CR4 Signaling Contributes to a DC-Driven Enhanced Immune Response Against Complement-Opsonized HIV-1

Abstract

Dendritic cells (DCs) possess intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. In turn, HIV-1 has evolved strategies to evade innate immune sensing by DCs resulting in suboptimal maturation and poor antiviral immune responses. We previously showed that complement-opsonized HIV-1 (HIV-C) was able to efficiently infect various DC subsets significantly higher than non-opsonized HIV-1 (HIV) and therefore also mediate a higher antiviral immunity. Thus, complement coating of HIV-1 might play a role with respect to viral control occurring early during infection via modulation of DCs. To determine in detail which complement receptors (CRs) expressed on DCs was responsible for infection and superior pro-inflammatory and antiviral effects, we generated stable deletion mutants for the α-chains of CR3, CD11b, and CR4, CD11c using CRISPR/Cas9 in THP1-derived DCs. We found that CD11c deletion resulted in impaired DC infection as well as antiviral and pro-inflammatory immunity upon exposure to complement-coated HIV-1. In contrast, sole expression of CD11b on DCs shifted the cells to an anti-inflammatory, regulatory DC type. We here illustrated that CR4 comprised of CD11c and CD18 is the major player with respect to DC infection associated with a potent early pro-inflammatory immune response. A more detailed characterization of CR3 and CR4 functions using our powerful tool might open novel avenues for early therapeutic intervention during HIV-1 infection.

Keywords: CD11b; CD11c; HIV-1; complement; dendritic cell.

FIGURE 1

THP1 monocytes can be differentiated into functional DCs. (A) WT THP1 cells were differentiated into DCs and expression of characteristic DC markers CD11c, CD11b, CD18, HLA-ABC, HLA-DR, CD86, DC-SIGN, CD1a, CD83, and HIV-1 receptors CD4, CCR5, and CXCR4 was analyzed using flow cytometry. A representative flow cytometry analysis is shown out of five independent experiments. (B) Phagocytosis of non- and complement-opsonized Beads (Beads, Beads-C) was analyzed in WT THP1 monocytes (blue), WT THP1-DCs (red), and monocyte-derived DCs (green). THP1-DCs (red) exerted similar phagocytosis capacities with respect to Beads and Beads-C compared to monocyte-derived DCs (green), while THP1 monocytes had a very low phagocytic activity (blue). Panel (B) illustrates a FACS analysis from one representative experiment (upper panel) and data from three independent experiments are summarized in panel (B) (lower panel).

FIGURE 2

CD11b-, CD11c-, and CD18 KO THP1 DCs comprise a novel tool to decrypt distinct functions of CR3 and CR4. (A) CD11b, CD11c, or CD18 were stably deleted by using the CRISPR/Cas9 genome editing technology. Top panel: WT THP1 cells differentiated to DCs were used as controls for monitoring expression of CD11b (blue), CD11c (red), and CD18 (green). Bottom panel: CD11b KO THP1-DCs do not express CD11b (blue), CD11c KO THP1-DCs are devoid of CD11c (red), and no CD18 is expressed on CD18 KO THP1-DCs (green). A representative flow cytometric analysis from one out of five experiments is illustrated. (B) Phagocytosis of C-opsonized Beads is hampered in CD11c- (yellow) and CD18 KO (green) THP1-DCs compared to WT (red) and CD11b KO (blue) THP1-DCs. Lowest phagocytosis using non-opsonized Beads was monitored in CD18 KO THP1 DCs (green). A representative flow cytometric analysis out of three independent experiments is depicted.

FIGURE 3

Complement-opsonized HIV-1 effectively infects WT- and CD11b KO THP1-DCs, while productive HIV-1 infection is impaired in CD11c KO THP1-DCs. (A,B) Upon infection of WT THP1 DCs with HIV or HIV-C (25 ng p24/ml) a significantly enhanced productive infection was monitored using HIV-C. Means ± SD from three independent experiments in duplicates are depicted in (A). Differences were analyzed using Unpaired Student’s t-test. In (B) a time-course of WT THP1-DC infection exposed to HIV (lime), HIV-C (orange), HIV-Vpx (green), and HIV-Vpx-C (red) is illustrated. Experiments were repeated three times in duplicates. (C,D) WT- (red), CD11b KO (blue), and CD11c KO (yellow) THP1-DCs were infected with HIV or HIV-C (C) and HIV-Vpx or HIV-C-Vpx. Uninfected and LPS-incubated THP1-DCs served as controls. Infection experiments were performed in three independent experiments performed in triplicates. Differences were analyzed using GraphPad Prism and one-way ANOVA with Bonferroni post-test.

FIGURE 4

Antiviral signaling pathways are impeded in CD11c-deleted THP1 DCs. (A) Relative p- p-IRF3 protein levels were assessed in WT- (red), CD11b KO (blue), and CD11c KO (yellow) THP1-DCs. Experiments were repeated four times independently and a summary as well as a representative immunoblot are depicted. UI, uninfected. (B) RT-PCR analyses of type I IFN (IFNB) levels in WT-, CD11b KO-, and CD11c KO-THP1 DCs after infection with HIV or HIV-C (BaL, YU-2). Non-infected iDC were used as controls. Data are mean ± SD from four different donors in duplicates. Differences were analyzed by using one-way ANOVA with Bonferroni post-test.

FIGURE 5

CD11c deletion significantly modifies the cytokine profile by decreasing IL-1β production and IL23 expression, while up-regulating IL10 levels. IL-1β production (A), IL6 (B), IL23A (C), and IL10 (D) expression levels were analyzed in WT-, CD11b KO-, CD11c KO-, and CD18 KO THP1-DCs as indicated following infection with HIV or HIV-C. UI cells served as controls. Experiments were repeated thrice in duplicates and RT-PCR analyses were performed. Differences were analyzed by using GraphPad Prism software and one-way ANOVA with Bonferroni post-test. *p < 0.01, **p < 0.001, ***p < 0.0001.