Effect Of Sensitization Induced By Macrobrachium Nipponense Tropomyosin On Macrophage Polarization

Macrobrachium nipponense,commonly known as river shrimp,is one of the common edible freshwater shrimps in China.Tropomyosin(TM)is a pan-allergenic protein of shrimp and crabs,which can easily cause allergic reactions in some sensitive population.Macrophages are involved in innate immunity and adaptive immunity,which have the functions of antigen presentation and activation of T cells.Macrophage could be polarized into different phenotypes in different microenvironments and perform different functions.1.Bone marrow-derived macrophages were induced by TM to different phenotypes of macrophages in vitro.The marker gene expressions of the different phenotype macrophage stimulated by TM were explored by fluorescence quantitative PCR to speculate the effect of TM on macrophage polarization.It was shown that the levels of M1 macrophage marker genes(iNOS,Fpr2 and TNF-α)in the TM group were significantly higher than those in the PBS group(P<0.05),while the levels of M2 macrophage marker genes(Arg1,CD206 and Dectin)were significantly lower than those in the PBS group(P<0.05).After stimulated with TM,the expression of iNOS and Fpr2 of M1 macrophages induced by IFN-γ and LPS was significantly up-regulated compared with the PBS group(P<0.01),the TNF-α level was slightly decreased,while the expressions of Arg1,CD206 and Dectin were were no significant difference.After stimulated with TM,the expressions of iNOS,Fpr2 and TNF-α of M2 macrophages induced by IL-4 were significantly increased compared with the PBS group(P<0.05),whereas the Arg1 and Dectin levels were significantly decreased(P<0.05),and the expression of CD206 was not significantly change.It indicated that TM can affect the polarization of macrophages in vitro,and TM may be promote the polarization of bone marrow-derived macrophages to the M1 subtype macrophage.2.Effect of TM on the polarization of macrophages was studied by co-culture of macrophage-basophil and macrophage-splenic lymphocyte.It was shown that,in the co-culture of KU812 cells and macrophages,the expression of iNOS in the coKM-TM group was significantly lower than that in the M-TM group(P<0.01),while the expression of Arg1 was significantly increased(P<0.01),indicating degranulation may inhibit the transformation of TM-induced macrophages to M1.Moreover,macrophages reduce the levels of TNF-α,IL-6,histamine and β-HEX expressed by KU812 cells,indicating macrophages may inhibit the degranulation of KU812 cells.In the co-culture of smacrophage-splenic lymphocyte,the expression of IL-12/23p40 in the coPM-TM group was significantly lower than that in the TM group(P<0.05),while the expressions of iNOS and Arg1 were significantly increased compared with those in the TM group(P<0.05),indicating spleen lymphocytes may polarize macrophages in different directions.Moreover,the expressions of IL-4 and TGF-β in the coPM-TM group increased when compared with those in the TM group,while the expression of IFN-γ decreased significantly(P<0.05),ingdicating that macrophages inhibited the secretion of Th1 cytokines from splenic lymphocytes.3.The relationship between macrophage polarization and food allergy in vivo was explored by TM-sensitized model,macrophage-depletion model and M1/M2 macrophages adoptive transfer model.It was shown that the levels of special antibodies(IgE,IgG),cytokines(IL-4,IL-5,IL-13),mMCP-1 and histamine in the TM,TDM1 and TDM2 groups were significantly higher than those in the PBS group(P<0.05).Regarding to the comparing with the TM group,the expressions of IgE,IL-4 and IL-5 were up-regulated in the TDM1 group and TDM2 group,and the expressions of IL-13,mMCP-1 and histamine were not significant difference,indicating that M1 macrophages and M2 macrophages may be involved in food allergic reactions.In addition,there were certain differences in the distribution of macrophages in different tissues in allergic mice.Moreover,th e expression of iNOS in the MLN of the TM group was significantly increased(P<0.001)when compared with the PBS group,and the expression of Arg1 was significantly decreased(P<0.001),indicating that the MLN in allergic mice may be an important location for the accumulation of M1 macrophages in the inflammatory response.The expressions of iNOS and Arg1 in the PP nodes of the TM group were significantly increased(P<0.001),and the expression of iNOS in the jejunum was significantly increased(P<0.05),indicating that there were more M1 and M2 macrophages in the PP nodes and jejunum.4.High-throughput sequencing of mouse bone marrow-derived macrophages stimulated with tropomyosin and cytokines was used to screen their differential genes and related signaling pathways.It was shown that 69 genes in the TM group were up-regulated and 154 genes were down-regulated when compared with the PBS group,and these genes were enriched to 180 pathways.Five immune-related signaling pathways including NOD-like receptor signaling pathway,Jak-STAT signaling pathway,NF-κB signaling pathway,Toll-like receptor signaling pathway and PI3K-Akt signaling pathway were selected according to the KEGG analysis,and the expressions of key differential genes in these signaling pathways were detected by fluorescence quantitative PCR.It was shown that the expressions of NOD2,TLR2,AKT3,NLRP3,Caspase-1,CD14,Myd88,NFKBIA,and STAT1 in the IFNLPS group were significantly higher than those of the PBS and IL4 groups,while Lat level was decreased significantly.It is consistent with the sequencing results,proving the accuracy of the sequencing results.The expressions of NOD2,TLR2,NLRP3,and STAT1 in the TM group were significantly higher than those in the PBS group.These genes are relevant to the four signaling pathway,including NOD-like receptor signaling pathway,Jak-STAT signaling pathway and Toll-like receptor signaling pathway,which may be involved in the process of TM-induced macrophage polarization.In conclusion,this study investigated the effect of M.nipponense tropomyosin on macrophage polarization at the cellular and animal levels,and preliminarily elucidated some of the mechanisms by which TM sensitization affects macrophage polarization.It provides a scientific basis for further revealing the relationship between food allergy and macrophage polarization.