The Functional Mechanism Of Mitochondrial Dynamics In The Antitumor Immune Response Mediated By Innate Immune Cells

Macrophages,dendritic cells and other myeloid immune cells play crucial roles in inflammatory responses and host defense against infection and tumor.Mitochondrial morphology swift leads to changes in a range of metabolic pathways and metabolites,but their function in myeloid innate immune cells and their mediated anti-tumor immune response remains unclear.In order to determine the trends of mitochondrial morphology during macrophage polarization and the critical regulatory molecules that mediate the process,we used LPS and IL-4 to treat wild-type(WT)bone marrow-derived macrophages respectively.The results of the confocal and electroscope images showed that LPS promotes mitochondrial division,while IL-4 promotes mitochondrial fusion.With real-time quantitative PCR(qPCR),we identified a series of key molecules involved in regulating mitochondrial morphology during macrophages polarization and confirm the change trends of their mRNA levels with stimulation.We found that the key molecule,Fam73b(also known as MIGA2),as one of outer membrane fusion protein of mitochondria,had a fundamental effect on mitochondrial morphology during macrophage polarization.To study the function of Fam73 b in the immune system,we first constructed a B16 melanoma transplant model using Fam73 b germline knockout(KO)mice.The results revealed that Fam73b-deficient mice significantly resist to the growth of melanoma and showed an elevated anti-tumor immune response(e.g.significant increases in the expression of IL-12 and IFN-?).To further clarify the essential role of Fam73 b in different immune cells,we constructed Fam73 b conditional knockout mice by crossing with variously specific Cre-mice,which led to Fam73 b T cells-and myeloid cell-conditional KO mice.The results indicated that Fam73 b myeloid cells-conditional mice showed similar antitumor characteristics with germline KO mice,while the Fam73 b T cellconditional KO mice performed comparable phenotypes with WT mice.To investigate the molecular mechanisms of Fam73 b in myeloid cells in regulating innate immune responses,we performed transcriptome analysis of WT and Fam73b-deficient macrophages.Together with the validation results of qRT-PCR,we confirmed that Fam73 b deficiency led to a significant increase in IL-12 induction.After the mitochondrial division inhibitor(Mdivi-1)treatment,the hyperproduction of IL-12 caused by the absence of Fam73 b was significantly restored.Consistently,specific upregulation of IL-12 was observed in various MEFs deficient with mitochondrial fusion proteins(Mfn1/Mfn2 and Opa1),suggesting the expression of IL-12 is regulated by mitochondrial morphology directly,not a specific function of one protein.To clarify the molecular mechanisms of mitochondrial morphological in regulation of IL-12 expression,we used conventional biochemical techniques together with IRF1 and PARKIN KO mice,and found that mitochondrial division increased the expression of Parkin and recruited it into the outer membrane of mitochondria.PARKIN on mitochondria reduces the level of monoubiquitinated CHIP through proteasome-dependent mechanism,which further inhibits the stability of IRF1,thus specifically regulated the production of IL-12.Our findings first reveal the relationship between mitochondrial dynamics and innate immune cell polarization,and promote the biological understanding of mitochondrial dynamics under stress conditions.This work not only identified the relevant mechanism and important role of mitochondrial dynamics in controlling the activation of immune cells and anti-tumor immune response,but also provided a potential target for cancer immunotherapy.