CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

(99% of HAND, n=116 vs. 55% in non-HAND sequences, n=51; p < 0.001). Also, nucleotide position 3137G (aa351, Gly) in the RT gene was associated with HAND phenotype (95% of HAND, n=125 vs. 51% in non-HAND sequences, n=51; p < 0.001). Furthermore, 7443A (aa407, Asn), 6676A (aa151, Lys) and 6656G (aa144, Ser) were significantly associated with HIV encephalitis (HIVE) phenotypes (p < 0.001). Despite majority of these individuals being on azidothymidine for at least 20 months, they developed HAND and HIVE phenotypes. Biologically, Env gene variants were associated with alteration in binding site to CCR5/CXCR4 coreceptor (7144T), gp41 (6979G), or development of drug resistance in RT gene (2754A and 3137G). Conclusion: Presence of unique Env and RT gene variants predicted the CNS phenotypes in HIV infected adults. These studies will help in developing advanced predictive models for identification of individuals at the risk of developing CNS phenotypes and in finding novel viral sequence targets for personalized therapeutics. 375 IFNγ-INDUCED PROTEASOMAL DEGRADATION OF ASTROCYTIC HO-1 IN HIV NEUROPATHOGENESIS Alexander J. Gill 1 , Colleen E. Kovacsics 1 , Suren A. Ambegaokar 2 , Benjamin B. Gelman 3 , Dennis L. Kolson 1 1 Univ of Pennsylvania, Philadelphia, PA, USA, 2 Ohio Wesleyan Univ, Delaware, OH, USA, 3 Univ of Texas Med Branch, Galveston, TX, USA Background: We previously demonstrated that heme oxgyenase-1 (HO-1) protein expression is significantly reduced within the prefrontal cortex of HIV+ individuals and correlated with neurocognitive dysfunction and central nervous system immune activation. We also demonstrated that HIV-infection of macrophages in vitro reduces HO-1 protein expression. Because macrophages are a small cellular component of the brain, the reduction of macrophage HO-1 expression likely accounts for a small part of the total brain HIV- associated HO-1 loss. We therefore investigated the potential contribution of astrocytes, the major reservoir of HO-1 protein in the brain, to this HIV-associated HO-1 loss. Methods: HO-1 protein and mRNA were quantified by Western blot and qPCR respectively, in two relevant tissue sources: primary human fetal astrocytes treated with interferon- gamma (IFNγ) and brain prefrontal cortex from HIV- and HIV+ brains (n =156) from subjects enrolled in the National NeuroAIDS Tissue Consortium. Pulse-chase HO-1 protein degradation experiments were performed in U251 astrocytic cells transiently expressing FLAG-HO-1. Results: HO-1 protein is decreased (p<0.01) and HO-1 mRNA is increased (p<0.01) in the prefrontal cortex of HIV+ individuals compared with HIV- controls. HO-1 protein correlates negatively with HO-1 RNA (p<0.05). This HO-1 protein reduction correlates with increased expression of immunoproteasome subunits (LMP7 and PA28α; p<0.05). Prolonged exposure of fetal astrocytes to IFNγ, an HIV-associated immune activator, selectively reduces HO-1 protein expression (p<0.05), increases immunoproteasome subunit (LMP2, LMP7, and PA28α; p<0.001) expression, while decreasing constitutive proteasome subunit (β1 and β2; p<0.001) expression. This IFNγ-mediated reduction of astrocyte HO-1 protein was associated with a non-significant increase in HO-1 RNA expression. Finally, prolonged IFNγ exposure reduced HO-1 protein half-life in astrocytic cells (p<0.001) and this HO-1 degradation was reduced by proteasome inhibitors. Conclusion: Immunoproteasome-mediated HO-1 degradation in astrocytes is a potential mechanism driving brain HO-1 loss in the HIV-infected brain, in addition to HIV-induced HO-1 loss in infected macrophages. Our data suggest unique causal links among HIV infection, IFNγ-mediated immunoproteasome induction, and enhanced HO-1 degradation, which likely contribute to the neuropathogenesis of HIV-associated neurocognitive disorders (HAND). 376 IFN-Β REDUCES MACROPHAGE HEME OXYGENASE-1 EXPRESSION: ROLE IN HIV NEUROPATHOGENESIS Fiorella P. Rossi 1 , Alexander J. Gill 1 , Rolando Garza 2 , Dennis L. Kolson 1 1 Univ of Pennsylvania, Philadelphia, PA, USA, 2 Univ of Texas at San Antonio, San Antonio, TX, USA Background: Chronic HIV infection induces persistent inflammation within the central nervous system (CNS). We have previously demonstrated a deficiency of the highly inducible anti-inflammatory enzyme heme oxygenase-1 (HO-1) in the brains of HIV+ individuals that correlates with markers of neuroinflammation and clinical neurocognitive impairment. Despite a reduction in HO-1 protein expression, HO-1 RNA is increased in the brains of HIV+ individuals. Additionally, we have shown that HIV infection decreases HO-1 protein expression within infected macrophages in association with increased release of neurotoxic levels of glutamate. We hypothesize that HIV-associated pro-inflammatory factors released from