Regulation of pattern recognition receptors by the apolipoprotein A-I mimetic peptide 4F

Abstract

Objective: The apolipoprotein A-I (apoA-I) mimetic peptide 4F favors the differentiation of human monocytes to an anti-inflammatory phenotype and attenuates lipopolysaccharide (LPS)-induced inflammatory responses. We investigated the effects of LPS on the Toll-like receptor (TLR) signaling pathway in 4F-differentiated monocyte-derived macrophages.

Methods and results: Monocyte-derived macrophages were pretreated with 4F or vehicle for 7 days. 4F downregulated cell-surface TLRs (4, 5, and 6) as determined by flow cytometry. 4F attenuated the LPS-dependent upregulation of genes encoding TLR1, 2, and 6 and genes of the MyD88-dependent (CD14, MyD88, TRAF6, interleukin-1 receptor-associated kinase 4, and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta) and MyD88-independent (interferon regulatory factor 3, TANK-binding kinase 1, and Toll-interleukin 1 receptor domain-containing adaptor-inducing interferon-β) pathways as determined by microarray analysis and quantitative reverse transcriptase polymerase chain reaction. Functional analyses of monocyte-derived macrophages showed that 4F reduced LPS-dependent TLR4 recycling, phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, activation and translocation of nuclear factor-κB and inhibited the secretion of tumor necrosis factor-α and interleukin-6 induced by LPS or lipoteichoic acid. These changes were associated with depletion of cellular cholesterol and caveolin, components of membrane lipid rafts.

Conclusions: These data suggest that disruption of rafts by 4F alters the assembly of TLR-ligand complexes in cell membranes and inhibits proinflammatory gene expression in monocyte-derived macrophages, thus attenuating the responsiveness of macrophages to LPS.

Figure 1. 4F treatment down-regulates cell-surface TLRs in MDMs

Expression of TLRs 1, 2, 4–6 was monitored in 4F- and vehicle-treated MDMs by flow cytometry. (A) Histograms showing surface expression of TLRs in MDMs treated with vehicle (black line) or 4F (dotted line). An isotype control (gray shaded) was run for each sample. The numbers indicate the MFI for each histogram. (B) 4F-treated cells (white bars) expressed reduced levels of TLRs 4 and 5 per cell as determined by MFI compared to vehicle-treated cells (black bars). (C) 4F-treated cells (white bars) expressed significantly reduced levels of TLRs 4–6 as determined by percent cells expressing TLR compared to vehicle-treated cells (black bars). Values are means ± SEM (n=3; *p<0.05 compared to vehicle treatment).

Figure 2. 4F treatment down-regulates LPS-mediated expression of genes of the MyD88-dependent and MyD88-independent TLR signaling pathway

MDMs were pre-treated with 4F (white bars) or vehicle (black bars) for 7days and then stimulated with LPS (1µg/ml) for 18hrs. RNA was extracted and transcriptional profiling was carried out using the Affymetrix Human Gene ST1.0 array. Absolute mRNA levels obtained were normalized to unstimulated MDMs, pre-treated with vehicle. Data are means ± SEM (n=4; p<0.05 across all treatments). (A) Effect of LPS on the expression of surface TLRs (B) Effect of LPS on adaptor molecules (C) Effect of LPS on enzymes of the TLR signaling pathway and (D) Effect of LPS on expression of genes of the MyD88-independent pathway. (E) mRNA levels of key genes in the TLR pathway in MDMs pretreated with vehicle (black bars) or 4F (white bars) for 7days and stimulated with LPS (1µg/ml) for 18hrs. The data are normalized to β-actin. Values are means ± SEM (n=4).

Figure 3. 4F pre-treatment decreases the activation of NF-κBp65 and nuclear translocation of activated NF-κBp50 in LPS-treated MDMs

(A) Western blots for IκBα and p-IκBα. 4F treatment decreased IκBα and inhibited LPS-stimulated p-IκBα. (B) Phosphorylation of NF-κBp65 in MDMs treated with LPS (1µg/ml) for 5min was determined by flow cytometry. Representative histograms show an increase in p-NF-κBp65 on LPS stimulation (black line) compared to resting cells (dotted line) in vehicle-treated MDMs (upper panel). Such an increase was not seen in 4F-treated cells (lower panel). Gray histograms represent cells treated with control antibody. (C) In the absence of LPS, p-NF-κBp65 was similar in MDMs pre-treated with vehicle (V) and 4F. Addition of LPS to vehicle pre-treated cells (V+LPS) revealed a significant (*p<0.05) increase in p-NF-κBp65 compared to vehicle alone. In contrast, p-NF-κBp65 was not increased by LPS in 4F pre-treated cells (4F+LPS). Results from five independent donors are presented. (D) Data from (B) expressed as MFI of pNF-κBp65. 4F-treated MDMs show significant decrease in MFI compared to vehicle treated MDMs (n=5, *p<0.05). (E) Nuclear transport of activated NF-κBp50 was quantitated by ELISA in MDMs treated with LPS (1µg/ml) for 60min. 4F pre-treatment significantly decreased nuclear translocation of activated NF-κBp50. Data are means ± SEM (n=5; *p<0.05 compared to unstimulated vehicle pre-treated cells, ^#p<0.05 compared to LPS-stimulated vehicle pre-treated cells). (F) and (G) MDMs pre-treated with 4F (white bars) or vehicle (black bars) were treated with LPS (1µg/ml) or LTA (1µg/ml) for 6hr. IL-6 (F) and TNF-α (G) levels in conditioned media were determined by ELISA. Data are means ± SEM (n=6; *p<0.05 compared to respective vehicle controls).

Figure 4. 4F decreases cell-associated cholesterol and caveolin-1, markers of lipid rafts, in MDMs

(A) 4F increases cholesterol efflux from MDMs. (B) 4F decreases cell-associated cholesterol (C) Immunoblot for caveolin-1 showing decreased caveolin-1 expression in 4F pre-treated MDMs compared to vehicle-treated (V) cells. (D) Analysis of band intensities revealed a significant reduction in caveolin-1 in 4F-treated MDMs. Data are means ± SEM (n=4, *p<0.05 compared to vehicle treatment).

Figure 5. 4F pre-treatment impairs LPS-induced TLR4 recycling

Vehicle (black circles) or 4F-treated (open circles) MDMs were stimulated with LPS (1µg/ml) for 0, 5, 15, 30 60 and 120min. The MFI for cell surface TLR4 was determined at each time point. MDMs from three different donors were examined. A representative graph of MFI for TLR4 as a function of time is shown. LPS stimulation of vehicle-treated MDMs shows the well established decrease of surface TLR4 (MFI) at early time points and an increase at later time points indicating TLR4 recycling. 4F treatment, however, not only decreases overall TLR4 (MFI) but also abolishes the cell’s capacity to recycle TLR4. **p=0.002 compared to vehicle treated MDMs.

Figure 6. Proposed mechanism for 4F-mediated inhibition of the TLR signaling pathway

LPS induces the recruitment of TLR4 to lipid rafts which forms a functional complex with CD14 leading to the activation of NF-κB. 4F treatment of cells disrupts lipid rafts thus preventing the formation of the TLR4-CD14 complex and activation of cells by LPS. LPS may still bind to phospholipids (PLs) in the membrane but due to disruption of the lipid rafts, a functional ligand-receptor complex formation is impaired.