NLRP3 Is Involved in the Maintenance of Cerebral Pericytes

Abstract

Pericytes play a central role in regulating the structure and function of capillaries in the brain. However, molecular mechanisms that drive pericyte proliferation and differentiation are unclear. In our study, we immunostained NACHT, LRR and PYD domains-containing protein 3 (NLRP3)-deficient and wild-type littermate mice and observed that NLRP3 deficiency reduced platelet-derived growth factor receptor β (PDGFRβ)-positive pericytes and collagen type IV immunoreactive vasculature in the brain. In Western blot analysis, PDGFRβ and CD13 proteins in isolated cerebral microvessels from the NLRP3-deficient mouse brain were decreased. We further treated cultured pericytes with NLRP3 inhibitor, MCC950, and demonstrated that NLRP3 inhibition attenuated cell proliferation but did not induce apoptosis. NLRP3 inhibition also decreased protein levels of PDGFRβ and CD13 in cultured pericytes. On the contrary, treatments with IL-1β, the major product of NLRP3-contained inflammasome, increased protein levels of PDGFRβ, and CD13 in cultured cells. The alteration of PDGFRβ and CD13 protein levels were correlated with the phosphorylation of AKT. Inhibition of AKT reduced both protein markers and abolished the effect of IL-1β activation in cultured pericytes. Thus, NLRP3 activation might be essential to maintain pericytes in the healthy brain through phosphorylating AKT. The potential adverse effects on the cerebral vascular pericytes should be considered in clinical therapies with NLRP3 inhibitors.

Keywords: Alzheimer’s disease; NLRP3; cerebral perfusion; neuroinflammation; pericyte.

Figure 1

NLRP3 deficiency reduces pericyte cell coverage and decreases protein levels of PDGFRβ and CD13 in the brain. (A) Nine-month-old mouse brains were co-stained for NLRP3 and PDGFRβ. PDGFRβ-immune reactive cell bodies (in green; marked with arrows) were stained by NLRP3-specific antibodies (in red). (B) Brain tissues from 9-month-old NLRP3-knockout (−/− and +/−) and wild-type (+/+) littermate mice were then co-stained for PDGFRβ (with anti-PDGFRβ antibodies, in green) and endothelial cells (with isolectin B4, in red). (C) The coverage of PDGFRβ-positive pericytes was calculated as a ratio of PDGFRβ/isolectin B4-positive area. One-way ANOVA followed by Tukey post hoc test, n = 4 per group. (D–G) Nine-month-old NLRP3, and 6-month-old MyD88 littermate mice with homozygous (−/−) and heterozygous (+/−) knockout, and wild-type (+/+) of nlrp3 and myd88 genes, respectively, were analyzed for protein levels of PDGFRβ and CD13 in isolated cerebral blood vessels. One-way ANOVA followed by Bonferroni post hoc test, n = 10, 10 and 8 for NLRP3 (+/+, +/− and −/−) mice in PDGFRβ detection and n = 9, 9 and 7 for NLRP3 (+/+, +/− and −/−) mice in CD13 detection; n = 8, 10 and 6 for MyD88 (+/+, +/− and −/−) mice in the detection of both PDGFRβ and CD13. *p < 0.05 and **p < 0.01.

Figure 2

NLRP3 deficiency reduces vasculature in the brain. (A) The brains of 9-month-old littermate mice with homozygous (−/−), heterozygous (+/−), and wild-type (+/+) of nlrp3 gene were stained for collagen type IV (Col IV). The blood vessels in the hippocampus were thresholded and skeletonized. The skeleton representation of vasculature is shown in red and branching points of blood vessels are in blue. (B,C) The total length and branching points of blood vessels were calculated and adjusted by area of analysis. One-way ANOVA followed by Bonferroni post hoc test, n = 8, 8, and 6 for NLRP3 (+/+, +/− and −/−) mice, respectively. **p < 0.01. (D) Nine-month-old NLRP3-deficient and wild-type mouse brains were further stained for mouse IgG and isolectin B4. We could not detect mouse IgG in the brain parenchyma.

