Oxidized low density lipoprotein induced caspase-1 mediated pyroptotic cell death in macrophages: implication in lesion instability?

Abstract

Background: Macrophage death in advanced lesion has been confirmed to play an important role in plaque instability. However, the mechanism underlying lesion macrophage death still remains largely unknown.

Methods and results: Immunohistochemistry showed that caspase-1 activated in advanced lesion and co-located with macrophages and TUNEL positive reaction. In in-vitro experiments showed that ox-LDL induced caspase-1 activation and this activation was required for ox-LDL induced macrophages lysis, IL-1β and IL-18 production as well as DNA fragmentation. Mechanism experiments showed that CD36 and NLRP3/caspase-1/pathway involved in ox-LDL induced macrophage pyroptosis.

Conclusion: Our study here identified a novel cell death, pyroptosis in ox-LDL induced human macrophage, which may be implicated in lesion macrophages death and play an important role in lesion instability.

Figure 1. Cleaved caspase-1 over-expressed in human atherosclerotic plaque.

A. Immunohistochemical analysis of cleaved caspase-1 in non-atherosclerotic vessel (control), EAL and AAL. In the left column, representative immunostaining for cleaved caspase-1(brown, arrow) in exemplary sections of non-atherosclerotic vessel (control), early atherosclerotic lesion (EAL) and advanced atherosclerotic lesion (AAL) were shown. High-power photomicrograph (×200) exhibited strong cleaved caspase-1 staining in AAL, as shown in the upper boxed area in low-power photomicrograph (×40). In the right column, bar graphs showed the relatively quantitative cleaved caspase-1 expression in control, EAL and AAL. *indicated vs control, ***p<0.001; ^#indicated vs EAL, ^### p<0.001. NC, necrotic core. B. Western blot analysis of caspase-1, mature IL-1β and IL-18 protein. Protein was extracted from 5 vessel tissues obtained from non-atherosclerotic vessel (control), EAL and AAL. β-actin was used for protein loading controls. C. Double staining of cleaved caspase-1 co-located with CD68 positive cells in AAL. In the right column, high-power photomicrograph (×400) showed strong cleaved caspase-1 staining (red) correlated with CD68 positive cell (green) around necrotic core (NC), as shown in the boxed area of HE staining and DAPI staining (blue) in the left column (×40). D. Double staining of cleaved caspase-1(red) co-located with TUNEL reaction (green) in AAL (×200).

Figure 2. Caspase-1 activated in ox-LDL induced human macrophages.

Human macrophages were cultured with PBS (control), with ox-LDL (100 µg/ml) or with native LDL (100 µg/ml) for 48 hours. After incubation, caspase-1 activity was measured by Ac-YVAD-pNA (A). Cells cultured with LPS (1 µg/ml) for 6 h followed by ATP (5 mM) for 30 min as a positive control. Data are presented as mean±SEM of at least three independent experiments. *indicated vs untreated cells (control), ***p<0.001. ^#indicated vs ox-LDL treated cells, ^## p<0.01, ^### p<0.001.NS, not significant differences (p>0.05). B. Protein of cleaved caspase-1, mature IL-1β and IL-18 in cell lysates was analyzed by western blot. β-actin was used for protein loading controls. C. Dose and time-dependent caspase-1 activity in HMDMs. Ox-LDL (5 µg/ml, 50 µg/ml, 100 µg/ml, and 200 µg/ml) were incubated with HMDMs for indicated time, and then caspase-1 activity was measured by Ac-YVAD-pNA. Data are presented as mean±SEM of at least three independent experiments. D. Ox-LDL ((5 µg/ml, 50 µg/ml, 100 µg/ml)) or native LDL (100 µg/ml) were incubated with HMDMs for 48 h, then protein of caspase-1, mature IL-1β and IL-18 protein in cell lysates were measured by western blot. β-actin was used for protein loading controls. E. Ox-LDL(100 µg/ml) were incubated with HMDMs for indicated duration, then cleaved caspase-1, mature IL-1β and IL-18 protein in cell lysates were measured by western blot. β-actin was used for protein loading controls.

Figure 3. Activated caspase-1 was required for ox-LDL induced macrophages death.

