Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages

Abstract

Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.

FIGURE 1. GX sPLA₂ enhances macrophage inflammatory responses

(A) Relative expression of TNFα, IL-6, and COX-2 mRNAs in peritoneal macrophages isolated from wild-type (WT) and GX sPLA₂ deficient (GX KO) mice following treatment with 100 ng/ml LPS for 6 h. Values are relative to WT cells treated with LPS after normalization internally to 18S RNA. (B) Relative expression of TNFα, IL-6, and COX-2 mRNAs in cells treated with 100 ng/ml LPS for 6 h; all values are relative to J774-C cells treated with LPS after normalization internally to 18S RNA. (C) Relative expression of TNFα, IL-6, and COX-2 mRNAs in J774 cells treated for 8 h with 100 ng/ml LPS in the absence (UT) or presence (+GX) of 0.1 μg/ml recombinant GX sPLA₂. Values are presented relative to control cells treated only with LPS after normalization internally to 18S RNA. (A-C) Data are means ± SEM (n = 4) and are representative of 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to control cells treated with LPS.

FIGURE 2. GX sPLA₂ enhancement of macrophage inflammatory responses is dependent on its catalytic activity

(A) TNFα concentrations in conditioned media from cells treated with 100 ng/ml LPS for 20 h. TNFα was not detected in media from untreated cells. (B) IL-6 concentrations in conditioned media from cells treated with 100 ng/ml LPS for 20 h. IL-6 was not detected in media from untreated cells. (C) TNFα concentrations in conditioned media from cells treated for 20 h with 100 ng/ml LPS in the presence of either vehicle or 20 μM Indoxam, a sPLA₂ inhibitor. (A-C) Data are means ± SEM (n = 4) and are representative of 3 independent experiments; **, p < 0.01; ***, p < 0.001.

FIGURE 3. GX sPLA₂ augments TLR4-dependent signaling in macrophages

(A) Relative expression of TNFα, IL-6 and COX-2 mRNAs in MPMs isolated from TLR4^+/+ and TLR4^−/− mice treated for 8 h with 100 ng/ml LPS in the absence (UT) or presence (+GX) of 0.1 μg/ml GX sPLA₂. Values are presented relative to TLR4^+/+ cells treated with LPS alone after normalization internally to 18S RNA. **, p < 0.01; ***, p < 0.001; ns, not statistically significant compared to cells treated with LPS alone. (B) Relative expression of TNFα, IL-6 and COX-2 mRNAs in MPMs isolated from MyD88^+/+ and MyD88^−/− mice treated for 8 h with 100 ng/ml LPS in the absence (UT) or presence (+GX) of 0.1 μg/ml GX sPLA₂. Values are presented relative to MyD88^+/+ cells treated with LPS alone after normalization internally to 18S RNA. Data are means ± SEM (n=4).*p <0.05; **, p < 0.01; ***, p < 0.001; ns, not statistically significant compared to cells treated with LPS alone. (C) Phosphorylation of Erk1/2 and JNK in WT and GX KO MPMs treated for 0 or 30 min with 100 ng/ml LPS after 18 h pretreatment with 0 or 100 ng/ml mGX sPLA₂, as indicated. Cell extracts (10 μg protein) were immunoblotted with the indicated antibodies.

FIGURE 4. GX sPLA₂ enhances macrophage free cholesterol and lipid raft content

(A) free cholesterol content of J774-C and J774-GX cells normalized to cell protein. (B) free cholesterol content of untreated J774 cells and cells treated with 0.1 μg/ml GX sPLA₂ for 20 h normalized to cell protein. (C) J774-C and J774-GX cells were labeled with Alexa Fluor 488-CT-B to visualize lipid rafts. (D) J774-C and J774-GX cells were labeled with Alexa Fluor 488-CT-B and analyzed by flow cytometry to determine mean fluorescence intensity of cells. (A,B, D) Data (means ± SEM; n = 4) are representative of 3 independent experiments. *, p < 0.05; ***, p < 0.001.

FIGURE 5. GX sPLA₂ mediated hyperresponsiveness to LPS is reversed by plasma membrane cholesterol depletion and repletion

(A) free cholesterol content of J774-C and J774-GX cells normalized to cell protein. Cells were incubated in the presence or absence of 10 mM MβCD at 37°C for 30 min to deplete cholesterol. Subsets of cholesterol-depleted cells were then incubated for 60 min with 1 mM MβCD -loaded cholesterol to replete cholesterol prior to LPS treatments. (B) TNFα in conditioned media from cells depicted in (A) after treatment with 100 ng/ml LPS for 8 h. (A, B) Data are means ± SEM (n > 3); **, p < 0.01; ***p < 0.001. The analysis of IL-6 secretion produced similar results.

FIGURE 6. LPS-induced cytokine production is significantly decreased in GX KO mice compared to WT mice

Plasma IL-6 (A), TNFα (B) and IL-1β (C) concentrations 3 h after administration of LPS (3μg/g body weight; n=6). Data are means ± SEM *, p < 0.05; **p < 0.01. Cytokines were undetectable in both genotypes after treatment with saline (not shown). Data are representative of 2 independent experiments.

FIGURE 7. Model for GX sPLA₂ regulation of macrophage inflammatory responses

GX sPLA₂ enhances macrophage free cholesterol and lipid raft content, most likely by suppressing the expression of LXR targets ABCA1 and ABCG1. Increased plasma membrane free cholesterol and lipid raft content enhances TLR4 signaling. Whether GX sPLA₂ hydrolyzes extracellular or intracellular phospholipid membranes, or both, is unclear.