Mechanisms of HIV entry into the CNS: increased sensitivity of HIV infected CD14+CD16+ monocytes to CCL2 and key roles of CCR2, JAM-A, and ALCAM in diapedesis

Abstract

As HIV infected individuals live longer, the prevalence of HIV associated neurocognitive disorders is increasing, despite successful antiretroviral therapy. CD14(+)CD16(+) monocytes are critical to the neuropathogenesis of HIV as they promote viral seeding of the brain and establish neuroinflammation. The mechanisms by which HIV infected and uninfected monocytes cross the blood brain barrier and enter the central nervous system are not fully understood. We determined that HIV infection of CD14(+)CD16(+) monocytes resulted in their highly increased transmigration across the blood brain barrier in response to CCL2 as compared to uninfected cells, which did not occur in the absence of the chemokine. This exuberant transmigration of HIV infected monocytes was due, at least in part, to increased CCR2 and significantly heightened sensitivity to CCL2. The entry of HIV infected and uninfected CD14(+)CD16(+) monocytes into the brain was facilitated by significantly increased surface JAM-A, ALCAM, CD99, and PECAM-1, as compared to CD14(+) cells that are CD16 negative. Upon HIV infection, there was an additional increase in surface JAM-A and ALCAM on CD14(+)CD16(+) monocytes isolated from some individuals. Antibodies to ALCAM and JAM-A inhibited the transmigration of both HIV infected and uninfected CD14(+)CD16(+) monocytes across the BBB, demonstrating their importance in facilitating monocyte transmigration and entry into the brain parenchyma. Targeting CCR2, JAM-A, and ALCAM present on CD14(+)CD16(+) monocytes that preferentially infiltrate the CNS represents a therapeutic strategy to reduce viral seeding of the brain as well as the ongoing neuroinflammation that occurs during HIV pathogenesis.

Figure 1. CD14⁺CD16⁺ Monocytes Increase in the Peripheral Blood of HIV(+) People and in Culture.

(A) PBMC from HIV(+) individuals were stained with CD14 APC and CD16 PE-Cy7-coupled antibodies. Forward and side scatter were used to determine monocyte gating. The CD14⁺CD16⁺ population represented a significant percentage of monocytes in HIV infected individuals as compared to uninfected individuals (see below). Data from 3 separate donors, representative of 5, are shown. (B–C) Monocytes from HIV(-) people were isolated from PBMC using CD14 magnetic beads and analyzed by flow cytometry. Data from a single donor representative of 57 independent individuals are shown. (B) Freshly isolated “Day 0” and (C) “Day 3” monocytes matured for 3 days in nonadherent culture with 10 ng/mL M-CSF were stained with isotype matched negative control antibodies or with CD14 APC and CD16 PE-Cy7-coupled antibodies. CD14⁺CD16⁺ monocytes represented only a small percentage of freshly isolated monocytes (B). Our culture system significantly increased their numbers (C) and modeled the expansion of the CD14⁺CD16⁺ population in HIV(+) individuals (A).

Figure 2. HIV Infection Significantly Increases Monocyte Transmigration Across the BBB in Response to CCL2.

“Day 3” monocytes from 8 separate individuals were infected with HIV or remained uninfected as described in the methods, added to our BBB model, and allowed to transmigrate for 24 hours to media alone or to 200 ng/mL CCL2. (A) The total number of CD14⁺CD16⁺ monocytes that transmigrated to media (circles) or CCL2 (squares) was determined. (B) The pooled average fold change in monocyte transmigration to media (white bars, set to 1) or CCL2 (black bars), was determined. HIV infected CD14⁺CD16⁺ monocytes transmigrated exuberantly in response to CCL2. This did not occur in the absence of the chemokine in response to media alone. Data are represented as mean ± standard error of the mean. Significance was determined using the Wilcoxon Signed Rank test. *p<0.01.

