Widespread use of combined antiretroviral therapy (cART) has greatly reduced central nervous system (CNS)-related morbidity associated with HIV-1 infection; nonetheless, HIV-associated neurocognitive disorder (HAND) remains common, affecting an estimated 20%–50% of people living with HIV (PLWH) and 19% of PLWH with well-suppressed HIV on cART.1,2 HAND represents a constellation of cognitive, motor, and behavioral symptoms with a wide spectrum of severity.3 Detection of cerebrospinal fluid (CSF) HIV RNA and elevated markers of inflammation in the blood and CSF during suppressive cART suggests that persistent reservoirs of HIV in the CNS and inflammatory cross-talk between the 2 compartments may be implicated in clinically relevant neuronal injury.4–6
Exosomes have emerged as potential mediators of long-term immune activation and CNS perturbation during HIV infection.7 Exosomes are cell-secreted lipid bilayer membrane microvesicles ranging from 30 to 150 nm in size that are released via exocytosis into a variety of body fluids, including the blood and CSF.8,9 Exosomes contain donor cell–derived lipids, proteins, messenger RNAs, and microRNAs that affect the functioning of target cells in the immediate microenvironment or at distant sites. Exosomal microRNAs (exo-miRNAs) regulate gene expression by post-transcriptionally controlling the translation and stability of their mRNA targets in recipient cells, thereby affecting cell proliferation, apoptosis, and differentiation.10,11
Pro-inflammatory microRNAs, encapsulated in exosomes, may traffic between the CNS and the peripheral circulation, with systemically derived exosomes contributing to neurological sequelae in people living with chronic HIV infection and/or CNS-derived exosomes in the plasma reflecting the state of ongoing CNS processes. CNS inflammation promotes the ability of exosomes to cross the blood–brain barrier, facilitating the transport of pathologic or pro-inflammatory molecules between the blood and CSF.12 Previous studies have implicated exosomal signaling in CNS pathologies. For example, pro-inflammatory exo-miRNAs have been shown to modulate microglial-mediated immune responses in neurodegenerative diseases including Parkinson and Alzheimer's diseases13,14 and can reflect disease status in multiple sclerosis.15 In studies of HIV, exosomes released from HIV-1 infected cells have been shown to induce quiescent CD4+ lymphocytes to produce HIV-1 and promote the release of pro-inflammatory cytokines from target monocyte-derived macrophages.16,17
Given that microRNAs are enriched in exosomes, in this study, we explore the association between exo-miRNAs and neurocognitive dysfunction during chronic HIV infection in people who have obtained viral suppression on cART. We hypothesized that there is a link between exosomal signaling patterns and neuropsychological testing (NP) performance as a clinical index of the effects of systemic inflammation on the brain as a target organ. This investigation of patterns of plasma exo-miRNAs in the pathogenesis of neurocognitive dysfunction during treated suppressed infection provides new potential targets for intervention for affected individuals.
MATERIALS AND METHODS
Study participants included: participants from the Primary Infection CNS Events Study (University of California, San Francisco) enrolled during primary HIV infection (PHI), defined as within 12 months of HIV acquisition (n = 19), participants from the HIV Associated Reservoirs and Comorbidities study (Yale University) who initiated cART during chronic HIV infection (n = 12), and HIV-uninfected participants without clinical diagnoses of neurologic disease who were recruited from the community [5 men, median age = 49 years, interquartile range (IQR) = 48–53 years]. For participants with HIV (n = 31), plasma, bloodwork, lumbar puncture, and NP testing were performed after at least 1 year of cART initiation and systemic viral suppression (plasma viral load ≤40 copies/mL) were documented.
