TLR4-dependant Immune Response, But Not Hepatitis B Virus Reactivation, Promotes Radiation-induced Liver Diseases Via Regulating Liver Microenvironment

Radiotherapy (RT) was recommended for most patients with inoperable and/or locally advanced hepatocellular carcinoma (HCC). However, radiation-induced liver disease (RILD) is a main complication which restricts the therapeutic effect of RT. Liver diseases, such as hepatitis B virus (HBV) infection and hepatic fibrosis, not only can result in HCC, but also might influence the severity of RILDs. Toll-like receptor 4 (TLR4) is an important trigger of the immune response against HBV infection and liver injuries. Theoretically, injured tissues induced by RT might generate damage-associated molecular patterns to stimulate TLR4, which would lead to activation of pro-inflammatory cytokines and further promoting radiation-induced deseases. In addition, as a special microenvironment around cells, liver tissue interstitial fluid (TIF) might play a vital role in cell communication during liver injuries. The radiosensitivity of hepatocytes would also be affected by the liver microenvironment. Therefore, how RILDs are influenced by TLR4 immunization or HBV reactivation, as well as liver TIF changes in these processes, will be further studied in this research.Part Ⅰ A radiation-induced liver disease model of tomotherapy-based stereotactic body radiation therapy according to normal tissue dose constraints in micePurpose:To define the exact doses resulting in mouse radiation-induced liver diseases (RILDs), define the dose constraints for the critical organs at risk (OARs), and construct RILD model in mice with the helical tomotherapy-based stereotactic body radiation therapy (HT-SBRT).Materials and Methods:Thirty mice treated with HT-SBRT were stratified according to escalating radiation doses to the livers. Signs, such as red or black tarry stools, mouse performance status (MPS), weight loss, serum aspartate aminotransferase (AST)/alanine aminotransferase (ALT)/albumin levels, and pathological differences were observed. The severity of radiation-induced disease was scored using systematic evaluation standards.Results:Mouse RILD models could be developed when livers were irradiated with the average doses>31.76±1.94 Gy in HT-SBRT (single fraction). Evaluation criteria of mouse abdominal radiotherapy signs were set up as follows:MPS≥1.5±0.56, weight loss>0≥0.89g, AST/ALT≥589.2±118.5/137.4±15.3 U/L, serum albumin ≤18.8±0.99g/L. Preliminary dose constraints of OARs had also been obtained, such as lungs:V20≤20.3%, kidneys:V10≤11.3%, livers:average dose≤26.36±1.71 Gy; gastrointestinal tract:maximum dose≤22.63 Gy.Conclusion:Mouse RILD models could be developed with HT-SBRT based on the dose constraints for OARs and evaluation criteria of abdominal radiotherapy signs, which would favor the study of further approaches of treating hepatocellular carcinoma and other abdominal cancers with HT-SBRT. stools, mouse performance status (MPS), weight loss, serum aspartate aminotransferase (AST)/alanine aminotransferase (ALT)/albumin levels, and pathological differences were observed. The severity of radiation-induced disease was scored using systematic evaluation standards.Part Ⅱ TLR4-dependant immune response, but not HBV reactivation, is important in radiation-induced liver diseasesPurpose:Toll-like receptor 4 (TLR4) is an important trigger of the immune response against hepatitis B virus (HBV) infection and liver injuries. The roles of HBV reactivation versus TLR4-dependant immune response may be critical factors in preventing radiation-induced liver diseases (RILDs) after liver radiotherapy.Methods and Materials:This study consists of 2 phases. In the primary phase, livers of TLR4 mutation (TLR4-) mice were irradiated with 30 Gy in either the absence or presence of HBV infection. The latter was done by introduction of plasmid pAAV/HBV 1.2. In the advanced phase, RILDs were compared in normal