Washington State University College of Pharmaceutical Science and Molecular Medicine Spokane, WA, United States
Paul Panipinto, Anil Singh, Farheen Sultan Shaikh and Salahuddin Ahmed, Washington State University, Spokane, WA
Background/Purpose: Gout disease flares occur when primed macrophages interact with and phagocytose monosodium urate crystals (MSU), activating IL-1β via the NLRP3 inflammasome. Abrogation of inflammasome activation holds the promise of preventing flares or reducing their severity. Therapeutic research in this area has focused on a variety of targets including direct inhibition of the inflammasome itself but targeting upstream signaling pathways may yield additional anti-inflammatory benefits. TAK1 is one of the most promising inflammatory signaling targets as it is positioned at the nexus of the NF-κB and MAPK pathways. Inhibition of TAK1 may reduce NF-κB-mediated cytokine production and disrupt NLRP3 inflammasome formation through inhibition of the intermediary c-Jun N-terminal kinase 1 (JNK1). This study investigates the efficacy of pentagalloyl glucose (PGG), a potent natural product with high tolerability and anti-inflammatory property, in inhibiting MSU-induced inflammation.
Methods: THP-1 macrophages were differentiated by treatment with 100 ng/mL PMA for 48h, followed by 72h rest. Macrophages were primed with 10 ng/mL lipopolysaccharide (LPS) for 4h and treated with PGG (0.1-100 μM) or signaling inhibitors for 2h prior to 100 μg/mL MSU treatment. Signaling was analyzed by Western blot analysis after 30 min of MSU stimulation, and ELISAs were performed on conditioned media collected after 24h of MSU stimulation. In vitro kinase assays were performed with recombinant TAK1 and PGG for 30 min in the presence of ATP, followed by Western blot analysis. Inflammasome formation was evaluated by immunofluorescence after pretreatment with inhibitors and MSU. One-way ANOVA with Dunnett's post-hoc test for multiple comparisons was used to determine significance (p< 0.05). All experiments use at least n=3 biological replicates.
Results: PGG significantly reduced production of the pro-inflammatory mediators in a dose-dependent manner. IL-8 was reduced (15-75%), MCP-1 (70-85%) and IL-1β (70-80%) in supernatants while demonstrating no toxicity in any treatment concentration. The efficacy of PGG was observed to be equally potent when compared to other selective TAK1 and JNK inhibitors. In vitro kinase assays show a dose-dependent (46-76%) decrease in phosphorylation of recombinant TAK1 when treated with PGG. Evaluation of the signaling pathways by Western blot showed significant inhibition of TAK1 (55-70%) and JNK1 (65-85%) phosphorylation, but not of ERK or p38 MAPKs or inhibition of pro-IL-1β production. Treatment with MSU induced inflammasome formation, which was abrogated by PGG, 5z -7-oxozeanol (TAK1 inhibitor) and MCC950 (NLRP3 inhibitor as shown by immunofluorescence experiments.
Conclusion: PGG inhibits MSU-induced production pro-inflammatory cytokines and chemokines by suppressing TAK1184/187 phosphorylation. Our findings suggest that there may be multiple signaling targets downstream of TAK1 that are inhibited by PGG preventing activation, but not production of pro-IL-1β. This suggests inflammasome formation may be abrogated – preventing NLRP3 derived inflammation potentially without compromising the function of other inflammasomes.
Disclosures: P. Panipinto, None; A. Singh, None; F. Shaikh, None; S. Ahmed, None.