For each PLWH, a 1.5-hour NP battery was performed. Participants were assessed across 5 common domains of neurocognitive functioning: motor (Timed Gait, Grooved Pegboard), executive function (Trail-Making A-B, Controlled Oral Word Association), processing speed (Digit Symbol, Stroop tests), memory (Figure Delay), and learning (Hopkins Verbal Learning Test, Rey Auditory Verbal Learning).18,19 Total z scores were derived by averaging individual test z scores across domains and externally normalizing individual raw test scores for age, gender, ethnicity, and years of education to the general population.20–27 Executive function z scores were derived similarly, using only executive function domain tests. Study participants were divided into 2 groups, higher- and lower-performing, based on normalized total z or executive function z score of >0 and <0, respectively. Other studies have used similar methods of studying participants by NP performance.28,29
Exosome Isolation From Plasma
The isolation of exosomes was performed using a solution of 20% polyethylene glycol (PEG, Mn 6,000; Sigma-Aldrich, St. Louis, MO) as previously reported.30–32 Briefly, the mixture of plasma and the solution (500 µL solution/500 µL plasma) was incubated at 4°C for 2 hours, followed by centrifugation at 13,000 rpm for 2 minutes to obtain the exosome pellet with 2 washes of 1× Dulbecco phosphate buffered saline (DPBS, Sigma-Aldrich).
Confirmation of Exosomes
Transmission electron microscopy (TEM) was performed to validate exosome morphology. DPBS-suspended exosomes were deposited on formvar carbon-coated electron microscopy grids. After negative staining with 2% uranyl acetate (pH 4), the grids were examined and imaged with a FEI TECNAI F20 FEG microscope running at 200 kV of accelerating voltage, the digital images were recorded with a FEI Eagle CCD camera (4k × 4k). Images of 100 representative vesicles were measured with ImageJ (https://imagej.nih.gov). Nanoparticle tracking analysis (NTA) was performed to measure exosomal size and concentration using a Nanosight LM10 instrument equipped with a 405-nm laser (NanoSight, Salisbury, United Kingdom, Malvern Instruments, Malvern, United Kingdom) at 21°C. The Brownian movement of particles was tracked by the NTA software (version 3.1, NanoSight). Quantitative ELISA assay was performed to measure exosome marker CD6333 using an ExoELISA-Ultra CD63 kit (System Biosciences, Inc., Palo Alto, CA, cat. EXEL-ULTRA-CD63-1) following the manufacturer's protocol.34 The total protein concentration of each exosome suspension was measured using NanoDrop_1000 spectrophotometer with a wavelength 280 nm. DPBS exosome suspensions (normalized to 100 μg of protein) were plated and run in duplicate. The absorbance of exosomal CD63 was determined using a Biotek spectrophotometric 96-well microplate reader with a wavelength of 450 nm.
RNA Preparation and Library Construction
Total RNA was extracted from purified exosomes using the SeraMir Exosome RNA purification kit (System Biosciences, Inc., cat. RA806A-1), and the quality and concentration of RNA were determined by the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). Small RNA libraries were prepared using an NEBNext multiplex small RNA library prep set for Illumina kit (New England BioLabs, Inc., Ipswich, MA) following the manufacturer's instructions.
Next-Generation Small RNA Sequencing and Analysis
Single-end deep sequencing was performed on all cDNA libraries with a 75-nucleotide (nt) read length using the HiSeq-2500 Genome Analyzer (Illumina, San Diego, CA). Adaptors and low quality regions were trimmed from raw sequences using btrim with options “-3 -P -l 15.”35 The trimmed sequences were mapped to the human genome (hg38) with Burrows-Wheeler Aligner.36 For microRNA annotation, miRBase v21 was used.37 Differential gene expression analysis was performed using the R package “DESeq2.”38 MicroRNAs with base mean expression <10 were excluded, and differential expression was defined as absolute log2(fold-change) >1.0.