Figure 3

NLRP3 inhibition attenuates cell proliferation in cultured pericytes. (A) The cell lysates collected from cultured pericytes with different passaging numbers were detected for NLRP3 with Western blot. (B) Pericytes were treated with NLRP3 inhibitor, MCC950, at 0, 25, 50, and 100 nM for 24 h and then detected for cleaved caspase-1. After inhibition of NLRP3, cleaved caspase-1 was nearly undetectable. (C) Cultured pericytes were treated with MCC950 with various doses and analyzed for proliferation with MTT assay every day for 7 days. Two-way ANOVA followed by Tukey post hoc test, n = 4 per group. **p < 0.01 and ***p < 0.001. (D–H) Pericytes were cultured and treated with MCC950 at indicated concentrations for 24 h. Cell lysates were detected for cleaved caspase-3, PCNA, and Ki-67 with a quantitative Western blot. As a positive control for cleavage of caspase-3, the brain lysate from neuronal ATG5-deficient mice was used. The result in (D) shows one experiment representative of four independent experiments. The inhibition of NLRP3 reduces protein levels of PCNA and Ki-67 in a dose-dependent manner. One-way ANOVA followed by Tukey post hoc test, n = 3 per group for PCNA, and n = 4 per group for Ki-67. *p < 0.05 and **p < 0.01. (I) cultured pericytes were further treated with recombinant IL-1β at 0, 5, 10, and 50 ng/ml and analyzed with MTT assay every 2 days for 7 days. Two-way ANOVA analysis did not show the effects of IL-1β on cell proliferation, n = 3 per group.

Figure 4

NLRP3 inhibition attenuates protein expression of PDGFRβ and CD13 and inhibits phosphorylation of AKT and ERK in cultured pericytes. Pericytes were cultured and treated with MCC950 at 0, 25, 50, and 100 nM for 24 h. (A,D,G) Western blot was used to detect PDGFRβ and CD13, as well as phosphorylated and total protein levels of AKT, ERK, and NFκB p65. (B,C,E,F) Inhibition of NLRP3 reduces protein levels of PDGFRβ and CD13 and inhibits phosphorylation of AKT and ERK with a dose-dependent pattern. one-way ANOVA followed by Tukey post hoc test, n = 4 per group. *p < 0.05, **p < 0.01 and ***p < 0.001. (H) Phosphorylation of NFκB p65 is not significantly changed by inhibition of NLRP3. One-way ANOVA, n = 3 per group.

Figure 5

IL-1β increases protein expression of PDGFRβ and CD13 in cultured pericytes. Pericytes were cultured and treated with recombinant human IL-1β at 0, 5, 10, and 50 ng/ml for 24 h. (A,D) Western blot was used to detect PDGFRβ and CD13, as well as phosphorylated and total protein levels of AKT and ERK. (B,C,E,F) Stimulation of IL-1β increases protein levels of PDGFRβ and CD13, and activates phosphorylation of AKT, but not ERK, in a dose-dependent manner. One-way ANOVA followed by Tukey post hoc test, n = 3 per group. *p < 0.05 and **p < 0.01.

Figure 6

Inhibition of AKT signaling pathway suppresses protein expression of PDGFRβ and CD13 and abolishes the effect of IL-1β in cultured pericytes. Pericytes were cultured and treated with AKT inhibitor at 0, 0.5, 1, and 5 μM for 24 h. (A–C) Western blot was used to detect phosphorylated and total AKT, and GSK3β, which shows that phosphorylation of both AKT and GSK3β was significantly inhibited by treatments with AKT inhibitors. One-way ANOVA followed by Tukey post hoc test, n = 4 per group. (D–F) The cell lysate from AKT inhibitor-treated pericytes were detected for protein levels of PDGFRβ and CD13 with a quantitative Western blot. The inhibition of AKT decreases protein levels of PDGFRβ and CD13 in a dose-dependent manner. One-way ANOVA followed by Tukey post hoc test, n = 3 per group. *p < 0.05, **p < 0.01 and ***p < 0.001. (G–I) Cultured pericytes were pre-treated with 1 μM AKT inhibitor and then incubated with IL-1β at 0, 5, 10, and 50 ng/ml, in the presence of AKT inhibitor for 24 h. AKT inhibition abolished the effect of IL-1β on the up-regulation of PDGFRβ and CD13 expression. one-way ANOVA followed by Tukey post hoc test, n = 3 per group. **p < 0.01.