Associations between selected single nucleotide polymorphisms and markers of inflammation and immune activation
To evaluate whether SNPs associated with kynurenine/tryptophan ratio might be related to additional immunologic pathways that have been associated with increased morbidity/mortality during treated HIV disease [4,11], we evaluated SNPs in relation to D-dimer, IL-6, sCD163, sCD14, and CD4+ and CD8+ T-cell activation levels during ART suppression (Supplementary Table 1, http://links.lww.com/QAD/A918). These biomarkers were only modestly correlated with each other (R = 0.09–0.45), with the exception of CD4+ and CD8+ T-cell activation (R = 0.67) (Supplementary Fig. 6, http://links.lww.com/QAD/A918). Several of the SNPs identified in the candidate gene analysis were associated with increased monocyte activation (sCD163 and sCD14) and increased IL-6 (Supplementary Table 1, http://links.lww.com/QAD/A918). SNPs identified in the genome-wide analysis were associated with sCD163, sCD14, D-dimer, and IL-6 (Supplementary Table 2, http://links.lww.com/QAD/A918).
Associations between selected single nucleotide polymorphisms and mortality
There were too few deaths in the cohort (35 deaths over a median of 7 years of follow-up) to directly perform a genome-wide analysis of predictors of mortality . Nonetheless, we were able to assess whether SNPs associated with kynurenine/tryptophan ratio were also associated with mortality. We observed statistically significant associations with mortality and rs13041834 [adjusted hazard ratio = 1.82, 95% confidence interval (CI) = 1.06, 3.13] and rs75257475 (adjusted hazard ratio = 2.05, 95% CI = 1.12, 3.76) (Supplemental Table 3, http://links.lww.com/QAD/A918, Supplementary Fig. 7, http://links.lww.com/QAD/A918).
Sensitivity analyses to evaluate potential effect of nongenetic factors during early antiretroviral therapy
We had limited data on nongenetic factors, such as coinfections. However, we were able to perform posthoc analyses excluding 39 individuals with adjudicated TB diagnoses and found no significant change in the effect estimates – though exclusion of these participants reduced the sample size and significance of some associations (Supplementary Table 4, http://links.lww.com/QAD/A918). We also compared the associations between SNPs and kynurenine/tryptophan ratios at months 6 and 12 of ART separately to evaluate potential nongenetic influences (i.e. inflammatory conditions) that might increase kynurenine/tryptophan ratio during early, but not later, ART timepoints. Of the 597 participants, 586 had kynurenine/tryptophan ratio measurements at month 6 and 538 had kynurenine/tryptophan ratio measurements at month 12 of ART. The effect estimates were largely unchanged at month 12 compared with month 6 of ART (Supplementary Table 5, http://links.lww.com/QAD/A918).
Attempt to replicate findings in a non-African sample of convenience
We lacked resources to be able to measure kynurenine/tryptophan ratio in an additional African cohort. However, we had access to existing data from ACTG studies that included participants with genome-wide array  and kynurenine/tryptophan ratio data (at 1 year of ART suppression)  (Supplemental Table 6, http://links.lww.com/QAD/A918). Although this ACTG cohort was smaller than our Ugandan sample (262 versus 597 participants), only assessed kynurenine/tryptophan ratio at a single timepoint (as opposed to two timepoints in our Ugandan study), and had 33% participants with African ancestry (Supplementary Table 6, http://links.lww.com/QAD/A918), we evaluated whether the SNPs associated with kynurenine/tryptophan ratio in our Ugandan cohort might be replicated – but were unable to confirm these findings (Supplementary Tables 7 and 8, http://links.lww.com/QAD/A918).
We performed a genome-wide study to identify potential host genetic determinants of kynurenine/tryptophan ratio in HIV-infected ART-suppressed Ugandans. A previous study in SIV-infected rhesus macaques of direct IDO and CTLA-4 inhibition led to severe autoimmune necrotizing pancreatitis in animals . However, several phase I and II cancer treatment trials of direct IDO inhibition are currently underway . The goal of the present study was to identify pathways that may modify IDO activity during treated HIV disease, potentially identifying targets for interventions other than IDO itself.
The specific candidate genes assessed in the study were selected by a priori knowledge of factors that modulate IDO in published in-vitro or animal model studies. IDO is upregulated in dendritic cells and phagocytes by proinflammatory cytokines including IFN-α, IFN-γ, TNF-α, and TGF-β [8,24]. Translocation of microbial products, including LPS, may stimulate Toll-like receptors, which then induce IDO activity . The relative importance of these regulators in vivo during treated HIV infection, however, remains unclear.
