Study On The Roles Of TLR2 Difficiency In Promoting Liver Fibrosis

ObjectLiver fibrosis is a scar repair response to liver injure caused by a variety of liver injury factors.Chronic liver diseases,such as cirrhosis,hepatocellular carcinoma,and virus-mediated immune pathogen infections,affect health of billions of people worldwide.These diseases are usually triggered by fibrosis.The main manifestation is the increase and activation of hepatic stellate cells(HSCs)into myofibroblasts,leading to excessive deposition of extracellular matrix(ECM).Activated myofibroblasts and hepatic stellate cells are the main producers of fibrotic scars.Their fibrotic activation and proliferation depend on complex interactions with other resident or recruited cells and their secreted factors.Due to the various side effects of antifibrotic therapy and the difficulty in diagnosing asymptomatic patients,effective drugs remain a urgent problem.In the process of liver fibrosis,hepatic stellate cells interact with a variety of immune cells to synthesize and release various cytokines,chemokines and other signal molecules to regulate the process of liver fibrosis.At present,the research on the phenotype of immune cells is becoming more and more detailed,and the related research on the impact of different stages of liver fibrosis is also deepening in controversy.TLR2,a member of the Toll-like receptor(TLR)family,initiates signal transduction by recognizing pathogen-associated molecular patterns and certain endogenous ligands,leading to the release of inflammatory mediators.Ligands for TLR2 include lipoproteins,lipopeptides,lipoic acid(LTA)arabinose(LAM).TLR2 plays different roles in different models of inflammatory diseases,and TLR2 immunoregulation is one of the hot spots in the field of immunity.However,the role of TLR2 in liver fibrosis and the effect of TLR2 on the progression of liver fibrosis in different cell subsets are still unclear.Therefore,in this study,the liver fibrosis mouse model induced by thioacetamide(TAA)was used to study the effect of TLR2 deficiency on liver fibrosis and its effect on liver parenchymal cells,hepatic stellate cells and immune cells,so as to provide a new target for the prevention and treatment of liver fibrosis.Aims1.To investigate the effect of TLR2 on the progression of liver fibrosis;2.To observe the effects of TLR2 on the number and function of hepatic stellate cells,hepatocytes,and immune cells.Methods1.Liver fibrosis models of WT mice and TLR2-/-mice were established by intraperitoneal injection of TAA once every three days for twelve times;2.These mice were killed,the liver and spleen were dissected out,photographed,weighed,and the ratio of organs to body weight was calculated.Mouse mononuclear cells were isolated and counted;3.Sirius red staining was used to observe the effect of TLR2 deficiency on collagen deposition;4.Western Blot and immunohistochemistry were used to analyze the protein expression level of alpha-SMA,a marker of hepatic stellate cell activation,in mice with TLR2 deficiency;5.The effect of liver fibrosis on TLR2 mRNA level in liver tissue was detected by real-time fluorescence quantitative PCR;6.The hepatic inflammation and scar formation in liver fibrosis were observed by H&E staining;7.Real-time fluorescence quantitative PCR was used to detect the effect of TLR2 deficiency on the mRNA levels of inflammatory factors and hepatic fibrosis-related molecules in liver tissue;8.Total hepatocytes of mice were isolated by in situ perfusion-enzymatic digestion,and hepatic stellate cells were isolated by gradient density superimposed centrifugation,then observed and cultured for identification and extraction;9.Real-time fluorescence quantitative PCR was used to detect the effect of TLR2 deficiency on the mRNA levels of inflammatory factors and liver fibrosis-related molecules in hepatic stellate cells and hepatic parenchymal cells;10.The total number of liver and spleen mononuclear cells,the proportion and function of macrophages,NK cells and T cells in the process of liver fibrosis were detected by flow cytometry.Results1.The expression levels of TLR2 decreased upon chronic liver fibrosisCompared with the control group,Sirius Red staining results showed the positive area of Sirus Red staining significantly increased by TAA treatment,and a large amount of collagen was deposited around the blood vessels.Western Blot experiments further displayed a significant increase in the expression of protein ?-SMA,which characterized the degree of fibrosis.Next,qPCR testing showed that the TLR2 level was reduced in TAA treated mice compared to the control group.These phenomena indicated that the liver fibrosis model was successfully constructed,and TLR2 was involved in liver fibrosis progression.2.The deficiency of TLR2 aggravated liver fibrosis in miceUntreated WT mice and TLR2-/-mice had smooth liver surfaces,no granulosities,soft and elastic texture,and there was no significant difference between the two groups.However,after TAA induction,TLR2-/-mice showed more granular sensation on the liver surface than WT mice.The Sirus Red staining results showed that the positive area of Sirius Red staining in the liver of TLR2-/-mice treated with TAA increased compared with WT mice,accompanied with the significant collagen deposition near the hepatic sinusoid.Immunohistochemical results also showed that the expression of-SMA in TLR2-/-mice treated with TAA was significantly higher than that of WT mice.3.The deficiency of TLR2 exacerbated liver damage in the miceBy H&E staining,compared with WT mice,the TLR2-/-mice treated with TAA showed more severe liver damage due to a large number of inflammatory cell infiltration and the formation of fibrous scar near the liver vessels.4.The deficiency of TLR2 up-regulated the expression of genes related to liver