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NLRP3 inflammasome: a novel link between lipoproteins and atherosclerosis

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Pattern recognition receptor-mediated signaling pathways have recently been elucidated to bridge the innate immune system and atherosclerosis. NLRP3 is a member of the NLR family. Upon activation, it initiates IL-1β and IL-18 processing, a key step in the inflammatory process of atherosclerosis.

Material and methods: We used three different types of lipoproteins, ox-LDL, ox-HDL, and HDL, in Thp-1 at the concentration of 50 mg/l, 100 mg/l, and 150 mg/l respectively. Using real-time polymerase chain reaction and western blot, ELISA detected the expression of NLRP3 and downstream cytokines. NLRP3 siRNA was constructed to down-regulate expression of the NLRP3 gene via the RNA interference technique. 150 mg/l of ox-LDL, ox-HDL and HDL was added to the Thp-1 cell line respectively. We observed the changes in the expression of caspase-1, IL-1β and IL-18 when the NLRP3 gene was down-regulated.

Results: Ox-LDL and ox-HDL addition not only increases the expression of NLRP3, but also activates the NLRP3 downstream cytokines and caspase-1 and induces IL-1β and IL-18 secretion. Moreover, the effects of activation and induction are shown to have a dose-dependent manner. Expression of NLRP3 and its downstream inflammatory cytokines is reduced in the presence of HDL (p < 0.05). Furthermore, our data demonstrated that NLRP3 siRNA downregulates NLRP3 expression in mononuclear cells, thus leading to a dramatic reduction in the expression of caspase-1, IL-1β and IL-18 (p < 0.05).

Conclusions: The data suggest that activation of the NLRP3 inflammasome is a critical step in caspase-1 activation and IL-1β and IL-18 secretion. Interference with the NLRP3 inflammasome can significantly inhibit the generation of cytokines, thus impeding the pathogenesis of inflammation.

No MeSH data available.


Ox-LDL, ox-HDL and HDL induced the protein expression of NLRP3 and its downstream cytokines in human mononuclear cellsThe sample order of Western blot from left to right: blank, ox-LDL-1, ox-LDL-2, ox-LDL-3, HDL-1, HDL-2, HDL-3, ox-HDL-1, ox- HDL-2, ox-HDL-3. Blank – blank control group, HDL-1 – group of HDL at the concentration of 50 mg/l; HDL-2 – group of HDL at the concentration of 100 mg/l, HDL-3 – group of HDL at the concentration of 150 mg/l, ox-LDL-1 – group of ox-LDL at the concentration of 50 mg/l, ox-LDL-2 – group of ox-LDL at the concentration of 100 mg/l, ox-LDL-3 – group of ox-LDL at the concentration of 150 mg/l, ox-HDL-1 – group of ox-HDL at the concentration of 50 mg/l, ox-HDL-2 – group of ox-HDL at the concentration of 100 mg/l, ox-HDL-3 – group of ox-HDL at the concentration of 150 mg/l. Each set of HDL, ox-LDL, ox-HDL concentration after processing, *p < 0.05, compared with the blank control group was statistically significantly different, #p < 0.05, compared with 50 mg/l group was statistically significantly different, Δp < 0.05, compared with 100 mg/l group was statistically significantly different.
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Figure 0002: Ox-LDL, ox-HDL and HDL induced the protein expression of NLRP3 and its downstream cytokines in human mononuclear cellsThe sample order of Western blot from left to right: blank, ox-LDL-1, ox-LDL-2, ox-LDL-3, HDL-1, HDL-2, HDL-3, ox-HDL-1, ox- HDL-2, ox-HDL-3. Blank – blank control group, HDL-1 – group of HDL at the concentration of 50 mg/l; HDL-2 – group of HDL at the concentration of 100 mg/l, HDL-3 – group of HDL at the concentration of 150 mg/l, ox-LDL-1 – group of ox-LDL at the concentration of 50 mg/l, ox-LDL-2 – group of ox-LDL at the concentration of 100 mg/l, ox-LDL-3 – group of ox-LDL at the concentration of 150 mg/l, ox-HDL-1 – group of ox-HDL at the concentration of 50 mg/l, ox-HDL-2 – group of ox-HDL at the concentration of 100 mg/l, ox-HDL-3 – group of ox-HDL at the concentration of 150 mg/l. Each set of HDL, ox-LDL, ox-HDL concentration after processing, *p < 0.05, compared with the blank control group was statistically significantly different, #p < 0.05, compared with 50 mg/l group was statistically significantly different, Δp < 0.05, compared with 100 mg/l group was statistically significantly different.

