Impact of Tryptophan Catabolism on CD4+ T Cell Recovery and Mortality in HIV-infected Ugandans Initiating ART Peter W. Hunt, Sheri Weiser, Yong Huang, Conrad Muzoora, Annet Kembabazi, Kathleen Ragland, John Bennett, Elvin Geng, Steven G. Deeks, David R. Bangsberg, Jeffrey N. Martin, and Joseph M. McCune

Background Immune activation persists in most HIV+ patients despite ART-mediated viral suppression This inflammatory state may contribute to morbidity/mortality during treated HIV infection. Demonstrating that specific inflammatory pathways predict subsequent clinical events is critical step in identifying therapeutic targets.

IDO-induced Tryptophan Catabolism IFN-γ and LPS stimulate Indoleamine 2,3-dioxygenase -1 (IDO) production in DCs/MØ Causes tryptophan catabolism Tryptophan depletion may impair T cell proliferation – Maternal tolerance of fetal antigens – Cancer evasion of immune response Catabolites may be neurotoxic – Neurodegenerative diseases, ADC Downstream catabolite 3- Hydroxyanthralinic Acid (HAA) causes Th17 depletion Favre, Mold et al, Science TM, 2010 IDO Kynurenine Tryptophan (K/T Ratio) = Marker of Tryptophan Catabolism

Impact of IDO Induction on HIV Pathogenesis IFN-γ LPS IDO Induction in DC/MØ Tryptophan T Cell Proliferative Defect Microbial Translocation HAA Th17 Depletion

Does the Extent of Tryptophan Catabolism Predict Subsequent Clinical Events During Treated HIV Infection? Cohort of 500 HIV-infected Ugandans starting first ART regimen (UARTO) Assessed plasma tryptophan and kynurenine levels through 1 st year of ART Assessed impact on subsequent CD4 recovery and mortality

Characteristics of HIV-infected Ugandans Starting ART Characteristic Median (IQR) N=435 Age, years34 (28-39) Female gender, %70 Pre-ART CD4+ T cell count, cells/mm (79-201) Pre-ART Plasma HIV RNA level, log 10 copies/ml5.0 ( ) Months of observation on ART38 (22-47) 8% Lost to follow up by month 36

Higher Baseline Tryptophan Catabolism Associated with Greater Early CD4 Recovery Though Largely Explained by Pre-ART VL No evidence for an association between K/T ratio and early CD4 recovery after adjustment for pre-ART VL (P=0.95)

But Higher Baseline Tryptophan Catabolism Predicts Diminished Late CD4 Recovery Higher pre-ART K/T ratio predicted diminished late CD4 recovery after adjustment for age, gender, and pre-ART VL and CD4 count (P=0.008)

Low Pre-ART CD4 Count Predicts Earlier Mortality

Higher Tryptophan Catabolism (K/T ratio) Predicts Earlier Mortality Each tertile increase in baseline K/T ratio associated with a 2.1-fold greater hazard of death after adjustment for pre-ART BMI and CD4 count (P=0.01).

Tryptophan Catabolism Decreases During ART Tryptophan LevelsK/T Ratio

Tryptophan Catabolism Continues to Predict Mortality during Suppressive ART (VL<400 at Month 6 of ART) Each tertile increase in month 6 K/T ratio associated with a 2.9-fold increased hazard of death after adjusting for BMI, CD4 count, and %CD38+HLA-DR+ CD8+ T cells (P=0.042).

Does dietary protein intake modify the relationship between tryptophan catabolism and mortality?

Low Protein Diet Might Exacerbate T Cell Proliferative Defects but Ameliorate Th17 Depletion Low Dietary Tryptophan IDO- induced Catabolism More Tryptophan Depletion More T Cell Proliferative Defects Less Microbial Translocation Less HAA Less Th17 Depletion

High Dietary Tryptophan IDO- induced Catabolism Less Tryptophan Deficiency Less T Cell Proliferative Defects More Microbial Translocation More HAA More Th17 Depletion High Protein Diet Might Ameliorate T Cell Proliferative Defects but Exacerbate Th17 Depletion Gal, J. Neurochemistry, 1978

Ugandan Diet is Low in Protein See also: Crawford et al, British Journal of Nutrition, 1970

Low Dietary Protein Intake Is Associated with Low Plasma Tryptophan Relationship remains significant even after adjustment for VL, CD4 count, gender, and BMI (P=0.021)

No Evidence for an Association Between Dietary Protein Intake and Mortality

Low Dietary Protein Does Not Appear to Exacerbate the Impact of Tryptophan Catabolism on Mortality

P for interaction = 0.34

Conclusions Among HIV-infected Ugandans, greater pre-ART tryptophan catabolism predicts a diminished rate of late CD4+ T cell recovery during suppressive ART. Tryptophan catabolism is a major independent predictor of mortality in HIV-infected Ugandans both pre-ART and during ART-mediated viral suppression. Interventions designed to block IDO induction (or the root causes of IDO induction) may hold promise in this setting.

Unanswered Questions Unclear if tryptophan catabolism is causally associated with disease or is simply another marker for monocyte and DC activation. – Testing of samples for other markers of monocyte and innate immune activation is ongoing to compare prognostic strength. Ultimately, the importance of IDO-mediated tryptophan catabolism in HIV pathogenesis will need to be established in clinical trials of specific IDO inhibitors.

Acknowledgements MUST-UARTO The participants Annet Kembabazi Derrick Musinga Simone Hausammann-Kigozi Bosco Bwana Conrad Muzoora UCSF - UARTO Elvin Geng Steven Deeks John Bennett Rain Mocello Jeffrey Martin MGH/Harvard - UARTO Anna Baylor Jessica Haberer Peggy Bartek David Bangsberg UARTO Team UCSF – Food Insecurity Kathleen Ragland Sheri Weiser UCSF – Laboratory Yong Huang Huyen Cao Joseph M. McCune Doris Duke CSDA, 1R21AI078774, R01MH054097