(A) (B)
(C)
(D) (E)
Figure 1. Effects of human nLDL, LDL(-) and PA on the production of IL-1β, TNF-a, and IL-6 in THP-1 macrophages.
(A) THP-1 macrophages were treated with 10, 20, or 40 g/mL human native LDL (nLDL)
for 6 h. (B) Macrophages were treated with 10, 20, or 40 g/mL human nLDL with 100
M palmitic acid (PA-BSA) for 6 h. Cell viability was determined by MTT assay. (C) Macrophages were treated with 10, 20, or 40 g/mL human nLDL alone (●), nLDL plus 100 M PA-BSA (○), 10, 20, or 40 g/mL human LDL(-) alone (▼) or human LDL(-) plus 100 M PA-BSA (∆) for 6 h. The levels of IL-1in the medium were determined by ELISA. D & EMacrophages were treated with 10 g/mL human nLDL alone or nLDL plus 100 M PA-BSA for 6 h.TNF-(D)and IL-6 (E) in the medium were determined by ELISA.
(A)
(B) (C)
Figure 2. Effects of rabbit LDL(-) on the production of IL-1β in THP-1 macrophages.
(A) A representative anion-exchange chromatography of LDL from rabbit fed with high-fat diet. (B) THP-1 macrophages were treated with 10, 20, or 40 g/mL rabbit LDL(-) for 6 h. Cell viability was determined by MTT assay. (C) Macrophages were treated with 5, 10, or 20 g/mL rabbit LDL(-) for 6 h (●) and 24 h (○). The levels of IL-1 in the medium were determined by ELISA. *p < 0.05, **p < 0.001, compared to the control cells; ‡p <
0.05; NS, not statistically significant as indicated. The results are the mean ± SD for three independent experiments.
(A) (B)
(C) (D)
Figure 3. Effects of PA on the production of IL-1β in THP-1 macrophages.
THP-1 macrophages were treated with 50, 100, or 150 PA-BSA for 6 h (A) or 24 h (B). Cell viability was determined by MTT assay. The results are the mean ± SD for three independent experiments. (C) Macrophages were treated with 50, 100, or 150 PA-BSA for 6 h (C) or 24 h (D). The levels of IL-1 in the medium were determined by ELISA.
(A) (B)
(C) (D)
(E)
(F)
Figure 4. Effects of rabbit LDL(-) plus PA and rabbit LDL(-) plus various FFAs on the production of IL-1β in THP-1 macrophages.
(A) THP-1 macrophages were treated with 10, 20, or 40 g/mL rabbit LDL(-) plus 100 M PA-BSA for 6 h. (B) Macrophages were treated with 10 g/mL rabbit LDL(-) alone or with 50, 100, 150, or 200 M PA-BSA for 6 h. Cell viability was determined by MTT assay. (C) Macrophages were treated with 10 or 20 g/mL rabbit LDL(-), 100 M PA-BSA, or 10 or 20 g/mL LDL(-) plus 100 M PA-BSA for 6 h. (D) Macrophages were treated with 10 g/mL rabbit LDL(-) alone or with 50, 100, 150, or 200 M PA-BSA for 6 h. (E) Macrophages were treated with 10
g/mL rabbit LDL(-) alone or with 100 M lauric acid (C12:0), myristic acid (C14:0),
Source of variation p value
Absent or present LDL(-) p < 0.001 Absent or present PA-BSA p < 0.001 Interaction between two factors p < 0.001
palmitic acid (C16:0), stearic acid (C18:0), oleic acid (cis-C18:1, n-9), elaidic acid (trans-C18:1, n-9), linoleic acid (C18:2, n-6), and docosahexaenoic acid (C22:6, n-3) for 6 h. (F) Macrophages were treated with 10 g/mL rabbit LDL(-), 100 M PA-BSA, or LDL(-) plus PA-BSA for 6 h. The levels of IL-1 in the medium were determined by ELISA. The results are the mean ± SD for at least three independent experiments. #p < 0.05, compared to the control cells; **p < 0.001; NS, not statistically significant in (C), ##p < 0.001, compared to the control cells; **p < 0.001, compared to the LDL(-)-treated cells; ‡p <
0.05; NS, not statistically significant in (D) **p < 0.001, analyzed by two-way ANOVA (Bonferroni t-test). The results of two-way ANOVA analysis are shown (F); two factors of variation are absent or present of LDL(-) and absent or present of PA-BSA.
(A)
(B)
mRNA level LOX-1
Source of variation p value
Absent or present LDL(-) p = 0.006
Absent or present PA-BSA p = 0.515
Interaction between two factors p = 0.678
(C)
Figure 5. LDL(-) and PA induce IL-1 production through a LOX-1 but not CD36, dependent pathway.
(A) THP-1 macrophages derived from shLOX-1 and shLuciferase knockdown THP-1 cells were treated with 10 g/mL rabbit LDL(-) alone or LDL(-) plus 100 mM PA-BSA for 6 h. The levels of IL-1 in the medium was determined by ELISA. *p < 0.05. (B) Macrophages were treated with 10 g/mL rabbit LDL(-) alone, 100 M PA-BSA alone, or LDL(-) plus PA-BSA for 6 h. The levels of LOX-1and CD36 mRNA were determined by real-time quantitative PCR (RT-qPCR), normalized to the levels of 36B4 mRNA, and expressed relative to the levels in control cells (RPMI) (relative value = 1). # p < 0.05, compared to the control cells (RPMI); NS, not statistically significant. The results of two-way ANOVA analysis are shown, two factors of variation are absent or present of LDL(-) and absent or present of PA-BSA. (C) Macrophages were treated with 10 g/mL rabbit LDL(-) alone, 100 M PA-BSA alone, or LDL(-) plus PA-BSA for 6 h or 24 h. The protein levels of LOX-1 and -actin in cell lysate were determined by western blot. The protein levels were quantitatively analyzed by ImageJ, and the levels of LOX-1 were normalized to that of -actin and expressed relative to the control cells (relative value = 1). The results are the mean ± SD for three independent experiments.
