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. 2021 Jul 1:11:535352.
doi: 10.3389/fcimb.2021.535352. eCollection 2021.

Human Beta-Defensin 2 and 3 Inhibit HIV-1 Replication in Macrophages

Jennifer P Bharucha  1 , Lingling Sun  1 , Wuyuan Lu  1   2 , Suzanne Gartner  1   3 , Alfredo Garzino-Demo  1   4   5
Affiliations

Human Beta-Defensin 2 and 3 Inhibit HIV-1 Replication in Macrophages

Jennifer P Bharucha et al. Front Cell Infect Microbiol. .

Abstract

Human beta-defensins (hBDs) are broad-spectrum antimicrobial peptides, secreted by epithelial cells of the skin and mucosae, and astrocytes, which we and others have shown to inhibit HIV-1 in primary CD4+ T cells. Although loss of CD4+ T cells contributes to mucosal immune dysfunction, macrophages are a major source of persistence and spread of HIV and also contribute to the development of various HIV-associated complications. We hypothesized that, besides T cells, hBDs could protect macrophages from HIV. Our data in primary human monocyte-derived macrophages (MDM) in vitro show that hBD2 and hBD3 inhibit HIV replication in a dose-dependent manner. We determined that hBD2 neither alters surface expression of HIV receptors nor induces expression of anti-HIV cytokines or beta-chemokines in MDM. Studies using a G-protein signaling antagonist in a single-cycle reporter virus system showed that hBD2 suppresses HIV at an early post-entry stage via G-protein coupled receptor (GPCR)-mediated signaling. We find that MDM express the shared chemokine-hBD receptors CCR2 and CCR6, albeit at variable levels among donors. However, cell surface expression analyses show that neither of these receptors is necessary for hBD2-mediated HIV inhibition, suggesting that hBD2 can signal via additional receptor(s). Our data also illustrate that hBD2 treatment was associated with increased expression of APOBEC3A and 3G antiretroviral restriction factors in MDM. These findings suggest that hBD2 inhibits HIV in MDM via more than one CCR thus adding to the potential of using β-defensins in preventive and therapeutic approaches.