TLR4 (TLR4+) versus TLR4- mice. HBV reactivation, TLR4 expression and severity of RILDs were studied in mice.Results:More HBV reactivation, without significant RILD, was observed in irradiated versus unirradiated TLR4- mice. However, RILD scores of TLR4+mice (2±0.27) were higher than TLR4- mice (0.5±0.19), P<0.05.Conclusion:Although RT in fact can activate HBV, RILDs correlate with TLR4-dependant immune response, but not with HBV reactivation. (TLR4+) versus TLR4- mice. HBV reactivation, TLR4 expression and severity of RILDs were studied in mice.Part Ⅲ TLR4-dependent immune response promotes radiation-induced liver diseases by changing the liver tissue interstitial microenvironment during mouse liver radiotherapyPurpose:To explore the roles of toll-like receptor 4 (TLR4)-dependent immune response and liver tissue interstitial fluids (TIFs) to prevent radiation-induced liver diseases (RILDs) after liver radiotherapy (RT).Methods and Materials:This study consisted of two phases. In the primary phase, the livers of TLR4 mutation (TLR4-) and normal (TLR4+) mice were irradiated with 30 Gy.TIF was obtained from mouse livers and assessed by mouse protein array analysis 20 days after completion of RT. The relationships among proteins in TIFs, TLR4, and RILDs were analyzed. In the validation phase, hepatocytes were isolated from TLR4+ or TLR4-mice, exposed to 8 Gy of irradiation, and then co-cultured with TIFs from mouse livers. Apoptosis of hepatocytes was measured using flowcytometry.Results:More serious RILDs, accompanied by higher expression of granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-related apoptosis inducing ligand (TRAIL), and vascular endothelial growth factor receptor 2 (VEGFR-2) in liver TIFs, were observed in TLR4+ mice compared with TLR4- mice (P< 0.05). In both TLR4+ and TLR4- hepatocytes, apoptosis after radiation therapy was increased to a significantly greater extent after co-culture in TIFs from TLR4+ mice that underwent liver RT, compared with TIFs from TLR4- mice with liver RT or TIFs from mice without RT (P< 0.05).Conclusion:TLR4-dependent immune response might promote RILDs by enhancing the expression of GM-CSF, VEGFR-2, and TRAIL in liver TIFs. significantly greater extent after co-culture in TIFs from TLR4+ mice that underwent liver RT, compared with TIFs from TLR4- mice with liver RT or TIFs from mice without RT (P< 0.05).Part Ⅳ Roles of HBV reactivation, TLR4 and TLR4-related proteins in radiation-induced liver diseases in clinicPurpose:To validate the roles of HBV reactivation, TLR4-dependant immune response and TLR4-related proteins during radiation-induced liver diseases (RILDs) in human, which were explored by the former studies in mice, and might favor us to fast use the achievements obtained by basic researches in clinic.Methods and Materials:Twenty-eight liver cancer patients, experienced liver radiotherapy before hepatectomy, were enrolled. Liver tissues near tumors embedded in paraffin, irradiated with 35-48 Gy, were used to construct tissue microarrays and assessed by immunohistochemistry. HBV reactivation, the expression levels of TLR4 or TLR4-related proteins, and the severity of RILDs were studied in human.Results:Serious RILDs tended to develop in patients with high TLR4 expression, but not in patients with low TLR4 or high HBV surface antigen (HBsAg) expression. High TLR4 expression was seen in only 2 of 12 HBV-reactive patients but in HBV-nonreactive patients it was seen in 6 of 9 (P<0.03). Moreover, companying with high TRAIL, VEGFR-2 and GM-CSF expression, serious RILDs tended to develop in patients with high than low TLR4 expression in livers (P< 0.05).Conclusion:In clinic, it is also TLR4-depended immune response, but not HBV reactivation, which plays a vital role in RILDs. TLR4-depended immune response might not only inhibit HBV reactivation post-RT, but also enhance liver TRAIL, VEGFR-2 and GM-CSF expressions to promote RILDs of liver cancer radiotherapy.