| Fish non-specific cytotoxic cells(NCC)are the evolutionary precursors of natural killer cells(NK),which have similar functions to NK cells.In addition to non-specific cytotoxic effects,NCC also has immunomodulatory functions.However,the NCC group and its mechanism of activity regulation remain unclear.In this study,(Oreochromis niloticus),an important freshwater aquaculture species in southern China,was studied in three aspects:first,the single cell transcriptional map of tilapia NCC was constructed by single cell sequencing,and four NCC subsets were identified.Second,a series of differentially expressed galectin(Gal)gene families were screened from transcriptome data,and the interaction between these proteins and NCC receptor protein I(NCCRP-1)was identified by yeast two-hybrid and GST Pull-down techniques.Third,functional studies were carried out on Gal-2 and Gal-8 related to the regulation of NCC activity.The main results are as follows:1.The immunocytogram and NCC cell subgroup information of teleost fish were obtained by high-throughput 10x single cell sequencing.The head kidney of tilapia was taken from tilapia and lymphocytes were isolated after 24 hours of Poly I:C immune stimulation.The transcripts of 24,062 lymphocytes were analyzed by single cell RNA sequencing(sc RNA-seq),and the lymphocyte map of tilapia was constructed.According to cell heterogeneity and known markers,head kidney lymphocytes were divided into four types:B cell,T cell,NCC and monocyte/macrophage(Mo/M?).WGCNA predicted the regulatory network of NCC population,identified four central genes(STBD1,VWF,PGP and GRN)and one transcription factor(Hvcn1).According to cell heterogeneity and intracellular functional gene expression,four NCC subsets of memory-like NCC,mature NCC,immature NCC and pre-NCC were further identified.2.Identification of Gals interacting with NCC.The main results are as follows:(1)the differentially expressed Gals were screened and cloned from the above single cell sequencing data,and five Gals were obtained:Gal-2(OnGal-2),Gal-3(OnGal-3),Gal-4(OnGal-4),Gal-8(OnGal-8),and Gal-9(OnGal-9).Sequence analysis showed that all genes had no signal peptide,and all of them contain at least one carbohydrate recognition domain(CRD)and carbohydrate recognition sites(V-N,H-N-R,and W-E-R).This structural feature is consistent with the sequence characteristics of Gal proteins of other species.(2)The yeast two-hybrid bait plasmids of Gal were constructed and verified with NCCRP-1,and the results showed that OnGal-2 could interact with NCCRP-1.(3)Prokaryotic expression vector of OnGals(GST-tag)was constructed,and NCC was isolated by percoll density gradient.The interacting protein was isolated from NCC cell lysate by GST Pull-down.Our results showed that the common intracellular interaction protein identified by r OnGal-2 and rOnGal-8 was matrix metalloproteinase(MMP);(4)subcellular localization analysis showed that On MMP and OnGal-2/OnGal-8 were co-located in the cell.3.The immunomodulatory function of NCCRP-1-dependent OnGal-2 and its regulation on NCC activity were analyzed.(1)firstly,the qRT-PCR showed that OnGal-2 was distributed in all tissues of healthy tilapia,in which the expression level of brain was the highest,followed by spleen,liver,intestine,heart,thymus,muscle,skin,head kidney and Gill.After S.agalactiae stimulation,the expression of OnGal-2 in thymus,head kidney,spleen and Mo/M?was significantly up-regulated,indicating that OnGal-2 was involved in the antibacterial immune response of tilapia.(2)The recombinant proteinr OnGal-2(His-tag)was subsequently prepared to carry out a series of studies on its function.In vitro study showed that rOnGal-2 could agglutinate and bind to G~+(Streptococcus agglutinus,Streptococcus dolphin,Staphylococcus pasteurus and Lactococcus gmelinii)and G~-bacteria(Escherichia coli,Vibrio parahaemolyticus,Vibrio parahaemolyticus and Vibrio alginolyticus).A variety of sugars(LPS,LTA,Gal,Man and Mal)could weaken the binding ability of r OnGal-2 to bacteria.OnGal-2 can promote the phagocytosis of Mo/M?to bacteria and enhance the expression and killing activity of NCC.(3)Dual luciferase reporter system showed that OnGal-2 can activate the NF-?B,IFN-1,STAT1 and ISRE signaling pathways,indicating that OnGal-2 can participate in the NCCRP-1 mediated NCC activity by binding to MMP regulation.(4)Further in vivo experiments showed that r OnGal-2 injection into tilapia significantly increased serum antibacterial activity(ALKP,ACP,LZM)and antioxidant capacity(CAT,POD,SOD).OnGal-2 could also reduce the bacterial load in head kidney,spleen and liver,reduce tissue damage,and regulate the relative expression of NCC effectors after S.agalactiae infection,thereby increasing the relative survival rate of tilapia.4.The immunomodulatory effect of OnGal-8 independent of NCCRP-1 was studied.(1)qRT-PCR analysis showed that OnGal-8 existed widely in all examined tissues,with the highest expression level in spleen.With S.agalactiae stimulation,the OnGal-8 transcription levels in spleen,head kidney and brain were significantly up-regulated.(2)The results of in vitro experiments showed that OnGal-8 recombinant protein(rOnGal-8)could agglutinate erythrocytes,S.agalactiae and A.hydrophila,and bind to S.agalactiae,A.hydrophila,and various PAMP(LPS,LPA,Poly I:C,PGN,Gal,Man and Mal).In addition,rOnGal-8 can regulate the expression of inflammatory factors,promote phagocytosis and respiratory burst of Mo/M?,as well as enhance the killing effect of NCC.(3)Double luciferase reporter gene detection showed that the overexpression of OnGal-8 inhibited the activity of NF-?B,IFN-1,STAT1 and ISRE signal pathway,indicating that OnGal-8 could participate in the regulation of NCC effectors through the interaction with MMP.(4)In vivo experiments showed that OnGal-8 overexpression could improve the survival rate of tilapia by regulating serum antibacterial activity(AKP,ACP and LZM)and antioxidant capacity(CAT,POD and SOD),reducing bacterial load in infected tissue,alleviating tissue damage and enhancing NCC effect gene expression after S.agalactiae infection. |
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