Circulating brain-derived extracellular vesicles expressing neuroinflammatory markers are associated with HIV-related neurocognitive impairment

Abstract

Background: Neurocognitive impairment remains prevalent in people with HIV (PWH) despite long term virological suppression by antiretroviral therapy (ART) regimens. Systemic and neuro-inflammatory processes are suggested to contribute to the complex pathology leading to cognitive impairment in this population, yet the underlying mechanisms remain unresolved. Extracellular vesicles (EVs) play a central role in intracellular communication and have emerged as key modulators of immunological and inflammatory responses. In this report, we examined the impact of EVs in PWH experiencing cognitive deficits to determine their relevance in HIV associated neuropathology.

Methods: EV phenotypes were measured in plasma samples from 108 PWH with either cognitive impairment (CI, n=92) or normal cognition (NC, n=16) by flow cytometry. Matched cerebrospinal fluid (CSF)-derived EVs were similarly profiled from a subgroup of 84 individuals who underwent a lumbar puncture. Peripheral blood mononuclear cells were assayed by flow cytometry to measure monocyte frequencies in a subset of 32 individuals.

Results: Plasma-EVs expressing CD14, CD16, CD192, C195, and GFAP were significantly higher in HIV-infected individuals with cognitive impairment compared to individuals with normal cognition. Increased CSF-EVs expressing GFAP and CD200 were found in the cognitive impairment group compared to the normal cognition group. Frequencies of patrolling monocytes correlated with plasma-EVs expressing CD14, CD66b, MCSF, MAP2, and GFAP. Frequencies of CD195 expression on monocytes correlated positively with plasma-EVs expressing CD41a, CD62P, and CD63. Expression of CD163 on monocytes correlated positively with CSF-EVs expressing GFAP and CD200. Finally, the expression of CD192 on total monocytes correlated with CSF-EVs expressing CD200, CD62P, and CD63.

Conclusions: EVs expressing monocyte activation and neuronal markers associated with HIV associated cognitive impairment, suggesting that distinct EV subsets may serve as novel biomarkers of neuronal injury in HIV infection. Further circulating platelet EV levels were linked to monocyte activation indicating a potential novel interaction in the pathogenesis of HIV-related cognitive impairment.

Keywords: extracellular vesicles; human immunodeficiency virus; monocytes; neurocognitive impairment; neurons.

Figure 1

Characterization of EVs in biological samples. (A) Schematic of the method for isolation and analysis of EVs for their absolute count and cell of origin. (B) Illustration of surface markers on EVs linked to neuronal cells, lymphoid cells, and myleoid cells. (C) SSC height (SSC-H) dot plot shows sensitivity to detect beads sized 100 to 1000 nm in diameter. Representative plots of EV gated from the threshold to the 1000 nm gate based on SSC and sorted according to surface markers from their cell of origin.

Figure 2

Circulating EVs expressing monocyte-associated markers and neuronal damage associate with cognitive impairment in HIV-infected individuals on stable ART. Scatter plots of EVs/µL numbers (log 10-transformed). (A) Plasma-EVs expressing monocyte-associated markers (CD14, CD16, CD192, and CD195) and neuronal damage marker (GFAP) were significantly higher in HIV+ adults with cognitive impairment (CI) compared to those with normal cognition (NC). (B) There was a significant increase in levels of CSF-EVs expressing GFAP+ and CD200+ in the CI compared to the NC group. P values were determined by using two-tailed Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Sensitive analysis of circulating EVs expressing monocyte-associated markers and neuronal damage associate with cognitive impairment in virally-suppressed HIV+ individuals. Scatter plots of total EV numbers and concentration of each subtype of EV are shown. (A) levels of EVs expressing CD14, CD16, and CD195 were elevated in the plasma of aviremic HIV-infected individuals with CI compared to the NC group. (B) There was no significant difference in levels of GFAP+EVs and CD200+EVs in the CSF of aviremic CI compared to the NC group. P values were determined by using two-tailed Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.001. NC, normal cognition; CI, cognitive impairment.

Figure 4

Relationship between all plasma-EV subtypes and circulating monocyte subsets. Spearman correlation matrix showing the association between all EV subtypes based on their cellular origin and circulating monocyte subsets in HIV-infected individuals on ART. Positive correlations are displayed in red shades and negative correlations are in blue shades.

Figure 5

Correlations between all CSF-EV subtypes and circulating monocyte subsets. Spearman correlation matrix showing the association between all CSF-EV subtypes and circulating monocyte subsets in HIV-infected individuals on stable ART. Positive correlations are displayed in red shades and negative correlations are in blue shades.

Figure 6

Diagrammatic representation of the biogenesis of EVs expressing inflammatory phenotypic markers in neurocognitive disorders. Scheme presents the healthy brain and the inflammatory environment of the cognitively impaired brain. EVs are generated reflecting the underlying surface proteins on their cell of origin.