Figure 3. HIV Infected Monocytes Have Highly Increased Sensitivity to CCL2.

Chemotaxis across a 5 µm polycarbonate membrane to increasing concentrations of CCL2 ranging from 1–500 ng/mL was examined in uninfected and HIV infected monocytes derived from eight independent donors. HIV infection (black bars) significantly increased monocyte chemotaxis to CCL2 relative to uninfected cells (white bars) at all concentrations. Uninfected cells did not chemotax to concentrations below 25 ng/mL, whereas HIV infected cells migrated even to very low levels of CCL2 (1 and 10 ng/mL). Data are represented as mean ± standard error of the mean. Significance was determined using a Wilcoxon Signed Rank test. ^#p<0.05 indicates significant chemotaxis relative to 0 ng/mL CCL2 for the uninfected cells. ^&p<0.01 indicates significant chemotaxis relative to 0 ng/mL CCL2 for the HIV infected cells. ^*p<0.05 and **p<0.01 indicate significant chemotaxis of HIV infected cells relative to uninfected cells. NS indicates no significant change as compared to 0 ng/mL CCL2 for the uninfected cells.

Figure 4. HIV Infection Increases Total and Surface Expression of CCR2 on CD14⁺CD16⁺ Monocytes.

(A) HIV infected and uninfected monocytes from 5 independent individuals, enriched for the CD14⁺CD16⁺ population, were examined for CCR2 by Western blot. HIV infection increased total CCR2 within monocytes. (B) Densitometric analysis was performed and the average pooled fold change of CCR2 normalized to GAPDH was determined for uninfected (white bar) and HIV infected (black bar) monocytes. Data are represented as mean ± standard error of the mean. Significance was determined using a Two-Tailed Paired T test. *p<0.01. (C) HIV infected and uninfected monocytes were analyzed by flow cytometry using antibodies specific to CCR2 (blue) or an isotype matched negative control (red). Representative histograms of the CD14⁺CD16⁺ monocytes from three separate individuals are shown. (D) After subtracting the fluorescence contribution from the isotype matched control, the average fold increase of CCR2 on the surface of HIV infected (black bar) CD14⁺CD16⁺ monocytes relative to uninfected cells (white bar, set to 1) was 1.6 fold. Data are represented as mean ± standard error of the mean. Significance was determined using Wilcoxon Signed Rank test. *p<0.05.

Figure 5. Surface Expression of Junctional Proteins Increases upon Monocyte Maturation.

The surface expression of JAM-A, ALCAM, CD99, and PECAM-1 was analyzed by flow cytometry on freshly isolated, “Day 0” and “Day 3” monocytes. (A) FACS data represented as histograms show the surface junctional protein expression from one representative HIV(-) individual. The mean fluorescent intensity (MFI) of each protein, or the MFI obtained with each isotype matched negative control antibody, is indicated on “Day 0” and “Day 3”. There was an increase in each junctional protein as the monocytes matured from “Day 0” (blue) to “Day 3” (green). (B) After subtracting the contribution of the isotype matched negative control antibodies, the MFI of JAM-A (red), ALCAM (blue), CD99 (yellow), and PECAM-1 (green) were analyzed on monocytes from 30 different HIV(-) individuals on “Day 0” (circles) and “Day 3” (squares). The extent to which each junctional protein increased after nonadherent culture for 3 days varied among donors. (C) The fold increase in the MFI of each junctional protein on “Day 3” monocytes (shaded bars) from 30 individual HIV(-) donors relative to “Day 0” (white bars, set to 1) was determined. (D) qRT-PCR was performed to determine the mRNA expression of junctional proteins. RNA was isolated from “Day 0” and “Day 3” monocytes from 4 independent donors. The fold change on “Day 3” (solid bars) relative to “Day 0” (white bars, set to 1) of JAM-A (red), ALCAM (blue), CD99 (yellow), and PECAM-1 (green) normalized to β-Actin was determined. Data are represented as mean ± standard error of the mean. Significance was determined using Wilcoxon Signed Rank test or a Two-Tailed Paired T test. ^*p<0.05**p<0.01. ALCAM was not significant due to high inter-donor variability, despite the large increases in mRNA expression (p=0.1).

