Cytokine Characterization Of Grass Carp Heat Shock Protein(HSP70) And Its Involvement In Inflammatory Regulation

Heat shock proteins(HSPs)belong to a highly conserved protein superfamily,which maintain cell homeostasis.In mammals,it is generally accepted that HSP70,as an induced heat shock protein with a molecular weight of 72 kDa,not only plays an important role as a molecular chaperone supporting protein folding,transmembrane transport,and translocation,but also works as a cytokine implicating in both innate and adaptive immunity.Teleost HSP70 gene has been cloned and identified from many fish species and is usually used as a biomarker of physiological emergency responses.Interestingly,extracellular HSP70 can be detected in the culture medium of grass carp peripheral blood lymphocytes(PBLs),indicating possible existence of soluble HSP70 in teleost.Moreover teleost HSP70 induces mRNA expression of pro-inflammatory cytokines,implying it may function as a cytokine.However,compared to its role in mammals,cytokine functions of HSP70 in teleost have not been fully understood,particularly the identification of its receptor,intracellular signal transduction pathways and its regulation on inflammatory responses etc.To address these issues,the functional role of HSP70 in grass carp will be extensively elucidated in this study,thereby providing clues for further study of the functions of HSP70 homologues in mammals,as well as revealing the mechanism of grass carp HSP70 involving the regulation of inflammatory response.These works will enrich the contents of fish immunity,and explore the potential of HSP70 in fish disease control.The results of this thesis are as follows:1.Induced expression profiles of grass carp HSP70(GcHSP70)mRNA and protein secretion.(1)Using grass carp head kidney leukocytes(HKLs)as a cell model,a doseand time-dependent increase of intracellular HSP70 mRNA expression were detected under lipopolysaccharide(LPS)stimulation.(2)It was found for the first time in fish that LPS induced cumulative secretion of HSP70 protein in the culture medium of HKLs,and this action was prior to its effect on the gene transcription of HSP70.(3)In in vivo studies,both HSP70 mRNA expression and protein synthesis were increased in the head kidney of grass carp infected by aeromonas hydrophila subsphydrophila(A.hydrophila);compared to the control group,a similar increase in the extracellular HSP70 contents in peripheral blood from grass carp infected by A.hydrophila was observed.These results suggest that grass carp HSP70 might participate in the inflammatory responses at the initial stage of immune stimulation,implying the possible role of GcHSP70 in immune regulation.2.Effects of recombinant grass carp HSP70 protein(rgcHSP70)on the mRNA expression and protein secretion of other cytokines in HKLs.(1)The rgcHSP70 promoted the mRNA expression of pro-inflammatory cytokines,including tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?).For anti-inflammatory cytokines,rgcHSP70 stimulated IL-10 but not transforming growth factor-?(TGF-?)mRNA expression,which was different from findings in mammals and showed the specificity of fish HSP70 function.(2)Moreover,rgcHSP70 also stimulated the secretion of TNF-?,IL-1?,and IL-10 in HKLs.However,the inductive secretion of TNF-? and IL-1? only occurred at the early stage of treatment whereas the induction IL-10 release was maintained for a long time.This is first time to reveal the function of HSP70 inducing cytokine secretion in fish.3.HSP70 regulated cytokine expression via different cellular signaling pathways in grass carp HKLs.(1)The rgcHSP70 stimulated TNF-? and IL-1? mRNA expression through NF-?B signaling pathway in HKLs;moreover,p38 MAPK signaling pathway was involved in the regulation of IL-1? gene expression,while JNK MAPK signaling pathway for TNF-?.(2)The p38 MAPK but not NF-?B signaling pathway mediated GcHSP70-triggered IL-10 gene expression.These results showed different signaling pathways for GcHSP70 regulation on cytokine expression.4.Preliminary identification of the grass carp HSP70 receptor.(1)Bioinformatics methods were used to analyze the possible interaction between GcHSP70 and grass carp toll-like receptor 4(GcTLR4).(2)Full length of GcTLR4 cDNA sequence was cloned;TLR4 eukaryotic expression plasmid was constructed and transfected into CIK cells,a cell strain from grass carp kidney.By detecting luciferase-based NF-?B activity,the results showed that GcHSP70 could promote NF-?B activity through GcTLR4.These findings suggested that TLR4 is the candidate receptor for HSP70 in fish,i.e.,the ligand of fish TLR4 is HSP70.5.The mechanisms of GcHSP70 participating in inflammation regulation.Here grass carp infected with A.hydrophila were selected as the model.(1)The rgcHSP70 had significantly effect aganist the death of grass carp caused by A.hydrophila.(2)The rgcHSP70 enhanced the activity of multiple antioxidant enzymes in vivo to increase the antioxidant ability of grass carp.(3)The rgcHSP70 rapidly regulated the expression of cytokines,improving fish immunity against bacterial pathogens.(4)The rgcHSP70 protected the fish through sustaining effect on the anti-inflammatory cytokine IL-10.All results revealed the possible mechanisms of rgcHSP70 mediating inflammation and its potent application in fish disease control.In conclusion,this study first explored functional role of HSP70 as a cytokine in grass carp,including its response to inflammatory stimuli,its regulation on cytokine expression and secretion,its specific receptor,as well as the mechanisms involving in inflammatory regulation.These results not only clarify the role of HSP70 in the immune system of the lower vertebrate,but also add novel information of fish immunity based on the regulatory mechanism of HSP70 in teleost,thereby providing new clues to facilitate immune defence against pathogens in fish.