The Protective Role Of LTα-55 In Radiation-induced Myeloid Injury And Its Potential Mechanism Based On NLRP3 Inflammasome

BackgroundResearchers of the US Genentech company found in 1996 that wild type lymphotoxin alpha（LTα）has a protective effect on mouse bone marrow injury caused by radiation,but the further preclinical animal testing confirmed that LTα can cause significant adverse reactions,such as severe capillary leakage,low blood pressure,chills and fever etc.,which hindered the in-depth study and application of LTα.We next developed the LT alpha derivative（LTα-27）with 27 amino acid residues shorter at N-terminal domain to reduce the toxic side effects of wild type LTα.In preclinical animal testing,LTα-27 combined with chemotherapy play a synergistic effect on killing the epithelial tumor cells,but do protect the bone marrow cells against the cytotoxicity of chemotherapeutic agents.However,70% of the subjects used LTα-27 in clinical I stage appeared serious chill and fever,which limited its further development and application.Further mechanism studies showed that LTα binding with tumor necrosis factor receptor 1（TNFR1）and TNFR2 binding to initiate downstream signaling pathways and play its biological function.The side effects of LTα were related to its combined with TNFR2.Therefore,we developed LTα-55 which has the specific binding of TNFR1 by computer aided design and experimental screening.Further experiments demonstrated that LTα-55 protected bone marrow from injury not only by chemotherapy drugs in rhesus monkeys but also by nuclear radiation in mice.Nuclear energy,as an efficient and clean energy,is widely used in the world due to environmental pollution and energy shortage,However,the security risks caused by nuclear leakage have a huge impact on human safety.The main cause of death of patients after radiation is the dysfunction of hematopoietic cells caused by bone marrow suppression and the decrease of the number of peripheral leukocytes.Therefore,the protection and recovery of bone marrow function is one of the key factors related to the prognosis of patients with radiation injury,but there is still lack of effective protection and treatment methods for bone marrow injury caused by nuclear radiation.Previous studies have shown that radiation mainly caused cell death of bone marrow hematopoietic cells,epidermal cells and epithelial cells those were active in cell division.Recent studies demonstrated that radiation significantly reduced peripheral mature immunecells（macrophages,T cells and B cells）and increased release of proinflammatory cytokine,thus damaged cells were repaired,but the immune function was inhibited.Among Macrophages play key roles in innate immunity and adaptive immunity,which can regulate cell function,growth factor secretion and phagocytosis of cell debris and other ways to promote the repair of tissue damage and reduce inflammation.It is found that macrophages are the earliest cell types in response to radiation damage in the innate immune system.In the study,we found that LTα55 significantly increased survival rate and accelerated the recovery of bone marrow hematopoietic function in mice under radiation in vivo.In vitro LTα-55 increased the secretion of IL-1β in human monocytic THP-1 cells,and the secretion of IL-1β is closely related to the activation of NLRP3 inflammatory bodies.Therefore,we hypothesized that NLRP3 inflammatory bodies may be involved in the protective effect of LTα-55 on the survival of mice.So we used three types of NLRP3 gene knockout mice(Nlrp3^-/-、Asc^-/-、Caspase-1^-/-)and human monocytic cell line THP-1to explore the potential role of NLRP3 inflammasome in the protective effect of bone marrow injury of LTα-55 under nuclear radiation in mice and its possible mechanism.MethodsIn vivo study: We observed the protective effect of LTα-55 on bone marrow injury induced by nuclear radiation in wild-type,Nlpr^3-/-,Asc^-/-and Caspase-1^-/-mice.Mice were injected with LTα-55 at a dose of 250 μg/kg via tail vein before radiation and two days ago,then after a dose of 9.5 Gy irradiation,survival rates were observed,or after a dose of 9 Gy irradiation,hemogram,pathology of bone marrow and major organs were observed.In vitro study: the effects of LTα-55 on cell proliferation,apoptosis,pyroptosis and ROS generation were observed in human monocytic cell line THP-1.Cell counting was used to detect cell proliferation.Cell cycle,cell apoptosis,pyroptosis and ROS were detected by flow cytometry.The activation of NLRP3 inflammasome in THP-1 cells was observed under electron microscope.The expressions of mRNA and protein were detected by realtime-PCR and western-blot,respectively.The changes of pyroptosis and apoptosis