Oxidized low-density lipoprotein stimulates dendritic cells maturation via LOX-1-mediated MAPK/NF-κB pathway

Abstract

Dendritic cells (DCs) play a crucial role as central orchestrators of immune system response in atherosclerosis initiation and progression. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in the immune maturation of DCs, but the underlying mechanisms remain unclear. We isolated mouse bone marrow progenitors and stimulated them with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 to induce immature DCs. We then treated DCs with oxidized low-density lipoprotein (oxLDL) to induce maturation. LOX-1 siRNA was used to investigate the modulation of LOX-1 on the development of DCs and the underlying signal pathways. CD11c-positive DCs were successfully derived from mouse bone marrow progenitors. OxLDL promoted the expressions of DCs maturation markers and pro-inflammatory cytokines. OxLDL also upregulated LOX-1 expression and activated MAPK/NF-κB pathways. LOX-1 siRNA could attenuate the expression of MAPK/NF-κB pathways and inflammatory cytokines. In conclusion, oxLDL induced the maturation of DCs via LOX-1-mediated MAPK/NF-κB pathway, which contributed to the initiation and progression of atherosclerosis.

Figure 1. Bone marrow dendritic cells (BMDC) culture and stimulation with oxidized low-density lipoprotein (oxLDL). A, Typical morphology of BMDCs at day 7. Scale bar=200 μm. B, Statistical results of the surface markers of mature BMDCs by flow cytometry. C, qRT-PCR analysis of pro- and anti-inflammatory cytokines in BMDCs after treatment by oxLDL. Data are reported as means±SD (n=3). *P<0.05 vs control, ***P<0.01 vs control (ANOVA).

Figure 2. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression and MAPK/NF-κB pathway in dendritic cells (DCs) stimulated by oxidized low-density lipoprotein (oxLDL) (A and B). C and D, Expression of p-p38, c-fos, and p-p105 in DCs after treatment by oxLDL. Data are reported as means±SD (n=3). *P<0.05 vs control, ***P<0.01 vs control (ANOVA).

Figure 3. Dendritic cells (DCs) transfected with siRNA-LOX-1 and LOX-1 expression. A, The fluorescence observation of DCs after transfection with siRNA-cy3. Scale bar=200 μm. B and C, Expression of LOX-1 in DCs after transfection with siRNA-LOX-1 and treatment by oxidized low-density lipoprotein (oxLDL). Data are reported as means±SD (n=3). ***P<0.01 vs control, ^###P<0.01 vs un-transfected groups (ANOVA).

Figure 4. Inhibition of LOX-1 attenuated MAPK/NF-κB pathways and inflammatory cytokines. A and B, Expression of p-p38 and p-p105 in dendritic cells (DCs) after transfection with siRNA-LOX-1 and treatment by oxidized low-density lipoprotein (oxLDL). C, qRT-PCR analysis of pro-inflammatory cytokines in DCs after transfection with siRNA-LOX-1 and treatment by oxLDL. Data are reported as means±SD (n=3). *P<0.05, ***P<0.01 vs control; ^#P<0.05, ^###P<0.01 vs un-transfected groups (ANOVA).