HIV-infected macrophages or other activated resident CNS cells drive the decrease in brain HO-1 protein expression. Methods: We stimulated human monocyte derived macrophages (MDMs) with HIV-associated proinflammatory factors including GM-CSF, TNF-alpha, IFN-beta, and IL-18 for 24 hours and 7 days to resemble acute and chronic exposure in the brain respectively. We determined protein and mRNA levels of HO-1 and heme oxygenase-2 (HO-2), a constitutive isoform of heme oxygenase, by western blot and qPCR, and quantified neurotoxicity by MAP2 ELISA. We also quantified glutamate levels in MDM supernatants using Amplex Red Glutamic acid/glutamate oxidase kit. Results: Chronic stimulation of MDMs with GM-CSF or TNF-alpha resulted in a marked reduction of HO-1 protein of -3.7 and -2.1 fold, respectively (p<0.001) and mRNA expression of -2.0 and -1.5 fold, (p<0.01) but not a significant difference in HO-2 expression (p>0.1). Surprisingly, while IFN-beta reduced HO-1 protein by 50% (p<0.0001), it increased HO-1 mRNA levels by 15.8 fold (p=0.1); suggesting a post-translational mechanism of HO-1 protein loss. Additionally, supernatants from IFN-beta stimulated MDMs had increased levels of glutamate and associated neurotoxicity (p<0.0001). Conclusion: Our results demonstrate that chronic stimulation with inflammatory cytokines can drive down HO-1 mRNA and protein in macrophages. However the mechanism of HO-1 protein loss in IFN-beta-stimulated macrophages differs from other inflammatory stimuli. These IFN-beta effects resemble our previous observations of HO-1 expression in the brains of HIV-infected individuals, where HO-1 protein loss was associated with increased HO-1 mRNA. This suggests that IFN-beta can play a major role in HIV neuropathogenesis by reducing HO-1 protein expression. 377 CNS IRON STATUS IS ASSOCIATED WITH NEUROCOGNITIVE FUNCTION OVER TIME IN HIV+ ADULTS Harpreet Kaur 1 , William S. Bush 2 , Scott Letendre 3 , Ronald J. Ellis 3 , Robert K. Heaton 3 , Stephanie M. Patton 4 , James R. Connor 4 , David C. Samuels 5 , Todd Hulgan 5 , Asha R. Kallianpur 1 1 Cleveland Clinic, Cleveland, OH, USA, 2 Case Western Reserve Univ, Cleveland, OH, USA, 3 Univ of California San Diego, San Diego, CA, USA, 4 Penn State, Hershey, PA, USA, 5 Vanderbilt Univ, Nashville, TN, USA Background: Dysregulated central nervous system (CNS) iron transport has been implicated in neurocognitive (NC) disorders. We hypothesized that cerebrospinal fluid (CSF) biomarkers of CNS iron status are associated with changes in NC function over time in HIV-infected (HIV+) adults. Methods: CSF iron, heavy-chain ferritin (H-ferritin) and transferrin were quantified in 403 participants in CHARTER, a prospective observational study of HIV+ adults who underwent comprehensive NC testing and assignment of a Global Deficit Score (GDS) at baseline and 6-month intervals up to 42 months (mos.). NC change status (improved/ stable/declined) was defined at specific visits or last follow-up compared to baseline. Non-parametric statistical tests were used to test associations at baseline with GDS as a continuous variable, HIV disease or demographic factors. Biomarker associations with NC change status were evaluated by analysis of variance (ANOVA) or logistic regression to adjust for potential confounders such as age, comorbidity, and zidovudine (ZDV) use, which impacts iron transport. Repeated-measures ANOVA was performed to assess biomarker-GDS associations at 30, 36, and 42 mos. Analyses stratified by age and APOE-ε4 carrier status were also explored. Results: Of 403 HIV+ adults with CSF biomarker data (22% aged≥50, 73% on ART, 68%with undetectable virus, 19%women), 157 completed follow-up at 30 mos., 131 at 36 mos., and 110 at 42 mos. CSF transferrin and H-ferritin were higher in men and participants aged≥50 years but were unrelated to APOE-ε4 status. Higher H-ferritin at baseline was associated with NC improvement at last follow-up in HIV+ adults aged<50, adjusting for age, comorbidity, ZDV use, and APOE-ε4 status, with relative risk 1.17 vs. stable status [p=0.01, 95% CI=1.03-1.33]. H-ferritin and transferrin were also associated with GDS differences at 30, 36 and 42 mos., adjusting for comorbidity [p<0.05 for both H-ferritin and transferrin; 0.5-1.1% of total GDS variance explained]. H-ferritin at baseline was also associated with better GDS in participants aged<50 at 30, 36, and 42 mos. (1.3% of GDS variance over 42 mos. explained). In APOE-ε4 carriers aged≥50 (N=10), CSF transferrin was associated with better GDS at 30 mos., explaining 11.6% of the total GDS variance. Conclusion: CSF transferrin and ferritin are independently associated with NC change status and longitudinal GDS differences in HIV+ adults. Larger studies, including more older HIV+ adults, are needed to confirm these findings.

Poster and Themed Discussion Abstracts

CROI 2017 151