related factors in bone marrow,spleen tissues were detected by immunofluorescence assay.Immune-fluorescence assay was used to detect the localization and expression of the related signaling molecules in bone marrow and spleen tissue.ResultsLTα-55 promoted the recovery of nuclear radiation damage in wild-type C57BL/6 mice in vivo.1.LTα-55 increased the survival rate of wild-type C57BL/6 mice after 9.5 Gy nuclear radiation.The mice in control group began to die on the 8th day,and died most in next two days,all died on the 12 th day.The female mice in LTα-55 group died on the 14 th day,the males died on the 18 th day,and the rest of the mice survived to the end of the observation period（28thday）.The weight of male mice of control group was significantly lower than that of LTα-55 group at 10 th day.2.LTα-55 promoted the recovery of red cells,white cells and platelet in irradiated mice.3.After the treatment of LTα-55,hemogram of the wild-type mice under irradiation of 9 Gy began to recover on the 7th day,and returned to normal on the 18 th day.The control mice did not begin to recover on the 18 thday.The pathology of bone marrow showed the same results.4.Spleen pathology showed that LTα-55 promoted the extramedullary hematopoiesis in the spleen of wild-type C57BL/6 mice after radiation.Compared with the control group,the proliferation of LTα-55 group was observed on the seventh day after irradiation,and there was obvious extramedullary hematopoiesis in the spleen at day tenth.Protective effect of LTα-55 on the damage of THP-1 cells induced by nuclear radiation in vitro.1.LTα-55 increased the proliferation of THP-1 cells and augmented the survival of the irradiated THP-1 cells.2.After 6 Gy 60Co-γ irradiation,THP-1 cells were blocked in G2 phase,and 50 g/mL LTα-55 completely reversed the G2 block at 48 hours.3.The rate of apoptosis of the irradiated THP-1 cells was decreased after the treatment of 50μg/mL LTα-55 for 48 hours.4.The level of ROS gradually increased in time-dependent manner after 10 Gy 60Co-γirradiation and 50μg/mL LTα-55 could reduce the level of ROS at 72 h.The effect of LT-55 on NLRP3 inflammatory in THP-1 cells1.LTα-55 increased the mRNA expression of NLRP3,Caspase-1and IL-1 beta,and promoted the secretion of IL-1 beta in THP-1 cells.2.Electron microscopy results showed that LTα-55 rapidly activated NLRP3 inflammatory body in THP-1 cells.NLRP3 inflammatory bodies（NLRP3,ASC,Caspase-1）were involved in the protective effect of LTα-55 on the radiation damage of C57BL/6 mice.1.Compared with wild-type mice,LTα-55 increased survival rate and promoted hemogram recovery slightly in NLRP3 inflammasome related gene knockout mice(Nlrp3^-/-、Asc^-/-、Caspase-1^-/-).2.Bone marrow smear results showed that after the treatment of LTα-55,gene knockout mice under 9 Gy radiation began to recovery at 14 th day,which was later than that of wild type mice.3.Spleen pathology showed that after the treatment of LTα-55,gene knockout mice under radiation began to extramedullary hematopoiesis in the spleen at 14 th day,which was later than that of wild type mice.LTα-55 activated NF-kappa B and NLRP3 inflammatory bodies in vivo1.LTα-55 promotes the nuclear translocation of the subunit of NF-kappa B p52 in wild-type C57BL/6 mice with immuno-fluorescence staining of sternum.Further studies confirmed that LT α-55 up-regulated the expression of IL-1 β in spleen tissue of wild-type mice.2.LTα-55 increased the protein expression of IL-1 beta and IL-18 in spleen in irradiated wide type mice,but the expression of IL-1 beta and IL-18 were very low in NLRP3 gene knockout mice.LTα-55 increased BCL-XL expression in the spleen of mice irradiated by nuclear radiation,and promoted the generation of platelets.LTα-55 increased the protein expression of BCL-XL in the spleen of mice at 7th day,maintain to the 18 th day,but the extend of the increase was obviously decreased in Nlrp3^-/-mice,which was consistent with the number of blood platelet.ConclusionLTα-55 has a significant protective effect on nuclear radiation-induced injury in vivo and in vitro,which is associated with the activation of inflammasome by LTα-55,then the release of ROS was reduced and the apoptosis and pyroptosis were inhibited.LTα-55 also inhibited the cell cycle arrest under irradiation and promote cell proliferation in THP-1cells.The activation of NF-κB（p52）by LTα-55 might be involved in the activation of NLRP3 inflammasome.LTα-55 also up-regulated the expression of Bcl-XL,which was not only related to reduce the apoptosis and pyroptosis,but also might be involved in the regeneration of platelets to promote the re-initiation of immunity.The mechanisms that LTα-55 protects mice from nuclear radiation are complicated.