Complement Potentiates Immune Sensing of HIV-1 and Early Type I Interferon Responses

Abstract

Complement-opsonized HIV-1 triggers efficient antiviral type I interferon (IFN) responses in dendritic cells (DCs), which play an important role in protective responses at the earliest stages in retroviral infection. In contrast, HIV-1 suppresses or escapes sensing by STING- and MAVS-associated sensors. Here, we identified a complement receptor-mediated sensing pathway, where DCs are activated in CCR5/RLR (RIG-I/MDA5)/MAVS/TBK1-dependent fashion. Increased fusion of complement-opsonized HIV-1 via complement receptor 4 and CCR5 leads to increased incoming HIV-1 RNA in the cytoplasm, sensed by a nonredundant cooperative effect of RIG-I and MDA5. Moreover, complement-opsonized HIV-1 down-modulated the MAVS-suppressive Raf-1/PLK1 pathway, thereby opening the antiviral recognition pathway via MAVS. This in turn was followed by MAVS aggregation and subsequent TBK1/IRF3/NF-κB activation in DCs exposed to complement- but not non-opsonized HIV-1. Our data strongly suggest that complement is important in the induction of efficient antiviral immune responses by preventing HIV-1 suppressive mechanisms as well as inducing specific cytosolic sensors. IMPORTANCE Importantly, our study highlights an unusual target on DCs-the α chain of complement receptor 4 (CR4) (CD11c)-for therapeutic interventions in HIV-1 treatment. Targeting CD11c on DCs mediated a potent antiviral immune response via clustering of CR4 and CCR5 and subsequent opening of an antiviral recognition pathway in DCs via MAVS. This novel finding might provide novel tools for specifically boosting endogenous antiviral immunity via CR4, abundantly expressed on multiple DC subsets.

Keywords: CR4; HIV-1; antiviral immunity; complement; complement receptors; cytosolic sensor; dendritic cell; dendritic cells; human immunodeficiency virus; type I IFN.

FIG 1

HIV-C initiates an efficient type I IFN response after HIV-1 infection in DCs. (a) Real-time RT-PCR analyses of APOBEC3G, IL-15, MX1, ISG15, ISG20, MX2, and RSAD2 mRNAs in moDCs after infection as indicated with various differentially opsonized (HIV and HIV-C) HIV-1 strains. RT-PCR was performed in duplicate for each sample, and data are means and standard deviations (SD) for cells from three donors exposed to HIV-1 BaL. Unpaired Student's t test was performed to analyze statistical significance between HIV and HIV-C. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (b) Microarray analyses of HIV- and HIV-C-exposed DCs after 6 h infection with different strains (BaL, 92BR030, and 92UG037). Cells from four donors exposed to either BaL and 93BR030 or BaL and 92UG037 were analyzed. (c) (Top) Representative immunoblot (IB) analyses of phosphorylated SAMHD1 and ERK1/2 and nonphosphorylated ERK1/2 as a loading control of DCs exposed for short times (5, 15, and 30 min) to differentially opsonized HIV-1 BaL (HIV and HIV-C). IB analyses were repeated in five independent experiments. (Bottom) Quantification of SAMHD1 and ERK1/2 phosphorylation at 4 h after HIV exposure using ImageJ for samples from five donors. (d) (Top) Representative IB analyses of phosphorylated PLK1 and c-Raf-1 (Ser338), and nonphosphorylated ERK1/2 as a loading control of DCs exposed for 4 h to differentially opsonized HIV-1 BaL (HIV and HIV-C). (Bottom) Quantification of PLK-1 and c-Raf-1 phosphorylation at 4 h after HIV exposure using ImageJ for samples from three donors. (e) c-Raf-1 phosphorylation was also analyzed in untreated and HIV-C-exposed CD11c-KO DCs. (Top) Relative c-Raf-1 phosphorylation levels in CD11c-KO DCs from four independent experiments; (bottom) representative IB of p-c-Raf-1 and ERK1/2 as a loading control. Unpaired Student's t test was performed to analyze statistical significance between controls or HIV and HIV-C in all analyses.

FIG 2

HIV-C mediates efficient type I IFN responses via a MAVS/IRF3/NF-κB signaling axis. (a) Representative IB analyses for one donor of full-length MAVS (FL) and mini-MAVS 9 h after infection of DCs with differentially opsonized HIV-1 (HIV and HIV-C) strains BaL and YU-2. (Top) Representative IB using ERK1/2 as a loading control for IB; (bottom) quantitative results from 3 donors. Statistical significance was analyzed using GraphPad Prism software with one-way ANOVA and Tukeýs posttest. (b) MAVS aggregation was shown under nonreducing conditions in cells from three independent donors using 8% acrylamide native gels in DCs left untreated (iDC) or exposed for 9 h to HIV or HIV-C. IB analyses of phosphorylated IRF3 and activated NF-κB, recognizing the NLS of human p65, are shown. Tubulin was used as a loading control. Quantitative analyses of activated NF-κB from 4 donors are also depicted, and statistical significance was analyzed using unpaired Student's t test. (c) (Left) Confocal microscopic analyses of MAVS (orange) and HIV (pink) in iDCs and DCs infected with HIV or HIV-C-mCherry for 9 h. Representative 3D overviews are presented, and the experiment was repeated three times independently. (Right) Spot analyses were performed using RMS spot analysis in the Harmony software (Perkin Elmer), and HIV/MAVS colocalizing spots are illustrated from 5 independent areas and 300 cells in total. Statistical analysis was performed using GraphPad Prism software and unpaired Student's t test. (d) RT-PCR analyses of type I IFN (IFN-β) levels after silencing (siRNA) of MAVS expression in moDCs (MAVS siRNA). A control siRNA and moDCs without siRNA served as controls. Data are means and SD for analyses with cells from 4 donors, done in duplicate. A highly significant reduction in IFN-β was observed in DCs treated with MAVS siRNA and infected with HIV-C (red) compared to controls (control siRNA and no siRNA). One-way ANOVA with Tukeýs posttest was performed (*, P < 0.05; ***, P < 0.001).