inflammation and liver fibrosis in miceLiver inflammation and fibrosis-related genes were detected by qPCR.Compared with WT mice,TLR2-/-mice treated with TAA significantly upregulated the fibrosis-related genes TGF-??collagen1?TIMP1.There was no obvious change in the pro-fibrogenic gene MMP2,and MMP9 was up-regulated.The level of inflammatory factor TNF-? increased,but IL-6 did not change significantly.5.The deficiency of TLR2 up-regulated inflammation and liver fibrosis related genes in hepatocytes and hepatic stellate cellsThe detection of qPCR found that there was no statistically significant difference in fibrosis-related gene TGF-? and the level of inflammatory factor IL-6 increased significantly in the hepatic stellate cell of TAA-treated TLR2-/-mice compared to WT mice.There was no significant change in IL-1??IL-18 while the level of fibrosis-related gene TGF-? was significantly increased,and there was no significant change in inflammatory factors IL-6 and IL-1? in hepatocytes.6.The influence of TLR2 deficiency on immune cells in liver and spleen of mice6.1 Deficiency of TLR2 promoted the liver infiltration of mononuclear cellsAfter TAA treatment,there is no significant difference in body weight,but the liver weight ratio and spleen weight ratio of TLR2-/-mice decreased slightly compared with WT mice,while the number of mononuclear cells in the liver increased.6.2 Deficiency of TLR2 dampened the responsibility of macrophages in the liver of miceTAA treatment significantly increased the proportion of M1-type macrophages and ly6Chi macrophages in the liver of WT mice,.Although compared with untreated TLR2-/-mice the proportion and number of macrophages in the liver of TAA-treated TLR2-/-mice were significantly increased.Compared with TAA-treated WT mice,the proportion and number of macrophages in the liver of TAA-treated TLR2-/-mice did not show significant change.However,Compared with untreated TLR2-/-mice,the percentage of Ly6Chi macrophages and Ly6Clo macrophages did not change significantly in TAA-treated TLR2-/-mice.Similarly,the percentage of iNOS+ type M1 macrophages and CD206+type M2 macrophages did not change significantly However,the ratio of iNOS+ macrophages to CD206+macrophages increased slightly.There was no significant difference in the number and proportion of macrophages and the up-regulation of MHC-II and CD80 was inhibited in the spleen.These results suggest that the deficiency of TLR2 leads to dampened responsibility of hepatic macrophages.6.3 Deficiency of TLR2 increased the percentage of LAG3+NK cells in liver upon chronic liver fibrosisAfter TAA treatment,the proportion and number of liver NK cells in WT mice and TLR2-/-mice,activator molecule CD69,killer molecule INF-?,CD 107a,perforin,inhibitory molecules TIGIT,LAG3 were not statistically different,but compared with untreated TLR2-/-mice,the expression of inhibitory molecule LAG3 increased in TAA-treated TLR2-/-mice.NK cells in spleen are similar to those in liver.However,compared with untreated WT mice,the expression of TIGIT and LAG3 in spleen NK cells of TAA-treated WT mice were significantly up-regulated,while the expression of TIGIT and LAG3 in NK cells of TAA-treated TLR2-/-mice were not significantly changed..Compared with TAA-treated WT mice,activation molecule CD69 increased in TAA-treated TLR2-/-mice.6.4 Deficiency of TLR2 affected the proportion of liver CD4+T cells and CD8+T cells upon chronic liver fibrosisFlow cytometry showed that compared with TAA-treated WT mice,there was no significant change in the total T cell proportion and number of livers in TAA-treated TLR2-/-mice;the proportion and number of CD4+T cells increased significantly and the activation molecule CD69,inhibitory molecule PD-1 has no significant effect;while the proportion and number of CD8+T cells decrease,the activation molecule CD69 and the inhibitory molecule PD-1 have no significant effect,but the degranulation marker CD 107a increases significantly;The ratio of CD4+T cells to CD8+T cells increased significantly.In spleen,the proportion of total T cells increased,but the number had no significant effect;CD4+T proportion,number,activation molecule CD69 and inhibitory molecules LAG3,PD-1 had no significant changes;CD8+T proportion increased slightly,but the number did not change significantly,The activation molecule CD69 was slightly increased,and the degranulation marker CD 107a was slightly decreased.The inhibitory molecules LAG3 and PD-1 had no significant effect;there was no statistical difference in the ratio of CD4+T cells to CD8+T cells.6.5 Deficiency of TLR2 did not significantly influence NKT cells in the liver of miceFlow cytometry analysis found that compared with TAA-treated WT mice,the proportion and number of liver NKT cells,activator CD69,killer molecules INF-?,CD 107a,perforin,inhibitory molecule LAG3 had no significant effect;In spleen the proportion of NKT cells increased slightly,but the total number decreased slightly.Activator molecule CD69,killer molecules INF-y,CD 107a,perforin,inhibitory molecules TIGIT,LAG3 had no significant effect.Conclusions1.This study found that TLR2 deficiency promoted liver fibrosis,accompanied with the increase of inflammatory factors and fibrosis-related molecules expression in liver tissue,liver parenchymal cells,and hepatic stellate cells.2.Deficiency of TLR2 dampened the responsibility of macrophages in the liver of mice and did not affect NKT cells in the liver of mice,but promoted the expression of LAG3 in NK cells and increased the proportion and number of CD4+T cells,but decreased the proportion and number of CD8+T cells,showing a immune microenvironment that promotes liver fibrosis.3.This study proved the role of TLR2 in inhibiting liver fibrosis,and provided an experimental basis for the development of new drugs for treating liver fibrosis.