HIV Impairs Alveolar Macrophage Function via MicroRNA-144-Induced Suppression of Nrf2

Abstract

Background: Despite anti-retroviral therapy, HIV-1 infection increases the risk of pneumonia and causes oxidative stress and defective alveolar macrophage (AM) immune function. We have previously determined that HIV-1 proteins inhibit antioxidant defenses and impair AM phagocytosis by suppressing nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Given its known effects on Nrf2, we hypothesize miR-144 mediates the HIV-1 induced suppression of Nrf2.

Methods: Primary AMs isolated from HIV-1 transgenic (HIV-1 Tg) rats and wild type littermates (WT) as well as human monocyte-derived macrophages (MDMs) infected ex vivo with HIV-1 were used. We modulated miR-144 expression using a miR-144 mimic or an inhibitor to assay its effects on Nrf2/ARE activity and AM functions in vitro and in vivo.

Results: MiR-144 expression was increased in AMs from HIV-1 Tg rats and in HIV-1-infected human MDMs compared to cells from WT rats and non-infected human MDMs, respectively. Increasing miR-144 with a miR-144 mimic inhibited the expression of Nrf2 and its downstream effectors in WT rat macrophages and consequently impaired their bacterial phagocytic capacity and H₂O₂ scavenging ability. These effects on Nrf2 expression and AM function were reversed by antagonizing miR-144 ex vivo or in the airways of HIV-1 Tg rats in vivo, but this protection was abrogated by silencing Nrf2 expression.

Conclusions: Our results suggest that inhibiting miR-144 or interfering with its deleterious effects on Nrf2 attenuates HIV-1-mediated AM immune dysfunction and improves lung health in individuals with HIV.

Keywords: Alveolar macrophage; HIV-1 transgenic rat; MIR-144; Nrf2.

Figure 1.. HIV-1 viral proteins induced miR-144 expression in macrophages.

(A) miR-144 was measured by RT-qPCR in the primary alveolar macrophages derived from WT or HIV-1 Tg rats, n=7 or (B) in human MDMs infected with HIV-1 for 8 days, n=4. Student’s t-test was used for the single comparisons shown in each panel using Prism (GraphPad, San Diego, CA). In each panel, the data are shown as mean ± SEM; *p<0.05 compared to WT (panel A) and to Control (panel B).

Figure 2.. miR-144 mimic decreased Nrf2 activity in the alveolar macrophages in vitro.

A. Primary alveolar macrophages (AM) were isolated from WT rats and transfected with 5 nM of either the miRNA mimic negative control or the miR-144 mimic for 3 days; n=3-4 wells. *P<0.05 compared to mimic negative control (p<0.05). B. NR8383 cells were transfected with 10 nM either the miRNA mimic negative control or the miR-144 mimic with the Cignal Reporter was also added. Forty-eight hours later the cells (n=12 wells in each condition) were lysed and Nrf2/ARE activity was quantified by luciferase activity. Student’s t-test was used for single comparisons using Prism (GraphPad, San Diego, CA). In each panel the data shown are the means ± SEM and *P<0.05 compared to cells treated with miRNA the mimic negative control.

Figure 3.. Increasing miR-144 impairs the alveolar macrophages function in the in vitro.

Primary alveolar macrophages (AM) were isolated from WT rats and treated with either miRNA mimic negative control or miR-144 mimic (10 nM). A. During the final 2 hours of culture, pHrodo Rad S. aureus BioParticles conjugates were added and images were captured by Olympus Fluorescent microscope and analyzed by Image J. Five images were taken from each well (150 – 160 cells per treatment); n=4-5 wells in each condition. B. H₂O₂ scavenging as quantified by the Amplex Red assay; n=4-5 wells in each condition. Student’s t-test was used for the single comparisons shown in each panel using Prism (GraphPad, San Diego, CA). Data shown as mean ± SEM; *P<0.05 compared to cells treated with miRNA mimic negative control.

Figure 4.. Delivering a miR-144 inhibitor into the airway of HIV-1 transgenic rats in vivo increases the expression of Nrf2 and Nrf2-dependent antioxidants in their alveolar macrophages.

Primary alveolar macrophages (AM) were isolated from HIV-1 Tg rats treated with either the miRNA inhibitor control (CTL) or the miR-144 inhibitor (50 nM ×3) for 1 week (2 female and 2 male rats in each group). AM were then analyzed for gene expression by qRT-PCR (panel A) and protein expression by western immunoblotting (panel B). Student’s t-test was performed for single comparisons. Data are presented as mean ± SEM (n=4 rats); *P<0.05 increased compared to AM from rats treated with the miRNA inhibitor control.

Figure 5.. Delivering a miR-144 inhibitor into the airway of HIV-1 transgenic rats in vivo restores the phagocytic function in their alveolar macrophages.

Primary alveolar macrophages (AM) were isolated from wild type (WT) rats and HIV-1 Tg rats treated with either miRNA inhibitor control or miR-144 inhibitor in vivo as in Figure 4. The AMs were then cultured for 2 hours with pHrodo Rad S. aureus BioParticle Conjugates and phagocytosis was assessed using an Olympus Fluorescent microscope and quantifying the percentage of AMs with visible bacterial internalization using ImageJ software. 10-15 images/4 rats with 30-50 cells/field were analyzed. One-way ANOVA with Newman-Keuls post-test was performed for multiple comparisons and the data are presented as mean ± SEM; *P<0.05 decreased compared to AM from WT rats and from HIV-1 Tg rats treated with the miR-144 inhibitor.

Figure 6.. miR-144 inhibition restored phagocytic function in alveolar macrophages from HIV-1 Tg rats, and this protection was blocked by silencing Nrf2 expression.

Primary alveolar macrophages (AMs) were isolated from WT and HIV-1 Tg rats. AMs from HIV-1 Tg rats were transfected ex vivo with either a control inhibitor or a miR-144 inhibitor ± Nrf2 RNA silencing (siNrf2) and then incubated with pHrodo Rad S. aureus BioParticles Conjugates as before. Twenty-four hours later, cells were extracted for analysis of Nrf2 mRNA expression (n=6 wells; panel A) and phagocytosis images (3-4 wells/treatment) were captured using an Olympus Fluorescent microscope and analyzed by Image J (150 – 200 cells per treatment; panel B). One-way ANOVA with Newman-Keuls post-tests was performed for these multiple comparisons. Data are presented as mean ± SEM; *P<0.05 decreased compared to AM from WT rats.