A consensus surface activation marker signature is partially dependent on human immunodeficiency virus type 1 Nef expression within productively infected macrophages

Abstract

Background: The high prevalence of HIV-associated comorbidities including neurocognitive disorder, high levels of residual inflammatory mediators in the plasma and cerebrospinal fluid and the resurgence of HIV replication upon interruption of antiviral treatment in HIV-1 infected individuals, strongly suggests that despite therapy HIV persists in its cellular targets which include T-lymphocytes and cells of the myeloid lineage. These reservoirs present a major barrier against eradication efforts. Knowledge of the molecular mechanisms used by HIV to modulate innate macrophage immune responses and impair viral clearance is quite limited. To explore the role of HIV in potentially modulating macrophage function through changes in protein expression, we used single-cell analyses with flow cytometry to determine whether, in unpolarized cultures, macrophage surface marker phenotype was altered by HIV infection in a manner that was independent of host genetic background.

Results: These analyses revealed that at several time points post-infection, GFP + HIV-infected macrophages were significantly enriched in the CD14+ fraction (3 to 5-fold, p = .0001) compared to bystander, or uninfected cells in the same culture. However, the enrichment and higher levels of CD14 on HIV expressing macrophages did not depend on the production of HIV Nef. Sixty to eighty percent of macrophages productively infected with HIV after day 28 post-infection were also enriched in the population of cells expressing the activation markers CD69 (2 to 4-fold, p < .0001) and CD86 (2 to 4-fold, p < .0001 ) but suppressed amounts of CD68 (3 to 10-fold, p < .0001) compared to bystander cells. Interestingly, there was no enrichment of CD69 on the surface of HIV producing cells that lacked Nef or expressed a variant of Nef mutated in its SH3-binding domain.

Conclusions: These findings suggest that HIV actively regulates the expression of a subset of surface molecules involved in innate and inflammatory immune signaling in primary human macrophages through Nef-dependent and Nef-independent mechanisms acting within productively infected cells.

Figure 1

Analysis of monocyte CD14/CD16 subpopulations in density gradient purified monocytes. Ficoll purified buffy coats from normal blood donors were subjected to a second percoll gradient to enrich for monocytes and analyzed by flow cytometry for CD14/CD16 subpopulations. (Top row): The forward (FSC) and side scatter (SSC) of leukocytes after gradient purification and gating with the isotype controls are shown. (Second row): The CD14 fraction of four different donors plotted against SSC is shown and the percentage of CD14+ monocytes is given. (Bottom row): The CD14/CD16 population of four different donors is shown. The percentage of CD14 + CD16+ monocytes is given and the mean fluorescent intensity is indicated in the top right corner of each panel.

Figure 2

Variable expression of typical receptors on CD14+ monocytes from normal blood donors. Monocytes were double-labeled for CD14 and the indicated markers and the percentage of positively labeled cells in the CD14+ fraction was quantified by flow cytometry. Two of the donors, Do4787 (brown) and Do3528 (deep blue) were used in the longitudinal analyses of HIV-infected macrophages (Figure 3).

Figure 3

Longitudinal analyses of monocyte-derived macrophages (MDM) with HIV-GFP. Monocytes from three different donors, Do4787, Do3528 and Do9432 were differentiated in RPMI 1640 complete medium without the addition of any exogenous cytokines. At day 7 post-differentiation, macrophages were infected with HIV-GFP and GFP fluorescence was quantified by flow cytometry at 7–8 day intervals. The forward (FSC) and side scatter (SSC) and GFP + versus SSC, is shown in the panels. Fluorescent images of representative HIV-GFP infected MDM cultures at the indicated time points are shown.

Figure 4

Quantification of GFP + HIV-infected macrophages and HIV receptor and coreceptor expression. (A) The percentage and mean fluorescent intensity (MFI) of GFP + macrophages for each donor (Do4787, Do3528 and Do9432) at the indicated time points after infection as quantified by flow cytometry is shown (mean and standard deviation). (B) The percentage of the macrophages expressing the HIV receptor CD4 and coreceptor CCR5 was determined at the indicated time points. ***p < .0001.

