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Updated: Dec 13, 2025
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
Beta-amyloid activates NLRP3 inflammasome via TLR4 in mouse microglia
1Department of Neurology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China; Institute of Neurological Diseases, Xuzhou Medical University, Xuzhou, China.
Abstract:
Beta-amyloid(Aβ)-induced inflammation plays a critical role in the pathogenesis of Alzheimer's disease (AD). Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) inflammasome is involved in the Aβ-induced inflammation. However, the mechanisms by which extracellular Aβ activates cytoplasmic NLRP3 inflammasome are poorly understood. Toll-like receptor 4(TLR4) acts as a sensor of Aβ and performs a key role in neuroinflammation. TLR4 is involved in activating the NLRP3 inflammasome in several diseases. In this study, the interaction between TLR4 and NLRP3 inflammasome in Aβ1-42-induced neuroinflammation was investigated. BV-2 microglia and primary microglia were primed with lipopolysaccharide (LPS) and then pretreated with TLR4 inhibitor CLI-095, followed by stimulation with Aβ1-42. The protein expression of NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 p 10 was detected by western blotting and immunostaining. The mRNA expression of inflammatory factors was measured by real-time PCR. The protein level of pro IL-1β and IL-1β was examined by ELISA. Activated microglia were examined by immunofluorescence staining for ionized calcium-binding adapter molecule-1 (Iba-1). Conditioned medium of BV-2 cells was collected to challenge HT-22 neurons. Cell viability was assessed with MTT assay. Assessment of HT-22 cell apoptosis was performed by Annexin V/PI staining and western blotting to detect the protein level of cleaved caspase 3. The results showed that Aβ1-42 activated and up-regulated the expression of NLRP3 inflammasome in BV-2 microglia, as indicated by increased activation of caspase-1 and secretion of IL-1β. Pharmacological inhibition of TLR4 by CLI-095 abolished Aβ1-42-induced NLRP3 inflammasome activation, which curbed the development of inflammation and exerted protective effect on HT-22 neurons. Furthermore, the inhibitory effects of CLI-095 on Aβ1-42-induced inflammation were reversed by NLRP3 activator ATP. Overall, our findings suggested TLR4 mediated Aβ1-42-induced NLRP3 inflammasome activation in mouse microglia. TLR4/NLRP3 pathway plays a critical role in Aβ1-42-induced neuroinflammation.
Insights
Toll-like receptor 4 (TLR4) mediates beta-amyloid (Aβ)-induced NLRP3 inflammasome activation in microglia, a key driver of Alzheimer's disease neuroinflammation. Inhibiting TLR4 reduces inflammation and protects neurons, highlighting the TLR4/NLRP3 pathway as a therapeutic target.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Beta-amyloid (Aβ) peptide accumulation and subsequent neuroinflammation are central to Alzheimer's disease (AD) pathogenesis.
- The Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) inflammasome is implicated in Aβ-induced inflammation, but its activation mechanism by extracellular Aβ remains unclear.
- Toll-like receptor 4 (TLR4) is recognized as a sensor for Aβ and plays a crucial role in initiating neuroinflammation.
Purpose of the Study:
- To investigate the interaction between Toll-like receptor 4 (TLR4) and the NLRP3 inflammasome in the context of Aβ₁₋₄₂-induced neuroinflammation.
- To elucidate the role of the TLR4/NLRP3 pathway in microglial activation and subsequent neuronal damage.
- To evaluate the therapeutic potential of targeting the TLR4 pathway in Aβ-induced neuroinflammation.
Main Methods:
- Utilized BV-2 and primary microglia models, primed with lipopolysaccharide (LPS) and pretreated with the TLR4 inhibitor CLI-095, followed by Aβ₁₋₄₂ stimulation.
- Assessed NLRP3 inflammasome components (NLRP3, ASC, caspase-1 p10), inflammatory cytokine mRNA and protein levels (IL-1β), and microglial activation (Iba-1) via western blotting, immunostaining, real-time PCR, and ELISA.
- Evaluated the impact of microglial conditioned medium on HT-22 neuronal viability (MTT assay) and apoptosis (Annexin V/PI staining, cleaved caspase 3 detection).
Main Results:
- Aβ₁₋₄₂ stimulation led to NLRP3 inflammasome activation in microglia, evidenced by increased caspase-1 activation and IL-1β secretion.
- Inhibition of TLR4 with CLI-095 effectively blocked Aβ₁₋₄₂-induced NLRP3 inflammasome activation, reducing inflammation and protecting HT-22 neurons.
- The protective effects of CLI-095 were reversible upon NLRP3 activation with ATP, confirming the pathway's involvement.
Conclusions:
- Toll-like receptor 4 (TLR4) critically mediates Aβ₁₋₄₂-induced NLRP3 inflammasome activation in mouse microglia.
- The TLR4/NLRP3 signaling pathway is a significant contributor to Aβ₁₋₄₂-induced neuroinflammation.
- Targeting the TLR4/NLRP3 pathway presents a promising therapeutic strategy for Alzheimer's disease.
