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. 2017 Jan 3:8:13997.
doi: 10.1038/ncomms13997.

Saturated palmitic acid induces myocardial inflammatory injuries through direct binding to TLR4 accessory protein MD2

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Saturated palmitic acid induces myocardial inflammatory injuries through direct binding to TLR4 accessory protein MD2

Yi Wang et al. Nat Commun. .

Erratum in

Abstract

Obesity increases the risk for a number of diseases including cardiovascular diseases and type 2 diabetes. Excess saturated fatty acids (SFAs) in obesity play a significant role in cardiovascular diseases by activating innate immunity responses. However, the mechanisms by which SFAs activate the innate immune system are not fully known. Here we report that palmitic acid (PA), the most abundant circulating SFA, induces myocardial inflammatory injury through the Toll-like receptor 4 (TLR4) accessory protein MD2 in mouse and cell culture experimental models. Md2 knockout mice are protected against PA- and high-fat diet-induced myocardial injury. Studies of cell surface binding, cell-free protein-protein interactions and molecular docking simulations indicate that PA directly binds to MD2, supporting a mechanism by which PA activates TLR4 and downstream inflammatory responses. We conclude that PA is a crucial contributor to obesity-associated myocardial injury, which is likely regulated via its direct binding to MD2.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Md2 knockout protects against PA-induced myocardial injury.
Wild-type (B6) or Md2−/− mice (KO) were challenged with intravenous (i.v.) injection of 5 mM PA, normal saline (NS) or 5% BSA (vehicle control) twice daily for 7 days; n=8. (a) Serum CK-MB values reported in U l−1. (b) Representative micrographs of heart tissue morphology showing hematoxylin- and eosin-stained sections; scale bars, 100 μm. (c) Representative micrographs of histochemical staining for connective tissue using Masson’s trichrome; scale bars=100 μM. (d) Quantification of trichrome staining of heart tissues of PA-challenged mice. (e) Serum TNF-α and IL-6 protein levels (**P<0.01, #P<0.01, compared with B6+PA group). (f) Protein levels of TNF-α, IL-6 and MCP-1 in heart tissue reported as μg g−1 myocardial protein. Data in a and df are reported as mean±s.e.m. and analysed by Student’s t-test, *P<0.05, **P<0.01, ***P<0.001, B6-PA group compared with either controls or KO groups; NS, not significant.
Figure 2
Figure 2. PA induces MD2-dependent inflammatory injury in cardiomyocytes.
(a,b) H9C2 cells were pretreated with 10 μM L6H9 before stimulation with 500 μM PA for 12 h. Representative images of cell morphology captured by phase microscopy (top panel) and F-actin distribution stained by rhodamine–phalloidin (bottom panel) are shown; n=3; scale bars=20 μm. Quantification of cell size is shown in b. (c) Effects of L6H9 pretreatment on apoptosis induced by 500 μM PA for 24 h in H9C2 cells. Quantification of flow cytometric data for apoptotic cells, determined by flow cytometric analysis of FITC-Annexin V and propidium iodide (PI); n=3. (d) Cytosolic and nuclear protein levels of IκB-α and NF-κB p65 subunit in cells pretreated with L6H9 before stimulation with 500 μM PA for 1 h (representative of n=3). Densitometric quantification of NF-κB pathway activation in cells treated with PA is shown below. (e) Cytokine expression in H9C2 cells following treatment with 500 μM PA for 6 h. Bar graphs showing relative mRNA values for TNF-α, IL-1β, ICAM-1 and VCAM-1 normalized with GAPDH (n=4). (f) Representative western blot with densitometric quantification of ICAM-1 and VCAM-1 in cells treated as in e (representative of n=3). (g) Knockdown of Md2 in H9C2 cells by siRNA transfection. Figure showing protein levels of MD2 in untransfected cells (Ctrl) and in cells transfected with either control/scrambled siRNA (siCtrl) or siRNA targeting Md2 (siMd2; n=4 experiments). (h) Effect of Md2 knockdown on pro-inflammatory gene expression in H9C2 cells stimulated with 500 μM PA for 6 h. Bar graphs showing relative mRNA for TNF-α, IL-6 and IL-1β (GAPDH used as housekeeping gene; n=4 experiments). Data in bh are reported as mean±s.e.m. and analysed by one-way analysis of varinace, #P<0.05, ##P<0.01, compared with dimethylsulphoxide, Ctrl or siCtrl group; *P<0.05, **P<0.01, compared with PA or siCtrl-PA group.