Functional Analysis of Predicted mRNA Targets of Differentially Expressed exo-miRNAs
To examine functional annotations of the mRNAs that the differentially expressed exo-miRNAs targets, KEGG pathway and Gene Ontology (GO) analyses were performed using the DiANA tool of mirPath v.3.39
Measurement of BDNF in Plasma
The concentration of brain-derived neurotrophic factor (BDNF) was measured as a read-out of exo-miRNA activity on axonal modeling pathways. The concentration of free BDNF in plasma was determined using a commercial ELISA assay (R&D Systems, Inc., Minneapolis, MN) following the manufacturer's protocol. Correlation coefficients were generated between BDNF and both miR-30a-5p and miR-206 levels, 2 reported inhibitors of BDNF translation.40,41
Fisher exact and Wilcoxon Rank Sum tests were used to compare clinical characteristics. To test for the ability of exo-miRNA expression to distinguish participants' NP status, principal component analysis and receiver operating characteristic (ROC) analysis were performed. ROC analysis was performed in SAS v9.4. All other statistical analyses were performed in R v3.5.0.
Characteristics for all study participants are summarized in Table 1. Of the 19 participants enrolled during PHI, the median time between estimated date of infection and treatment initiation was 0.6 years (IQR 0.2–1.8). Patients enrolled during PHI had median age 43 years (IQR 35.5–48), whereas patients enrolled during chronic infection had median age 58.5 years (IQR 51.75–62.5, P = 0.0001). Based on total z scores on NP testing, 13 participants with HIV were sorted into the NP higher-performing group (NP-higher, median total z = 0.3) and 18 into the NP lower-performing group (NP-lower, median total z = −0.7). Based on executive function z scores, 14 participants with HIV were sorted into executive function higher-performing (median z-score = 0.44, 64% enrolled during PHI) and 16 into executive function lower-performing (median z-score = −0.49%, 56% enrolled during PHI). Seven patients enrolled during PHI had incomplete learning and memory NP data, 1 patient had incomplete executive function data, and 2 patients enrolled during chronic infection had incomplete motor NP data; missing domains were not included in the total z-score calculations for these patients. Participants enrolled during PHI were more prevalent in the NP-higher group, whereas most of the participants enrolled during chronic infection were in the NP-lower group (P = 6.5 × 10−6). NP-lower participants had an overall longer duration of cART treatment (P = 0.035), fewer years of education (P = 6.0 × 10−4), and lower CSF protein level (P = 0.009) relative to NP-higher.
Characterization of Circulating Exosomes in Plasma
TEM images showed that the morphology of PEG-purified exosomes was vesicular with diameters ranging from 20 to 70 nm (Fig. 1A). NTA, which is based on the principle of Brownian motion of particles in liquid, demonstrated that most purified vesicles ranged 110–210 nm in diameter and were approximately 1010–1011 particles per mL of plasma (Fig. 1B). The smaller sizes of exosomes measured under TEM versus NTA may be because of sample preparation and dehydration for TEM imaging and lower sensitivity of NTA to detect vesicles in the 20–60 nm range.42,43 ELISA assay confirmed that all purified exosomes were CD63-positive and revealed no significant difference in exosome abundance, quantified by the CD63 ELISA signal, between the NP-higher (median = 1.56 × 1010) versus NP-lower groups (median = 1.73 × 1010, P = 0.569) or between cohorts enrolled during PHI (median = 1.55 × 1010) versus chronic infection (median = 1.78 × 1010, P = 0.371).
Differential exo-miRNA Expression Between Neuropsychological Higher- and Lower-Performance Groups
Expression levels of exo-miRNAs identified by NGS were compared between NP-higher and NP-lower groups (see Table 1, Supplemental Digital Content, http://links.lww.com/QAI/B380). Principal component analysis performed on the exo-miRNA expression data demonstrated that NP-higher and NP-lower separated into distinct groups, particularly along the second component (Fig. 2A). After correcting for multiple comparisons using false discovery rate (FDR < 0.1), we found 11 exo-miRNAs were significantly differentially expressed between the groups (miR-206, miR-193b-5p, miR-193a-5p, miR-30a-5p, miR-216b-3p, miR-499a-5p, miR-499b-3p, miR-708-3p, miR-1183, miR-375, and miR-483-5p). All 11 differentially expressed exo-miRNAs were upregulated in NP-lower relative to NP-higher (Fig. 2B). Exo-miRNA expression was also compared between PLWH with higher-versus lower-executive function performance. Three exo-miRNA (miR-216b-3p, miR-148a-3p, and miR-504-5p) were significantly upregulated in participants with lower executive function performance relative to higher performance by P < 0.05.
