HBV Upregulated Triggering Receptor Expressed On Myeloid Cells-1 (TREM-1) Expression On Monocytes Participated In Disease Progression

Objective:Chronic inflammation and fibrosis of the liver caused by persistent infection with Hepatitis B virus virus(HBV)is an important factor in end-stage liver disease in China,host immune cells play an important role in the balance between anti-viral immunity and the control of over-immune response.As one of the antigen presenting cells,monocytes not only promote the activation of Hapten,but also participate in the maintenance of the balance of immune microenvironment.Myeloid triggered receptor1(TREM-1)is a type of transmembrane receptor mainly expressed in myeloid cells.The activation of this receptor can promote the response of Toll-like receptor 4(TLR-4)signaling pathway,further amplifies the inflammatory response to participate in the occurrence and development of a variety of diseases.At the same time,more and more studies show that the occurrence of cell pyrosis leads to the release of cell contents,and the increase of proinflammatory factors is related to the progression of liver inflammation and fibrosis.TNF-α is also one of the important cytokines involved in inducing cell Tumor necrosis factors,as the activation of the TREM-1 pathway promotes the expression and release of TNF-α.It is not clear whether this phenomenon is related to the increased expression of TREM-1 in mononuclear cells because of the possibility of cell scorch death in hepatocytes,macrophages and iNKT cells.Therefore,this study intends to investigate whether the virus participates in the regulation of TREM-1 expression in monocytes during HBV infection,and through the activation of TREM-1 signal to promote the occurrence of cell scorch in the liver and thus participate in the pathogenesis of liver disease Materials and Methods:Objective to detect the expression of TREM-1 in peripheral blood mononuclear cells of healthy controls,patients with chronic HBV infection and patients with HBV infection related cirrhosis,so as to determine whether the expression of TREM-1 in mononuclear cells is involved in the progression of HBV infected liver diseases;2)recombinant human hepatitis B surface antigen(HBs Ag)was obtained from the culture supernatant of Hep G2.2.15 cells Anti,HBs Ag antigen peptide and recombinant human hepatitis B e antigen(HBe Ag,HBe Ag antigen peptide stimulates peripheral blood mononuclear cells(PBMC)of healthy people at different concentrations Flow cytometry was used to detect the expression of TREM-1 in PBMC at different concentrations and time points to determine whether HBV can up regulate the expression of TREM-1 in PBMC;3)bioinformatics was used to predict that NF-κB and AP-1 may up regulate the expression of TREM-1,si RNA was used to interfere the expression of NF-κB and AP-1 in THP-1 cells,and HBV was used to stimulate THP-1 cells,Flow cytometry was used to detect the expression of TREM-1 in THP-1 cells;T-5224 and BAY11-7082 were used to inhibit the expression of AP-1 and NF-κB in primary monocytes,respectively;HBs Ag and HBe Ag were used to stimulate the monocytes pretreated with inhibitors,and the expression of TREM-1 in monocytes was detected by flow cytometry;4)LP17,an inhibitor of TREM-1,was used to pretreat THP-1 cells,and the expression of TREM-1 was detected by flow cytometry Lx-2 cells were co cultured and stimulated with HBV for 24 hours.The m RNA expression levels of proliferation and activation related genes of LX-2 cells were detected;5)Methods:the expression of TREM-1 on THP-1 cells was knocked down by si RNA interference,and the expression of downstream inflammatory factors(TNF-α,IL-6 and IL-1 β)was detected by chemiluminescence detection after stimulated by HBV;THP-1 cell lines overexpressing TREM-1 were constructed by lentivirus,and the expression of the above cytokines was detected after stimulated by HBV;6)THP-1 cells and Hep G2.2.15 cells were separated by Transwell chambers were co cultured to detect the expression of inflammasome on THP-1 cells and pyrolytic protein on Hep G2.2.15 cells.6)Flow cytometry was used to detect the number and subsets of iNKT cells in peripheral blood of the patients mentioned above.Primary iNKT cells from healthy people were amplified and sorted.THP-1 cells with TREM-1 overexpression and THP-1 cells in control group were co cultured with Hep G2.2.15 cells to stimulate the sorted iNKT cells.m RNA and protein of iNKT cells were collected and IFN-γ of iNKT cells was detected,The expression of IL-4 and IL-17 m RNA was detected,and the expression of pyrolytic protein in iNKT cells was detected.Results:1)In the study of clinical patients,it was found that the mean fluorescence intensity(MFI)of monocyte TREM-1 expression gradually increased in each group(HC:1240.8±413.0;CHB: 1456.5±391.2;LC: 1729.3±615.0,P=0.004).The expression of TREM-1 on monocytes was positively correlated with indicators