266 - Suppression of IL-1β production in alveolar macrophages by the thioredoxin reductase inhibitor auranofin

Abstract

Inflammation via alveolar macrophage production of IL-1β plays a major role in the development of acute lung injury (ALI). Previous work in our lab has established that thioredoxin reductase-1 (TXNRD1) inhibition is protective in ALI mouse models. This response is largely driven by nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation. We hypothesize that TXNRD1 inhibition decreases IL-1β production and increases Nrf2 dependent antioxidants and anti-inflammatory cytokines in alveolar macrophages. MH-S cells were cultured in the presence or absence of 0.05 µg/ml lipopolysaccharide (LPS) and 0.5 µM auranofin (AFN). AFN inhibited TXNRD1 activity by 90% and resulted in nuclear Nrf2 accumulation. Within 2hrs, IL-1β mRNA levels, assessed by qRT-PCR, were 200-fold greater in the presence of LPS alone; an effect that was attenuated by 50% when co-treated with AFN. The same trend was observed for IL-1β protein levels, assessed by ELISA at 6h. IL-1β levels were 18-fold higher than control in LPS-treated cells but only 10-fold greater in the presence of AFN. Glutathione (GSH) levels, measured at 24h by Tietze recycling assay, were 2.5-fold greater in AFN and LPS/AFN-treated cells compared to control treated cells. LPS alone resulted in a 1.4-fold increase in GSH levels. Cell viability was assessed by trypan blue exclusion at 24h. Compared to control-treated cells, two way ANOVA analyses revealed an independent effect of LPS but not AFN on cell viability. Viability was not different between cells treated with LPS or LPS/AFN. These results indicate that TXNRD1 inhibition attenuates LPS mediated increases IL-1β in alveolar macrophages. Along with the suppression of IL-1β, a robust increase in endogenous antioxidant responses likely contributes increased survival in our ARDS model. This work implicates alterations in macrophage anti-inflammatory and antioxidant biology as a mechanism by which TXNRD1 inhibition is protective in ALI.