To evaluate the ability of differentially expressed circulating exo-miRNAs to distinguish NP performance groups, we conducted ROC analyses with the 11 identified exo-miRNAs. The 11 differentially expressed exo-miRNAs between NP-higher and NP-lower showed high area under the curve with a value of 0.93 [95% confidence interval (CI): 0.83 to 1.00] in distinguishing the performance groups (Fig. 2C).
Biological Role of Differentially Expressed exo-miRNAs
To better understand the biological relevance of the differentially expressed microRNAs between NP performance groups, we performed canonical KEGG pathway and GO enrichment analyses using the 11 differentially expressed exo-miRNAs. Axon guidance ranked highest of the enriched KEGG pathways (Fig. 3), with 9 of the 11 identified exo-miRNAs targeting genes in this pathway.
GO analysis demonstrated that genes involved in organelle function, ion binding, cellular nitrogen compound metabolic process, cellular protein modification, biosynthesis, and neurotrophin tyrosine receptor kinase (TRK) signaling were predicted targets of the 11 differentially expressed exo-miRNAs (Fig. 4A). Two identified exo-miRNAs, miR-30a-5p and miR-206, are known inhibitors of BDNF translation. Measurement of BDNF in the 31 plasma samples revealed a significant negative correlation between both exosomal miR-206 and miR-30a-5p expression and BDNF levels (Fig. 4B). Pearson correlation coefficients were −0.50 [95% CI: (−0.72 to −0.16); P value = 0.004] and −0.47 [95% CI: (−0.91 to −0.13); P value = 0.008], respectively. There was no difference in BDNF levels between the NP-higher (7.64, range 1.43–15.4) and NP-lower (5.57, range 0.64–39.0) groups (P = 0.514). There was no significant correlation between BDNF levels and CD4+/CD8+ cell count ratios (P = 0.16) or CD8+ cell counts (P = 0.60) across all participants.
Differential exo-miRNA Expression Between NP Higher- and Lower-Performing Groups in the PHI Group Alone
Our analysis of differentially expressed exo-miRNAs between the NP-higher and NP-lower groups encompassed 2 distinct populations: participants from the San Francisco, USA, area, treated during early HIV infection, and HIV Associated Reservoirs and Comorbidities participants from the New Haven, USA, area, initially treated during chronic infection. To ensure that our findings were not driven by demographic differences between these 2 populations, differential exo-miRNA expression analyses were performed between NP-higher and -lower performing participants within the PHI study alone, where participants were distributed more evenly between NP-higher and -lower than the chronic infection group.
Using the same threshold of total z-score greater than or less than 0, 12 participants were classified as NP-higher (median total z = 0.4) and 7 participants were classified as NP-lower (median total z = −0.5) within the PHI study alone. There was no significant difference in clinical characteristics between the NP groups (Table 1).