Mentions: To better understand the potential role of NLRP3 in atherosclerotic inflammation, we also determined the expression of NLRP3 and its downstream inflammatory cytokines as they function as a multi-protein complex. The data clearly indicated that the expression of NLRP3 is enhanced in ox-LDL and ox-HDL groups, and the concentration dependence is more pronounced in the ox-LDL group. However, the level of NLRP3 expression is only increased at the high concentration of ox-HDL (100 mg/l and 150 mg/l) as compared to the control, and there is no significant difference in the expression of NLRP3 between these two concentrations. In contrast, the expression of NLRP3 is diminished in the presence of HDL in a concentration-dependent manner. NLRP3-induced activation of caspase-1 is more pronounced in the presence ox-LDL or ox-HDL. In particular, there is a remarkable increase in the expression of caspase-1 at a high concentrations of ox-LDL (100 mg/l and 150 mg/l) as compared to the control. However, there is no statistically significant difference between these two concentrations. In the group of ox-HDL, the caspase-1 expression can only be induced at the high concentration of 100 mg/l or 150 mg/l, and no statistically significant difference is observed between these two concentrations. In contrast, the level of caspase-1 expression is reduced in the presence of HDL, and this inhibitory effect is also concentration dependent. Activation of caspase-1 can further promote the splicing and modification of its downstream cytokines, IL-1β and IL-18 precursor and induce the maturation and secretion of IL-1β and IL-18. We also determined the expression of IL-1β and IL-18 via Western blotting analysis. It showed that the IL-1β expression is only increased at the concentration of ox-LDL (100 mg/l, 150 mg/l) as compared to the control, but there is a statistically significant difference between these two concentrations. No change in the expression of IL-1β at the concentrations of 50 mg/l and 100 mg/l of ox-HDL was observed as compared to the control. However, at the concentration of 150 mg/l, the expression of IL-1β is dramatically enhanced. In contrast, the expression of IL-1β is inhibited in the presence of HDL as compared to the control group. However, no statistically significant difference was observed between the concentrations of 100 mg/l and 150 mg/l of HDL. The expression of IL-18 is significantly increased in the presence of ox-LDL although there is no statistically significant difference between the concentration of 50 mg/l and 100 mg/l. However, the ox-LDL induction of IL-18 is more evident at the concentration of 150 mg/l. The IL-18 expression is remarkably enhanced in an ox-HDL concentration dependent manner. In comparison, HDL addition has a statistically significant inhibitory effect on the expression of IL-18 as compared to the control. However, no statistically significant difference was detected between 100 mg/l and 150 mg/l of HDL (Figure 2).


