ABCA1 protein enhances Toll-like receptor 4 (TLR4)-stimulated interleukin-10 (IL-10) secretion through protein kinase A (PKA) activation

Abstract

Background: ABCA1 is known to suppress proinflammatory cytokines.

Results: ABCA1 activates PKA and up-regulates anti-inflammatory cytokine IL-10. Elevated PKA transforms macrophages to M2-like phenotype. Disrupting lipid rafts by statins MCD, and filipin recuperates ABCA1 phenotype and likely functions downstream of ABCA1.

Conclusion: By modulating cholesterol, ABCA1 activates PKA. This generates M2-like macrophages.

Significance: ABCA1 does not simply suppress inflammatory response. It promotes M2-like activation and facilitates resolution. Nonresolving inflammatory response from macrophages is a major characteristic of atherosclerosis. Macrophage ABCA1 has been previously shown to suppress the secretion of proinflammatory cytokine. In the present study, we demonstrate that ABCA1 also promotes the secretion of IL-10, an anti-inflammatory cytokine critical for inflammation resolution. ABCA1(+/+) bone marrow-derived macrophages secrete more IL-10 but less proinflammatory cytokines than ABCA1(-/-) bone marrow-derived macrophages, similar to alternatively activated (M2) macrophages. We present evidence that ABCA1 activates PKA and that this elevated PKA activity contributes to M2-like inflammatory response from ABCA1(+/+) bone marrow-derived macrophages. Furthermore, cholesterol lowering by statins, methyl-β-cyclodextrin, or filipin also activates PKA and, consequently, transforms macrophages toward M2-like phenotype. Conversely, cholesterol enrichment suppresses PKA activity and promotes M1-like inflammatory response. As the primary function of ABCA1 is cholesterol removal, our results suggest that ABCA1 activates PKA by regulating cholesterol. Indeed, forced cholesterol enrichment in ABCA1-expressing macrophages suppresses PKA activation and elicits M1-like response. Collectively, these findings reveal a novel protective process by ABCA1-activated PKA in macrophages. They also suggest cholesterol lowering in extra-hepatic tissues by statins as an anti-inflammation strategy.

FIGURE 1.

ABCA1 expression in primary mouse BMDM increases IL-10 secretion and decreases proinflammatory cytokine secretion. WT and ABCA1^−/− primary mouse BMDM were induced with 10 μm T0901317 overnight followed by 100 ng/ml LPS treatment for 6 h. A, A mouse cytokine array (see supplemental data) was used to determine the levels of cytokine secretion from WT and ABCA1^−/− BMDM. C and B, IL-10 and TNFα levels in the medium from primary mouse BMDM (B) and RAW 264.7 macrophages (C) were measured by mouse cytokine ELISAs. Data are presented as average of duplicated samples with S.D.

FIGURE 2.

CREB phosphorylation is increased with the expression of functional ABCA1. BHK cells were treated overnight with 5 nm mifepristone. BMDM and RAW macrophages were treated overnight with T0901317. A–C, cell lysates were immunoblotted for ABCA1, p-CREB and total CREB in BHK cells (A), BMDM (B), and RAW macrophages (C). Hsp70 was used as loading control. Results are representative of at least three independent experiments.

FIGURE 3.

PKA phosphorylation is higher with ABCA1-expressing cells. Cells were prepared as in Fig. 2. A–C, cell lysates were immunoblotted (IB) for phospho-PKA substrates of BHK cells (A), BMDM (B), and RAW macrophages (C). D, immunofluorescence staining of BHK cells with anti-ABCA1 and anti-phosphorylated PKA substrate antibodies (anti-P-PKA-S). Na/K-ATPase, Parp-1, and Hsp70 were used as loading control, respectively. E, fluorescent intensities per cell (FI/cell) as a correlation between the level of phospho-PKA substrates and level of ABCA1 expression in individual cells.

FIGURE 4.

PKI suppresses IL-10 secretion but increases TNF-α release in ABCA1^+/+ BMDM, without significant effect on ABCA1^−/− BMDM. Primary mouse ABCA1^+/+ and ABCA1^−/− BMDM were treated with 10 μm T0901317 overnight. BMDM were then pretreated with 50 μm PKI for 30 min before LPS challenge (100 ng/ml) for 6 h. A and B, IL-10 (A) and TNFα (B) levels in the medium were measured by mouse cytokine ELISA and presented as average of duplicates ± S.D.

FIGURE 5.

Cholesterol depletion increases p-CREB. A–C, RAW macrophages were treated with 0, 1, or 5 mm MCD for 30 min (A), 3 μg/ml filipin for 1 h (B), or 3, 5, or 7 μm statins (compactin and simvastatin) for 48 h (C). Cell lysates were immunoblotted for p-CREB and total CREB with Hsp70 as loading control. Bar graphs represent averages from at least two independent experiments with standard deviations. LPDS, lipoprotein-deficient serum.

FIGURE 6.

Cholesterol depletion increases PKA-phosphorylated proteins. A and B, RAW macrophages were pretreated with or without 50 μm PKI for 1 h and subsequently treated 30 min with 5 mm MCD or 10 μg/ml filipin (A) or incubated with statins in 10% lipoprotein-deficient serum (LPDS) medium for 48 h (B). Whole cell lysates were analyzed by immunoblotting for phospho-PKA substrates with Hsp70 as loading control. Bar graphs represent averages from multiple independent experiments with standard deviations.

FIGURE 7.

Cholesterol depletion promotes IL-10 secretion but suppresses TNF-α release. A, RAW macrophages were treated with 10 μm T0901317 overnight and followed by 100 ng/ml LPS treatment in combination with MCD or filipin for 6 h. B, some of the cells were preincubated with 50 μm PKI and then treated as above. Medium IL-10 and TNF-α levels were measured by ELISA. Data are presented as average of duplicated samples with S.D.

FIGURE 8.

Cholesterol loading decreases p-CREB and promote proinflammatory response. RAW macrophages were incubated with 50 μg/ml acetylated LDL (AcLDL) or without for 24 h and then 10 μm T0901317 overnight. Some of the cells were then treated with 100 ng/ml LPS for 6 h. A, cell lysates were immunoblotted for p-CREB, total CREB, and PKA-phosphorylated proteins with Hsp70 as loading control. B and C, IL-10 (B) and TNFα (C) levels in the medium were measured by ELISA. Data are presented as average of duplicated samples with S.D.