Figure 3
Figure 3. PA induces MD2/TLR4 complex formation and signalling.
(a,b) Effects of MD2 inhibitor L6H9 on secretion of TNF-α (a) and IL-6 (b) by mouse primary macrophages stimulated with 100 μM PA for 24 h. Cytokine concentrations in the condition medium were measured by ELISA and reported as pg ml−1 (n=3). (c,d) Effects of PA stimulation (100 μM for 24 h) on cytokine secretion from primary macrophages isolated from Md2−\− or wild-type mice, KO-MPM or WT-MPM, respectively. Graphs show relative amounts of TNF-α (c) and IL-6 (d) (n=3). (e,f) Effects MD2 blockade on formation of the MD2/TLR4–MyD88 complex in macrophages stimulated with 100 μM PA for 5 min. MD2 blockade was achieved by MD2-neutralizing antibody (anti-MD2, 100 ng ml−1) or MD2 inhibitor (L6H9, 10 μM). Shown are representative (n=3) western blots (IBs) from the co-precipitation (IP) studies for MD2/TLR4 complex formation (e) and TLR4/MyD88 complex formation (f). (g,h) Effects of MD2 blockade on NF-κB activity in mouse macrophages stimulated with 100 μM PA (30 min); MD2 blockade was achieved by anti-MD2 or 10 μM L6H9. Shown are (g) representative western blot for p-IKKβ and IκB-α (n=3), and (h) EMSA analysis for NF-κB DNA-binding activity (n=3). (i) Effects of MD2 inhibition through knockdown or L6H9 pretreatment (10 μM) on MAPK pathway activation in macrophages stimulated with 100 μM PA for 30 min. Shown are representative western blots of phosphorylated JNK, ERK and P38 (densitometric quantification for gi from three separate determinations were shown in Supplementary Figs 11a,b and 12a, respectively). Data are reported as mean±s.e.m. and analysed by Student’s t-test, *P<0.05, **P<0.01 compared with PA or WT-MPMs; NS, not significant.
Figure 4
Figure 4. PA interacts directly with MD2 to activate TLR4 signalling.
(a) Flow cytometric analysis showing the effects of MD2 blockade on the binding of FITC-PA (50 μM) to the cell surface of mouse macrophages. MD2 was blocked with either anti-MD2 (100 ng ml−1) or L6H9 (1 and 10 μM). (b,c) Representative graphs from surface plasmon resonance spectroscopy analysis showing the binding of increasing concentrations of PA with human recombinant MD2 (b) or TLR4 (c). The Ka and KD values are indicated on the upper left. (d) Binding of biotin-PA with rhMD2 pre-absorbed in the ELISA plate (OD values reported as mean±s.e.m. and analysed by Student’s t-test; *P<0.05, **P<0.01, compared with biotin-PA group). n=4 for all in vitro experiments.
Figure 5
Figure 5. Md2 knockout reduces myocardial injury in HFD.
Wild-type (B6) and Md2−/− (KO) mice were fed a HFD for 4 months, and blood and heart tissue collected for evaluation of myocardial inflammatory injury. (a) Levels of FFA in 20mg heart tissue of each mouse fed a control (Ctrol) diet or HFD; FFA reported as mean ± s.e.m. and analysed by Student’s t-test, **P<0.01 compared with Ctrol diet; n=8). (b,c) Serum markers of cell injury indicated by CK-MB and LDH (n=8). (d) Upper panel shows representative micrographs of heart tissue stained with hematoxylin and eosin; lower panel shows histochemical staining for connective tissue using Masson’s trichrome; n=4; scale bars, 100 μm. (e) Quantification of fibrosis indicated by trichrome staining, n=3. (fj) RT–qPCR determination of inflammatory genes and adhesion molecules in heart tissue (n=4). Figure showing TNF-α (f), IL-6 (g), IL-1β (h), ICAM-1 (i) and VCAM-1 (j). mRNA was normalized to housekeeping gene β-actin, and reported relative to B6-Con. (k) Representative immunohistochemical analysis of TNF-α and CD68 (immunoreactivity=brown); n=4; scale bars, 100 μm. Data in b,c and ej are reported as mean±s.e.m. and analysed by Student’s t-test; *P<0.05, **P<0.01, B6-HFD group compared with control (Con) or KO groups; NS, not significant.

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