NLRP3 inflammasome: a novel link between lipoproteins and atherosclerosis
Ox-LDL, ox-HDL and HDL induced the protein expression of NLRP3 and its downstream cytokines in human mononuclear cellsThe sample order of Western blot from left to right: blank, ox-LDL-1, ox-LDL-2, ox-LDL-3, HDL-1, HDL-2, HDL-3, ox-HDL-1, ox- HDL-2, ox-HDL-3. Blank – blank control group, HDL-1 – group of HDL at the concentration of 50 mg/l; HDL-2 – group of HDL at the concentration of 100 mg/l, HDL-3 – group of HDL at the concentration of 150 mg/l, ox-LDL-1 – group of ox-LDL at the concentration of 50 mg/l, ox-LDL-2 – group of ox-LDL at the concentration of 100 mg/l, ox-LDL-3 – group of ox-LDL at the concentration of 150 mg/l, ox-HDL-1 – group of ox-HDL at the concentration of 50 mg/l, ox-HDL-2 – group of ox-HDL at the concentration of 100 mg/l, ox-HDL-3 – group of ox-HDL at the concentration of 150 mg/l. Each set of HDL, ox-LDL, ox-HDL concentration after processing, *p < 0.05, compared with the blank control group was statistically significantly different, #p < 0.05, compared with 50 mg/l group was statistically significantly different, Δp < 0.05, compared with 100 mg/l group was statistically significantly different.
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Figure 0002: Ox-LDL, ox-HDL and HDL induced the protein expression of NLRP3 and its downstream cytokines in human mononuclear cellsThe sample order of Western blot from left to right: blank, ox-LDL-1, ox-LDL-2, ox-LDL-3, HDL-1, HDL-2, HDL-3, ox-HDL-1, ox- HDL-2, ox-HDL-3. Blank – blank control group, HDL-1 – group of HDL at the concentration of 50 mg/l; HDL-2 – group of HDL at the concentration of 100 mg/l, HDL-3 – group of HDL at the concentration of 150 mg/l, ox-LDL-1 – group of ox-LDL at the concentration of 50 mg/l, ox-LDL-2 – group of ox-LDL at the concentration of 100 mg/l, ox-LDL-3 – group of ox-LDL at the concentration of 150 mg/l, ox-HDL-1 – group of ox-HDL at the concentration of 50 mg/l, ox-HDL-2 – group of ox-HDL at the concentration of 100 mg/l, ox-HDL-3 – group of ox-HDL at the concentration of 150 mg/l. Each set of HDL, ox-LDL, ox-HDL concentration after processing, *p < 0.05, compared with the blank control group was statistically significantly different, #p < 0.05, compared with 50 mg/l group was statistically significantly different, Δp < 0.05, compared with 100 mg/l group was statistically significantly different.
Mentions: To better understand the potential role of NLRP3 in atherosclerotic inflammation, we also determined the expression of NLRP3 and its downstream inflammatory cytokines as they function as a multi-protein complex. The data clearly indicated that the expression of NLRP3 is enhanced in ox-LDL and ox-HDL groups, and the concentration dependence is more pronounced in the ox-LDL group. However, the level of NLRP3 expression is only increased at the high concentration of ox-HDL (100 mg/l and 150 mg/l) as compared to the control, and there is no significant difference in the expression of NLRP3 between these two concentrations. In contrast, the expression of NLRP3 is diminished in the presence of HDL in a concentration-dependent manner. NLRP3-induced activation of caspase-1 is more pronounced in the presence ox-LDL or ox-HDL. In particular, there is a remarkable increase in the expression of caspase-1 at a high concentrations of ox-LDL (100 mg/l and 150 mg/l) as compared to the control. However, there is no statistically significant difference between these two concentrations. In the group of ox-HDL, the caspase-1 expression can only be induced at the high concentration of 100 mg/l or 150 mg/l, and no statistically significant difference is observed between these two concentrations. In contrast, the level of caspase-1 expression is reduced in the presence of HDL, and this inhibitory effect is also concentration dependent. Activation of caspase-1 can further promote the splicing and modification of its downstream cytokines, IL-1β and IL-18 precursor and induce the maturation and secretion of IL-1β and IL-18. We also determined the expression of IL-1β and IL-18 via Western blotting analysis. It showed that the IL-1β expression is only increased at the concentration of ox-LDL (100 mg/l, 150 mg/l) as compared to the control, but there is a statistically significant difference between these two concentrations. No change in the expression of IL-1β at the concentrations of 50 mg/l and 100 mg/l of ox-HDL was observed as compared to the control. However, at the concentration of 150 mg/l, the expression of IL-1β is dramatically enhanced. In contrast, the expression of IL-1β is inhibited in the presence of HDL as compared to the control group. However, no statistically significant difference was observed between the concentrations of 100 mg/l and 150 mg/l of HDL. The expression of IL-18 is significantly increased in the presence of ox-LDL although there is no statistically significant difference between the concentration of 50 mg/l and 100 mg/l. However, the ox-LDL induction of IL-18 is more evident at the concentration of 150 mg/l. The IL-18 expression is remarkably enhanced in an ox-HDL concentration dependent manner. In comparison, HDL addition has a statistically significant inhibitory effect on the expression of IL-18 as compared to the control. However, no statistically significant difference was detected between 100 mg/l and 150 mg/l of HDL (Figure 2).

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Pattern recognition receptor-mediated signaling pathways have recently been elucidated to bridge the innate immune system and atherosclerosis. NLRP3 is a member of the NLR family. Upon activation, it initiates IL-1&beta; and IL-18 processing, a key step in the inflammatory process of atherosclerosis.

Material and methods: We used three different types of lipoproteins, ox-LDL, ox-HDL, and HDL, in Thp-1 at the concentration of 50 mg/l, 100 mg/l, and 150 mg/l respectively. Using real-time polymerase chain reaction and western blot, ELISA detected the expression of NLRP3 and downstream cytokines. NLRP3 siRNA was constructed to down-regulate expression of the NLRP3 gene via the RNA interference technique. 150 mg/l of ox-LDL, ox-HDL and HDL was added to the Thp-1 cell line respectively. We observed the changes in the expression of caspase-1, IL-1&beta; and IL-18 when the NLRP3 gene was down-regulated.

Results: Ox-LDL and ox-HDL addition not only increases the expression of NLRP3, but also activates the NLRP3 downstream cytokines and caspase-1 and induces IL-1&beta; and IL-18 secretion. Moreover, the effects of activation and induction are shown to have a dose-dependent manner. Expression of NLRP3 and its downstream inflammatory cytokines is reduced in the presence of HDL (p &lt; 0.05). Furthermore, our data demonstrated that NLRP3 siRNA downregulates NLRP3 expression in mononuclear cells, thus leading to a dramatic reduction in the expression of caspase-1, IL-1&beta; and IL-18 (p &lt; 0.05).

Conclusions: The data suggest that activation of the NLRP3 inflammasome is a critical step in caspase-1 activation and IL-1&beta; and IL-18 secretion. Interference with the NLRP3 inflammasome can significantly inhibit the generation of cytokines, thus impeding the pathogenesis of inflammation.

No MeSH data available.