HIV-Derived ssRNA Binds to TLR8 to Induce Inflammation-Driven Macrophage Foam Cell Formation

Even though combined anti-retroviral therapy (cART) dramatically improves patient survival, they remain at a higher risk of being afflicted with non-infectious complications such as cardiovascular disease (CVD). This increased risk is linked to persistent inflammation and chronic immune activation. In this study, we assessed whether this complication is related to HIV-derived ssRNAs inducing in macrophages increases in TNFα release through TLR8 activation leading to foam cell formation. HIV ssRNAs induced foam cell formation in monocyte-derived macrophages (MDMs) in a dose-dependent manner. This response was reduced when either endocytosis or endosomal acidification was inhibited by dynasore or chloroquine, respectively. Using a flow cytometry FRET assay, we demonstrated that ssRNAs bind to TLR8 in HEK cells. In MDMs, ssRNAs triggered a TLR8-mediated inflammatory response that ultimately lead to foam cell formation. Targeted silencing of the TLR8 and MYD88 genes reduced foam cell formation. Furthermore, foam cell formation induced by these ssRNAs was blocked by an anti-TNFα neutralizing antibody. Taken together in MDMs, HIV ssRNAs are internalized; bind TLR8 in the endosome followed by endosomal acidification. TLR8 signaling then triggers TNFα release and ultimately leads to foam cell formation. As this response was inhibited by a blocking anti-TNFα antibody, drug targeting HIV ssRNA-driven TLR8 activation may serve as a potential therapeutic target to reduce chronic immune activation and inflammation leading to CVD in HIV+ patients

Dependence of foam cell formation in MDMs on TLR8 activation by HIV ssRNA.

<p>(<b>A</b>) Intracellular expression of TLR8 in MDMs. MDMs which were incubated with PE-conjugated anti-TLR8 or isotype antibody control. Intracellular expression was determined by flow cytometry. Representative profiles were similar in four independent experiments (n = 4 subjects). (<b>B</b>) Functional silencing of human TLR8 leads to diminution of foam cell formation in MDMs. BODIPY staining of MDMs after pretreatment with TLR8 siRNA or nonsilencing control. Cells were challenged with HIV ssRNA or ssRNA control for 24 h. Bar graph shows integrated fluorescence intensities of BODIPY per cell of the confocal micrographs. Results are representative of four independent experiments with similar results. **p<0.01 compared to NS siRNA + ssRNA41 control.</p

HIV replication and foam cell formation in MDM.

<p><b>A.</b> MDMs were exposed to HIV particles, Ba-L strain (10 ng/0.1 Gag p42/10⁶ cells for 3 h and washed. p24 levels were assayed by ELISA. <b>B.</b> MDMs were infected with HIV for 4 days and foam cell formation was analyzed by BODIPY staining. Data shown is a representative experiment with similar results from four different healthy uninfected subjects. *p<0.01 compared to mock infected control.</p

Foam cell formation is induced by HIV vmiR99 in alveolar macrophages in a dose-dependent manner and is inhibited by antagomir99.

<p>Alveolar macrophages were plated and pretreated with different doses of antagomir99 followed different doses of HIV vmiR99 and incubated for 24/503 and DAPI. Results are representative of four independent experiments with similar results.</p

Induction of foam cell formation in MDMs by HIV ssRNA is dependent on MyD88.

<p>MDMs were pretreated with MyD88 siRNA and nonsilencing control. Cells were challenged with HIV ssRNA or ssRNA control and incubated for 24α by ELISA (<b>A</b>) and BODIPY staining (<b>C</b>). Western blot analysis of MyD88 after gene silencing with the use of MyD88 siRNA and nonsilencing siRNA control. β-actin was used to monitor loading after stripping the membrane (<b>B</b>). Bar graph shows integrated fluorescence intensities of BODIPY per cell of the confocal micrographs (<b>D</b>). A representative blot shows results from one experiment with similar results obtained in four independent experiments.</p

HEK-TLR8 cells take up ssRNA40 (flow cytometry with dual laser excitation).

<p>(<b>A</b>) HEK cells expressing TLR8 were treated with ssRNA40-Alexa 546 followed by fixation, permeablization and staining for TLR8 using anti-TLR8 Alexa 488 conjugate or isotype control IgG1/κ Alexa 488. TLR8 detection is shown along the horizontal axis, and ssRNA40 Alexa 546 detection is shown along the vertical axis. Direct excitation (488 and 561 nm lasers) of both fluorophores (Alexa 488 and Alexa 546) allowed quantitation of cells staining positive for TLR8 (anti-TLR8-Alexa488) and cells that had taken up ssRNA40-Alexa 546. Single-stranded RNA40 binds TLR8 (flow cytometry FRET assay). (<b>B</b>) HEK cells expressing TLR8 were unlabeled or labeled with isotype control antibody (IgG1), anti-TLR8 Alexa 488 conjugate (anti-TLR8) and ssRNA40-Alexa 546 (ssRNA40). Upon excitation at 488 nm, horizontal axes depict Alexa 488 emission (530 nm), and vertical axes show FRET emission (575 nm). Detection of TLR8 is shown in green. Detection of ssRNA binding by TLR8 was measured using emission by the FRET acceptor, Alexa 546 (red). Alexa 546 fluorescence was also observed (D–F, gold). A representative experiment is shown from three independent experiments with similar results.</p