CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

the TCR-signalling pathway. Although a preferential miRNA downregulation was also observed when we compared stimulated to the respective resting samples, VP presented a differential miRNA expression pattern. VP showed a downregulation of both hsa-miR-155 and hsa-miR-181a whereas in the other groups, we observed either an upregulation or no differences, respectively. Overall, functional enrichment analysis revealed that the predicted target genes were involved in metabolic regulation, apoptosis, immune response and signal transduction pathways. Conclusions: Resting CD8+ T-cells do not exhibit a differential miRNA expression between HIV-infected individuals but they do differ from non-infected individuals. Moreover, a specific miRNA pattern is present in stimulated CD8+ T-cells from VP which probably reflects a detrimental pattern in terms of CD8+ T cell immune response. 249 P2X7 Purinergic Receptors Are Required for HIV-1 Infection and Inflammation Talia Swartz ; Meagan K. O’Brien; Natasha D. Durham; Anthony M. Esposito; Nina Bhardwaj; Benjamin K. Chen Icahn Sch of Med at Mount Sinai, New York, NY, USA Background: HIV-1 infection is incurable and causes a chronic inflammation which causes multiple comorbidities, even with virologic suppression. The mechanism of this inflammation is not clearly understood. Purinergic receptors are known to be mediators of inflammatory responses and can contribute to pro-inflammatory cytokine production and lymphocyte cell death. Purinergic receptor signaling is important for HIV-1 infection and we have recently found that inhibition of the P2X subtype purinergic receptors potently blocks HIV-1 productive infection at the level of membrane fusion. This study examines whether virus-induced purinergic signaling is responsible for inflammatory cytokine release during HIV-1 infection. Methods: We use fluorescent constructs of HIV-1 to evaluate productive infection by flow cytometry in CD4 T cell lines and primary cells in the presence or absence of purinergic inhibitors. Infected supernatants can be subjected to multiplex bead capture assays to test for an array of human cytokines. We have tested the effect of HIV-1 infection on peripheral blood mononuclear cells and observed levels of pro-inflammatory cytokine production. Results: We observe that HIV-1 productive infection in CD4 T lymphocytes is potently blocked by P2X7 selective inhibitors. Exposure of peripheral blood mononuclear cells to HIV-1 results in induction of pro-inflammatory cytokines and levels are reduced with P2X7 inhibition. This suggests that P2X7 inhibitors can block both HIV-1 productive infection and associated inflammation. Conclusions: Our findings distinguish purinergic receptors, specifically P2X7, as key signaling mediators of HIV-1 infection and inflammation. We believe that these drugs could be used as adjunctive antiretroviral therapy that could serve to reduce the morbidity and mortality associated with HIV-1 chronic inflammation. 250 Characterization of Plasma Exosome Protein Cargo in HIV Patients on ART Sukrutha Chettimada ; Lorenz David;Vikas Misra; Dana Gabuzda Dana-Farber Cancer Inst, Boston, MA, USA Background: Exosomes are microvesicles originating frommany cell types including immune cells. Prior studies suggest exosomes play a role in HIV pathogenesis and some comorbidities. The relationship of exosome cargo in peripheral blood to HIV infection, immune responses and comorbidities has potential applications for biomarker discovery and new treatment strategies. Here, we perform a cross-sectional study to characterize protein cargo of circulating exosomes in HIV patients and its relationship to virological and immunological markers. Methods: Plasma exosomes were isolated from 53 subjs (n=30 HIV+ from NNTC, age 38-54, 66%male, 53% black, on ART with suppressed viral load [VL<1500 copies] & n=23 HIV- controls matched for age, gender, race). Exosome quality was assessed by dynamic light scattering, transmission electron microscopy & immunoblotting (WB) for exosome markers (HSP70, CD9, CD63, CD81). Proteomic analysis was by LC/MS/MS. Following bioinformatic analysis of hits, proteins were confirmed by WB Results: Circulating exosomes were increased in HIV+ subjects compared to controls based on WB for exosomal HSP70 & CD9 (p<0.01). HSP70, CD9 & CD63 were also detected in exosomes released by PBMCs treated with hemin (24 hrs). Exosomal HSP70 & CD9 levels correlated with plasma VL & kynurenine:tryptophan ratio (K:T ratio, a marker of immune activation) in HIV+ subjects (p<0.01). CD81+ exosome numbers (by ELISA) & exosome-associated NOTCH4 (by WB), but not HSP70 & CD9 levels, were higher in HIV+ subjects using cocaine compared to non-users, despite similar VL & K:T ratios (p<0.05). LC/MS/MS proteomics suggested plasma exosomes were mainly derived frommyeloid cells (CD33, CD11A, CD11B, CSF1R) & revealed proteins related to exosomes (EXOSC10, BST2, SYNE1, VPS), immune activation/inflammation (CD69, CRP, TNFRSF10, CSF1R, chemokines), Wnt signaling (E-Cadh, DDR1, LRP5, NOTCH4), & metabolism (ADIPOQ); many of these were also detected in exosomes released by heme-treated PBMC. Treatment of THP-1 cells with HIV+ patient-derived exosomes induced modest increases in CXCL10 & other IFN-induced genes, indicating pro-inflammatory effects Conclusions: This study demonstrates associations between exosome proteins & disease markers in HIV patients on ART. Circulating exosomes in HIV+ individuals on ART are mainly derived frommyeloid cells, carry protein cargo related to immune responses, inflammation, Wnt signaling and may have pro-inflammatory effects during pathogenesis. 251 A Model of CD4 TCM Cell Death in HIV Infection Based on a Gene Expression Signature Gustavo Olvera-García 1 ;Tania Aguilar-García 1 ; Ivan Imaz-Rosshandler 2 ; Claudia Rangel-Escareño 2 ; Lorena Orozco 2 ; Irma Aguilar-Delfín 2 ; JoelVázquez-Pérez 1 ; Joaquín Zúñiga 1 ; Santiago Pérez-Patrigeon 3 ; Enrique Espinosa 1

