CCR2 on CD14⁺CD16⁺ monocytes is a biomarker of HIV-associated neurocognitive disorders

Standard protocol approvals, registrations, and patient consents.

Blood samples from HIV-seropositive participants were obtained through the Manhattan HIV Brain Bank (MHBB; U01MH083501, U24MH100931) in New York, NY and the Women's Interagency HIV Study (WIHS; U01AI35004, U01 A142590) in Bronx, NY. All patients gave written informed consent for the provision of blood for the purpose of HIV research. The protocol under which these samples were obtained is approved by the Institutional Review Board at the Montefiore Medical Center, Albert Einstein College of Medicine, and the Mount Sinai Program for the Protection of Human Subjects Institutional Review Board. HIV-infected patient demographic and virologic information is listed in table e-1 at Neurology.org/nn. The inclusion criterion for participants in the study was HIV-positive individuals older than 18 years. An additional inclusion criterion for the MHBB participants was the agreement to be an organ donor for research purposes upon death. HIV-seropositive individuals with persistently low CD4 counts and neuropsychological impairment likely due to causes other than HIV were excluded from the study. The study was performed between June 25, 2012 and October 4, 2013, during which time samples were obtained from 60 participants that met the inclusion/exclusion criteria. The participants in this study were predominantly of a black and Hispanic racial/ethnic background and female, and the majority had undetectable viral loads. These individuals are representative of the population in East Harlem and the Bronx, NY, where the cohorts are located; however, our participants are oversampled with regard to female sex.

HIV-seropositive individuals underwent extensive neuromedical and psychiatric assessments, as described previously.^12–14 The neuromedical examination included the collection of a detailed medical history and a structured medical evaluation. Collected blood samples were tested for liver diseases (hepatitis B and C antibodies), CD4⁺ lymphocyte counts, and HIV load. Diabetes status was ascertained through patient interview or review of the medical record and was confirmed in most cases by the documentation of oral or injectable hypoglycemic agents in the patient's medication list. Urine toxicology screened for amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, opiates, phencyclidine, methadone, and propoxyphene (illicit and prescribed).

Blood from HIV-seronegative participants was obtained from deidentified leukopaks from the New York Blood Center in accordance with protocols established at the Albert Einstein College of Medicine.

Neuropsychological testing.

MHBB participants were administered a battery of neuropsychological tests and functional status questionnaires that assessed a broad range of cognitive abilities sensitive to HIV impairment.¹⁵ Specific tests and their normative references can be found in Byrd et al.¹⁴ All individual tests were grouped according to the theoretically derived domains indicated in table e-2.¹⁵ Raw scores from all tests were converted to demographically adjusted T scores, which adjusted performance for the effects of age, education, sex, and ethnicity, where appropriate. Domain scores were derived from the mean T scores of the individual tests in that particular domain, and clinical ratings of severity of impairment (range = 1–9) were assigned from these.¹⁵ HAND diagnoses (HIV-associated dementia [HAD] and minor cognitive motor disorder [MCMD]) were assigned according to a modified American Academy of Neurology algorithm published by the Dana Consortium.¹⁶ Asymptomatic neurocognitive impairment (ANI) was diagnosed according to the clinical ratings system published by Woods et al.¹⁵ in 2004 and reflects the presence of cognitive impairment in the absence of functional impairment.

For the purposes of this study, cognitive data for the WIHS participants were extracted for those tests that were identical to those from the MHBB battery: Grooved Pegboard, Hopkins Verbal Learning Test-Revised, Controlled Oral Word Association Test F-A-S, Wechsler Adult Intelligence Scale-III (WAIS-III) Digit Symbol, WAIS-III Symbol Search, and WAIS-III Letter Number Sequencing (see table e-2). Raw scores were converted to standard T scores using the same normative data applied to the MHBB test scores (see above). Functional status data were not available for these participants. Thus, HAND diagnoses could not be assigned. The presence of cognitive impairment in WIHS participants was determined by application of the cognitive criteria of the diagnostic algorithms referenced above.¹⁶

Statistical analysis.

Statistical analyses were performed using Prism 6.02 software (GraphPad Software, Inc., San Diego, CA) and the Statistical Package for Social Sciences (version 20.0 for Windows; IBM, Armonk, NY). Mann–Whitney tests were used to determine statistical significance for figures 1, A–C, 2A, and 2B (p ≤ 0.05). Linear regression was used to determine statistical significance for figure 2, E and F (p ≤ 0.05). Wilcoxon signed rank test was used to determine statistical significance for figure 3, B–D (p ≤ 0.05). Paired 2-tailed t test was used to determine statistical significance for figure 4, A–C (p ≤ 0.05). A logistic regression was performed to test the strength of comorbid clinical factors (presence of each condition coded as “1”) and cognitive status for the prediction of CCR2 high and low group classifications based on expression levels above 45 (p ≤ 0.05).

Details on methods regarding cell isolation, monocyte identification by flow cytometry, CCR2 analysis by flow cytometry, and transmigration assays across the BBB model are provided in appendix e-1.

Participant characteristics.

As summarized in table e-1, the study consisted of 60 HIV-seropositive individuals from the MHBB (27 people) and the WHIS (33 people) cohorts. The participants in the cohorts did not differ with respect to race/ethnicity, CD4⁺ T-cell count, plasma viral load, the frequency of viral hepatitis, or rates of cognitive impairment. The WIHS cohort was comprised of women who were significantly younger than the MHBB cohort (49 ± 7 years compared to 55 ± 6 years, p < 0.001), a portion of whom were naive to cART.

CCR2 is increased on CD14⁺CD16⁺ monocytes in HIV-seropositive individuals with cognitive impairment.

