No abstract

The Krebs cycle-derived metabolite itaconate is highly upregulated in inflammatory macrophages and exerts immunomodulatory effects through cysteine modifications on target proteins. The NLRP3 inflammasome, which cleaves IL-1β, IL-18, and gasdermin D, must be tightly regulated to avoid excessive inflammation. Here we provide evidence that itaconate modifies NLRP3 and inhibits inflammasome activation. Itaconate and its derivative, 4-octyl itaconate (4-OI), inhibited NLRP3 inflammasome activation, but not AIM2 or NLRC4. Conversely, NLRP3 activation was increased in itaconate-depleted Irg1 −/− macrophages. 4-OI inhibited the interaction between NLRP3 and NEK7, a key step in the activation process, and “dicarboxypropylated” C548 on NLRP3. Furthermore, 4-OI inhibited NLRP3-dependent IL-1β release from PBMCs isolated from cryopyrin-associated periodic syndrome (CAPS) patients, and reduced inflammation in an in vivo model of urate-induced peritonitis. Our results identify itaconate as an endogenous metabolic regulator of the NLRP3 inflammasome and describe a process that may be exploited therapeutically to alleviate inflammation in NLRP3-driven disorders.

SignificanceThe molecular clock provides an anticipatory mechanism, allowing organisms to prepare and respond to daily changes in the external environment. The response of the innate immune system to pathogenic threats is dependent on time of day; however, the molecular mechanisms underlying this have yet to be fully uncovered. We observe that the core molecular clock component, BMAL1, is crucial in promoting an antioxidant response in myeloid cells. Deletion of Bmal1 in macrophages disrupts NRF2 activity, facilitating accumulation of reactive oxygen species and the proinflammatory cytokine, IL-1β. Thus the molecular clock directly controls NRF2 transcriptional activity and antioxidant capacity to regulate IL-1β in myeloid cells.

PGE has been shown to increase the transcription of pro-IL-1β. However, recently it has been demonstrated that PGE can block the maturation of IL-1β by inhibiting the NLRP3 inflammasome in macrophages. These apparently conflicting results have led us to reexamine the effect of PGE on IL-1β production. We have found that in murine bone marrow-derived macrophages, PGE via the cAMP/protein kinase A pathway is potently inducing IL-1β transcription, as well as boosting the ability of LPS to induce IL-1β mRNA and pro-IL-1β while inhibiting the production of TNF-α. This results in an increase in mature IL-1β production in macrophages treated with ATP. We also examined the effect of endogenously produced PGE on IL-1β production. By blocking PGE production with indomethacin, we made a striking finding that endogenous PGE is essential for LPS-induced pro-IL-1β production, suggesting a positive feedback loop. The effect of endogenous PGE was mediated by EP2 receptor. In primary human monocytes, where LPS alone is sufficient to induce mature IL-1β, PGE boosted LPS-induced IL-1β production. PGE did not inhibit ATP-induced mature IL-1β production in monocytes. Because PGE mediates the pyrogenic effect of IL-1β, these effects might be especially relevant for the role of monocytes in the induction of fever. A positive feedback loop from IL-1β and back to PGE, which itself is induced by IL-1β, is likely to be operating. Furthermore, fever might therefore occur in the absence of a septic shock response because of the inhibiting effect of PGE on TNF-α production.

No abstract