We found that SNPs in TNF, IFNGR1, and TLR4 were strongly associated with kynurenine/tryptophan ratio. Several of these SNPs localize to potential functionally relevant regions – for example, rs17200810 (TNF) lies in a region predicted to bind to several transcription factors involved in host immunoregulation, including the response to HIV (e.g. RELA , IRF4 , and JUND ). Both rs276565 (IFNGR1) and rs270148 (TLR4) lie in active H3K27Ac histone mark regulatory regions, loci that exhibit increased enhancer activity . Interestingly, rs276565 (IFNGR1) also lies near IL20RA, which encodes for the IL-20 receptor (IL-20RA) and IL22RA2, which encodes for the soluble form of the IL-22 receptor (IL-22BP) (Supplementary Fig. 4B, http://links.lww.com/QAD/A918). Both IL-20 and IL-22 are members of the IL-20 subfamily of cytokines, which plays a critical role in enhancing the host innate immune response such as during viral infection or with intestinal epithelial homeostasis .
In the genome-wide analysis, we identified additional pathways that might plausibly be involved in IDO immunoregulation. PTPRN2 and PTPRM are both protein tyrosine phosphatases (PTPs), which, with protein tyrosine kinases, rapidly modulate signaling processes to maintain cell proliferation, differentiation, and gene transcription [31,32]. Based on ENCODE data, rs6950107 (PTPRN2) is predicted to decrease binding to SMAD3 , and rs75257475 (PTPRM) lies in a region that binds FOLS2 , a transcription factor that interacts with SMAD3 . SMAD3 is a key intracellular effector of TGF-β signaling , and via formation of the activator protein 1 transcription factor complex  and binding to Foxp3 , plays a critical role in the development, function, and survival of regulatory T cells.
A second potential pathway identified from the genome-wide analysis is the vitamin D pathway. CYP24A1 (rs13041834) encodes the key enzyme catalyzing the metabolism of active vitamin D. In mouse models, vitamin D inhibits T-cell proliferation , suppresses Th17 production , and induces regulatory T-cell expansion [41,42] – immunologic effects strikingly similar to those of IDO. Mice administered vitamin D also generates tolerogenic mature dendritic cells with enhanced TGFB and IDO mRNA expression, and increased IL-10 production with regulatory T-cell expansion . In HIV-infected ART-suppressed patients, vitamin D supplementation is associated with decreased T-cell activation . Of note, this SNP also lies downstream of BCAS1 and MIR4756. However, the clinical significance of these genes and their association with IDO signaling are unknown.
We identified one genome-wide significant SNP, but to our knowledge, neither chondroitin sulfate proteoglycan a5 (CSPG5) nor elongator acetyltransferase complex subunit (ELP6) is biologically relevant molecules in IDO signaling. The SNP does not lie in a putative regulatory site, polymorphisms closer to these two genes were not associated with kynurenine/tryptophan ratio, and this SNP was not associated with other markers of immune dysfunction.
Our data are in contrast to a recent GWAS, which did not find any polymorphisms significantly associated with markers of immune activation and microbial translocation (sCD14 and i-FABP levels) . However, only untreated HIV-infected individuals were included in the study, potentially making it more difficult to overcome additional variation due to the extent of disease progression and genetic and nongenetic contributors to viral load setpoint (factors that might confound the relationship between host genetics and sCD14). Participants were also restricted to those with higher CD4+ T-cell counts, possibly excluding those with the most microbial translocation. Our study minimizes potential confounding by plasma HIV RNA levels by only evaluating ART-suppressed individuals. By sampling two different timepoints per participant, we also minimize within-subject variability and increase our power to detect an association with host genetic factors.