A. Cell was cultured as described previously. Cells death was visualized by ETHD-III (red) and calcein AM (green) staining (×100). B. Qualitative analysis of cell death expressed as a percentage of LDH release by Triton X-100 detergent or a percent of ETHD-III positive cells in the total cells. *indicated vs untreated cells, *p<0.05; **p<0.01; ***p<0.001. NS, not significant differences (p>0.05). C. Cells were pretreated for 1 hour with vehicle (DMSO), with inhibitors of caspase-1 (Ac-YVAD-CHO), caspase-3 (Ac-DEVD-CHO), caspase-4 (Ac-LEVD-CHO), caspase-6 (Ac-VEID-CHO), caspase-8 (Z-IETD-CHO), caspase-9 (Ac-LEHD-CHO), or with pan-caspase (z-VAD-CHO), and then cells were cultured with ox-LDL (100 µg/ml) for 48 h. Cell death was determined by LDH release. Inhibitors concentration was 100 µM in all cases. *indicated vs cells pre-cultured with DMSO.*p<0.05; **p<0.01; ***p<0.001. NS, not significant differences (p>0.05). D. Cells were transfected with non-targeting siRNA (control) or siRNA specific for caspase-1. After transfection, inhibition rate of siRNA was measured by RT-PCR. Transfected cells were cultured for 48 h with or without ox-LDL (100 µg/ml). Cell death was determined by LDH release and ETHD-III/calcein AM staining (×100). ***p<0.001. NS, not significant differences (p>0.05). Data are presented as mean±SEM of at least three independent experiments.

Figure 4. Caspase-1 activation was required for cytokines production.

Cells were transfected with non-targeting siRNA (control), siRNA specific for caspase-1, caspase-3, caspase-8 and caspase-9. After transfection, cells were cultured for 48 h with or without ox-LDL (100 µg/ml). Cytokines production of IL-1β, IL-18, IL-33, TNF-α, IL-6 and MCP-1 was determined by ELISA. *p<0.05; ***p<0.001. NS, not significant differences (p>0.05). Data are presented as mean±SEM of at least three independent experiments.

Figure 5. Involvement of activated caspase-1 in ox-LDL induced DNA fragmentation.

A. Cells were cultured with PBS (control), with ox-LDL (100 µg/ml, 48 h), LPS (1 µg/ml, 6 h) followed by ATP (5 mM, 30 min), or with STS (1 Μm, 4 h).After incubation, DNA fragmentation was measured by TUNEL staining (green). Qualitative analysis of DNA fragmentation by randomly counting 10 fields of the section and were expressed as a percentage of the total nuclei population. * indicated vs untreated cells (control), *p<0.05; ***p<0.001. B. Cells were transfected with non-targeting siRNA (control) or siRNA specific for caspase-1. Then cells were cultured for 48 h with or without ox-LDL (100 µg/ml). DNA fragmentation was determined by TUNEL positive counting. *p<0.05. NS, not significant difference (p>0.05). C. Cells were pretreated for 1 hour with vehicle (DMSO) or with corresponding caspase inhibitors (100 uM), and then cells were cultured with ox-LDL (100 µg/ml) for 48 h. DNA fragmentation was determined by TUNEL positive counting. *indicated vs control, *p<0.05; **p<0.01; ***p<0.001. NS, not significant difference (p>0.05). D. Protein of cleaved caspase-1 and ICAD in cell lysates were measured by western blot. β-actin was used for protein loading controls. Data are presented as mean±SEM of at least three independent experiments.

Figure 6. Role of inflammasome components ASC and NLRP3 in ox-LDL induced caspase-1 activation.

A. Cells were cultured with PBS (control) or with ox-LDL (100 µg/ml, 48 h). Gene expression of inflammasome components was measured by RT-PCR. * indicated vs control, *p<0.05. NS, not significant differences (p>0.05). Cells were transfected with non-targeting siRNA (control) or siRNA specific for NLRP3 (B) or ASC (C). After transfection, inhibition rate of siRNA was measured by RT-PCR. Transfected cells were then cultured for 48 h with or without ox-LDL (100 µg/ml, 48 h). Caspase-1 activity was measured by Ac-YVAD-pNA. ***p<0.001. NS, not significant differences (p>0.05). D. Cells were transfected and cultured as described above. Protein of cleaved caspase-1 was measured by western blot. Un-transfected human macrophages cultured with ox-LDL was used as control. β-actin was used for protein loading controls. Data are presented as mean±SEM of at least three independent experiments.

Figure 7. Involvement of CD36 in ox-LDL induced caspase-1 activation, LDH release and IL-1β and IL-18 production.

A. Cells were pretreated with vehicles (control), NAC (10 mM, 24 h) or VitC (100 mM, 24 h), or with CD36 blocking antibody (5 ug/ml or 20 ug/ml), then cells were cultured with ox-LDL (100 µg/ml, 48 h). Intracellular ROS in HMDMs were assessed by DCFH2-DA. B. Cleaved caspase-1 was measured by western blot. β-actin was used for protein loading controls. C. Caspase-1 activity was measured by Ac-YVAD-pNA. ^###p<0.001. NS, not significant differences (p>0.05). D. Cell death was measured by the percentage of LDH release. E. Cytokine production of IL-1β and IL-18 was measured by ELISA. *indicated vs cells cultured with ox-LDL, *p<0.05; **p<0.01; ***p<0.001. ^#indicated vs cells cultured with control. ^### p<0.001. NS, not significant differences (p>0.05). Data are presented as mean±SEM of at least three independent experiments.