FlaA N/C attenuates radiation-induced lung injury by promoting NAIP/NLRC4/ASC inflammasome autophagy and inhibiting pyroptosis.

Background: Radiation-induced lung injury (RILI) is the most common complication experienced by patients with thoracic tumors after radiotherapy. Among patients receiving thoracic tumor radiotherapy, 14.6-37.2% develop RILI. RILI is characterized by an acute inflammatory response; however, the exact mechanism remains unclear and an ideal drug is still lacking. In this study, we investigated the protective effects of flagellin A with linked C- and N-terminal ends (FlaA N/C) against the development of RILI.

Methods: Mice and bronchial epithelial cells were exposed to radiation (15 Gy) after FlaA N/C treatment. Lung injury, bronchial epithelial cell injury, and RILI were assessed by histological evaluation in vivo and cell viability and cell death detection in vitro. Pyroptosis was assessed by western blotting (WB), immunofluorescence (IF), and immunohistochemistry (IHC). To explore the molecular mechanisms by which FlaA N/C inhibits RILI, conditional Beclin 1 (Beclin1) and NLR family CARD domain-containing protein 4 (Nlrc4)-knockout (Nlrc4) mice were generated. An autophagy inhibitor was used for in vitro cell assays, and pyroptosis indicators were detected. Data were analyzed using one-way analysis of variance.

Results: FlaA N/C attenuated radiation-induced lung tissue damage, pro-inflammatory cytokine release, and pyroptosis in vivo and cell viability, cell death, and pyroptosis in vitro. Mechanistically, FlaA N/C activated the neuronal apoptosis inhibitory protein (NAIP)/NLRC4/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) inflammasome, which was then degraded during Beclin 1-mediated autophagy. Deletion of the FlaA N/C D0 domain reversed the inhibitory effect of FlaA N/C on radiation-induced pyroptosis in vivo and in vitro. Similarly, Nlrc4-knockout in vivo or inhibition of autophagy in vitro eliminated the protective effects of FlaA N/C against radiation-induced pyroptosis.

Conclusions: These results indicate that FlaA N/C attenuates RILI by promoting NAIP/NLRC4/ASC inflammasome autophagy and inhibiting pyroptosis. This study provides a potential approach for RILI intervention.