Abstract Body

Exosomes are nanovesicles released from most 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.

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.

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 1011 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.

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.