Poster Abstracts

1 Natl Inst of Respiratory Diseases, Mexico City, Mexico; 2 Natl Inst of Genomic Med, Mexico City, Mexico; 3 Natl Inst of Nutrition, Mexico City, Mexico Background: Considering that direct cytopathicity of HIV cannot completely explain HIV pathogenesis, that central memory CD4 T cells (T CM

) are functionally altered along HIV

infection, and given the critical homeostatic role of T CM

in chronic HIV infection, we asked if T CM

from HIV + patients have an activation-related genetic expression pattern closer to

effector memory CD4 T cells (T EM

), and if differentially expressed genes entail a decrease in their proliferative and survival capacities.

Methods: We analyzed mRNA expression patterns (with gene expression microarrays) of 3 CD4 T naive, 3 T CM , and 3 T EM

healthy controls’ purified cell samples, plus 3 CD4 T naive

and 3 T

CM cell samples from HIV-1 + patients (purity >90%). Log 2

FC ≥|0.5| and Log (odds)>0 defined differential expression. Gene expression patterns were compared by 2-way

hierarchical clustering. Differentially expressed genes discriminating patients’ and controls’ T CM

cells were subject to functional enrichment analysis (IPA, GSEA, DAVID). 75 genes of

T CM signature genes were validated by qPCR using a qPCR array. Results: Hierarchical clustering grouped each CD4 T cell differentiation subpopulation from patients with its corresponding subpopulation from controls. The expression of differentiation-related genes did not differ between patients and controls, and increased or decreased progressively as follows: T naive à T CM à T EM , ruling out T EM -like differentiation of T CM in HIV infection. Comparing T CM cells from patients and controls we found 210 differentially expressed genes, According to functional enrichment analysis this HIV-related T CM gene expression signature indicated movement to cell cycle phases G1 and S (increased CCNE1, MKI67, IL12RB2, ADAM9, decreased FGF9, etc.), but also arrest in G2/M (increased CHK1, RBBP8, KIF11, etc.). Unexpectedly CSTA, RNASEL, and NR4A2 expression patterns from this signature, among others, predict decreased caspase-mediated apoptosis (Fig. 1). 75 qPCR validated genes independently reproduced the enrichment analysis results. Results also suggested increased IL-1β, IFN-γ, TNF, and RANTES activities upstream of the T CM signature, agreeing with the demonstrated milieu in HIV, and compatible with a non-apoptotic death pathway. Conclusions: Our findings support a model where CD4 T CM cell progressive loss in chronic HIV-1 infection can be driven by increased cell cycle entry followed by mitotic arrest, leading to a non-apoptotic death pathway, possibly contributing to increased turnover.


CROI 2016

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