Figure 6. JAM-A and ALCAM are Critical for Transmigration of CD14⁺CD16⁺ Monocytes Across the BBB.

“Day 3” monocytes highly enriched for CD14⁺CD16⁺ cells were added to our in vitro model of the human BBB in the presence of blocking antibodies to JAM-A, ALCAM or irrelevant isotype matched negative control antibody and allowed to transmigrate for 24 hours to 200 ng/mL CCL2. Monocytes were stained with CD14 APC and CD16 PE-Cy7-coupled antibodies. (A) Dot plots show the transmigration assay for a representative individual. Before transmigration, the “Day 3” monocytes consisted of both CD14⁺CD16^- and CD14⁺CD16⁺ cells. After transmigration across our human BBB model, the population consisted primarily of CD14⁺CD16⁺ cells. (B–C) Transmigration assays with monocytes from at least 6 independent individuals were performed and the number of CD14⁺CD16⁺ monocytes that transmigrated was quantified by FACS (B–C left). Depicts the total number of CD14⁺CD16⁺ monocytes that transmigrated for each experiment (B–C, right). Shows the average fold change in monocyte transmigration, relative to the baseline transmigration that occurred to media alone. CCL2 (red) promoted significantly increased transmigration compared to media alone (grey). The CCL2-mediated increase in transmigration was completely inhibited by antibodies (green) to JAM-A (B) or ALCAM (C). An isotype matched negative control antibody (blue) had no affect on transmigration. Data are represented as mean ± standard error of the mean. Significance was determined using Wilcoxon Signed Rank test. ^*p<0.05**p<0.01.

Figure 7. JAM-A and ALCAM Increase on CD14⁺CD16⁺ Monocytes Upon HIV Infection.

The surface expression of JAM-A (A) and ALCAM (B) was analyzed by flow cytometry on HIV infected or uninfected monocytes from 20 independent individuals. After subtracting the contribution of the isotype matched negative control antibodies, the fold increase in the MFI of each junctional protein on the monocytes that were cultured nonadherently and were either HIV infected (diamonds) or remained uninfected (triangles), relative to the freshly isolated monocytes (circles, set to 1) was determined. The majority of the individuals (11/20) had an additional increase in JAM-A and ALCAM after HIV infection over that which had already occurred during monocyte maturation (see Figure 5). Monocytes from the same 11 individuals had an increase in both JAM-A and ALCAM. Data are represented as mean ± standard error of the mean. Significance was determined using Wilcoxon Signed Rank test. **p<0.01.

Figure 8. Blocking JAM-A and ALCAM Inhibits CCL2-Mediated Increase in HIV Infected Monocyte Transmigration Across the BBB.

HIV infected or uninfected monocytes from 4 independent individuals were added to the BBB model and transmigrated in response to CCL2 in the presence of blocking antibodies to (A) JAM-A or (B) ALCAM. The number of CD14⁺CD16⁺ monocytes that transmigrated was analyzed by flow cytometry. CCL2 (horizontal hatching) promoted increased transmigration compared to media alone (white bars) for both uninfected and HIV infected cells. (A–B) HIV infected monocytes transmigrated in significantly greater numbers relative to uninfected monocytes in response to CCL2. Blocking antibodies (vertical hatching) to (A) JAM-A and (B) ALCAM decreased monocyte transmigration similarly for both HIV infected and uninfected monocytes. An isotype matched negative control antibody (diagonal hatching) did not affect CCL2-mediated transmigration. Data are represented as mean ± standard error of the mean. Significance was determined using a Two-Tailed Paired T test. ^*p<0.05**p<0.01.