Keywords: APOBEC3A; APOBEC3G; CCRs; HIV-1; human β-defensin 2; macrophages.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Human β-defensins inhibit HIV-1 replication in macrophages in vitro. MDM were infected with HIV-1BaL. After virus removal and washing, hBD1, hBD2, and hBD3 (4.7 µM) (A) or increasing concentrations of hBD2 (0-23.3 µM) (B) were added to cultures. Cells were pretreated with AZT as control. Infection was monitored by assaying supernatants for HIV p24 production by ELISA at the times indicated. Data are presented as mean ± SEM of triplicates. Representative experiment, n=3. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 between treatment and control groups determined with unpaired two-tailed t test. (C) ß-defensins are not toxic to macrophages at the concentrations that inhibit HIV-1. Cells treated with hBDs were tested using MTS assay. Cells were cultured in triplicate in 96-well plates for 3 days in the presence or absence of β-defensins; MTS mix was added and incubated 1 to 4 hrs prior to spectrophotometric absorbance readings at 490 nm. Triplicate readings were averaged (± SEM) and percentage OD ratios of treated/control cells were calculated. (D) hBD2 inhibit infection of MDM with a transmitted-founder HIV strain. MDM were infected with transmitter-founder virus AD17. After virus removal and washing, hBD2 at concentrations indicated above were added to cultures. HIV p24 release in supernatants was monitored by ELISA at the time indicated, and % inhibition was calculated as % of HIV p24 production from untreated MDM. Data are presented as mean ± SEM of triplicates, N=3.
Figure 2
Figure 2
hBD2 does not alter surface expression of HIV receptors on macrophages. MDM were cultured in the absence (black solid lines) or presence (black dotted lines) of hBD2 for different times. The surface expression of CD4, CCR5, and CXCR4 was assessed by flow cytometry as described in Materials and Methods. Data analyzed using FlowJo software. Left, middle, and right panels show staining of CD4, CCR5, and CXCR4, respectively. Isotype-matched control antibodies are shown in grey. The x-axis and y-axis show fluorescence intensity and cell count, respectively. Representative experiment, n=2.
Figure 3
Figure 3
hBD2 suppresses HIV-1 at an early post-entry stage. (A) Single-cycle infection of MDM. Cells were infected with HIV-luciferase pseudotyped with AMLV envelope. After infection, cells were incubated 3 days in presence or absence of hBD2. Subsequently, cells were lysed and luciferase activity was measured. Percentage of inhibition was calculated for treated infected cells in reference to untreated infected cells. Data are presented for independent experiments from 4 different donors. (B) hBD2 inhibits accumulation of early reverse transcription products of HIV-1. MDM were challenged with DNase I-treated HIV-1BaL. Post-infection, hBD2 was added to the cultures. Total cellular DNA was isolated at the indicated time points and copies of LTR/RU5 products of reverse transcription were measured in triplicate by real-time PCR. Readings were averaged ± SEM and are presented as copies per million cells; log-scale graph. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 between treatment and control groups determined with unpaired two-tailed t test. Data are presented for independent experiments from 4 different donors and (C) summary graph of the average inhibition for the four donors shown in 3B was determined as the percentage of HIV-1 DNA copies in treated infected cells in reference to untreated infected cells.
Figure 4
Figure 4
hBD2 inhibits HIV-1 in macrophages post-entry via Gi-protein-mediated signaling. MDM were infected with single-cycle HIV-luciferase virus pseudotyped with AMLV envelope followed by pretreatment with or without PTx (100 ng/ml). Cells were then incubated 3 days in presence or absence of hBD2. Subsequently, cells were lysed and luciferase activity was measured. Percentage of inhibition was calculated for treated infected cells in reference to untreated infected cells. Data are presented for independent experiments from 3 different donors.
Figure 5
Figure 5
CCR6 is expressed on macrophages. Untreated, uninfected MDM were harvested and stained for flow cytometry analysis of CCR6 as described in Materials and Methods. Data analyses were performed using FlowJo software. Forward scatter dot plots show the fluorescence and percentage of cells positive for CCR6 as compared to the respective isotype-matched control. A representative of each type (A) CCR6-, (B) CCR6+/-, and (C) CCR6+ is shown. (D) Median Fluorescence Intensity (MFI) values for CCR6 surface expression (as compared to MFI for isotype control) on MDM from different donors designated as either CCR6-, CCR6+/-, or CCR6+. Data are presented as median and range of MFI values. Each dot represents one donor. ****P < 0.0001 between CCR6+/- and CCR6- or CCR6+ and CCR6- determined with unpaired two-tailed t test. (E) Immunoblot analysis of CCR6 on MDM from 3 donors, 1 day and 10 days of tissue culture without treatment. JKT-FT CCR6 GFP cell line lysate in first lane from left is used as a positive control; the second lane is empty; subsequent lanes are donors #1-3, at day 1 and day 10 of tissue culture.
Figure 6
Figure 6
hBD2 can signal via more than one receptor type on macrophages. (A) Neutralization of CCR2 rescues HIV-1 infection. MDM were infected with HIV-1BaL. Cells were pretreated with AZT as control. Post-infection, infected untreated cells were pretreated with pharmacological antagonist RS102895 or DMSO control for 2 hrs followed by culture in presence or absence of hBD2 to the cultures. Infection was monitored by assaying supernatants for HIV p24 production by ELISA at the times indicated. Data are presented as mean ± SEM of triplicates. *P < 0.05 between treatment and control infection determined with unpaired two-tailed t test. Representative experiment, n=2. (B) hBD2 signals via an as yet unidentified receptor. MDM, that were CCR2- CCR6- (by FACS staining), prior to start of infection, were infected with HIV-1BaL. Cells were pretreated with AZT as control. Post-infection, cells were cultured in presence or absence of hBD2. Infection was monitored by p24 ELISA. Data are presented as mean ± SEM of triplicates. **P < 0.005, ****P < 0.0001 between treatment and control infection determined with unpaired two-tailed t test. (C) CCR2 and CCR6 surface expression levels vary with time. Uninfected, untreated cells from the donor used in (B) were harvested and stained for flow cytometry analysis as described in Materials and Methods. Data analyzed using FlowJo software. Forward scatter dot plots show the fluorescence and percentage of cells positive for CCR2 and CCR6 at Day 0 (grey) and Day 23 (black) as compared to the respective isotype-matched controls.
Figure 7
Figure 7
hBD2 upregulates APOBEC3G and/or APOBEC3A in macrophages. (A) APOBEC3G and APOBEC3A expression in response to hBD2. MDM were treated with hBD2 for indicated times and mRNA levels were assessed by quantitative real-time RT-PCR. The data was normalized to 18S ribosomal RNA. Triplicate measurements were used to calculate fold change as described in Materials and Methods. Data are presented as fold change in treated samples compared to untreated samples at matched time points. Data are for independent experiments from different donors. Analyses of APOBEC3G (B) and APOBEC3A (C) protein levels in response to hBD2. MDM were treated with hBD2 for various times and cell lysates were used to detect APOBEC3G and APOBEC3A proteins by western blotting. ß-actin and GAPDH serve as load controls. Representative experiment, n=3.
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