(A) (B)
(C)
Protein level p-I-B/I-B pro-IL-1
Source of variation P value
Absent or present LDL(-) p = 0.003 p = 0.001
Absent or present PA-BSA p = 0.699 p = 0.558
Interaction between two factors p = 0.398 p = 0.427
(D)
Figure 6. Effects of rabbit LDL(-) and PA on NF-B activation in THP-1 macrophages.
THP-1 macrophages were treated with 10 g/mL rabbit LDL(-), 100 M PA-BSA, or LDL(-) plus PA-BSA for 6 h. (A) The levels of phospho-I-BtotalI-B(B) pro-IL-1and-actin in the cell lysate were determined by western blot. The protein levels were quantitatively analyzed by ImageJ and the levels of phospho-I-B were normalized to the levels of total I-B(A, bottom) the levels of pro-IL-1 were normalized to the levels of -actin (B, bottom), and expressed relative to the control cells (relative value = 1). The results are the mean ± SD for three independent experiments. #p < 0.05, compared to the control cells (RPMI); NS, not statistically significant. The results of two-way ANOVA analysis are shownC, two factors of variation are absent or present of LDL(-) and absent or present of PA-BSA. (D) Macrophages were pretreated with DMSO, 10 M Bay11-7082, 10 M U0126, 10 M SB203580, 15 M SP600125, or 0.5 M L-JNKi for 1 h, and then 10 g/mL rabbit LDL(-) and 50 M SA-BSA were added and incubated for 24 h. The levels of IL-1 in the medium were determined by ELISA. The results are the mean ± SD for three independent experiments. ##p < 0.001, compared to the control cells (RPMI); *p < 0.05, **p < 0.001, compared to the DMSO-pretreated cells.
(A)
mRNA level IL-1 TNF- IL-6
Source of variation p value
Absent or present LDL(-) p < 0.001 p = 0.007 p = 0.002
Absent or present PA-BSA p = 0.320 p = 0.440 p = 0.543
Interaction between two factors p = 0.188 p = 0.471 p = 0.410
(B)
Source of variation p value
Absent or present LDL(-) p < 0.001
Absent or present PA-BSA p = 0.020
Interaction between two factors p = 0.024
(C)
Figure 7. Effects of rabbit LDL(-) and PA on expressions of NF-B downstream genes, IL-1TNF-and IL-6, and their protein levels in THP-1 macrophages.
THP-1 macrophages were treated with 10 g/mL rabbit LDL(-), 100 M PA-BSA, or LDL(-) plus PA-BSA for 6 h. (A) The levels of IL-1TNF-and IL-6 mRNA were determined by real-time quantitative PCR (RT-qPCR), normalized to the levels of 36B4 mRNA, and expressed relative to the levels in the control cells (RPMI) (relative value = 1). #p < 0.05, compared to the control cells (RPMI); NS, not statistically significant. (B) The levels of TNF-and (C) IL-6 in the medium were determined by ELISA. The results are the mean ± SD for at least three independent experiments. **p < 0.001, analyzed by two-way ANOVA (Bonferroni t-test). The results of two-way ANOVA analysis are shown; two factors of variation are absent or present of LDL(-) and absent or present of PA-BSA.
Source of variation P value
Absent or present LDL(-) p < 0.001
Absent or present PA-BSA p = 0.066
Interaction between two factors p = 0.102
(A) (B)
Figure 8. PA enhances LDL(-)-induced IL-1 production is not through phagocytosis and lysosomal degradation.
THP-1 macrophages were pretreated (A) 10 M cytochalasin D (inhibits actin polymerization) or (B) 25 M chloroquine (a lysosome inhibitors) for 1 h, and then treated with 10 g/mL rabbit LDL(-) alone or LDL(-) plus 100 M PA-BSA for 6 h. The levels of IL-1 in the medium were determined by ELISA. The results are the mean ± SD for three independent experiments.NS, not statistically significant.
(A) (B)
(C) (D)
Figure 9. PA enhances LDL(-)-induced IL-1 production is possibly through potassium efflux.
(A) THP-1 macrophages were treated with 10 mg/mL rabbit LDL(-) alone or LDL(-) plus 100 M PA-BSA in RPMI medium or potassium free buffer. (B) Macrophages were pretreated treated with 50 or100 mM tetraethylammonium chloride (TEA, a non-selective potassium channel blocker), (C) 5 or 15 mM 4-aminopyridine (4-AP, a non-selective potassium channel blocker), or (D) 22 or 50 M A 438079 (competitive P2X7 receptor
antagonist) for 1 h, and then treated with LDL(-) alone or LDL(-) plus PA-BSA for 6 h.
The levels of IL-1 in the medium were determined by ELISA. ##p < 0.001, compared to the control cells; **p < 0.001, compared to the LDL(-)-treated cells; NS, not statistically significant.