Oxidized Low-Density Lipoprotein (OxLDL)-Treated Dendritic Cells Promote Activation of T Cells in Human Atherosclerotic Plaque and Blood, Which Is Repressed by Statins: microRNA let-7c Is Integral to the Effect

Abstract

Background: Activated T cells and dendritic cells (DCs) are colocalized in atherosclerotic plaques in association with plaque rupture. Oxidized low-density lipoprotein (oxLDL) promotes immune activation and inflammation. We studied the effects of statins (atorvastatin and simvastatin) on human DC maturation and T-cell activation.

Methods and results: Human peripheral blood monocytes were differentiated to DCs and stimulated with oxLDL. T cells were isolated from carotid endarterectomy specimens from patients undergoing carotid endarterectomy or from healthy individuals. Naïve T cells were cocultured with pretreated DCs. The effects of statin were studied. OxLDL induced DC maturation and T-cell activation. OxLDL induced atherogenic heat shock proteins (HSP) 60 and 90 and decreased potentially atheroprotective heat shock protein 27, effects restored by atorvastatin. T cells exposed to oxLDL-treated DCs produced interferon-γ and interleukin (IL)-17. Atorvastatin and simvastatin suppressed the DC maturation showing lower expression of CD80, CD83, and CD86, and limited their production of tumor necrosis factor-α, IL-1β and IL-6, and increased transforming growth factor-β and IL-10 secretion. Statin-treated DCs inhibited Th1 and/or Th17 polarization by downregulation of transcriptional factors T-bet and RORγt expression, and induced T regulatory cells with IL-10 production. OxLDL-induced miRNA let7c and phosphorylation of Akt and ERK were repressed by statins. Let-7c had a pivotal role in mediating effect of oxLDL. Experiments on T cells derived from carotid atherosclerotic plaques or healthy individuals showed similar results.

Conclusions: Statins repress human DC maturation induced by oxLDL, limit T-cell activation, and repress an atherogenic heat shock protein profile and promote induction of T regulatory cells. MicroRNA let-7c is integral to the effects.

Keywords: T cells; atherosclerosis; dendritic cells; immune system; microRNA; oxidized low‐density lipoprotein; statin.

Figure 1

Dendritic cell (DC) maturation, proinflammatory cytokine production, and the subsequent T‐cell proliferation induced by oxidized low‐density lipoprotein (OxLDL) were suppressed by statins. DCs at day 6 were treated with 10 μg/mL oxLDLfor 24 hours. For atorvastatin treatment, an aliquot of DCs on day 5 were treated with 2 μmol/L atorvastatin (AVA) for 24 hours, then stimulated with 2 μmol/L atorvastatin in the presence or absence of 10 μg/mL oxLDL and/or 100 μmol/L mevalonate (Mev) for another 24 hours. A, The expression of CD80, CD86, CD83, and HLA‐DR on day 7 DCs was analyzed by flow cytometry. A representative of 10 independent experiments is shown. B, Cytokine production tested by ELISA. DC supernatants were collected at day 7. Results represent the mean concentration±SEM of 8 independent experiments. **P<0.01, DC+OxLDL vs DC; ^## P<0.01, DC+AVA+oxLDL vs DC+OxLDL. C, T‐cell proliferation. After the indicated treatment, DCs were washed and cocultured in triplicate for 5 days with autologous T cells. BrdU (bromodeoxyuridine), 10 μmol/L, was present in the last 16 hours. Proliferative response was evaluated as OD value. Results show mean±SD (n=6). **P<0.01, DC+OxLDL vs DC; ^## P<0.01, DC+AVA+oxLDL vs DC+OxLDL. IL indicates interleukin; TGF, transforming growth factor; OD, optical density; TNF, tumor necrosis factor.

Figure 2

Simvastatin suppressed oxidized low‐density lipoprotein (oxLDL)‐induced dendritic cell (DC) maturation, cytokine production, and the subsequent T‐cell proliferation. DCs at day 6 were treated with 10 μg/mL oxLDLfor 24 hours. An aliquot of DCs on day 5 were treated with 5 μmol/L simvastatin (Simva) for 24 hours, then stimulated with 5 μmol/L simvastatin in the presence or absence of 10 μg/mL oxLDL and/or 100 μmol/L mevalonate (Mev) for further 24 hours. A, The expression of CD80, CD86, CD83, and HLA‐DR on DCs were detected by flow cytometry. A representative of 4 independent experiments is shown. B, Cytokine production was tested by ELISA. Supernatants of DC were collected at day 7. Results represent the mean concentration±SD of 5 independent experiments. **P<0.01, DC+oxLDL vs DC; ^## P<0.01, DC+Simva+oxLDL vs DC+oxLDL. C, T‐cell proliferation. After the indicated treatment, DCs were washed and cocultured in triplicate for 5 days with autologous T cells. BrdU (bromodeoxyuridine), 10 μmol/L, was present in the last 16 hours. Proliferative response was evaluated as OD value. Results show mean±SD (n=4). **P<0.01, DC+oxLDL vs DC; ^## P<0.01, DC+Simva+oxLDL vs DC+oxLDL. AVA indicates atorvastatin; OD, optical density.