Our study has several limitations that deserve mention. First, we had limited data on nongenetic factors, such as coinfections. We were able to adjudicate potential TB diagnoses  and found little change in effect estimates after excluding these individuals. We also performed posthoc analyses of SNPs in relation to months 6 and 12 kynurenine/tryptophan ratios separately. These analyses did not demonstrate any bias toward a stronger relationship at month 6, which might suggest potential confounding by coinfection or other inflammatory conditions. Second, we had too few deaths in this cohort to directly perform a genome-wide analysis of mortality. Due to the lack of a dedicated mortality tracking system in Uganda, we also did not have information on the exact cause of death, for example, AIDS-related death. However, we did identify two SNPs that were significantly associated with mortality, suggesting that the genetic determinants of this pathway may be clinically meaningful. Third, though we included nearly 600 participants, based on a median of two observations per participant, we only had 80% power to detect SNPs that explain approximately 5% of the variance in plasma log10 kynurenine/tryptophan ratio (assuming a log-additive model, two-tailed Type I error rate of 5.0 × 10−8 and MAF ≥ 1%). This difference would be equivalent to a 2.0-fold difference in kynurenine/tryptophan ratio, corresponding to an estimated 3.7-fold increase in mortality risk . Thus, it is likely that we lacked sufficient power, particularly for less common SNPs, to detect effect sizes that might be most clinically relevant. Finally, we did not have the resources to be able to measure kynurenine/tryptophan ratio in an African replication cohort. We instead analyzed existing ACTG data but were unable to confirm the associations with kynurenine/tryptophan ratio observed in our Ugandan study. Based on the above estimates of power and the small number of participants with African ancestry in the replication cohort, however, we cannot definitively exclude the associations observed in our Ugandan study at this time.
In conclusion, we identified several polymorphisms associated with kynurenine/tryptophan ratio during treated HIV disease. Several of these SNPs lie in biologically plausible regions that could modulate the IDO pathway; these include polymorphisms in genes related to proinflammatory cytokines such as IFN-γ, TNF-α, and TGF-β, as well as genes involved in Toll-like receptor signaling and vitamin D metabolism. Although the SNP associations identified herein are plausible, in a small, primarily white cohort from a resource-rich region, we were unable to confirm these findings. The immunologic pathways predicting mortality among HIV-infected Ugandans may be different than those described in resource-rich settings; immune recovery (i.e. CD4+ T-cell count) after ART initiation is significantly slower in resource-limited compared with resource-rich regions . There may be nongenetic differences between regions that could plausibly affect kynurenine/tryptophan ratio such as viral clade (e.g. A and D in Uganda versus B in the United States) and prevalent coinfections. Further studies are needed to confirm the role of these SNPs in IDO-mediated gut epithelial immunity, especially among African populations.
The authors wish to acknowledge the participation of all the study participants who contributed to this work as well as the clinical research staff of the UARTO and ARKS cohorts who made this research possible. The authors would also like to acknowledge significant contributions by David R. Bangsberg, Yusuke Nakamura, Tricia H. Burdo, Kenneth C. Williams, Russell P. Tracy, Yap Boum, Miriam Laker-Oketta, Leslie W. Chinn, and Janine E. Micheli. Chin and Micheli coordinated DNA sample collection and extraction for genome-wide genotyping and assisted with initial genetic data collection. Bangsberg secured funding for and coordinated the design and operations of the UARTO cohort and Boum oversaw its operations locally. Laker-Oketta oversaw the local operations of the ARKS study. Nakamura coordinated the collection of the genotyping data at the RIKEN center. Burdo, Williams, and Tracy generated the additional biomarker data.
This work was supported in part by the National Institutes of Health (R56AI100765, R21AI078774, K24MH087227, T32AA007240, R01MH054907, UM1CA181255, P30AI27763, P01AI076174, and U01GM061390), the Doris Duke Charitable Foundation (P.W.H., Clinical Scientist Development Award 2008047), the Sullivan Family Foundation, HIV Translational Research Training Grant (T32AI060530) (S.A.L.), a Center for AIDS Research HIV Mentored Scientist Award (S.A.L.), Bristol-Myers Squibb Virology Fellowship Award (S.A.L.), and the NIH Pharmacogenomics Research Network-RIKEN Center for Genomic Medicine Global Alliance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
S.A.L., P.W.H., and D.L.K. conceived and designed the study. J.N.M., P.W.H., and H.B. coordinated collection, management, and quality control processes for the Ugandan cohort clinical data and specimens. D.W.H., P.J.M., and P.W.H. coordinated collection of the ACTG replication cohort data. S.A.L., D.L.K., T.M., and M.K. coordinated generation of the genotyping data. Y.H. conducted the laboratory kynurenine/tryptophan ratio measurements. P.W.H. coordinated the acquisition of additional biomarker measurements. S.A.L. analyzed the data. J.A.M. and J.S.W. provided key data management and analysis support. S.A.L. wrote the report. All authors provided critical feedback in finalizing the report.
