Immunomodulatory Effects And Related Mechanisms Of Interleukin-12 Isoforms In Grass Carp (Ctenopharyngodon Idellus)

Interleukin-12(IL-12)is a heterodimeric cytokine composed of p40 and p35 subunits through an inter-chain disulfide bond.IL-12 plays an extensive and important role in the immune system and it has been considered a bridge between innate and adaptive immunity.Teleost IL-12 has been preliminarily identified in fugu,common carp,grouper and rainbow trout.Unlike in mammals,owing to teleost-specific whole-genome duplication(TS-WGD)events generating multicopy genes in teleosts,some teleosts possess multiple p35 and p40 paralogues,thereby probably producing IL-12 isoforms.As teleost IL-12 isoforms are homologous polymorphic immune molecules,their expression and function may reflect teleost immunity corresponding to the evolution and complex environment.Until now,the inductive expression pattern,isoform composition,and specific receptors of teleost IL-12 remain unclear.Besides,whether the immunological function of teleost IL-12 is completely consistent with the mammalian ortholog is also worth investigating.To address these questions,using grass carp as a model,this dissertation investigated whether and how IL-12 isoforms are produced and isolated.Then characterized the IL-12 specific receptors and revealed classical and new immunological functions of IL-12 isoforms,especially exploring IL-12's role in skin wound repair and related mechanisms.These works conducted systematic research on the production,receptor signaling and functional role and implication of grass carp IL-12(gc IL-12)isoforms.The dissertation was divided into the following four research sections:Firstly,molecular composition and immunological function characterization of grass carp IL-12 isoforms.Grass carp has two p35 paralogues(p35a and p35b)and three p40paralogues(p40a,p40 b and p40c).In this study,p35 a and p35 b genes were isolated from grass carp and found they might come from the same ancestral gene.Subsequently,the inducible expression patterns of p35 a and p35 b were uncovered.In concrete terms,poly I:C was able to induce p35 a gene expression through the JNK,ERK1/2 and NF-?B pathways,but did not affect p35 b expression in grass carp monocytes/macrophages.In the same cell model,IFN-? could enhance p35 b gene expression through p38 and NF-?B pathways but did not affect p35 a expression.Although two p35 paralogues and three p40 paralogues can theoretically encode six IL-12 isoforms in grass carp.However,the nonreducing electrophoresis experiments demonstrated that the main composition forms of gc IL-12 isoforms were IL-12AB(p35a + p40b)and IL-12BB(p35b + p40b).Furthermore,recombinant grass carp IL-12AB(rgc IL-12AB)and IL-12BB(rgc IL-12BB)proteins were obtained using the CHO eukaryotic expression system.Consistent with the classical function of mammalian IL-12,these two recombinant grass carp IL-12 isoforms exhibited extensive regulation of IFN-? expression in the head kidney leukocytes(HKLs),lymphocytes and monocytes/macrophages.Accidentally,it was found that grass carp IL-12 isoforms could induce IL-17A/F1 expression through STAT3/ROR?t signal in lymphocytes,while TGF-?1 negatively regulated this stimulation by inhibiting STAT3 activation.In addition,under the condition of Aeromonas hydrophila(A.hydrophila)infection intraperitoneally,IL-12 could increase the individual survival rate,reduce the number of A.hydrophila in the head kidney and improve the histopathological changes in the head kidney and intestine of grass carp.These works unveiled specific expression patterns and the exact composition of grass carp IL-12 isoforms,confirmed their classical role and explored novel aspects of teleost IL-12 in immune and inflammatory regulation.Secondly,the isolation and identification of grass carp IL-12 receptors.In this section,three grass carp IL-12 receptor candidates(named gc IL-12R?1,gc IL-12R?2a and gc IL-12R?2b)were cloned,and locally installed BLAST and gene synteny analysis uncovered that three gc IL-12 receptors were single-copied genes.Compared with the orthologues of other species,gc IL-12R?1 and gc IL-12R?2a shared good gene collinearity and clustering,similar structural characteristics and expression profiles.Additionally,gc IL-12R?2b was more similar to IL-27 Ra