjusted 0.0021.49 (1.16-1.91)

Adjusted* 0.0071.42 (1.10-1.84)

sTNFr-I

Unadjusted <.0011.99 (1.49-2.66)

Adjusted* <.0011.94 (1.45-2.60)

sTNFr-II

Unadjusted <.0011.88 (1.44-2.46)

Adjusted* <.0011.81 (1.38-2.38)

Soluble CD14

Unadjusted <.0011.74 (1.29-2.35)

Adjusted* <.0011.67 (1.23-2.27)

D-Dimer

Unadjusted <.0012.41 (1.78-3.27)

Adjusted* <.0012.38 (1.75-3.25)

CD8+ %DR+38+

Unadjusted 0.5161.06 (0.88-1.28)

Adjusted* 0.8630.98 (0.80-1.20)

0.50 1.00 4.00

Pre-event Marker P ValueOdds Ratio per 1 IQR increase

*Adjusted by CD4 c

OR at baseline for:

Death CA MI/Stroke

20.9** 3.1** 2.2**19.9** 2.9** 2.1**

1.9 1.7* 1.7*

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

**

**

**

**

**

**

**

**

**

**

**

**

Tenorio AR et al J Infect Dis 2014 May ahead of print

Death Ca AMI/stroke Pre-event marker Odds ratio 1 IQR increase p value OR at baseline for

IL-6

IP-10

sTNFR-I

sTNFR-II

sCD14

D-dimer

CD38+DR+CD8

• Increased innate immune inflammatory markers are strongly associated with non-AIDS 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

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

HIV-

HIV+

•Mϕ accumulate in gut mucosa * in untreated HIV

Further drive pro-inflammatory cytokine production

• 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

Data from multiple sources &reviews including Deeks SG et al; Immunity; 2013: 39 :633

Gut-derived bacterial products translocate to the systemic circulation

Increased gut permeability & microbial

translocation

Innate immune activation & systemic inflammation

(monocytes, mϕ)

LPS

Liver

End organ disease

ribosomal DNA

unmethylated CpG

DNA

peptidoglycan

lipo-techoic

acid

Altered gut microbiome+

Portal vein

Gut-derived bacterial products

Activation of Kynurenine/IDO pathway may be involved in the pathogenesis of non-AIDS events

Increased gut permeability & microbial

translocation

Innate immune activation & inflammation

(monocytes, mϕ)

LPS

End organ disease

activation of IDO pathway

Altered gut microbiome(dysbiosis)

+

Quinolinic acid

Tryptophan

Pro-inflammatory cytokine production (TNF, IL-6)

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;

Byakwaga H, et al J infect Dis 2014 August

Activation of the IDO pathway is also linked to premature mortality

Tacke F& Zimmerman H J Hepatol 2014;60:1090; Balagopal A et al Gastroenterology 2008 135:226

Multi-pronged liver attack by HIV, co-pathogens and gut-derived bacterial products

Increased gut permeability & microbial

translocation

Activation of innate pro-inflammatory &

pro-fibrotic pathways

LPS

Kupffer mϕ& hepatic

stellate cell activation

End organ diseaseIncluding

liver fibrosis

ribosomal DNA

unmethylated CpG

DNA

peptidoglycan

lipo-techoic

acid

Portal veinLiver damage

clearanceLPS

Gut-derived bacterial products

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

Activation of innate pro-inflammatory &

pro-fibrotic pathways

LPS

Kupffer mϕHepatic

stellate cellactivation

End organ diseaseeg

liver fibrosis

ribosomal DNA

unmethylated CpG

DNA

peptidoglycan

lipo-techoic

acid

Portal vein Impaired hepatic function

clearanceLPS, etc

Gut-derived bacterial products

CoinfectionEg HCV

Redd AD et al AIDS Res Hum Retroviruses 2013:29:1026

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

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)

Redd AD et al AIDS Res Hum Retroviruses 2013:29:1026

How do monocytes contributeto the development of cardiovascular disease in HIV+?

