Pulsed-Ultrasound Irradiation Induces the Production of Itaconate and Attenuates Inflammatory Responses in Macrophages

BACKGROUND: Itaconate is a key metabolite in the innate immune system and exerts strong anti-inflammatory effects in macrophages. For the production of itaconate in macrophages, immune-responsive gene 1 (IRG1) is an imperative enzyme, and activating the IRG1-itaconate pathway is reported to alleviate inflammatory diseases by upregulating nuclear factor-erythroid 2-related factor 2 (NRF2). However, there are very few reports on strategies to increase itaconate production. Ultrasound therapy is a widely used intervention for anti-inflammatory and soft-tissue regeneration purposes. Here we show the effect of ultrasound irradiation on the production of itaconate in macrophages. METHODS: Murine bone marrow-derived macrophages (BMDMs) were exposed to pulsed ultrasound (3.0 W/cm(2)) for 5 minutes. Three hours after irradiation, the intracellular levels of metabolites and mRNA expression levels of Irg1 and Nrf2 were measured using CE/MS and qPCR, respectively. To evaluate macrophage inflammation status, 3 h after irradiation, the cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) for 1.5 h and the mRNA expression levels of pro-inflammatory factors (Il-1β, Il-6, and Tnf-α) were measured. Student’s t-test, one-way ANOVA and Tukey’s multiple comparison test were used for statistical processing, and the significance level was set to less than 5%. RESULTS: Ultrasound irradiation significantly increased the intracellular itaconate level and the expression levels of Irg1 and Nrf2 in BMDMs. Upregulation of Il-1β, Il-6, and Tnf-α by LPS was significantly suppressed in BMDMs treated with ultrasound. Ultrasound irradiation did not affect cell viability and apoptosis. CONCLUSION: Ultrasound irradiation induces the production of itaconate by upregulating Irg1 expression and attenuates inflammatory responses in macrophages via Nrf2. These results suggest that ultrasound is a potentially useful method to increase itaconate production in macrophages.