Figure 3

Th1 and Th17 polarization induced by oxidized low‐density lipoprotein (oxLDL)‐treated dendritic cells (DCs) were inhibited, while T regulatory cells were induced by atorvastatin. A, T‐cell polarization‐related transcription factors. Quantitative PCR data represent mean±SD of 6 independent experiments. B, Intracellular staining of cytokine production in T cells. Naïve CD4⁺ T cells were cultured with oxLDL‐ or atorvastatin (AVA)+oxLDL–treated autologous DCs for 13 days. After stimulation, T cells were activated with anti‐CD3 monoclonal antibody and intracellular staining of cytokine production. Percentages of interferon‐γ (IFN‐γ)‐, interleukin (IL)‐17‐, IL‐10‐producing cells, and FoxP3‐positive cells in T (DC), T (DC [oxLDL]), or T (DC [AVA+oxLDL]) cells are presented. Figure depicts a representative experiment of 5. C, Frequency of T‐cell subsets is summarized. Results show mean±SD (n=5). D, T (DC [AVA+oxLDL]) cells suppressed primary T‐cell proliferation. T‐cell proliferation was measured by CFSE staining. Naïve CD4⁺ T cells were stimulated with mature DCs alone or in the presence of T (DC), T (DC [oxLDL]), or T (DC [AVA+oxLDL]) cells at a 1:1 ratio. Results show a representative experiment of 4. **P<0.01, T (DC [oxLDL]) vs T (DC); ^## P<0.01, T (DC [AVA+oxLDL]) vs T (DC [oxLDL]). CFSE indicates carboxyfluorescein succinimidyl ester; PCR, polymerase chain reaction.

Figure 4

MicroRNA (miRNA) let‐7c and PI3k/ERK signaling pathways were involved in the effect of oxidized low‐density lipoprotein (oxLDL) and statins. A, Expression of miRNAs in dendritic cells (DCs) was tested by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). The value was normalized as fold change to that of nontreated DC samples. Results represent the mean±SEM of 7 experiments. *P<0.05, **P<0.01, DC+oxLDL vs DC; ^# P<0.05, ^## P<0.01, DC+ atorvastatin (AVA)+oxLDL vs DC+oxLDL. B, Phosphorylation of Akt and ERK induced in oxLDL‐treated DCs were confined by AVA. OxLDL‐induced phosphorylation of Akt and ERK were tested by phospho‐flow using specific antibody anti‐Akt (pS473) and anti‐MEK1 (pS298). Results show 1 representative experiment of 5. LY294002 (PI3k inhibitor) and U0126 (MEK inhibitor) are used as control. C, Inhibition of let‐7c in DCs was tested by qRT‐PCR. DCs at day 6 were treated with 10 μg/mL oxLDL for 24 hours. For inhibition of let‐7c, inhibitor of miRNA let‐7c, mimic control was transfected with Lipofectamine^® RNAiMAX (Invitrogen) in a 20 nmol/L concentration at day 6. oxLDL was added 6 hours after transfection, and cells were further incubated for 24 hours. The value was normalized as fold change to that of nontreated DC samples. Results represent the mean±SD of 4 experiments. **P<0.01, DC+oxLDL vs DC; ^## P<0.01, DC+inhibitor+oxLDL vs DC+mimic+oxLDL. D, Downregulation of let‐7c restricted the phosphorylation of Akt and ERK in the oxLDL‐treated DC. One of 4 experiments is shown. E, Expression of heat shock proteins (HSPs) in DCs was tested by qRT‐PCR. Results represent the mean±SD of 4 experiments. *P<0.05, **P<0.01, DC+oxLDL vs DC; ^# P<0.05, ^## P<0.01, DC+AVA+oxLDL vs DC+oxLDL. F, Expression of B lymphocyte–induced maturation protein‐1 (BLIMP1) in DCs was tested by qRT‐PCR showing the mean±SD of 4 experiments, and flow cytometry showing a representative of 4 experiments. **P<0.01, DC+oxLDL vs DC; ^## P<0.01, DC+AVA+oxLDL vs DC+oxLDL. MFI indicates median fluorescence intensity.

