Lipopolysaccharide-induced autophagy participates in the control of pro-inflammatory cytokine release in grass carp head kidney leukocytes

Microtubule-associated protein 1 light chain 3B (LC3B) is a known marker of autophagy in mammals. In the present study, we isolated and identified grass carp LC3B (gcLC3B) cDNA and found its inductive expression in response to bacterial infection in vivo. To assess the occurrence of autophagy in immune response, the role of gcLC3B as an autophagy marker in grass carp was characterized. Accordingly, grass carp kidney cells (CIKs) with stable expression of GFP-gcLC3B were established and GFP-gcLC3B puncta were counted under a confocal fluorescence microscope. Results showed that starvation, a conventional inducer of autophagy, significantly enhanced GFP-gcLC3B puncta number, indicating the existence of gcLC3B-linked autophagy in fish cells. Moreover, a commercial antibody recognizing gcLC3B and 3-methyladenine (3-MA) were validated in grass carp CIKs, and used to evaluate autophagy in grass carp head kidney leukocytes (HKLs) in response to LPS. Western blotting assay showed that LPS significantly induced the conversion of gcLC3B protein, providing the evidence for autophagy induced by LPS in fish immune cells. Importantly, autophagy inhibition by 3-MA enhanced grass carp IL-1β and TNF-α secretion, indicating the involvement of autophagy in pro-inflammatory cytokine production. Besides, 3-MA could amplify LPS-induced IL-1β and TNF-α release, implying that autophagic induction may drive a mechanism for controlling inflammatory response in fish. Thus, our data highlight the role of autophagy in fish immunity and provide new insight into the mechanism for the regulation of inflammation in fish.