α1-antitrypsin mitigates NLRP3-inflammasome activation in amyloid β1-42-stimulated murine astrocytes

Abstract

Background: Neuroinflammation has an essential impact on the pathogenesis and progression of Alzheimer's disease (AD). Mostly mediated by microglia and astrocytes, inflammatory processes lead to degeneration of neuronal cells. The NLRP3-inflammasome (NOD-like receptor family, pyrin domain containing 3) is a key component of the innate immune system and its activation results in secretion of the proinflammatory effectors interleukin-1β (IL-1β) and interleukin-18 (IL-18). Under physiological conditions, cytosolic NLRP3-inflammsome is maintained in an inactive form, not able to oligomerize. Amyloid β_1-42 (Aβ_1-42) triggers activation of NLRP3-inflammasome in microglia and astrocytes, inducing oligomerization and thus recruitment of proinflammatory proteases. NLRP3-inflammasome was found highly expressed in human brains diagnosed with AD. Moreover, NLRP3-deficient mice carrying mutations associated with familial AD were partially protected from deficits associated with AD. The endogenous protease inhibitor α1-antitrypsin (A1AT) is known for its anti-inflammatory and anti-apoptotic properties and thus could serve as therapeutic agent for NLRP3-inhibition. A1AT protects neurons from glutamate-induced toxicity and reduces Aβ_1-42-induced inflammation in microglial cells. In this study, we investigated the effect of Aβ_1-42-induced NLRP3-inflammasome upregulation in primary murine astrocytes and its regulation by A1AT.

Methods: Primary cortical astrocytes from BALB/c mice were stimulated with Aβ_1-42 and treated with A1AT. Regulation of NLRP3-inflammasome was examined by immunocytochemistry, PCR, western blot and ELISA. Our studies included an inhibitor of NLRP3 to elucidate direct interactions between A1AT and NLRP3-inflammasome components.

Results: Our study revealed that A1AT reduces Aβ_1-42-dependent upregulation of NLRP3 at the mRNA and protein levels. Furthermore, A1AT time-dependently mitigated the expression of caspase 1 and its cleavage product IL-1β in Aβ_1-42-stimulated astrocytes.

Conclusion: We conclude that Aβ_1-42-stimulation results in an upregulation of NLRP3, caspase 1, and its cleavage products in astrocytes. A1AT time-dependently hampers neuroinflammation by downregulation of Aβ_1-42-mediated NLRP3-inflammasome expression and thus may serve as a pharmaceutical opportunity for the treatment of Alzheimer's disease.

Keywords: Alpha 1-antitrypsin; Alzheimer’s disease; Amyloid β; Astrocytes; Inflammasome; NALP3; NLRP3; Neuroinflammation.

Fig. 1

Experimental setting. a Cortices from BALB/c mice were prepared and seeded on flasks 2 weeks prior to stimulation. After a week, when cell confluence was about 80%, flasks were shaken to remove microglia and oligodendrocytes from astrocytes. At passage 2, astrocytes were seeded on experimental plates 48 h prior to stimulation. After short-term stimulation (3 h/ 6 h) with two concentrations of Aβ_1–42 (4, 10 μM) or LPS (1 μg/mL) and treatment with A1AT (4 mg/mL) immunocytochemistry, viability assays, caspase 1 assay, RNA and protein isolation were performed. For our control experiments, pretreatment with MCC950 (1 μM) was performed 1 h prior to stimulation. b Astrocyte purity was assessed by ICC using GFAP and Iba1 combined with Hoechst for DNA staining

Fig. 2

Effect of Aβ_1–42 stimulation and A1AT treatment ± MCC950 on cell viability at 3 h and 6 h. Treatment with Aβ_1–42 [4 μM], A1AT [4 mg/mL], and MCC950 [1 μM] did not affect LDH-release at 3 h and 6 h stimulation, whereas LPS-stimulation [1 μg/mL] significantly increased LDH-release (a, c). Cell lysis defined maximal (100%; 1.0) release of LDH. MTT-assay (b, d) revealed a trend towards decrease in cell metabolism after 3 h and 6 h Aβ_1–42-stimulation, which was not significant. Treatment with MCC950 led to an increase of cell metabolism in each treatment group at 3 h and 6 h, which was not significant. *^/ap < 0.05; **^/aap < 0.01; ^***/aaap < 0.001, ns not significant compared to untreated cell control (ctr)

Fig. 3

Treatment with A1AT abrogated Aβ_1–42-induced upregulation of NLRP3 mRNA and protein in primary astrocytes. a As quantified by RT-PCR treatment with 4 μM and 10 μM of Aβ_1–42 significantly increased mRNA levels of NLRP3 in astrocytes. In addition, co-treatment with 4 mg/mL of A1AT blocked this increase in NLRP3 mRNA expression significantly. Data of n = 6 in triplicate represent mean ± SD. b–c Densitometric analysis of western blots confirmed an Aβ_1–42-induced significant increase of NLRP3 protein levels at 3 h. Treatment with 4 mg/mL of A1AT significantly attenuated this increase at 10 μM Aβ_1–42. Data of n = 3 in triplicate represent mean ± SD. d–e Astrocytes treated with 10 μM of Aβ_1–42 showed a significant increase of NLRP3-fluorescence intensity, whereas co-treatment with A1AT significantly reduced NLRP3-expression. Fluorescence images of each experiment were taken using the exact same microscope settings and fluorescence intensity was measured by ImageJ (USA). Data of n = 4 in triplicate represent mean ± SD. *^/ap < 0.05; **^/aap < 0.01; ^***/aaap < 0.001, ns not significant compared to untreated cell control (ctr)

