Increasing autophagy ameliorates all--retinal-activated NLRP3 inflammasomes in human THP-1 macrophages.

OBJECTIVE

Severe inflammation, mediated by innate immune sensors, can be observed in the retina and is considered to play an important role in the pathogenesis of retinal degeneration caused by all--retinal (atRAL). However, the underlying mechanism thereof remains elusive. This study investigated the effects of atRAL on the macrophage cell line THP-1 and determined the underlying signaling pathway through pharmacological and genetical manipulation.

METHODS

The cytotoxicity of atRAL in THP-1 macrophage cells was assessed using the cell counting kit-8 (CCK-8) assay, and mature IL-1β was detected by enzyme-linked immunosorbent assay (ELISA). We measured levels of NLRP3 and cleaved caspase-1 by western blotting to evaluate the activation of NLRP3 inflammasomes. Oxidative stress was validated by measuring mitochondria-associated reactive oxygen species (ROS) with MitoSOX Red staining. Autophagy was assessed with the LC3BII turnover assay and tandem mCherry-eGFP-LC3B fluorescence microscopy.

RESULTS

The maturation and release of IL-1β were regulated by the activation of the NLRP3 inflammasome. Mitochondria-associated ROS were involved in the regulation of NLRP3 inflammasome activation and caspase-1 cleavage. In addition, atRAL functionally activated autophagy in THP-1 cells, and atRAL-induced NLRP3 inflammasome activation was suppressed by autophagy.

CONCLUSIONS

atRAL activates both the NLRP3 inflammasome and autophagy in THP-1 cells, and the increasing level of autophagy leads to the inhibition of excessive NLRP3 inflammasome activation. These findings shed new light on the pathogenesis of age-related retinal degeneration.