Conflicts of interest
There are no conflicts of interest.
1. Lohse N, Hansen AB, Pedersen G, Kronborg G, Gerstoft J, Sorensen HT, et al. Survival of persons with and without HIV infection in Denmark, 1995–2005
. Ann Intern Med
2. Hunt PW. HIV and aging: emerging research issues
. Curr Opin HIV AIDS
3. Hunt PW, Sinclair E, Rodriguez B, Shive C, Clagett B, Funderburg N, et al. Gut epithelial barrier dysfunction and innate immune activation predict mortality in treated HIV infection
. J Infect Dis
4. Tenorio AR, Zheng Y, Bosch RJ, Krishnan S, Rodriguez B, Hunt PW, et al. Soluble markers of inflammation and coagulation but not T-cell activation predict non-AIDS-defining morbid events during suppressive antiretroviral treatment
. J Infect Dis
5. Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism
6. Werner ER, Fuchs D, Hausen A, Jaeger H, Reibnegger G, Werner-Felmayer G, et al. Tryptophan degradation in patients infected by human immunodeficiency virus
. Biol Chem Hoppe Seyler
7. Stone TW, Darlington LG. Endogenous kynurenines as targets for drug discovery and development
. Nat Rev Drug Discov
8. Pallotta MT, Orabona C, Volpi C, Vacca C, Belladonna ML, Bianchi R, et al. Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells
. Nat Immunol
9. Baban B, Chandler PR, Sharma MD, Pihkala J, Koni PA, Munn DH, et al. IDO activates regulatory T cells and blocks their conversion into Th17-like T cells
. J Immunol
10. Favre D, Mold J, Hunt PW, Kanwar B, Loke P, Seu L, et al. Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 alters the balance of TH17 to regulatory T cells in HIV disease
. Sci Transl Med
11. Byakwaga H, Boum Y 2nd, Huang Y, Muzoora C, Kembabazi A, Weiser SD, et al. The kynurenine pathway of tryptophan catabolism, CD4+ T-cell recovery, and mortality among HIV-infected Ugandans initiating antiretroviral therapy
. J Infect Dis
12. Huengsberg M, Winer JB, Gompels M, Round R, Ross J, Shahmanesh M. Serum kynurenine-to-tryptophan ratio increases with progressive disease in HIV-infected patients
. Clin Chem
13. Lee SA, Byakwaga H, Boum Y, Burdo TH, Haberer JE, Tracy RP, et al.. Immunologic pathways that predict mortality in HIV+ Ugandans initiating ART [abstract ∼ 317]
. 22nd Conference on Retroviruses and Opportunistic Infections
, 2015; Seattle, WA.
14. Huang Y, Louie A, Yang Q, Massenkoff N, Xu C, Hunt PW, et al. A simple LC–MS/MS method for determination of kynurenine and tryptophan concentrations in human plasma from HIV-infected patients
15. Geng EH, Odeny TA, Lyamuya RE, Nakiwogga-Muwanga A, Diero L, Bwana M, et al. Estimation of mortality among HIV-infected people on antiretroviral therapy treatment in east Africa: a sampling based approach in an observational, multisite, cohort study
. Lancet HIV
16. Byakwaga H, Hunt PW, Laker-Oketta M, Glidden DV, Huang Y, Bwana BM, et al. The kynurenine pathway of tryptophan catabolism and AIDS-associated Kaposi sarcoma in Africa
. J Acquir Immune Defic Syndr
17. Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, Reich D. Principal components analysis corrects for stratification in genome-wide association studies
. Nat Genet
18. Moskvina V, Schmidt KM. On multiple-testing correction in genome-wide association studies
. Genet Epidemiol
19. Pereyra F, Jia X, McLaren PJ, Telenti A, de Bakker PI, Walker BD, et al. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation
20. Beardsley DI, Kowbel D, Lataxes TA, Mannino JM, Xin H, Kim WJ, et al. Characterization of the novel amplified in breast cancer-1 (NABC1) gene product
. Exp Cell Res
21. Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. miRBase: microRNA sequences, targets and gene nomenclature
. Nucleic Acids Res
22. Boasso A, Vaccari M, Fuchs D, Hardy AW, Tsai WP, Tryniszewska E, et al. Combined effect of antiretroviral therapy and blockade of IDO in SIV-infected rhesus macaques
