Soluble MULT1 Overexpressing In Mice And Its Effect On Liver Fibrosis Of Schistosomiasis Japonica

Objective:Hepatic fibrosis is the main pathological injury due to Schistosoma japonicum infection,where by,the hepatic stellate cell is the key triger.As a powerful stimulatory receptor,Natural killer group 2 member D Receptor（NKG2D）is involved in eliciting Natural killer（NK）cells cytolysis against hepatic stellate cells（HSCs）and inhibiting the progression of liver fibrosis.As a soluble form of NKG2D ligand,soluble Murine ULBP-like transcript 1（s MULT1）can up-regulate the expression of NKG2D on the surface of NK cells and enhance the immune function of NK cells.This study is aimed to construct and characterize a genetic modified murine line with s MULT1 overexpression,then,with the newly established s MULT1 overexpressing mice,to explore the effect of s MULT1 overexpression on progress of schistosomiasis associated hepatic fibrosis.Methods:1)A CAG-loxp-stop-loxp s MULT1 coding sequence was introduced into mice to obtain Loxp-stop-Loxp s MULT1 mice using CRISPR/Cas9 gene editing technology（technically copleted by Vendor）.Plasmid pc DNA3.1-Cre was injected into Loxp-stop-Loxp s MULT1 mice by tail vein to make the mice overexpress s MULT1.Control mice were injected with the same dose of pc DNA3.1-GFP plasmid.One month after the plasmid injection,the concentration of serum s MULT1 was measured by a double antibody sandwich microsphere method;H&E histochemical staining was used to analyze the pathological changes of the mouse liver,spleen and kidney;the frequency of CD4⁺T,CD8⁺T and NK cells in the liver and spleen were detected by flow cytometry;and the expression of NKG2D and CD69 were also detected by flow cytometry;the levels of serum IFN-γ,IL-6,IL-10,IL-12P70,MCP1and TNF were measured by Cytometric Bead Array（CBA）.2)Based on genetyping,mice with both Alb-Cre-ERT and Loxp-stop-Loxp s MULT1 genes were selected as the experimental group while else litter-mated siblings of the same sex as controls.Alb-Cre⁺/s MULT1^CKI+mice and WT mice were infected with 18±2 cercariaes through skin.Tamoxifen was intraperitoneally injected to mice once at the 4th week post infection,and the end point of the animal experiment was set at the 8th week.Body weights were recorded;the concentration of s MULT1 in the mice serum,prepared on the day 9,16,23,and 27 after tamoxifen injection,were caculated by the double antibody sandwich microsphere method;H&E histochemical staining was used to analyze the pathological changes of mouse liver,spleen,kidney and colon;MASSON histochemical staining was employed in evaluating collagen deposition;the m RNA levels of liverα-Smooth muscle actin（α-SMA）,Collagen1（col1a-1）,Transforming growth factor-β（TGF-β）,Connective tissue growth factor（CTGF）and Desmin were determined by quantitative real-time PCR;the serum Alanine aminotransferase（ALT）level was calculated by alanine aminotransferase（ALT/GPT）microplate method;the frequency of CD4⁺T,CD8⁺T,NK and NKT cells in the liver and spleen as well as the expression of NKG2D and IFN-γwere analyzed by flow cytometry;the levels of serum IFN-γ,IL-6,IL-10,IL-12P70,MCP1 and TNF were measured by CBA.Results:1)Compared with the control group,the expression of s MULT1 in Loxp-stop-Loxp s MULT1 mice was significantly increased by pc DNA3.1-Cre plasmid administration（P=0.0016,n≥5）,indicating that the s MULT1 overexpressing transgenic mice were successfully constructed.H&E staining demonstrated no significant histological difference between the two groups except more spleen Reed-Sternberg-like cells were revealed in the s MULT1 overexpressing mice than in the control mice.Meanwhile,upon s MULT1 overexpression under normal physiological conditions,no significant change either in lymphocytes composition（CD4⁺T,CD8⁺T and NK cells in the spleen and CD8⁺T and NK cells in the liver）or in the surface NKG2D and CD69 level of interested lymphocytes were detected（P>0.05,n≥5）,with an exception that the frequency of CD4⁺T cells in the spleen was significantly decreased（P=0.0391,n≥5）.No significant change in the serum level of IFN-γ,MCP-1,IL-6,IL-10,IL-12P70 or TNF was detected either（P>0.05,n≥5）,in response to s MULT1 overexpression.2)During investigating the effect of s MULT1 overexpression on progress of schistosomiasis associated hepatic fibrosis,significantly increased serum s MULT1 in Alb-Cre⁺/s MULT1^CKI+mice with Schistosoma infection was detected upon tamoxifen injection（P>0.05,n≥3）.No significantly change of the degree of infection between the s MULT1 overexpressing mice and the control group（P values were 0.9180）.Compared with the control group,the area of egg granuloma and collagen deposition in the liver of s MULT1 overexpressing mice were both decreased.However,there were more Reed-Sternberg-like cells in the spleen and a little lymphocyte proliferation was observed in the renal capsule of infected mice of s MULT1overexpressing than of the control mice.The m RNA levels ofα-SMA,TGF-β,CTGF and Desmin in the liver tissues of s MULT1 overexpressing mice were significantly reduced（P values were 0.0441,0.0002,0.0041 and 0.0066,respectively,n≥3）and the serum ALT level was significantly decreased（P=0.0313,n≥3）.The frequencies of CD4⁺T,CD8⁺T,NK and NKT cells in the liver and spleen of the infected mice were close without significant difference between the two groups（P>0.05,n≥3）.In the S.j infected mice,the expression of NKG2D on NK and NKT cells both in the spleen（P values were respectively<0.0001 and 0.0024,n≥3）and in the liver（P values were respectively 0.0070 and 0.0005,n≥3）were significantly increased inresponse to s MULT1 overexpression.In consistance,the proportion of NKG2D⁺NK1.1⁺cells in the spleen and liver were increased（P values were 0.0249and 0.0327,n≥3）.No significantly change of IFN-γexpression in CD4⁺T,CD8⁺T,NK or NKT cells either in the liver or in the spleen was detected in the s MULT1overexpressing mice with S.j infection comparing to their non-overexpressing counterparts（P>0.05,n≥3）.Neither obvious histomorphological change in the colon nor significant change of serum inflamentery cytokines,including IFN-γ,MCP-1,IL-6,IL-10,IL-12P70 and TNF,was observed in the S.j infected mice,because of s MULT1 overexpression（P>0.05,n≥3）.Conclusion:Overexpression of sMULT1 in vivo has very little effect,if any,on immune homeostasis and does not elicit detectable inflammation in C57BL/6 mice of physiological condition,nor was it observed that NKG2D-positive immune cells were activated,indicating the s MULT1 overexpressing mice can be used to study the immunological mechanism of schistosomiasis.Under the pathogenical circumstances of schistosomiasis,s MULT1 overexpression can dramatically augment surface NKG2D levels on NK1.1⁺cells and attenuate schistosomiasis associated hepatic fibrosis.