FIG 3

HIV-C mediates efficient type I IFN responses via TBK1 phosphorylation. IB analyses of phosphorylated TBK1 after infection of DCs with differentially opsonized HIV-1 (HIV and HIV-C). (Left) Representative IBs from experiments using BaL or YU-2 for infection with α-tubulin as a loading control for IB; (right) quantitative results from 4 donors infected with BaL and YU-2. One-way ANOVA with Tukey’s posttest was performed (**, P < 0.01; ***, P < 0.001).

FIG 4

Incoming viral RNA is the PAMP recognized in DCs upon HIV-C infection. (a and b) Real-time RT-PCR analyses of IFN-β mRNA in moDCs after infection with HIV or HIV-C (BaL and YU-2) or differentially opsonized beads of similar size (a) or VLPs (b). Data are means and SD for analyses of cells from four donors, performed in duplicate. One-way ANOVA with Tukeýs posttest was performed (***, P < 0.001; ****, P < 0.0001) (c) RT-PCR analyses of IFN-β mRNA in moDCs after pretreatment with AZT or efavirenz prior exposure to HIV or HIV-C (BaL and YU-2), VLP, or VLP-C. Data are means and SD for analyses of cells from four donors, performed in duplicate. One-way ANOVA with Tukey’s posttest was performed (****, P < 0.0001). (d) RT-PCR analyses of type I IFN (IFN-β) levels after pretreatment with the cGAS inhibitor RU.521 (20 μM) prior exposure to HIV or HIV-C (BaL and YU-2). Data are means and SD for analyses of cells from three donors, performed in duplicate. One-way ANOVA with Tukey’s posttest was performed (****, P < 0.0001). (e) RT-PCR analyses of IFN-β levels after silencing RIG-I, MDA-5, or NOD2 expression in moDCs (RIG-I siRNA, MDA5 siRNA, and NOD2 siRNA). A control siRNA and moDCs without siRNA served as controls. Data are means and SD for analyses of cells from 4 donors done in duplicate. A highly significant reduction in IFN-β was observed in DCs treated with MAVS siRNA and infected with HIV-C (red) compared to controls. One-way ANOVA with Tukey’s posttest was performed (*, P < 0.05; ****, P < 0.0001). IB analyses of FL and mini-MAVS or RIG-I after DC infection with HIV or HIV-C followed by immunoprecipitation using a MAVS Ab directed against another epitope. MAVS pulldown is shown on the left; input is on the right. The pulldown was repeated three times independently.

FIG 5

CR4 and CCR5 act in concert via rafts to enhance early type I IFN responses in HIV-C-exposed DCs. (a) BlaM-Vpr analyses of HIV and HIV-C in moDCs after 4 h and 24 h infection. Noninfected DCs served as controls. Data are means and SD from four independent experiments using cells from different donors. Unpaired Student's t test was used to characterize differences between HIV and HIV-C (**, P < 0.01). (b) (Left) Representative confocal microscopic analyses (top, xyz stack; bottom, 3D analysis) of HIV (pink), CD11c (green), CCR5 (orange), and nucleus (blue) in DCs infected 1 h with HIV or HIV-C–mCherry. (Right) Quantitative analyses of DC numbers (white plots) and numbers of HIV-CD11c-CCR5 colocalizing spots (gray plots) are illustrated. For each condition, at least 350 cells were analyzed, and data were generated using the RMS spot analysis of Harmony software (Perkin Elmer). (c) (Left) RT-PCR analyses of type I IFN (IFN-β) levels after blocking CCR5 using maraviroc in LPS-, HIV-, HIV-C, VLP- or VLP-C-exposed moDCs. Data are means and SD for analyses of cells from 4 donors, done in duplicate. In HIV-C-exposed DCs, type I IFN levels were highly significantly down-modulated upon CCR5 blocking (**, P < 0.01; ****, P < 0.0001), and this too was associated with a reduced RIG-I signal (middle and right) after IP (MAVS pulldown) and IB using MAVS and RIG-I Abs. Tubulin was used as loading control. A representative IB after IP (middle) and the quantification of the RIG-I signal from three independent experiments (right) are depicted. (d) RT-PCR analyses of type I IFN (IFN-β) levels after blocking CR3 using CD11b or CR4 using CD11c in HIV-infected, HIV-C-infected, or noninfected moDCs. Data are means and SD for analyses of cells from 4 donors in duplicate. One-way ANOVA with Tukey’s posttest was performed (****, P < 0.0001). (e) RT-PCR analyses of type I IFN (IFN-β) levels after disrupting rafts using β-cyclodextrin in HIV-infected, HIV-C-infected, or noninfected moDCs. Data are means and SD for analyses with cells from 4 donors, done in duplicate. One-way ANOVA with Tukey’s posttest was performed (**, P < 0.01; ****, P < 0.0001).

FIG 6

Knocking out CR4 eliminates efficient early type I IFN responses. IB analyses of IRF3 and NF-κB phosphorylation after infection of WT, CD11b-KO, and CD11c-KO THP1 DCs with differentially opsonized HIV-1 (HIV and HIV-C) strains BaL and YU-2. (Left) Quantitative results; (right) representative IB with α-tubulin as a loading control for IB. Data are means and SD for 4 experiments. **, P < 0.01.