Mechanisms Of Lactic Acid-Induced Interleukin-1 Beta Release

ME Edye, D Brough, SM Allan. University of Manchester, Manchester, UK

INTRO. Lactic acidosis is a clinical consequence and contributor to many major diseases. Inflammation, and in particular the pro-inflammatory cytokine interleukin-1 beta (IL-1β), contributes to the development and exacerbation of multiple diseases; including epilepsy, Alzheimer’s disease and stroke. IL-1β is synthesised as an inactive precursor which is subsequently cleaved to a 17kDa active form by caspase-1. To cleave IL-1β, caspase-1 also requires prior activation. This occurs through association with a large protein complex termed the ‘inflammasome’, of which the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is the most studied. To determine whether acidosis affects IL-1β processing, here we examined the effect of lactic acid on IL-1β release from primary mouse glial cells.

METHODS. Mixed glial cultures were grown from wild type C57BL/6 and NLRP3 knock-out mice as described previously (Nguyen 2011). Pro- and mature IL-1β release was measured using ELISA and Western blot following 8h treatment with 25mM lactic acid or vehicle (PBS) with, or without, pre-treatment with inhibitors/vehicle. The caspase-1 inhibitor, YVAD-CHO (100μM; Calbiochem) or DMSO vehicle or cathepsin D inhibitor, pepstatin A (50µM; Sigma-Aldrich) or methanol vehicle was added for 15min prior to lactic acid treatment. Statistical analysis to compare two groups of data used unpaired t-tests, and for 3 or more groups, one way analysis of variance (ANOVA) with Bonferroni multiple comparisons test post hoc was performed. P≤0.05 was considered significant.

RESULTS. Lactic acid (25mM) induced the release of IL-1β from mouse glial cultures (n=6, p<0.001 vs vehicle). IL-1β release was not significantly altered by pre-treatment with 100µM YVAD-CHO (n=6, p>0.05). Lactic acid-induced IL-1β release also occurred in NLRP3 knock-out cultures (n=6, p<0.01 vs vehicle). The lactic acid-induced IL-1β release is distinct from the caspase-1 dependent process since the mature IL-1β present in treated supernatants is predominantly 20kDa as opposed to 17kDa. Pre-treatment with the cathepsin D inhibitor pepstatin A (50µM) significantly reduced lactic acid-induced IL-1β release (n=6, p<0.05).

CONCLUSION Lactic acid induced IL-1β release from glial cells and this release was independent of the classical NLRP3 inflammasome/caspase-1 pathway. Cathepsin D is likely to mediate lactic acid-induced IL-1β release. Further investigation of this caspase-1-independent IL-1β pathway may in future provide novel targets for the treatment of inflammatory disease.

This work was supported by BBSRC CASE Award with Pfizer.