CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

216 HIV UNSPLICED RNA EXPRESSION INDUCES INNATE IMMUNE ACTIVATION AND T CELL DYSFUNCTION Hisashi Akiyama , Caitlin Miller, Chelsea R. Ettinger, Anna C. Belkina, Jennifer Snyder-Cappione, Rahm Gummuluru Boston University, Boston, MA, USA Background: A hallmark of HIV-1 infection in vivo is systemic chronic immune activation, which has been postulated to lead to HIV-associated non-AIDS complications (HANA) and dysfunction of T cells. While many factors have been hypothesized to cause aberrant immune activation, recent studies suggest chronic low-level production of type I interferons (IFN-I) as the driving force for chronic inflammation and T cell exhaustion in vivo. In this study, we demonstrate that persistent expression of HIV-1 unspliced viral RNAs in myeloid cells is the trigger that induces IFN-I responses and contributes to T cell dysfunction. Methods: Monocyte-derived macrophages (MDMs) were derived from CD14+ cells purified from PBMCs. MDMs were infected with HIV-1, and expression of CD169, a myeloid cell specific interferon stimulated gene (ISG), and IP-10 was measured as markers of immune activation. Autologous PBMCs were co-cultured with HIV-1-infected MDMs and expression of a panel of inhibitory receptors (IRs) on T cells was analyzed. Finally, production of IFNγ from IR+ T cells upon stimulation was measured by intracellular cytokine staining. Results: Establishment of productive HIV-1 infection in MDMs resulted in robust upregulation of CD169 and IP-10, which was abrogated upon treatment of MDMs with HIV-1 entry, reverse transcription, integration or viral transcription inhibitors or upon treatment with an IFN-I neutralizing reagent (B18R), suggesting that activation of MDMs was dependent on de novo viral gene expression and is mediated by soluble IFN-I. Infection of MDMs with a panel of HIV-1 mutants revealed that Rev-dependent nuclear export of unspliced RNA (usRNA), but not viral protein expression was necessary for ISG induction and MDM activation. Interestingly, PBMCs co-cultured with HIV-1-infected MDMs up-regulated IRs on both CD4+ and CD8+ T cells, and IFNγ production from IR+ T cells upon stimulation was significantly reduced. Importantly, this T cell exhaustion phenotype was not observed when nuclear export of viral usRNA was inhibited in HIV-infected MDMs or upon initiation of co-cultures in the presence of B18R. Conclusion: Our findings suggest that persistent expression of HIV-1 usRNA in macrophages contributes to chronic immune activation and impairment of effector T cell functions and that use of HIV RNA expression inhibitors as adjunct therapy might abrogate aberrant inflammation and restore immune function in HIV-infected individuals on HAART. 217 INSULIN-LIKE GROWTH FACTOR 1 INVERSELY RELATES TO MONOCYTE ACTIVATION MARKERS IN HIV Lindsay T. Fourman , Natalia Czerwonka, Sofia D. Shaikh, Takara L. Stanley, Background: Monocyte activation is increased among people living with HIV (PLWH), and may contribute to the development of HIV complications including atherosclerosis and neurocognitive dysfunction. Thus, strategies that dampen monocyte activity in HIV are critically needed. Insulin-like growth factor 1 (IGF-1) is an endogenous peptide that exerts endocrine and autocrine/paracrine effects. IGF-1 is low in chronic inflammatory states, whereas augmentation of IGF-1 reduces monocyte-specific inflammation in animal models of atherosclerosis and inflammatory bowel disease. Here, we investigate for the first time the association of IGF-1 with monocyte activation markers in PLWH and uninfected controls. Methods: 131 PLWH (46.7±8.2 years, 67%men, CD4 count 556 [392,816] cells/ mm^3, undetectable VL 75%, ART use 81%) and 65 well-matched controls (45.4±7.1 years, 62%men) without known cardiac disease or viral hepatitis were recruited previously. IGF-1 and inflammatory markers were measured. Variables were compared between HIV and non-HIV groups using Student’s two-tailed t-test if normally distributed, Wilcoxon rank-sum test if not normally distributed, and chi-square test if categorical. IGF-1 z-score was related to log- transformed inflammatory markers in HIV and non-HIV groups using Pearson correlation. Markers associated with IGF-1 z-score were tested in multivariable models controlling for factors that may affect inflammation. Results: PLWH had higher sCD163 (1081 [711,1564] vs. 820 [591,1054] ng/ mL, P = 0.0002) and MCP-1 (261 [179,359] vs. 223 [166,271] pg/mL, P = 0.01) than uninfected controls. sCD14 also tended to be greater among PLWH (431 Kathleen V. Fitch, Janet Lo, Steven K. Grinspoon Massachusetts General Hospital, Boston, MA, USA

