The Regulation Of Liver ILC1 By Transcription Factor RORa

The liver is not only a metabolic organ,but also acts as a critical immune organ.Owing to the large amounts of innate immune cells and molecules,the liver is in an innate immune predominant status.Innate lymphoid cells(ILCs)are classified into three groups and contribute to control of infections,inflammatory responses and maintaining mucosal homeostasis.Group 1 ILCs consist of two different subsets,conventional natural killer(cNK)cells and ILC1s.Group 1 ILCs in the mouse liver are defined as CD45+CD3-NK1.1+NKp46+.Recently,our studies identified the marker 'CD49a' as the characteristic phenotype of ILC1s.Thus,group 1 ILCs are devided into two subsets,CD49b+CD49a-cNK cells and CD49a+CD49b-ILC1s.As the tissue residency of liver ILC1s,these cells are also called Liver-resident NK(LrNK)cells.Up to now,the development,homeostasis maintenance and function of cNK cells are well known.However,the knowledge of liver ILC1s is poor.In this study,we investigated the transcription factors of liver ILC1s,and had found the transcription factor ROR? was required for the maintenance and function of liver ILC1s.The results are as follow:1.Liver ILCls highly express ROR?.To compare the transcriptional difference between cNK cells and ILC1s,we performed RNA-seq analysis of these two subsets.Liver ILC1s expressed Rora at a much higher level compared to cNK cells.We also tested the Rora expression on liver ILC1s,cNK cells,CD4+T cells,CD8+T cells and CD19+B cells by qPCR.Western blot analysis further confirmed highly expression of RORa protein.These data showed a relatively high level of ROR? expression in liver ILC1s.2.ROR? regulates the amount of liver ILC1s.We overexpressed Rora in hematopoietic progenitors by retrovirus infection before transferring these cells to lethally irradiation mice.The results showed that liver ILC1s increased in Rora-overexpression bone marrow chimeric mice.While Rora knocked down in hematopoietic progenitors by lentivirus infection led to reduction of liver ILC1s.These data indicated that ROR? had positive effects on liver ILC1 cellularity.Depletion of ROR? in hematopoietc cells caused reduction of liver ILC1s.We generated Rorafl/fl mice and crossed them with Vavcre mice to get mice which RORa was deleted in hematopoietic cells.Liver ILC1s decreased significantly in these mice.However,the progenitors related to ILC development in the bone marrow was not affected in the VavcreRorafl/fl mice.These results suggested that ROR? deficiency caused a defect in liver ILC1 cellularity.Depletion of ROR? in NKp46+ cells also caused decreased liver ILC1s.We crossed Rorafl/fl mice with Vavcre mice to get mice which ROR? was deleted in NKp46+cells.We examined the amount of liver ILC1s in Ncr1creRorafl/fl mice and found a similar reduction with that in VavcreRorafl/fl mice.The data indicated that ROR? played a role in liver ILC1 maturation state.In addition,the frequency and absolute number of liver ILCls increased via RORa agonist SR1078 stimulation.These data demonstrated that ROR? was required for liver ILC1 maintenance.3.RORa is important for liver ILC1 survival.Liver ILC1s were prone to apoptosis in Ncr1creRorafl/fl mice than Rorafl/fl control mice.In addition,SR1078 stimulation resulted in less apoptotic of liver ILC1s.However,the proliferation of these cells were not influenced.These findings indicated that ROR?played an important role in the liver ILC1 survival.4.ROR? promotes effector molecule expression by liver ILCls.Liver ILC1s in the Ncr1creRorafl/fl mice showed lower expression of activation receptors CD226 and NKG2D,as well as cytokine receptor IL-18R? compared to the Rorafl/fl control mice.While the expression of activation receptor CD226 and cytokine receptor IL-18R? was higher on liver ILC1s of WT mice after SR1078 stimulation.In addition,the expression of effector molecules IFN-?,TNF-? and Perforin on liver ILC1s of WT mice was higher after SR1078 stimulation.Therefore,we found that RORa promoted liver ILC1 function.5.ROR? is required for the tumoricidal activity of liver ILC1s.We generated a CRC liver metastasis model and found that tumor grew faster in Ncr1creRorafl/fl mice than Rorafl/fl control mice.We also found decreased liver ILC1s and a reduction in cytokine expression of these cells in Ncr1creRorafl/fl mice.In addition,liver ILC1s expressed lower activation receptor CD226 and cytokine receptor IL-18R?and higher suppression receptors Tigit and PD-1 in Ncr1creRorafl/fl mice than Rorafl/fl control mice.The data showed the impaired tumoricidal activity of liver ILC1s in the absence of ROR?.These results suggested that ROR? promoted anti-tumor immunity of liver ILC1s.6.ROR? agonist therapy restrains tumor progression.We used ROR? agonist SR1078 to treat CRC liver metastasis in mice.The tumor burden was relieved and the overall survival was prolonged significantly after SR1078 treatment.Meanwhile,the amount of liver ILCls increased,as well as the enhanced effector functions,including the higher expression of activating receptors CD226 and NKG2D and the lower expression of suppressing receptors Tigit and PD-1.SR1078 treatment also induced also expressed higher expression of effector molecules IFN-?,TNF-? and Granzyme B of Liver ILC1s.These data suggested that ROR? agonist was a promising therapeutic approach to cure cancer patientsin the future.Conclusion:In this study,we firstly reported that ROR? was crucial for liver ILC1 maintenance.ROR? deficiency led to increased apoptosis and impaired function of liver ILC1s.We also portray the importance of ROR? in enhancing tumoricidal activity of liver ILC1s for the first time.More importantly,we found that ROR? activation shed new light on HCC treatment by igniting ILC1 functions,as well as suppressing tumor cells directly.