Characterization of monocyte maturation/differentiation that facilitates their transmigration across the blood-brain barrier and infection by HIV: implications for NeuroAIDS

Abstract

The prevalence of human immunodeficiency virus 1 (HIV) associated neurocognitive disorders resulting from infection of the central nervous system (CNS) by HIV continues to increase despite the success of combination antiretroviral therapy. Although monocytes are known to transport HIV across the blood-brain barrier (BBB) into the CNS, there are few specific markers that identify monocyte subpopulations susceptible to HIV infection and/or capable of infiltrating the CNS. We cultured human peripheral blood monocytes and characterized the expression of the phenotypic markers CD14, CD16, CD11b, Mac387, CD163, CD44v6 and CD166 during monocyte/macrophage (Mo/Mac) maturation/differentiation. We determined that a CD14(+)CD16(+)CD11b(+)Mac387(+) Mo/Mac subpopulation preferentially transmigrates across our in vitro BBB model in response to CCL2. Genes associated with Mo/Mac subpopulations that transmigrate across the BBB and/or are infected by HIV were identified by cDNA microarray analyses. Our findings contribute to the understanding of monocyte maturation, infection and transmigration into the brain during the pathogenesis of NeuroAIDS.

Fig. 1

Schematic representation of monocyte maturation/differentiation into a macrophage in vivo and in vitro. Our culture system is represented in the top panel of the schematic. The in vivo system it was developed to mirror is represented in the bottom panel. Monocyte maturation/differentiation in the peripheral circulation in vivo is represented as a maturing monocyte, and in our culture system in vitro, this stage is represented by monocytes cultured non-adherently for 3 days. Upon adhesion to the BBB, monocyte maturation/differentiation in vivo is represented as an intermediate macrophage (MΦ) (transitional cell), and in our culture system in vitro, this stage is represented by monocytes cultured adherently for 3 days. In the CNS parenchyma, monocyte maturation/differentiation in vivo is represented as a macrophage (MΦ), and in our culture system in vitro, this stage is represented by monocytes cultured adherently for 6 days.

Fig. 2

Cell surface CD14 expression by freshly isolated monocytes as analyzed by flow cytometry. CD14⁺ cells were isolated from PBMC by positive selection using anti-CD14 magnetic beads and then stained with anti-CD14-PE or IgG2a-PE, an isotype matched negative control antibody. (A) A representative dot plot of side scatter (SSC) and forward scatter (FSC) with the R1 gate used for analysis of cell immunofluorescence, (B) negative control IGg2a immunofluorescence, and (C) CD14 immunofluorescence. Values in each quadrant represent the percentage of cells.

Fig. 3

Loss of cell surface CD14 after culture with MCSF for 3 days is not due to shedding and these monocytes maintain intracellular levels of CD14. (A) Supernatants from monocytes cultured for 3 days with MCSF were assayed for soluble CD14 (sCD14) by ELISA. The percentage of cells expressing cell surface CD14, as analyzed by FACS, and the levels of sCD14 (ng/ml) in the supernatants are shown. (B) Cell surface CD14 is analyzed by FACS in freshly isolated monocytes (Day 0) and monocytes cultured for 3 days non-adherently with MCSF (Day 3) and intracellular CD14 is analyzed in cells permeabilized before staining with anti-CD14-FITC.

Fig. 4

CD14⁺ monocytes, both freshly isolated and after 3 days of culture with MCSF, express cell surface CCR2. A representative histogram of PE immunofluorescence in freshly isolated CD14⁺ monocytes (Day 0) and in monocytes cultured for 3 days non-adherently with MCSF (Day 3) incubated with CCR2-PE or IgG2b-PE (isotype matched negative control) antibodies.

Fig. 5

A CD14⁺/CD16⁺/CD11b⁺/Mac387⁺ monocyte subpopulation preferentially transmigrates across the BBB. CD14⁺ monocytes cultured non-adherently with MCSF for 3 days were designated the start population and analyzed by four color FACS with CD14-APC, CD16-PE-Cy7, Mac387-FITC and CD163-PE or CD11b-PE antibodies. A representative dot plot of CD14-APC and CD16-PE-Cy7 immunofluorescence is shown, with 100% of gated CD14⁺CD16⁺ cells exhibiting Mac387-FITC immunofluorescence. All CD14⁺CD16⁺ cells were also CD11b⁺ with minimal CD163 immunofluorescence (not shown). Start population cells were added to BBB cocultures with CCL2 for 24 h and transmigrated monocytes were also analyzed by four color FACS. A representative dot plot of CD14-APC and CD16-PE-Cy7 immunofluorescence shows that the vast majority of transmigrated monocytes were CD14⁺CD16⁺. All of the transmigrated CD14⁺CD16⁺ cells were also Mac387⁺ and CD11b⁺ (not shown), with minimal CD163 immunofluorescence (not shown). Values in each quadrant represent the percentage of cells (representative of five independent experiments).

Fig. 6

A gene network generated from microarray analyses of monocytes that transmigrated across the BBB compared to the start population of CD14⁺ monocytes cultured with MCSF for 3 days. One of the top five gene networks identified after analysis of the differentially regulated genes using IPA tools (Ingenuity) was the antigen presentation, cell-mediated immune response, humoral immune response network. Genes are represented as nodes and the intensity of the node color indicates the degree of up (red) or down (green) regulation. Interactions are shown by lines. Lines with arrows represent direct interactions and lines without arrows indicate binding only. Solid lines show reported direct interactions and broken lines show indirect interactions. Functions are indicated by shapes: enzymes (diamonds), cytokines (squares), kinases (triangles), transcription regulators (horizontal ovals), transmembrane receptors (vertical ovals), transporters (trapezoids), complexes (concentric circles), and others (circles). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

HIV infection of freshly isolated monocytes as compared to infection of monocytes cultured for 3 days with MCSF. CD14⁺ monocytes were either infected immediately after isolation (Day 0), or cultured for 3 days non-adherently and then infected (Day 3). Media was collected daily from each culture for 7 days and assayed for p24 by ELISA (representative of three independent experiments).

Fig. 8

A gene network generated from microarray analyses of freshly isolated monocytes (Day 0) as compared to Day 3 non-adherent monocytes. One of the top gene networks identified after analysis of the differentially regulated genes using IPA tools (Ingenuity) was the cell–cell signaling/interactions, cellular movement, immune cell trafficking, cell morphology and neurologic disease network.

Fig. 9

Western blot analysis of enolase 2 and neuropilin in freshly isolated monocytes (Day 0) and Day 3 and Day 6 non-adherent and adherent monocytes. Protein lysates of freshly isolated monocytes and monocytes cultured non-adherently and adherently for 3 and 6 days with MCSF were prepared and analyzed by Western blot for (A) enolase 2 and (B) neuropilin. The blots were stripped and analyzed for GAPDH and densitometric analysis of enolase 2 (C) and neuropilin (D) normalized to GAPDH was performed.