[223,1692] vs. 327 [162,1222] ng/mL, P = 0.08). CRP, IL-6, LPS, and IGF-1 were similar between HIV and non-HIV groups. Among PLWH, IGF-1 inversely related to sCD163 (r = -0.28, P = 0.002) and sCD14 (r = -0.29, P = 0.002). There was no association of IGF-1 with MCP-1, CRP, IL-6, or LPS in PLWH, or between IGF-1 and any inflammatory marker in controls. The relationship of IGF-1 with sCD163 and sCD14 remained significant among PLWH in multivariable models accounting for age, sex, smoking, BMI, visceral fat, statin use, VL, and ART. For every 1-unit decline in IGF-1 z-score, sCD163 increased by 14% (95%CI 0.23%, 29%), and sCD14 increased by 29% (95%CI 1.4%, 63%). Conclusion: In PLWH, there was a robust inverse association of IGF-1 with monocyte activation markers. Interventional studies are needed to examine IGF-1 as a novel therapy to reduce monocyte activation in HIV.

Poster Abstracts

218 EXOSOMES ARE ASSOCIATED WITH IMMUNE ACTIVATION AND OXIDATIVE STRESS IN HIV PATIENTS Sukrutha Chettimada , David Lorenz, Vikas Misra, Dana H. Gabuzda Dana–Farber Cancer Institute, Boston, MA, USA Background: Exosomes are nanovesicles released frommost cell types including immune cells. Prior studies suggest exosomes play a role in HIV pathogenesis, but little is known about exosome cargo in relation to immune responses and oxidative stress. Here, we characterize protein and RNA cargo of circulating exosomes in HIV patients and examine their relationship to immunological and oxidative stress markers. Methods: Plasma exosomes were isolated from 92 subjects (n=51 HIV+, age 37-60 years, 70%male, on ART with suppressed or low viral load [undetectable or <2500 HIV RNA copies/ml, respectively] and n=41 HIV- controls matched for age, gender, race). Exosomes were characterized by electron microscopy, nanoparticle tracking analysis (NTA), and immunoblotting for exosome markers. The plasma metabolome was characterized by LC-MS/MS to examine inter- relationships between plasma exosomes and metabolite changes related to immune activation and oxidative stress. Exosomal protein cargo was assessed by LC-MS/MS proteomics and RNA cargo by small RNA sequencing. Results: Plasma exosomes were more abundant in HIV-positive subjects compared to controls based on immunoblotting for exosome markers and NTA (median 6.76 vs. 3.41 X 10 ∧ 11 particles/ml, respectively, p=.038). Plasma exosome markers correlated positively with oxidative stress markers (cystine, oxidized cys-gly, p<0.05) and inversely with PUFA (DHA, EPA, DPA, p<0.05). Untargeted proteomics detected markers of exosomes (CD9, CD63, CD81), immune activation and inflammation (CD14, CRP, HLA-A, HLA-B, CSF1R, LILRB1), and oxidative stress (CAT, PRDX1, PRDX2, TXN, SEPP1) in plasma exosomes. Small RNA-seq analysis of exosomal RNA cargo identified several classes of small RNAs, including microRNA (10%), snoRNA (8%), tRNA (20%), and piRNA (60%). MiRNA target enrichment analysis suggested these exosome-associated miRNAs could have potential functional roles in pathways involved in HIV infection, Wnt and Notch signaling, inflammation, and stress responses. Conclusion: HIV-positive individuals on ART have higher abundance of plasma exosomes compared to HIV-negative controls, and this increase correlates with markers of oxidative stress. Exosome cargo includes proteins related to immune activation, inflammation, and oxidative stress, and may have pro-inflammatory and redox effects during pathogenesis. Exosomal small RNA cargo may also influence pathogenesis and stress responses.

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CROI 2018