of non-fish species.Furthermore,the recombinant protein of the gc IL-12R?1 extracellular region was prepared.In vitro protein binding experiments showed that gc IL-12R?1 could bind to three gc IL-12 isoforms which contain different p40 genes(p40a,p40 b and p40c)with the same affinity.Moreover,the extracellular region of IL-12R?1 significantly antagonized the gc IL-12 isoforms-induced expression of IFN-? in grass carp monocytes/macrophages,providing functional evidence that newly cloned gc IL-12R?1 is the ortholog of mammalian IL-12R?1.Subsequently,ectopic expression of gc IL-12 receptors in HEK293 cells was performed to determine the receptor signaling for gc IL-12,and the results showed the co-expression of gc IL-12R?1 and gc IL-12R?2a could transmit gc IL-12 signal,while that of gc IL-12R?1 and gc IL-12R?2b did not do it.Taken together,these results functionally characterized gc IL-12R?1 and gc IL-12R?2a as the specific receptors of gc IL-12 isoforms,and fish IL-12R?2b could be the IL-27 Ra orthologues in non-fish species.It is worth mentioning that despite multiple IL-12 isoforms existing in grass carp,their signals were transmitted through the same combination of receptors(gc IL-12R?1 and gc IL-12R?2a),thereby suggesting the receptors are regarded as a possible regulatory point in mediating IL-12 isoforms signaling in fish.Thirdly,functional identification and mechanism exploration of gc IL-12 in skin wound healing.To strengthen the physiological significance of teleost IL-12 function,the role of teleost IL-12 in skin wound repair was explored in this section.In agreement with this notion,both the gene and protein expression of gcp35 b were significantly induced in the wounded skin.Combined with the previous data of high expression of gcp40 b at the skin wound site,it was speculated that gc IL-12BB(p35b + p40b)might play a role in skin wound healing.Subsequently,injection treatment with rgc IL-12 BB at the wound site showed that rgc IL-12 BB significantly improved wound healing and increased individual survival.Moreover,rgc IL-12 BB was effective in promoting epithelial repair,epithelial cell proliferation and migration,and reducing bacterial invasion.Additionally,rgc IL-12 BB significantly decreased the gene expression of IL-1? and TNF-? and enhanced the transcription of i NOS and IFN-? in the wound site.These results indicated that gc IL-12 facilitates skin wound repair by consolidating the integrity of the epithelial barrier,reducing bacterial invasion and inflammatory response,thereby highlighting the role and mechanism of gc IL-12 in promoting skin wound repair in fish.Fourthly,the mechanism of the gc IL-12/IFN-? signaling in promoting skin wound repair.Considering that IFN-? is the main effector of IL-12,it's worth investigating whether and how IFN-? participates in skin wound repair.Actually,the results showed that IFN-? was involved in gc IL-12BB-mediated skin inflammation control and bacterial clearance during wound healing according to the Immunoneutralization assay using a gc IFN-?-specific antibody.Moreover,the use of 4-ABAH(MPO inhibitor for limiting neutrophil function)proved that neutrophils were also involved in bacterial clearance during the same process.These findings were in line with the in vitro experiments in which IFN-? could accelerate bacterial clearance through the autophagy pathway in grass carp neutrophils.Guanylate-binding proteins(GBPs)in the interferon-stimulated genes(ISGs)family are closely associated with autophagy.Therefore,GBPs were selected to explore whether they are involved in the above regulatory effects of IFN-?.In fact,IFN-? significantly induced GBP1 expression in grass carp neutrophils,suggesting that GBP1 might be involved in the regulation of IFN-?.In accordance with this notion,GBP1 participated in IFN-?-induced autophagy through its interaction with autophagy-related proteins LC3 B and P62.Interestingly,GBP1 also directly bound A.hydrophila,acting as a pattern-recognition receptor targeting anchor pathogens.In summary,these data have elucidated the bactericidal efficacy and related autophagy mechanism of IL-12 effector molecules IFN-? and GBP1,strengthening the important role of IL-12-mediated IFN-?signaling and enriching the content of bacterial clearance mechanism in IL-12-facilitating skin healing.