Class B Scavenger Receptor CD36 as a Potential Therapeutic Target in Inflammation Induced by Danger-Associated Molecular Patterns

Abstract

The class B scavenger receptor CD36 is known to bind and mediate the transport of lipid-related ligands and it functions as a pattern recognition receptor (PRR) for a variety of pathogens, including bacteria and viruses. In this study, we assessed CD36's role as a PRR mediating pro-inflammatory effects of several known Danger-Associated Molecular Patterns (DAMPs) used either as a single preparation or as a combination of DAMPs in the form of total cell/skeletal muscle tissue lysates. Our data demonstrated that multiple DAMPs, including HMGB1, HSPs, histone H3, SAA, and oxPAPC, as well as cell/tissue lysate preparations, induced substantially higher (~7-10-fold) IL-8 cytokine responses in HEK293 cells overexpressing CD36 compared to control WT cells. At the same time, DAMP-induced secretion of IL-6 in bone marrow-derived macrophages (BMDM) from CD36-/- mice was markedly (~2-3 times) reduced, as compared to macrophages from normal mice. Synthetic amphipathic helical peptides (SAHPs), known CD36 ligands, efficiently blocked CD36-dependent inflammatory responses induced by both cell and tissue lysates, HMGB1 and histone H3 in CD36+ cells. IP injection of total cellular lysate preparation induced inflammatory responses that were assessed by the expression of liver and lung pro-inflammatory markers, including IL-6, TNF-α, CD68, and CXCL1, and was reduced by ~50% in CD36-deficient mice compared to normal mice. Our findings demonstrate that CD36 is a PRR contributing to the innate immune response via mediating DAMP-induced inflammatory signaling and highlight the importance of this receptor as a potential therapeutic target in DAMP-associated inflammatory conditions.

Keywords: CD36; DAMPs; HMGB1; SAA; heat shock proteins; histones; inflammatory markers; oxPAPC.

Figure 1

Dose-dependent IL-8 secretion induced by HeLa cell and skeletal muscle CF in WT and CD36-overexpressing HEK293 cells. WT and CD36-overexpressing HEK293 cells were incubated with increasing concentrations of the CF preparations (see Section 2) from HeLa cells, c-CF (A) or murine skeletal muscle, SM-CF (B) for 20 h. IL-8 levels were quantified in duplicate samples of cell culture supernatants by ELISA. Data represent one of three separate experiments that yielded similar results.

Figure 2

Dose-dependent IL-8 secretion induced by the HMGB1 and LPS in WT and CD36-overexpressing HEK293 cells. Effect of heat treatment on the cytokine-inducing activity of HMGB1 and LPS. WT and CD36-overexpressing HEK293 cells were incubated with increasing concentrations of recombinant HMGB1 (A) or LPS (B) for 20 h. IL-8 levels were quantified in duplicate samples of cell culture supernatants by ELISA. Data represent one of three separate experiments that yielded similar results. The IL-8 levels were determined using duplicate samples of cell culture supernatants collected after a 20 h incubation of cells with either intact or heat-treated (100 °C for 45 min) HMGB1 (C) and LPS (D). The data presented are from one of two separate representative experiments.

Figure 3

Effects of SAHPs on IL-8 secretion induced by the CF preparations and HMGB1 in CD36-overexpressing HEK293 cells. Cells were preincubated with or without increasing doses of L-37pA, ELK-B, or L3D-37pA for 1 h before a 20 h treatment with 1% c-CF (A) or 0.3% SM-CF (B). Cells were preincubated for 1 h with or without 10 µg/mL of L-37pA, ELK-B, or L3D-37pA before a 20 h treatment with increasing doses of HMGB1 (0.25, 1, and 5 µg/mL). IL-8 levels were determined in cell culture supernatants in duplicate (C). Data are from one of at least two representative experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns—nonsignificant, versus no peptide.

Figure 4

Dose-dependent IL-8 secretion induced by the histone H3 in WT and CD36-overexpressing HEK293 cells. Effect of L-37pA on hH3-induced IL-8 secretion in CD36-HEK293 cells. WT and CD36-overexpressing HEK293 cells were incubated with increasing concentrations of histone H3 for 20 h. IL-8 levels were quantified in duplicate samples of cell culture supernatants by ELISA (A). Data represent one of three separate experiments that yielded similar results. CD36-HEK293 cells were preincubated for 1 h with 0, 10 µg/mL, and 25 µg/mL of L-37pA or L3D peptides before a 20 h treatment with 25 µg/mL of histone H3. IL-8 levels were determined in cell culture supernatants in duplicate (B). * p < 0.05, ** p < 0.01, and ns—nonsignificant, versus histone H3 alone. Data are from one of at least two representative experiments.

Figure 5

DAMPs-induced dose-dependent pro-inflammatory responses in WT and CD36-overexpressing HEK293 cells. WT and CD36-overexpressing HEK293 cells were incubated with increasing concentrations of HSP60 (A), HSP70 (B), oxPAPC (C), and SAA (D) for 20 h. IL-8 levels were quantified in duplicate samples of cell culture supernatants by ELISA. Data represent one of two separate experiments that yielded similar results.

Figure 6

Pro-inflammatory responses induced by the various DAMPs in BMDM from WT and CD36-knockout mice. BMDM isolated from WT and CD36−/− mice were incubated with increasing doses of cCF (A), HMGB1 (B), histone H3 (C), and LPS (D) for 20 h. IL-6 levels were quantified in duplicate samples of cell culture supernatants by ELISA. Data represent one of two separate experiments that yielded similar results.

Figure 7

Effects of signaling pathways Inhibitors on CF-induced IL-6 secretion in BMDM from WT and CD36-knockout mice. BMDM isolated from WT and CD36−/− were pre-incubated for 1 h with increasing doses of PD98059 (A), SP600125 (B), SB202190 (C), or PP2 (D). Following pre-incubation, cells were incubated with CF preparation for the next 20 h. IL-6 levels were quantified in duplicate samples of cell culture supernatants by ELISA. Data represent one of two separate experiments that yielded similar results.

Figure 8

Hepatic gene expression of inflammatory markers in WT and CD36-knockout mice IP injected with a TCL preparation. Livers were collected for mRNA extraction and qRT-PCR assay as described in Materials and Methods. Expression levels of IL-6 (A), TNF-α (B), CD68 (C), and CCL2 (D) were normalized by GAPDH expression and are presented as the fold change relative to the PBS-treated WT control mice. Values shown are the means ± STD (n = 12–15 for the WT group, n = 20 for the CD36-KO group). * p < 0.05, versus WT TCL-treated mice.

Figure 9

Pulmonary gene expression of inflammatory markers in WT and CD36-knockout mice IP injected with a TCL preparation. Lungs were collected for mRNA extraction and qRT-PCR assay as described in Materials and Methods. Expression levels of IL-6 (A), TNF-α (B), and CXCL1 (C) were normalized by GAPDH expression and are presented as the fold change relative to PBS-treated WT control mice. Values shown are the means ± STD (n = 10 for the WT group, n = 14 for the CD36-KO group). * p < 0.05, versus WT TCL-treated mice.