Differential expression analyses revealed 15 exo-miRNAs that were significantly differentially expressed (P < 0.05) between NP-higher and -lower within this subset of participants. 5 exo-miRNAs (miR-454-3p, miR-548k, let-7a-5p, let-7e-5p, and let-7f-5p) were upregulated in NP-higher, whereas 10 exo-miRNAs (miR-30d-3p, miR-125b-5p, miR-193a-5p, miR-4742-3p, miR-4755-3p, miR-141-3p, miR-125b-2-3p, miR-205-5p, miR-1183, miR-708-3p) were upregulated in NP-lower (see Figure 1 and Table 2, Supplemental Digital Contents, http://links.lww.com/QAI/B380 and http://links.lww.com/QAI/B380, respectively). 3/10 exo-miRNAs that were upregulated in NP-lower (miR-193a-5p, miR-1183, and miR-708-3p) within the PHI-only group were also upregulated in NP-lower in the combined PHI and chronic infection group analysis. These 3 exo-miRNAs, differentially expressed in both analyses, demonstrated area under the curve of 0.86 (95% CI: 0.73 to 0.99) in distinguishing NP performance groups within the PHI cohort.
KEGG pathway analysis revealed that the axon guidance pathway was again significantly enriched for the 15 differentially expressed exo-miRNAs between the NP-higher and -lower groups in this PHI group, targeting 54 genes in the pathway (see Table 3, Supplemental Digital Content, http://links.lww.com/QAI/B380). The 3 exo-miRNAs differentially expressed in both analyses were specifically implicated in the phosphatidylinositol signaling (P = 0.01) and glycan degradation (P = 0.0016) KEGG pathways and were predicted to target genes involved in nervous system development (P = 1.5E-06), neurotrophin TRK signaling (1.6E-04), and ion binding (P = 6.69E-20) by GO analysis.
Differential exo-miRNA Expression Between Individuals With and Without HIV Infection
In an exploratory analysis, we compared exo-miRNA expression between a small group of HIV-negative individuals (n = 5) and participants with HIV (n = 31) to investigate whether the 11 exo-miRNA associated with NP status would differentiate by infection status. 25 exo-miRNAs were found to be differentially expressed (P < 0.05) between the participants with and without HIV infection (see Table 4, Supplemental Digital Content, http://links.lww.com/QAI/B380). miR-375, which was found to be up-regulated in NP-lower HIV-infected participants was also found to be 5.7-fold higher in HIV-infected participants relative to HIV-uninfected participants. Otherwise, the differential exo-miRNA expression between infection status did not overlap with those that were found when stratifying groups by NP testing.
In this study, we demonstrated that PLWH with higher versus lower neuropsychological performance have different circulating exo-miRNA content. Our analysis of exo-miRNA transcriptomic data revealed that the expression of 11 exo-miRNAs in plasma is associated with lower neuropsychological performance in PLWH, and certain processes, including axon guidance, Epidermal growth factor receptor signaling, and TRK receptor signaling, are predicted targets of these 11 microRNAs. Three of these exo-miRNAs, along with the axon guidance KEGG pathway, were further validated in the exo-miRNA expression analysis within the group of PHI participants alone, suggesting that these exo-miRNA may be implicated in cognitive function during chronic HIV infection that is not specific to the timing of ART initiation.
Several of the differentially expressed exo-miRNAs have been implicated in inflammatory pathways, suggesting that exo-miRNAs may play a role in regulating inflammation during chronic HIV infection. For example, miR-483-5p and miR-30a-5p were up-regulated in NP-lower individuals. miR-483-5p has been shown to attenuate the host antiviral immune response following hepatitis C infection through down-regulation of NF-kB, whereas miR-30a-5p has been shown to enhance antiviral inflammation via up-regulation of IFN-1.44,45 The interplay between anti- and pro-inflammatory exo-miRNAs may contribute to damaging, prolonged inflammation in PLWH and downstream clinical sequelae such as HAND, although this was not reflected by a difference in blood neopterin concentration by the NP group.
