Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative disorder that contributes to approximately 60-70% of the incidence of dementia worldwide. Inflammation in AD is thought to accelerate neuronal cell degeneration and synapse loss, and this inflammatory CNS phenotype can contribute to the aggregation of Aβ oligomers and the worsening of disease severity. Activation of microglial Toll-like receptor 4 (TLR4) by AD-specific damage-associated molecular patterns (DAMPs) leads to the activation of the p38 MAPK and subsequent upregulation of pro-inflammatory mediators such as IL-6 and TNF-α. In the AD brain, p38 MAPK activation is increased and thus has been suggested as a potential therapeutic target. Here, we investigated ex vivo stimulated human peripheral blood mononuclear cells (PBMCs) as an assay for screening p38 MAPK inhibitors. PBMCs were isolated from the whole blood of healthy donors (n=5) and stimulated ex vivo for 24 hours with 10ng/ml of the TLR4 agonist lipopolysaccharide (LPS; endotoxin). Prior to LPS stimulation PBMCs were treated with either vehicle, the TLR4 inhibitor TAK-242 (0.1 uM; positive control) or one of 5 concentrations of the p38 inhibitor SB239063 (0.001 – 10 uM). Analysis of the cytokines TNF-α, IL-1β, IL-6, IL-8 and IL-10 in the cell culture supernatant was performed using a MesoScale Diagnostics assay. A significant increase in the expression of all cytokines was observed following LPS stimulation. Pre-treatment with TAK-242 significantly inhibited the expression of all cytokines analysed. SB239063 produced a concentration-dependent reduction in the LPS-induced TNF-α, IL-1β, IL-8 and IL-10 expression, but not the expression of IL-6. Concentration-response curves fitted using non-liner regression yielded the following maximum inhibition (%) and IC50 (nM) values: TNF-α (67.4%; 47.8nM), IL-1β (92.1%; 26.1nM), IL-6 (16.9%; 39.1nM), IL-8 (55.1%; 102.1nM) and IL-10 (92.1%; 26.1nM). 

Using primary human PBMCs, we have established a cost effective, semi-high-throughput assay for efficacy testing of novel pipeline p38 MAPK inhibitors under investigation for the treatment of AD-associated innate immune activation and inflammation. PBMCs isolated from AD patients are reported to exhibit altered innate immune activity in comparison to aged-matched controls, thus, future work aims to establish this assay in patient-derived PBMCs.