Fig. 4

Treatment with A1AT abrogated Aβ_1–42-induced upregulation of NLRP3 mRNA and protein in primary astrocytes. Western blot confirmed an Aβ_1–42-induced significant increase of NLRP3 protein levels at 3 h (a) and 6 h (b). Co-treatment with A1AT significantly attenuated this increase at 3 h (a) and 6 h (b) stimulation time. Addition of MCC950 did not alter NLRP3-protein levels in all treatment groups. Treatment with Aβ_1–42 significantly increased mRNA levels of NLRP3 in astrocytes at 3 h (c) and 6 h (d). Co-treatment with A1AT significantly blocked this increase in NLRP3 mRNA expression. MCC950-pretreatment had no effects on NLRP3 mRNA expression at 3 h and 6 h. Data of n = 3 in triplicate represent mean ± SD. *^/ap < 0.05; **^/aap < 0.01; ^***/aaap < 0.001, ns not significant compared to untreated cell control (ctr)

Fig. 5

ASC was not regulated by Aβ_1–42 and A1AT at 3 h and 6 h stimulation. (a) Quantitative RT-PCR demonstrated ASC was not regulated by Aβ_1–42 or A1AT. Data of n = 6 in triplicate represent mean ± SD. (b–c) Western blot revealed no regulation of ASC by Aβ_1–42 or A1AT. Data of n = 3 in triplicate represent mean ± SD. (e–f) RT-PCR demonstrated that also at 6 h stimulation time such as MCC950-pretreatment did not regulate ASC mRNA expression. Data of n = 3 in triplicate represent mean ± SD. *^/ap < 0.05; **^/aap < 0.01; ^***/aaap < 0.001, ns not significant compared to untreated cell control (ctr)

Fig. 6

A1AT time-dependently blocked Aβ_1–42-induced upregulation of caspase 1 mRNA in primary astrocytes. (b–c) As quantified by RT-PCR treatment with 4 μM Aβ_1–42 significantly increased mRNA levels of caspase 1 in astrocytes at 6 h, but not at 3 h. Co-treatment with A1AT blocked this increase in caspase 1 mRNA expression significantly at 6 h, but not at 3 h. Addition of MCC950 had no effects on caspase 1 mRNA expression at both stimulation times. Data of n = 3 in triplicate represent mean ± SD.*^/ap < 0.05; **^/aap < 0.01; ^***/aaap < 0.001, ns not significant compared to untreated cell control (ctr)

Fig. 7

A1AT mitigated caspase activity in Aβ_1–42-stimulated astrocytes. (a, c) For convenience, we have only illustrated the overlay images of all treatment groups ± MCC950. Counting of caspase active cells (b, d) revealed a significant increase of caspase active cells with 4 μM Aβ_1–42-stimulation at both stimulation times. Co-treatment with A1AT significantly blocked this effect at both stimulation times. In MCC950-treated and Aβ_1–42-stimulated cells caspase activity significantly declined at 3 h. Additive treatment by MCC950 to A1AT and Aβ_1–42-stimulated astrocytes did not significantly change caspase activity. Data of n = 3 in triplicate represent mean ± SD. *^/ap < 0.05; **^/aap < 0.01; ^***/aaap < 0.001, ns not significant compared to untreated cell control (ctr)

Fig. 8

A1AT mitigated Aβ_1–42-induced upregulation of IL-1β mRNA in primary astrocytes. (a–b) As quantified by RT-PCR stimulation with 4 μM Aβ_1–42 significantly increased mRNA levels of IL-1β in astrocytes at 3 h and 6 h. Co-treatment with A1AT blocked this increase significantly at 3 h and 6 h. Addition of MCC950 had no significant impact on gene expression at 3 h and 6 h. (c) Aβ_1–42-stimulation such as A1AT-treatment did not affect IL-18 mRNA expression at 3 h. (d) Stimulation with Aβ_1–42 significantly increased mRNA levels of IL-18 in astrocytes at 6 h. Co-treatment with A1AT did not block this increase. MCC950 had no effects on gene expression of IL-18 at 3 h and 6 h. Data of n = 3 in triplicate represent mean ± SD. *^/ap < 0.05; **^/aap < 0.01; ^***/aaap < 0.001, ns not significant compared to untreated cell control (ctr)

Fig. 9

Treatment with A1AT time-dependently mitigated Aβ_1–42-induced upregulation of IL1β-precursor protein in primary astrocytes. Western blot confirmed an Aβ_1–42-induced significant increase of of IL1β-precursor protein at 3 h (a) and 6 h (b). Co-treatment with A1AT significantly attenuated this increase at 3 h and 6 h stimulation time. Addition of MCC950 did not alter levels of IL1β-precursor protein in Aβ_1–42-stimulated astrocytes. A1AT + in Aβ_1–42 in the presence of MCC950 did not alter IL1β-precursor protein levels at both 3 h and 6 h. (c–d) IL-1β-ELISA revealed a time-dependent upregulation of IL-1β protein levels in Aβ_1–42-stimulated astrocytes, significant at 6 h stimulation. Co-treatment with A1AT significantly decreased protein expression of IL-1β time-dependently, at 6 h stimulation time. The presence of MCC950 in Aβ_1–42-stimulated decreased IL-1β-expression significantly at both 3 h and 6 h. Further, there is a trend towards a decrease of IL-1β-protein levels in the presence of MCC950 in nearly all treatment groups, though not significant. Co-stimulation of Aβ_1–42 and A1AT in MCC950-pretreated astrocytes did not affect IL-1β-expression. Data of n = 3 in triplicate represent mean ± SD. *^/ap < 0.05; **^/aap < 0.01; ^***/aaap < 0.001, ns not significant compared to untreated cell control (ctr)