Figure 5

MicroRNA let‐7c mediated the effect of oxidized low‐density lipoprotein (oxLDL) on dendritic cell (DC) maturation, cytokine production, and T‐cell proliferation. A, let‐7c was involved in the DC maturation. The expression of CD86 and CD83 were analyzed by flow cytometry. Results show 1 representative of 6 independent experiments. B, Cytokine production in DC cultures after let‐7c downregulation. DC supernatants were collected at day 7, and tested by ELISA. Results represent the mean concentration±SEM of 6 independent experiments. **P<0.01, DC+OxLDL vs DC; ^## P<0.01, DC+inhibitor+oxLDL vs DC+OxLDL. IL indicates interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor. C, T‐cell proliferation. After the indicated treatment, DCs were washed and cocultured with autologous T cells in triplicates for 5 days. Bromodeoxyuridine (BrdU) 10 μmol/L was present in the last 16 hours. Proliferative response was evaluated as OD value. Results show mean±SD of 6 independent experiments. **P<0.01, DC+oxLDL vs DC; ^## P<0.01, DC+inhibitor+oxLDL vs DC+mimic+oxLDL. OD indicates optical density.

Figure 6

Dendritic cell (DC) maturation, proinflammatory cytokine production, and the subsequent T‐cell proliferation from plaque patients induced by oxidized low‐density lipoprotein (oxLDL) were suppressed by statins. DCs were generated from peripheral blood monocytes of patients who underwent carotid endarterectomy. The same protocol was used as that for DCs of normal donors. Briefly, DCs at day 6 were treated with 10 μg/mL oxLDL for 24 hours. An aliquot of DCs at day 5 were treated with 2 μmol/L atorvastatin for 24 hours, and continued with 2 μmol/L atorvastatin in the presence or absence of 10 μg/mL oxLDL for an additional 24 hours. A, The expression of CD80, CD86, CD83 and HLA‐DR was tested on day 7 DCs derived from patient blood. Figure depicts the expression of CD86 and CD83 tested by flow cytometry. A representative of 6 independent experiments is shown. B, Cytokine production in DC cultures derived from patient blood. Culture supernatants were collected after 24 hours treatment. Cytokine level was determined by ELISA. Results represent the mean±SD from 6 independent experiments. IL indicates interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor. C, Percentage of CD4, CD8, CD45RO, CD45RA, CD25, and CD69/71 positive cells in plaque T cells are presented. Results represent the mean±SD generated from 5 plaques. Plaque mononuclear cells were isolated from single cell populations using the standard Ficoll‐Paque PLUS, and followed by T‐cell purification using Dynabeads^® Untouched^™ Human T Cells. D, Plaque T‐cell proliferation induced by oxLDL‐treated DC was suppressed by atorvastatin (AVA). After the indicated treatment for 24 hours, DCs derived from patient peripheral blood monocyte were washed and cocultured with autologous plaque T cells in triplicates for 5 days. Bromodeoxyuridine (BrdU) was present in the last 16 hours. **P<0.01, DC+oxLDL vs DC and DC+AVA+oxLDL. ^## P<0.01, DC+ (AVA)+oxLDL vs DC+oxLDL.

Figure 7

Downregulation of let‐7c in dendritic cells (DCs) abrogated the plaque T‐cell proliferation. A, Expression of let‐7c in patient DCs was tested by quantitative reverse transcription polymerase chain reaction, with normal dendritic cell (DC) samples as controls. The value was normalized as fold change to that of nontreated normal DC samples. Results represent the mean±SD of 4 experiments. **P<0.01, DC+ oxidized low‐density lipoprotein (oxLDL) vs DC; ^## P<0.01, DC+ atorvastatin (AVA)+oxLDL vs DC+oxLDL. B, Downregulation of let‐7c abrogated the plaque T‐cell proliferation. After downregulation of let‐7c, oxLDL‐treated DCs were washed and cocultured in triplicates for 5 days with autologous plaque T cells. Bromodeoxyuridine (BrdU) was present in the last 16 hours and proliferative response was evaluated as OD value. **P<0.01 compared to that of mock‐treated samples. OD indicates optical density.