To examine the monocyte subpopulations present in the peripheral blood of HIV-seropositive and seronegative individuals, peripheral blood mononuclear cells (PBMC) were isolated and analyzed by flow cytometry. Forward and side scatter characteristics were used to identify monocytes in the PBMC population (figure e-1A). Surface CD14 and CD16 were used to discriminate further among the CD14⁺CD16⁻, CD14⁺CD16⁺, and CD14^lowCD16⁺ monocyte subsets (figure e-1B). We characterized CCR2 on the cell surface of each monocyte subset present in HIV-positive individuals to determine the mechanisms that may be mediating transmigration across the BBB and contributing to the neuropathogenesis of HIV. Multicolor flow cytometry was performed and CCR2 was characterized on the surface of each monocyte population from 16 seropositive donors with normal cognition and 44 individuals with HAND. CCR2 was present on CD14⁺CD16⁺ (figure 1A) and CD14⁺CD16⁻ (figure 1B) monocytes isolated from those with normal cognition, but was minimally expressed on CD14^lowCD16⁺ cells (figure 1C). There was a significant increase in the mean fluorescence intensity (MFI) of CCR2 on CD14⁺CD16⁺ monocytes from individuals with HAND (65.6 ± 4.8) compared to seropositive people with normal cognition (37.9 ± 2.5, p < 0.001), suggesting that it may be used to identify those at risk of developing cognitive deficits (figure 1A). There was no relationship between the presence of CCR2 on the other monocyte subsets and cognitive status (figure 1, B and C).

There was a “threshold of impairment” above which CCR2 was indicative of HAND. The MFI of CCR2 on CD14⁺CD16⁺ monocytes was below 45 for 88% of those with normal cognition (figure 1A). Conversely, the MFI of CCR2 on these cells was above 45 for 84% of individuals with HAND. While the majority of the MFIs of CCR2 were above the “threshold of impairment” for individuals with HAND, the expression pattern for the chemokine receptor was variable among donors and was not normally distributed. Future studies will evaluate host genetic contributors to the variability in CCR2 and the functional consequences of this heterogeneity. The expression of CCR2 on CD14⁺CD16⁺ monocytes was not significantly associated with cART status (figure 2A), viral load (figure 2B), the severity of cognitive deficits in the MHBB (figure 2C) or WIHS (figure 2D) participants, and nadir (figure 2E) or current (figure 2F) CD4 T-cell counts.

CCR2 on CD14⁺CD16⁺ monocytes is not associated with substance use, liver disease, or diabetes.

HAND may be present with increased incidence or severity due to comorbid conditions that often affect a large number of HIV-infected individuals. To examine the degree to which comorbid conditions may have influenced CCR2 expression, a logistic regression was performed with CCR2 group status above or below the threshold value of 45 as the outcome variable and the following as predictor variables: urine toxicology positive for substance use (see Methods for details), diabetes status, liver disease status, and cognitive impairment status. Results indicate that the overall model was significant (model χ² = 20.584, p < 0.01), with cognitive status emerging as the only significant predictor variable associated with CCR2 (table 1). None of the comorbid conditions significantly predicted CCR2 status (all p > 0.05). These analyses suggest that CCR2 is predictive of cognitive impairment in HIV-infected individuals even in the context of comorbid conditions, and they provide further support for CCR2 on CD14⁺CD16⁺ monocytes as a potential prognostic biomarker of HAND.

CCR2 facilitates increased transmigration of CD14⁺CD16⁺ monocytes from individuals with HAND across the BBB in response to CCL2.

To determine whether the elevated amounts of CCR2 on CD14⁺CD16⁺ monocytes had functional consequences, PBMC from 9 HIV-infected individuals with normal cognition and 22 people with cognitive impairment were added to our in vitro model of the human BBB and allowed to transmigrate for 24 hours in response to media alone or CCL2. After the indicated time, the cells that transmigrated were recovered and stained with CD14, CD16, and CCR2 antibodies, and the monocyte populations were characterized and enumerated by flow cytometry. The CD14⁺CD16⁺ subset comprised the majority of monocytes that crossed the BBB (68.5% of transmigrating cells) (figure 3A). The migration of the other populations of monocytes was much lower in comparison. CD14⁺CD16⁻ and CD14^lowCD16⁺ cells comprised only 22.0% and 6.8% of transmigrating cells, respectively (figure 3A).

The CCL2-mediated transmigration of CD14⁺CD16⁺ monocytes across the BBB was significantly higher in participants with HAND (3- ± 0.3-fold higher than media alone, set to 1) compared to those with normal cognition (1.4- ± 0.1-fold higher than media alone, set to 1, p < 0.001) (figure 3B). This transmigration was not dependent on the severity of impairment, as cells isolated from individuals with ANI, MCMD, and HAD all responded similarly to CCL2 (data not shown). In contrast, there was no difference in the CCL2-mediated transmigration of CD14⁺CD16⁻ (2.3- ± 0.2-fold compared to 2.1- ± 0.4-fold, p > 0.05) and CD14^lowCD16⁺ (1.2- ± 0.1-fold compared to 1.1- ± 0.1-fold, p > 0.05) monocytes from seropositive individuals with HAND and those with normal cognition (figure 3, C and D).

CCR2 was characterized on each monocyte population after transmigration and compared to that present on the cells prior to migration. CCR2 was significantly increased on CD14⁺CD16⁺ monocytes following transmigration across the BBB (74.5 ± 10.5) compared to prior to migration (33.3 ± 4.5, p < 0.05) (figure 4A). There was no difference in CCR2 after transmigration for the other monocyte populations (figure 4, B and C). This suggests a preferential selection for CD14⁺CD16⁺ cells with high levels of CCR2, underscoring its importance in facilitating the entry of these cells into the brain.