Figures 5

Longitudinal analyses of surface marker expression in mock, HIV-GFP infected and bystander macrophages. Mock- and HIV-GFP infected macrophages were harvested at 7–8 day intervals and stained for flow cytometry for the indicated markers. The day 0 time point is given only for the Mock sample and represents the day of infection. The uninfected or GFP negative cells in the HIV-GFP culture were designated as the bystander MDM (BYSTD). For each group the combined mean percentage of positively stained cells and standard deviations from all three donors is shown. Statistical comparisons were determined by one-way ANOVA with Tukey’s test for multiple comparisons and significance of p < .05. For the receptors shown, no significant differences were found. For the day 43 pi time point, values shown are from a single donor, Do9432.

Figure 6

Flow cytometric analyses for CD14, CD69, CD86 and CD68 expression on the surface of HIV-GFP infected MDM. Representative scatter plots are show for the MDM donor data summarized in Figure 7. Left column. Forward (FSC) and side scatter (SSC) for mock-infected and HV-GFP gated for the live cells (R1) is shown and the percentage of live cells indicated. Second column from left. MDM in the live cell gate (R1) were analyzed by SSC and in the FL1 channel to identify the GFP + HIV-infected MDM (gate R2) and the GFP- or bystander cells (gate R3). Mock-infected MDM stained with isotype control antibodies were used for gating as indicated in the top row. The cells in gates R2 and R3 were then analyzed in the FL4 channel for the indicated surface markers. The percentage of positive cells and the mean fluorescent intensity of staining are given.

Figure 7

Surface markers expression on mock, HIV-GFP + or bystander macrophages from Do4787, Do3528 and Do9432. Mock- and HIV-GFP infected macrophages were harvested at 7–8 day intervals and stained for flow cytometry for CD14, CD68, CD69, and CD86. The day 0 time point is given only for the Mock sample and represents the day of infection. The uninfected or GFP negative cells in the HIV-GFP culture were designated as the bystander MDM (BYSTD). For each group the combined mean percentage of positively stained cells and standard deviations from all three donors is shown. Statistical comparisons were determined by one-way ANOVA with Tukey’s test for multiple comparisons and significance of p < .05.

Figure 8

Flow cytometric analyses for CD14, CD69, CD86 and CD68 expression on the surface of MDM infected with the HIV-Nef+, HIV-Nef- and HIV-Nef^P7480 GFP reporter viruses. Representative scatter plots are show for the MDM donor data summarized in Figure 9. For each panel A-D: Top row: mock-infected MDM stained with isotype controls or CD14-APC antisera. Left column. Forward (FSC) and side scatter (SSC) for the indicated infected MDM gated for the live cells (R1) is shown and the percentage of live cells indicated. Second column from left. MDM in the live cell gate (R1) were analyzed by SSC and in the FL1 channel to identify the GFP + HIV-infected MDM (gate R2) and the GFP- or bystander cells (gate R3). The percentage of GFP + cells is indicated in the lower right corner and of bystander cells in the upper left as indicated by the arrow. Third and fourth columns from left. The cells in gates R2 and R3 were then analyzed in the FL4 channel for CD14 (Figure 8A), CD69 (Figure 8B), CD86 Figure 8C and CD68 (Figure 8D). The percentage of positive cells and the mean fluorescent intensity of staining are given.

Figure 9

HIV Nef-dependent and Nef-independent modulation of HIV-GFP macrophage cell surface phenotype. Nine donor MDM infected with HIV-GFP, HIV-GFP-Nef^-, HIV-GFP-Nef P7480, or mock-treated were harvested between day 15–28 post-infection and analyzed by flow cytometry with anti-CD14-PE and CD69-APC or CD68-PE and CD86-APC as shown in the panels at the upper left. Statistical comparisons were determined by one-way ANOVA with Tukey’s test for multiple comparisons and significance of p < .05, *** < .0001. Images of macrophage monolayers of nine donors at day 15 pi is shown. Figure 9A-I.