related to liver damage(serum total bilirubin: r=0.421,P=0.006;serum glutamate aminotransferase:r=0.398,P=0.005;aspartame Acid aminotransferase-platelet ratio APRI: r=0.552,P<0.0001),which is negatively correlated with liver synthesis related indicators(serum albumin: r=-0.470,P=0.001);monocyte TREM-1 expression As the patient’s serum ALT expression level(P=0.017)and APRI(P=0.036)increase,it gradually increases;2)In vitro experiments show that HBV and antigen peptides HBs Ag and HBe Ag can induce the expression of monocyte TREM-1 in peripheral blood PBMC,and it can last for at least 72 hours;3)Bioinformatics suggests that p65(NF-κB)and cFOS(AP-1)are transcription factors that may regulate the expression of TREM-1.In vitro experiments have found that HBV can up-regulate the expression of p65 and cFOS on THP-1 cells through si RNA After interfering with the expression of p65 and cFOS on THP-1 cell line,HBV stimulation can be given again to reduce the expression of TREM-1 on THP-1 cells;the small molecule inhibitor T-5224 inhibits primary monocyte AP-1 Afterwards,it can reduce the expression and regulation of HBs Ag on monocyte TREM-1;after the small molecule inhibitor BAY 11-7082 inhibits primary monocyte NF-κB,it can inhibit the effect of HBe Ag or HBs Ag on monocyte TREM-1 Expression regulation4)In the co-culture system of THP-1 cells and LX-2 cells,using LP17 to inhibit the TREM-1 signaling pathway on THP-1 cells,and then adding HBV stimulation to the co-culture system can significantly reduce LX-2 cells’ proliferate and reduce the m RNA expression levels of α-SMA and COL1α1 on LX-2 cells;5)Interfering with the expression of TREM-1 on THP-1 cells can significantly reduce the inflammation-related cytokine IL-1β of monocytes after HBV stimulation(control group VS si RNA group: 3165.6±1116.9 pg/ml VS 2211.1±979.5 pg/ml P =0.0009),IL-6(control group VS si RNA group: 5450.7±1266.3 pg/ml VS 3507.1±625.8 pg/ml P=0.0017)and TNF-α(control group VS si RNA group: 60944.4±4111.3 pg/ml VS35611 ±4548.9 pg/ml P<0.0001);THP-1 cells overexpressing TREM-1 can significantly increase the downstream cytokine IL-1β(control group VS overexpression group: 2533.3 ± 76.4 pg/ml VS 4976.0 ± 340.5 pg/ml P=0.0094),IL-6(control group VS overexpression group: 1621.0±82.4 pg/ml VS 2949.0±444.4 pg/ml P=0.0077),IL-6(control group VS overexpression group: 24366.7± 802.1 pg/ml VS54378.1±12850 pg/ml P=0.0011)expression;6)In the co-culture system of THP-1 cells and Hep G2.2.15 cells,interference with TREM-1 can significantly reduce the expression of NLRP3 on THP-1 cells,and overexpression of TREM-1 can significantly increase the expression of NLRP3 on THP-1 cells.expression.7)The activation of TREM-1 signaling pathway on THP-1 cells can increase the expression of AIM2,caspase-1/p20 and GSDMD/GSDMD-N in Hep G2.2.15 cells in the co-culture system,and promote the occurrence of hepatocyte pyroptosis.8)In clinical studies,the number of iNKT cells in the peripheral blood of patients with HBV infection decreased with the progression of the disease [HC: 0.17(0.08-0.31)%;CHB: 0.071(0.039-0.180)%;LC: 0.043(0.018-0.084)%;P=0.001],and CD4+iNKT cell subsets increase with the progression of the disease(HC: 30.5±17.8%;CHB:48.5±22.8%;LC: 65.9±23.6%,P=0.001);patients Peripheral blood iNKT percentage is negatively correlated with APRI(r=-0.378,P=0.002);9)Using the supernatant of TREM-1 overexpressing THP-1 cells and Hep G2.2.15 cells to stimulate primary iNKT cells,which can promote the expression of caspase-1 and GSDMD proteins in iNKT cells,reduce the number of iNKT cells,and promote The m RNA expression of IL-17 in iNKT cells increased.Conclusion:1)The expression of TREM-1 in peripheral blood mononuclear cells of patients with HBV infection gradually increases with the increase of serum ALT expression and APRI index,which is closely related to the degree of liver inflammation and fibrosis in patients.2)HBV can up-regulate the expression of TREM-1 by promoting the activation of NF-κB or AP-1 transcription factors in monocytes for at least 72 hours,which may be an important factor in maintaining the chronic inflammatory environment of the liver.3)The activation of monocyte TREM-1 can promote the proliferation and activation of stellate cells by promoting the release of inflammatory factors to participate in the progress of liver fibrosis;4)The activation of TREM-1 on monocytes can up-regulate the expression of monocyte inflammasome NLRP3,and it also participates in the pyroptosis of hepatocytes,iNKT cells and iNKT cell activation,thereby mediating liver inflammation The maintenance of the response and the progression of chronic fibrosis.5)The expression of monocyte TREM-1 has potential clinical application value in the evaluation of liver chronic inflammation and fibrosis progression after HBV infection.At the same time,it may also have certain clinical application prospects in the treatment of fibrosis in targeted patients.