Toll And Starvation Signaling Block ILS To Induced Antibacterial Peptide Expression And Autophagy Response In Insect Cells

Innate immunity is the first line of defense against pathogen infection in insect,antibacterial peptide(AMPs)expression regulated by Toll and IMD pathway is the key process.Recent studies have showed that insulin-like signaling(ILS)pathway and autophagy are involved in insect innate immunity.Autophagy widely exists in eukaryotic cells,and one of its functions is to play immune autophagy to swallow invasion microbial,such as xenophagy participates in insect innate immunity.Between innate immunity signaling pathway,ILS and autophagy there may be unclear pathway that could switch different signaling,but there is no clear experimental evidence until now.Exploring and studying this scientific issue has important scientific implications for insect development and immunological research.According to the literature and related research before this article,we believed that between the Toll pathway,the ILS and the insect immune autophagy response there may be an unclear pathway that link these different signaling.So that,we supposed a signaling pathway model that starvation and Toll signaling could inhibit ILS to regulate antibacterial peptide expression and autophagy response and studied on five aspects:(1)starvation up-regulated AMPs expression,(2)ILS induced AMPs expression mediated by FoxO transcription factor,(3)insect innate immunity signaling(Tolls-Spzs)induced autophagy,(4)insect innate immunity signaling inhibited ILS to regulated autophagy response,(5)NF-?B transcription factor activated the promoter of autophagy related gene DmAtg8a.The mainly achievements as follows:1.The expression of AMP genes,BmCecB6,BmAtt1,BmLeb3 and BmDefB,was up-regulated after starvation in silkworm fat body and Bm-12 cells.InR and Brummer were target genes of FoxO,the transcription factor in down-stream of ILS,and their expression was also up-regulated after starvation.These results showed that starvation could induce AMPs expression.2.The phosphorylation of Akt was inhibited by starvation in fat body and Bm-12 cells.Moreover,starvation induced the transcription factor FoxO to translocate into nucleus.Activated FoxO could recognize the1^st,4^th,5^thand 6^th binding site in BmDef B promoter and up-regulate the expression of BmDef B.ILS transduction inhibited by InR RNAi interfere also could induce AMPs expression.These results showed that FoxO could regulate AMPs expression as a key transcription factor in the downstream of ILS.3.LC3 fused with GFP and RFP was expressed and stable transfection S2 cell line was constructed.LC3-II band and puncta increased after starvation detected by western blot and fluorescence,indicated that the cell line can be used for monitoring autophagy.4.Autophagy was induced by overexpression of Toll receptor,Toll-7 receptor and their ligands Spz1,2,5 in S2 cells,and showed immunity signaling could activate autophagy.Furthermore,VSV could activate autophagy response in S2 cells that overexpressed Toll or Toll-7 receptor,and Toll-7 was more sensitive for VSV infection.5.Toll or Toll-7 receptor mediated immunity signaling transduction decreased the phosphorylated level of Akt and induced autophagy response.The results of Myd88 RNAi showed that Toll receptor mediated innate immunity and autophagy both dependent on Myd88,Toll-7 mediated innate immunity but not autophagy dependent on Myd88.6.Overexpress NF-?B transcription factors,TIR1 or TIR7 could activate the promoter of DmAtg8a through Dual-luciferase assay.In summary:Starvation could activate FoxO transcription factor and up-regulate AMPs expression through ILS.There were multiple immunity signalings(Tolls-Spzs)to induce autophagy in S2 cells.Toll or Toll-7 mediated immunity signaling inhibited ILS,but there may have different intracellular signaling pathway to regulate autophagy through Toll receptor or Toll-7 receptor.As two conserved signaling pathway,the innate immunity signaling pathway and insulin-like pathway may have a node to switch their signaling transduction,indicate that ILS could mediated Toll and starvation signaling induced AMPs expression and autophagy response in insect cells.