. J Immunol
23. Vacchelli E, Aranda F, Eggermont A, Sautes-Fridman C, Tartour E, Kennedy EP, et al. Trial watch: IDO inhibitors in cancer therapy
24. Mellor AL, Munn DH. IDO expression by dendritic cells: tolerance and tryptophan catabolism
. Nat Rev Immunol
25. O’Connor JC, Lawson MA, Andre C, Moreau M, Lestage J, Castanon N, et al. Lipopolysaccharide-induced depressive-like behavior is mediated by indoleamine 2,3-dioxygenase activation in mice
. Mol Psychiatry
26. Coiras M, Lopez-Huertas MR, Rullas J, Mittelbrunn M, Alcami J. Basal shuttle of NF-kappaB/I kappaB alpha in resting T lymphocytes regulates HIV-1 LTR dependent expression
27. Carbone A, Gloghini A, Larocca LM, Capello D, Pierconti F, Canzonieri V, et al. Expression profile of MUM1/IRF4, BCL-6, and CD138/syndecan-1 defines novel histogenetic subsets of human immunodeficiency virus-related lymphomas
28. Kagnoff MF, Roebuck KA. Human immunodeficiency virus type 1 (HIV-1) infection and expression in intestinal epithelial cells: role of protein kinase A and C pathways in HIV-1 transcription
. J Infect Dis
1999; 179 (Suppl 3):S444–447.
29. Creyghton MP, Cheng AW, Welstead GG, Kooistra T, Carey BW, Steine EJ, et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state
. Proc Natl Acad Sci USA
30. Rutz S, Wang X, Ouyang W. The IL-20 subfamily of cytokines – from host defence to tissue homeostasis
. Nat Rev Immunol
31. Tonks NK. Protein tyrosine phosphatases: from genes, to function, to disease
. Nat Rev Mol Cell Biol
32. Alonso A, Sasin J, Bottini N, Friedberg I, Osterman A, Godzik A, et al. Protein tyrosine phosphatases in the human genome
33. Kheradpour P, Kellis M. Systematic discovery and characterization of regulatory motifs in ENCODE TF binding experiments
. Nucleic Acids Res
34. Dunham I, Kundaje A, Aldred SF, Collins PJ, Davis C, Doyle F, et al. An integrated encyclopedia of DNA elements in the human genome
35. Wang J, Sun D, Wang Y, Ren F, Pang S, Wang D, et al. FOSL2 positively regulates TGF-beta1 signalling in nonsmall cell lung cancer
. PLoS One
36. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling
37. Zhang Y, Feng XH, Derynck R. Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription
38. Tone Y, Furuuchi K, Kojima Y, Tykocinski ML, Greene MI, Tone M. Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer
. Nat Immunol
39. Rigby WF, Noelle RJ, Krause K, Fanger MW. The effects of 1,25-dihydroxyvitamin D3 on human T lymphocyte activation and proliferation: a cell cycle analysis
. J Immunol
40. Cantorna MT, Yu S, Bruce D. The paradoxical effects of vitamin D on type 1 mediated immunity
. Mol Aspects Med
41. Daniel C, Sartory NA, Zahn N, Radeke HH, Stein JM. Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile
. J Pharmacol Exp Ther
42. Cantorna MT, McDaniel K, Bora S, Chen J, James J. Vitamin D, immune regulation, the microbiota, and inflammatory bowel disease
. Exp Biol Med
43. Ferreira GB, Gysemans CA, Demengeot J, da Cunha JP, Vanherwegen AS, Overbergh L, et al. 1,25-Dihydroxyvitamin D3 promotes tolerogenic dendritic cells with functional migratory properties in NOD mice
. J Immunol
44. Fabre-Mersseman V, Tubiana R, Papagno L, Bayard C, Briceno O, Fastenackels S, et al. Vitamin D supplementation is associated with reduced immune activation levels in HIV-1-infected patients on suppressive antiretroviral therapy
45. Perkins MR, Bartha I, Timmer JK, Liebner JC, Wollinsky D, Gunthard HF, et al. The interplay between host genetic variation, viral replication, and microbial translocation in untreated HIV-infected individuals
. J Infect Dis
antiretroviral therapy; genome-wide association study; HIV; kynurenine; tryptophan
Supplemental Digital Content
Copyright © 2016 Wolters Kluwer Health, Inc.