Contributing factors to CVD in HIV+ patients

Traditional risk factors

cART toxicityCo-infection with eg CMV

Cardiovascular disease

monocyte & mϕactivation

chronic inflammation

other pro-inflammatory & pro-coagulant pathways

Role of monocytes in atheromatous plaque development

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

Slide produced by G Martin

Adhesion m’cules

HIV promotes monocyte foam cell formation in vitro

• 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;93:2202. Maisa A et al submitted 2014

monocyte macrophage Foam cell

How does monocyte metabolism augmentinflammationin 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

• Glut1 is rate-limiting transporter in mΦ

• (murine & human)

• Murine MΦ with high Glut1 expression express high levels of pro-inflammatory cytokines

Freemerman AJ et al J Biol Chem 2014 289:7884

IL-6 TNF

Glut1LOW

Glut1HIGH

Glut1HIGH

Glut1LOW

Glut1 Glut2 Glut3 Glut4

p = .018 p = .006

Fol

d ex

pres

sion

Glucose uptake is linked to pro-inflammatory

responses of mΦ

%G

lut1

+ c

ell

s o

nm

on

oc

yte

su

bp

op

ula

tio

ns

NC I C NC I C NC I C0

20

40

60

80

100

HIV- HIV+/naïve HIV+/cARTn= 15 14 8

p= 0.047p=0.021

p=0.018p=0.002

HIV increases glucose metabolic activity in monocytes, linked to inflammation

Palmer C , Anzinger J et al submitted, 2014See POSTER MOPE-006 & TODAY WEPE-009

• The increased Glut1 expression is on intermediate and non-classical (NC) monocytes in HIV+• Not restored by ART

%C

D14

+G

lut1

+

HIV-

HIV+/

naïve

HIV+/

cART

0

5

10

15

2030405060

p=0.005 p= 0.48

p=0.008

n= 16 17 11

% G

lut1

+ m

onoc

ytes

% G

lut1

+ m

onoc

ytes

NC I C NC I C NC I C HIV- HIV+ naïve HIV+cART+

HIV- HIV+ naïve HIV+cART+

N = 18 17 11

• Increase in proportion of Glut1+ monocytes in HIV+• Not restored by ART

• Correlates with monocyte activation & D-dimer expression

Does prematureInnate immune senescence contribute topremature risk of non-AIDS morbidities ?

Ageing HIV

CVD, cancers, kidney, bone & liver disease,

neurocognitive impairment

:

Shared immunologic features between HIV infection & ageing

Immune senescence

Chronic immune activation/inflamm

ation

Immunologic changes

Shortened telomeres in young HIV+ and in healthy elderly

Hearps A et al AIDS 2012; 26: 843

• Telomeres shorten during each cell division

• As telomeres shorten, cells age

• Telomere length is a classical marker of immune ageing

Median age: 28 72 29 28 72 29 yrsRange (yrs): 20-32 70-82 27-45

Telomere length in monocytes

Young Elderly Young Young Elderly Young0

5

10

15

CD14+ monocytes CD16+ monocytes

HIV+ HIV+

p=0.001

p=0.03

p=0.008

p=0.02

Mo

no

cyte

tel

om

ere

len

gth

(%

of

con

tro

l)

Telomere length is shorter in healthy elderly and

young HIV+ on cART with VL<50

Young Aged Young HIV+

Young Aged Young HIV+

CD16+ monocytesClassical monocytes

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;24:843-53Martin G et al 2013;8(1):e55279;

Palmer CS & Crowe SM AIDS Res Human Retro 2014 30:335

• 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

Summary

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

Anthony JaworowskiAnna HearpsGenevieve MartinClovis PalmerTom AngelovichAnna MaisaGregor LichtfussWan-Jung ChengJingling ZhouTim SpelmanM Gouillou

Infectious DiseasesSharon LewinJenny HoyJulian ElliottKate CherryJanine Trevillyon

Cardiovascular MedicineAnthony DartLiz DewarSofie Karapanagiotidis

Dmitri Sviridov

Alan Landay

The HaCH Study volunteers

Acknowledgements

William Muller

Funding

Univ West indies, Jamaica

Josh Anzinger

Clare Westhorpe

“The Ageing Team” Also with grateful thanks to colleagues who contributed slides for this presentation, some

not included because of time limitations

Mike McCune