Dual-parallel inhibition of IL-10 and TGF-β1 controls LPS-induced inflammatory response via NF-κB signaling in grass carp monocytes/macrophages

•LPS stimulated the production of IL-10 and TGF-β1 in grass carp monocytes/macrophages.•Grass carp IL-10 and TGF-β1 attenuated the pro-inflammatory effects of LPS in the same cell model.•IL-10 and TGF-β1 impaired NF-κB activation by blocking LPS-induced IκBα protein degradation.•IL-10 and TGF-β1 might perform their anti-inflammatory function in an independent manner.•Our data demonstrate an intrinsic mechanism of limiting excessive inflammation in teleost.

In fish, the knowledge on the regulation of inflammatory responses is limited. In the present study, LPS rapidly increased the mRNA levels of grass carp pro-inflammatory factors, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxides synthase (iNOS) and IL-8 in monocytes/macrophages, indicating the occurrence of innate inflammatory responses in fish as seen in mammals. Intriguingly, the gene expression and protein secretion of grass carp IL-10 (gcIL-10) and TGF-β1 (gcTGF-β1) were induced by LPS in the same cell model, promoting us to clarify their roles in regulating inflammatory response. Results revealed that grass carp IL-10 polyclonal antibody (anti-gcIL-10 pAb) and grass carp TGF-β1 monoclonal antibody (anti-gcTGF-β1 mAb) could amplify the stimulation of LPS on the mRNA levels of tnfα, il1β, inos and il8, suggesting the inhibitory tone of endogenous IL-10 and TGF-β1 in LPS-challenged immune responses. This notion was further supported by the fact that recombinant grass carp IL-10 (rgcIL-10) and recombinant grass carp TGF-β1 (rgcTGF-β1) attenuated LPS-stimulated tnfα, il1β, inos and il8 gene expression in monocytes/macrophages. Further study revealed that rgcIL-10 and rgcTGF-β1 impaired NF-κB activation by blocking LPS-induced grass carp IκBα (gcIκBα) protein degradation in the cells. In addition, the correlation between gcIL-10 and gcTGF-β1 in this regulation was examined by immunoneutralization, unveiling that anti-gcTGF-β1 mAb and anti-gcIL-10 pAb were unable to alter the inhibitory effects of rgcIL-10 and rgcTGF-β1 on pro-inflammatory factors expression in grass carp monocytes/macrophages, respectively. This dual and parallel effect of gcIL-10 and gcTGF-β1 strengthened their importance in controlling inflammatory responses. Taken together, our findings shed a light on the functional role, regulatory mechanism and relationship of fish IL-10 and TGF-β1 in regulating inflammatory response.