The sesquiterpene lactone estafiatin exerts an anti-inflammatory effect against Mycobacterium abscessus infection by regulating interleukin-1 beta production.

BACKGROUND

Mycobacterium abscessus, a species of non-tuberculous mycobacteria, is known to induce chronic pulmonary inflammation in humans. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays a critical role in the maturation of interleukin-1 beta (IL-1β) and the initiation of pyroptosis. However, excessive activation of the NLRP3 inflammasome during M. abscessus infection results in the overproduction of IL-1β, leading to subsequent tissue damage. Estafiatin (EST), a sesquiterpene lactone, has previously exhibited anti-inflammatory properties in lipopolysaccharide (LPS)-stimulated macrophages and mouse models. Based on these findings, we investigated whether EST could attenuate M. abscessus-induced inflammation by regulating NLRP3 inflammasome activation.

PURPOSE

This study aimed to evaluate the anti-inflammatory effects of EST in the context of M. abscessus infection and explore the involvement of IL-1β signaling, thereby establishing foundational evidence for the potential therapeutic application of EST in managing M. abscessus infection.

METHODS

Murine bone marrow-derived macrophages and those primed with LPS, were pretreated with various concentrations of EST for 2 h before M. abscessus infection. Phagocytosis activity and intracellular bacterial growth were evaluated by colony-forming units (CFU) assays. The expression of NLRP3-mediated genes and the production of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1β were measured by quantitative PCR and ELISA, respectively. The components of NLRP3 inflammasome were also evaluated by Western blot analysis. In the in vivo experiment, the effects of EST on bacterial clearance, lung histopathology, cytokine production, and IL-1β-positive alveolar macrophage population were assessed in wild-type and IL-1β-deficient mice following pulmonary infection with M. abscessus.

RESULTS

EST treatment significantly decreased TNF-α, IL-6, and IL-1β production by macrophages in response to M. abscessus infection, without affecting intracellular bacterial growth. Additionally, EST specifically reduced IL-1β production and cleavage of caspase-1 and IL-1β induced by M. abscessus in LPS-primed macrophages. In a mouse model of M. abscessus infection, intraperitoneal administration of EST alleviated the inflamed area and histopathological alteration in the lungs in a dose-dependent manner, but it did not influence bacterial clearance. EST treatment also decreased TNF-α, IL-6, IL-1β, and interleukin-17 (IL-17) levels in lung homogenates and reduced the protein levels of the cleaved forms of caspase-1 and IL-1β. Flow cytometry analysis revealed a reduced number of IL-1β-positive cells among alveolar macrophages in EST-treated mice. Notably, EST treatment did not affect the histopathology of major organs, including the liver, spleen, and kidney, indicating that the doses used did not induce toxicity. The protective effects of EST against M. abscessus-induced pulmonary inflammation disappeared in IL-1β-deficient mice, suggesting that EST may exert an anti-inflammatory effect by regulating IL-1β signaling.

CONCLUSIONS

This study demonstrated that EST inhibits NLRP3 inflammasome activation in M. abscessus-infected macrophages and mitigates excessive pulmonary inflammation during M. abscessus infection in mice, without compromising bacterial clearance. These findings support its potential as a novel host-directed therapeutic candidate for M. abscessus infection.