Regulation Mechanism Of IL-6 Mediated JAK/STAT3 Pathway On Innate Immune Response In Megalobrama Amblycephala

Interleukin-6(IL-6)is a pleiotropic inflammatory cytokine,which plays a critical role in innate immune response.IL-6 could bind with membrane IL-6 receptor(m IL-6R)or soluble IL-6 receptor(s IL-6R),which turn on IL-6 classical signaling or transsignaling,respectively.Studies in mammals have shown that IL-6 binds IL-6R and glycoproteins 130(gp130)gp130 to activate the janus kinase/signal transducer and activator of transcription 3(JAK/STAT3)pathway,thereby regulates the expression of downstream genes.However,the function of IL-6,especially the regulatory mechanism of IL-6 classical signaling and trans-signaling in innate immune response remains largely unclear in teleost.Blunt snout bream(Megalobrama amblycephala)is one of the major freshwater aquaculture species in China.Due to the high breeding density,germplasm degradation,and deterioration of the breeding environment,various diseases caused by pathogenic bacteria frequently occur,among which bacterial sepsis caused by Aeromonas hydrophila infection is the most serious.In teleost,it has been confirmed that IL-6 plays a critical role in host defense against pathogens bacteria.Therefore,taking M.amblycephala as the research object,this study focused on the regulation mechanism of IL-6-mediated JAK/STAT3 pathway in innate immune response.Bioinformatics,quantitative real-time PCR(q PCR),western blot,pathway inhibitor treatment,transcriptome sequencing and other techniques were used to screen the key downstream signaling pathways of IL-6,analyze the effects of activation or inhibition of IL-6/JAK/STAT3 on immune and iron metabolism gene expression,and elucidate the differential activation of different ma STAT3 isoforms by IL-6 classical signaling and trans-signaling,aiming to throw light on the regulatory role of IL-6/JAK/STAT3 in innate immune and provide a theoretical basis for fish disease resistance breeding.The main results are as follows:1.Regulation of downstream signaling pathways and genes by M.amblycephala IL-6 classical signaling and trans-signalingIn this study,q PCR detection showed that il-6 in the head kidney lymphocytes of M.amblycephala were significantly up-regulated after LPS stimulation,indicating that IL-6 was involved in the immune response.The recombinant IL-6 protein and soluble IL-6R protein of M.amblycephala(rma IL-6 and rmas IL-6R)were obtained by prokaryotic expression system.Stimulation of rma IL-6 or rmas IL-6R induced the expression of immune related genes(il-1β,il-6 and socs3a)and the STAT3 phosphorylation in L8824 cells,indicating their biological activity.Subsequently,western blot results showed that rma IL-6 treatment could significantly up-regulate the phosphorylation of STAT3 and ERK1/2 in L8824 cells,while rma IL-6 in combination with rmas IL-6R(rma IL-6+rmas IL-6R)only induced the STAT3 phosphorylation,but with stronger effect.Therefore,the IL-6 classical signaling and trans-signaling of M.amblycephala mainly function by activating the JAK/STAT3 pathway rather than the MEK/ERK pathway.To further investigate the regulation of IL-6 trans-signaling through the JAK/STAT3 pathway,transcriptome analysis was performed in L8824 cells after stimulation with STAT3 inhibitor(c188-9),rma IL-6+rmas IL-6R,and both.The enrichment analysis of GO and KEGG showed that rma IL-6 trans-signaling is mainly involved in stress and inflammation response,and the activation of STAT3 is mainly related to cell proliferation,apoptosis and immune regulation.The q PCR results showed that c188-9 could significantly inhibit the up-regulation trend of immune related genes(cebpd,fra2,mrp1,rsk2 a,sash3,socs3 a and upar)induced by rma IL-6+rmas IL-6R.Furthermore,treatment of JAK2 inhibitor(TG101348)could also inhibit the expression of the above immune-related genes.These results implied that IL-6trans-signaling induces the expression of immune related genes through the JAK2/STAT3 pathway,and then participates in the innate immune regulation of fish.2 Regulation of iron metabolism genes by M.amblycephala IL-6 through JAK/STAT3 pathwayInflammation immune responses are closely related to iron homeostasis.In order to explore whether IL-6 is involved in the regulation of iron metabolism,this study used rma IL-6 or rma IL-6+rmas IL-6R to stimulate the hepatocytes of M.amblycephala,and found that the iron metabolism related genes(ferritin H subunit,fth;transferrin receptor,tfr;hepcidin,hepc and ferroportin 1,fpn1)and the STAT3 phosphorylation were significantly up-regulated.This indicated that IL-6 regulated the expression of iron metabolism related genes in M.amblycephala.In order to further analyze the role of JAK/STAT3 pathway,the hepatocytes were treated with c188-9 or TG101348,and the phosphorylation of STAT3 and the expressions of fth,tf,hepc and fpn1 were detected.The results showed that both c188-9 and TG101348 could significantly inhibit the STAT3 phosphorylation and the up-regulated expression of fth,tf and hepc genes induced by rma IL-6 and rma IL-6+rmas IL-6R.This indicated that the IL-6 classical signaling and trans-signaling could induce the expression of iron metabolism genes through the JAK/STAT3 pathway,thereby participate in the iron metabolism process.3 Expression of four ma STAT3 isoforms and differential activation by IL-6classical signaling and trans-signalingAs an important member of the JAK/STAT3 pathway,STAT3 has been cloned and identified in several species of teleost,but studies on its function is still limited.In this study,through sequence alignment and q PCR verification,four STAT3 isoforms(ma STAT3α1,ma STAT3α2,ma STAT3β1 and ma STAT3β2)were identified from M.amblycephala.Sequence analysis showed that the four ma STAT3 isoforms were produced by splicing of the same gene,and the amino acid sequences were highly similar in teleost.The q PCR results showed that the four mastat3 isoforms were widely expressed in 10 tissues including the liver,spleen,and head kidney of healthy M.amblycephala,but the expression patterns were different.After A.hydrophila challenge,the expression treads of mastat3a1,mastat3a2 and mastat3β2 were similar in the liver and middle kidney,and all of them could be significantly up regulated in a short time.However,the expression of mastat3β1 was significantly up-regulated in the liver,and down-regulated in the middle kidney and head kidney.To further analyze the activation differences of the four ma STAT3 isoforms by IL-6 stimulation,EPC cells transfected with ma STAT3α1,ma STAT3α2,ma STAT3β1 or ma STAT3β2 were treated with rma IL-6+rmas IL-6R.The results showed that the four ma STAT3 isoforms could be significantly induced by rma IL-6+rmas IL-6R,but the induction trend was different.Meanwhile,only ma STAT3α1 and ma STAT3α2 could be significantly induced by rma IL-6.This suggests that the four ma STAT3 isoforms are different in response to IL-6 stimulation,and ma STAT3α1 and ma STAT3α2 might be the main function executors of IL-6.In conclusion,the functions of the four ma STAT3 isoforms in response to pathogen infection or IL-6 stimulation have overlaps and differences,which may be beneficial to the survival of fish against pathogen invasion.