Deep sequencing of exo-miRNAs allowed us to take an unbiased approach to detecting biologic processes that are differentially regulated in PWLH with different NP performance. Interestingly, a high proportion of exo-miRNAs in both the full group and subset group analyses were predicted to target genes in the axon guidance KEGG pathway. Integrated transcriptomic analyses of mRNAs and microRNAs in the frontal cortex have suggested that dysregulated axon guidance plays a key role in HIV-mediated neurodegeneration.46 These results suggest that exo-miRNAs may traffic between the periphery and CNS, interfering with neuron repair pathways or reflecting ongoing degenerative processes associated with HAND. We were particularly interested in the executive function NP domain because of the known involvement of the frontal cortex in HIV-mediated neurodegeneration. We found that 3 exo-miRNA, including miR-148a-3p, which has been shown to promote apoptosis by targeting Bcl-2, were more highly expressed in PLWH with lower executive function performance.47 miR-216b-3p was upregulated in both NP-lower and in the cohort with lower executive function, suggesting that this exo-miRNA may specifically contribute to deficits in executive function in PLWH. In addition, processing speed and motor function, 2 domains tested by our standardized neuropsychological exams, are commonly compromised in participants with HAND because of damage to myelinated white matter tracts.48,49 Defects in axon guidance would impede maintenance and normal functioning of these tracts.
Exo-miRNAs up-regulated in NP-lower participants were also predicted to be involved in neurotrophin TRK signaling by GO analysis, and 2 of these microRNAs, miR-30a-5p and miR-206, have previously been shown to specifically inhibit translation of an important TRK ligand, BDNF.50–53 We were able to corroborate this association by showing that there were significant negative correlations between plasma BDNF and both exosomal miR-30a-5p and miR-206. BDNF and its high-affinity receptor, tropomysin-related kinase B, are important mediators of axon guidance and synaptic plasticity. Functional suppression of BDNF leads to the deficits of long-term synaptic potentiation, a major cellular mechanism underlying learning and memory.54 Therefore, up-regulation of miR-30a-5p and miR-206 observed in NP-lower participants may cause neuropsychological deficits in PLWH by impeding BDNF signaling, and in turn, axon guidance.
In studies not focused on CNS deficits, it has been consistently shown that HIV infection alters the microRNA content of exosomes.45,55,56 We similarly found significant differences in circulating exo-miRNA expression between individuals with and without HIV infection. These differences motivated our choice to focus on HIV-infected participants alone for our study of neuropsychological function. Elucidating whether the 11 exo-miRNA associated with neurocognitive status in PLWH are specific to HIV infection requires a larger, controlled study of uninfected volunteers with neuropsychological testing.
This study was an effort to investigate the relationship of exo-miRNA signaling and clinical sequelae of chronic HIV after viral suppression, and there are limitations to our analyses. Primarily, a higher proportion of participants who initiated cART during chronic infection sorted into the NP lower-performing group compared with those who started cART during early infection. Although NP tests have been standardized and validated to control for education level, age, and other demographic variables, performing differential exo-miRNA analysis in the more demographically homogenous PHI cohort alone was an important validation step. It is highly suggestive that 3 differentially expressed exo-miRNAs in both analyses are implicated in pathways of inflammation and neuronal function; however, repeating this analysis in a larger cohort of demographically homogenous individuals would strengthen this finding. In addition, our study included only one female patient, and studying a more balanced cohort would increase the generalizability of these results. Second, without enriching for neuronal-derived exosomes in the plasma, we cannot know that the exosomes examined in this study would traffic to or from the CNS. Several studies have investigated NDEs specifically in the circulation as read-outs of CNS biology in neurocognitive disorders which could be used in future work.28,57,58 Nonetheless, this study provided a snapshot of biologic processes occurring in PLWH that may have widespread effects across various organs.
In summary, we found there was a distinct exo-miRNA pattern that distinguished lower versus higher NP performance groups in PLWH. The differentially expressed exo-miRNAs were predicted to be involved in inflammation and neurodegeneration pathways. These findings suggest that circulating exo-miRNAs may reflect processes ongoing in the CNS in PLWH in the setting of durable viral suppression. Exo-miRNA content may serve as a useful diagnostic tool for individuals with chronic HIV infection on cART and provide further insight into potential targetable mechanisms of NP sequelae in this population.
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