Molecular Characterization And Biological Effects On Monocytes/macrophages Of Two IL-4/13 Homologues In Large Yellow Croaker (Larimichthys Crocea)

Monocytes/macrophages(MO/M(ps)are innate immune cells with well-established roles in the primary response to pathogens,inflammation,resolution and repair.As anti-inflammatory Th2 cell cytokines,interleukin-4(IL-4)and interleukin-13(IL-13)also mediate the alternative activation of MO/M?s.However,the exact function of fish IL-4/13 homologs in MO/M?s polarization remains unclear.In this study,we identified two IL-4/13 homologues from large yellow croaker(Larimichthys crocea),LycIL-4/13A and LycIL-4/13B,which share low amino acid sequences identity to the previously reported teleost IL-4/13 homologues.Phylogentic analysis showed that LycIL-4/13A is evolutionarily closely related to Dicentrarchus labrax IL-4/13 A,and LycIL-4/13B is evolutionarily closely related to Takifugu rubripes IL-4/13B.The LycIL-4/13A and LycIL-4/13B mRNA were constitutively expressed in all examined tissues and immune cells,although at different levels.The mRNA expression of LycIL-4/13A was highest in gills,and that of LycIL-4/13B was high in heart,gills,spleen,peripheral blood lymphocytes(PBL)as well as head kidney-derived MO/Mcps.Upon inactivated trivalent bacterial vaccine induction,mRNA expression levels of LycIL-4/13A and LycIL-4/13B were up-regulated in the head kidney,climbing to the peak at 24 h and 48 h after induction,respectively.Recombinant LycIL-4/13 proteins(rLycIL,4/13A and rLycIL-4/13B)were produced in Escherichia coli and used for functional investigation.When in vitro cultured head kidney-derived MO/M(ps from large yellow croaker were treated by different concentrations of rLycIL4/13A or rLycIL4/13B,the superoxide production(O2-)and reactive oxygen species(ROS)in MO/M?s was gradually decreased,and reached the bottom at dose of 1000 ng/mL and 100 ng/mL,respectively.Moreover,nitrogen oxide(NO)production was also reduced in MO/M?s when treated with rLycIL-4/13 for 6 h.The phagocytosis of MO/M?s was inhibited to varying degrees by treatment with different concentrations of rLyc IL-4/13 A or rLycIL-4/13B as well.At 12 h,24 h and 48 h after being treated by 1000 ng/mL of rLycIL-4/13A or 100 ng/mL of rLycIL-4/13B,the mRNA transcriptional levels of pro-inflammatory cytokines(IL-1?,TNF-a)and iNOS were all down-regulated in MO/Mcps,while that of growth factors(VEGF,TGF-?)and arginase-2 increased.These results reveal that although LycIL-4/13A and LycIL-4/13B exhibit different molecular characteristics,both of them could promote MO/M?s towards alternative activation.