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

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

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

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

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 2009 83:3719-3733

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

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

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 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

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

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%

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 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 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

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

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;

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 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

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

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

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 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 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

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;

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

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 2008 135:226

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 2008 135:226

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

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

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

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

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

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

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 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 2014 30:335

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-6 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 2014 289:7884

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 = 18 17 11 Palmer C , Anzinger J et al submitted, 2014 See POSTER MOPE-006 & TODAY WEPE-009

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

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

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: 28 72 29 28 72 29 yrs Range (yrs): 20-32 70-82 27-45 Hearps A et al AIDS 2012; 26: 843

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 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 10-14 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;

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

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”