Molecular Cloning And Preliminary Study On The Function Of Macrophage Migration Inhibitory Factor Family In Japanese Sea Bass(Lateolabrax Japonicas)

Macrophage migration inhibitory factor family members are composed of macrophage migration inhibitory factor(MIF)and D-dopachrome tautomerase(DDT).MIF family members have pluripotent effects in inflammation and immune responses.As a primary member of MIF family,MIF has been studied for more than 50 years since its first report.Unlike most other cytokines,MIF is constitutively expressed and stored in intracellular pools within the cytoplasm.MIF is produced by a variety of immune and non-immune cells and has a broad range of activities including inhibition of macrophage migration,and activation of T cells.As a key upstream medium of innate and adaptive immune response,MIF has attracted extensive attention of researchers.It is associated with many infectious,inflammatory and immune diseases,including septic shock,colitis,malaria,rheumatoid arthritis,atherosclerosis and tumorigenesis.However,its functions have not been well studied in fish.DDT is the only known homologue of MIF with similar structure and exerts several overlapping functions with MIF.In mammals,DDT is systemically expressed and its circulating levels correlate with MIF and the severity of inflammatory disease and malignancy.In addition,among all known cytokines,DDT and MIF have a unique ability to antagonize the immunosuppressive effects of glucocorticoids.The macrophage migration inhibitory factor family members also play a vital role in the immune response of fish.However,studies on fish MIF and DDT are limited.In consequence,an exhaustive research on the roles of fish MIF and DDT immunomodulation properties will provide theoretical references for the application of MIF family proteins in the field of aquatic disease prevention and control.In this study,we identified an MIF molecule from Japanese sea bass(Lateolabrax japonicus;LjMIF).Multiple sequence alignment showed that LjMIF has the typical structural features of MIFs.Phylogenetic tree analysis revealed that LjMIF is most closely related to those of the yellowfin tuna(Thunnus albacares),large yellow croaker(Larimichthys crocea),and red drum(Sciaenops ocellatus)homologs.Constitutive mRNA expressions of LjMIF were detected in all tested tissues,with the highest level in the liver.Upon Vibro harveyi infection,LjMIF transcripts were upregulated in the tested tissues,including the liver,spleen,and head kidney.Subsequently,we prepared recombinant LjMIF(rLjMIF)and the corresponding antibody(anti-LjMIF antibody).The in vitro study showed that rLjMIF inhibited the trafficking of Japanese sea bass monocytes/macrophages(MO/M?)and lymphocytes,but not of neutrophils,while anti-LjMIF antibody had the opposite effect.rLjMIF also enhanced phagocytosis and intracellular killing of V.harveyi by MO/M?,while anti-LjMIF antibody only inhibited phagocytosis of MO/M?.The in vivo study showed that rLjMIF aggravated the course of V.harveyi infection in Japanese sea bass,but anti-LjMIF antibody increased the survival rate of the fish and decreased the bacterial burden.In conclusion,our observation revealed that LjMIF is closely involved in the immune responses combating V.harveyi infection of Japanese sea bass.In this study,we also identified a DDT homolog gene(LjDDT)from Japanese sea bass.Sequence analysis showed that LjDDT had typical sequence features of known DDT and MIF homologs and was most closely related to DDT of rock bream(Oplegnathus fasciatus).LjDDT transcripts were also detected in all tested tissues of healthy Japanese sea bass,with the highest expression in the liver.Upon infection with V.harveyi,LjDDT transcripts were significantly downregulated in three tested tissues including the liver,spleen,and head kidney.Recombinant LjDDT(rLjDDT)and the corresponding antibody(anti-LjDDT antibody)were subsequently prepared.The administration of 100 ?g/g anti-LjDDT antibody had a statistically significant protective effect on the survival of V.harveyi-infected fish.Moreover,rLjDDT was able to induce the migration of MO/M? and lymphocytes both in vitro and in vivo,but without significant influence on the migration of neutrophils.rLjDDT exhibited chemotactic activity for lipopolysaccharide(LPS)-stimulated M1-type MO/M? in vitro,but not for c AMP-stimulated M2-type MO/M?.Furthermore,the knockdown of Japanese sea bass major histocompatibility complex,class II invariant chain(LjCD74),but not Japanese sea bass C-X-C motif chemokine receptor 4(LjCXCR4)significantly downregulated the rLjDDT-enhanced migration of MO/M?,and relieved the rLjMIF-inhibited migration of MO/M?.These results indicated that LjCD74 is possibly the major chemotatic receptor of LjDDT and LjMIF on Japanese sea bass MO/M?.The combined rLjDDT and rLjMIF treatment had no significant effect on the migration of mismatch si RNA(Msi RNA),LjCD74si-and LjCXCR4si-treated MO/M? compared to the control group,suggesting that the roles of LjDDT and LjMIF are antagonistic.Our study demonstrates for the first time that DDT might also play a role in the immune response of fish against bacterial infection through chemotactic recruitment of MO/M? via the mediation of CD74 as an antagonist of MIF.In conclusion,our data suggest that LjMIF and LjDDT exert antagonistic regulatory effects on MO/M? and lymphocyte migration.The method of neutralizing endogenous proteins by preinjection of anti-LjMIF or anti-LjDDT antibodies can play an immunological protective role against V.harveyi infection in Japanese sea bass.These results extended our understanding of immunomodulatory effects of macrophage migration inhibitory factor family members in fish,and provide basic materials for the application of MIF and DDT in the prevention and control of aquaculture diseases.