Investigation Of The Mechanism Of Action Of Interfering With Mitochondrial Dynamics To Inhibit M2 Polarization Of BMDMS To Promote Inflammatory Response

Background:Chronic inflammation is a risk factor for metabolic diseases such as diabetes and its complications,gout,and hypertension,and the persistent activation of this inflammation is closely related to the dysregulation of anti-inflammatory responses.Macrophages are deeply involved in the regulation of chronic inflammation due to their extensive plasticity.The different polarization states of macrophages are the main markers of their plasticity.Macrophages can be polarized into the M1 type,which is involved in maintaining the pro-inflammatory state,and the M2 type,which exerts anti-inflammatory effects.Therefore,regulating the polarization status of macrophages is an effective therapeutic approach to ameliorate the inflammatory milieu.This remodeling of macrophage polarization is dependent on intracellular metabolic reprogramming:whereas M1-type macrophages rely predominantly on aerobic glycolysis,M2-type macrophages utilize oxidative phosphorylation pathways.This suggests that mitochondria,the hub of cellular energy metabolism,are involved in macrophage activation and plasticity by influencing metabolic reprogramming.Mitochondria are susceptible to oxidative damage due to their frequent exposure to high levels of reactive oxygen species（ROS）.For this reason,mitochondria have evolved several quality control systems to ensure that the necessary number of functional mitochondria are present to meet cellular needs.Mitochondrial quality control systems include the mitochondrial unfolded protein response（UPRmt）,mitochondrial dynamics,and mitochondrial autophagy.Mitochondria exhibit highly variable shapes,sizes,and positions through division,fusion,and to meet the dynamic needs of the cell,collectively referred to as mitochondrial dynamics.In the presence of abnormal protein accumulation,mitochondria can help maintain mitochondrial homeostasis by degrading misaccumulated proteins through UPRmt-mediated signaling cascades of nuclear transcriptional chaperone proteins and upregulation of proteases.Studies have shown that dysregulation of mitochondrial quality control is closely associated with inflammatory diseases.High temperature requirement factor A2（Htr A2/Omi）is a nuclear encoded mitochondrial serine protease located in the mitochondrial membrane space.Previous studies have focused on its potential regulatory role in neurodegenerative diseases,sarcopenia and tumors.Recent studies have shown that Htr A2/Omi proteins are also important regulators of mitochondrial molecular quality control.Htr A2/Omi proteins can participate in the degradation of misaccumulated proteins in the UPRmt to maintain normal mitochondrial function through the enzyme activity that they possess,as well as participate in the interactions of mitochondrial kinetic-related proteins to maintain mitochondrial morphology,etc.,suggesting that Htr A2/Omi proteins may play an important role in mitochondrial quality control,suggesting that Htr A2/Omi proteins may play an important role in mitochondrial quality control.Therefore,the present study used the Htr A2/Omi protein as an entry point to investigate the role of mitochondrial quality control in the regulation of inflammation associated with macrophage polarization.In this study,we used bone marrow-derived macrophages（BMDMs）from Htr A2/Omi mutant mice to first investigate the role of Htr A2/Omi protein in mitochondrial quality control of BMDMs in mice,and then to observe the effect of mitochondrial quality control on the shift of macrophage polarization metabolic mode,and finally to observe the effect on mitochondrial function to further elucidate the potential role of mitochondrial quality control in chronic inflammatory diseases such as diabetes mellitus.Method:1.Wild-type WT,Htr A2/Omi mutant mice Htr A2^{mnd2（+/-）},and Htr A2^{mnd2（-/-）}were used as experimental subjects to observe the changes of physiological indices in mice,while Western blot method was used to detect the expression of Htr A2/Omi proteins in BMDMs of mice of each genotype.2.The expression levels of mitochondrial dynamin-related protein-1（DRP1）,mitofusin-1（MFN1）,optic atrophy-1（OPA1）,and metalloproteinase OMA1,which are involved in mitochondrial fusion and division,were detected by Western blot in BMDMs from mice of all genotypes in the presence of lipopolysaccharide（LPS）and interleukin-4（IL-4）,respectively.And changes in the expression levels of the UPRmt-associated mitochondrial protein molecular chaperones heat shock protein 60（HSP60）and heat shock protein 90（HSP60）to assess the effect of Htr A2/Omi proteins on mitochondrial quality control of BMDMs in mice.3.The q PCR method was used to detect changes in the transcript levels of the M2polarization marker genes arginase-1（Arg1）,mannose receptor C-type1（Mrc1）,and chitinase-like proteins（YM1）,and the M1 polarization marker genes interleukin-1α（IL-1α）,interleukin-1β（IL-1β）,and interferon-γ（IFN-γ）in the BMDMs of mice of all genotypes in the presence of IL-4 transcript levels.We also determined the effects of IL-4 on ATP production,glucose uptake,and lactate production in BMDMs of each genotype,as well as changes in extracellular oxygen consumption rate（OCR）and extracellular acidification rate（ECAR）to explore the polarization tendency and altered metabolic patterns of BMDMs of each genotype.4.Changes in the number of mitochondria in BMDMs from mice of different genotypes under the action of IL-4 using immunofluorescence and mt DNA copy number assays.5.Changes in mitochondrial ROS levels,levels of 8-hydroxydeoxyguanosine（8-OHDG）,a marker of oxidative stress damage,and mitochondrial membrane potential were examined in BMDMs from mice of all genotypes under the influence of IL-4 using flow cytometry and immunofluorescence to assess mitochondrial function.Result:1.There was no significant difference in the physiological performance of Htr A2^{mnd2（+/-）}compared to WT,whereas Htr A2^{mnd2（-/-）}mice showed growth arrest at approximately two weeks of age,progressive weight loss over time,and finally death at approximately 30 days,accompanied by tremors and dyskinesia during this period.Htr A2/Omi protein expression was slightly elevated in BMDMs from Htr A2^{mnd2（+/-）}mice,and Htr A2/Omi protein levels were significantly reduced in BMDMs from Htr A2^{mnd2（-/-）}mice compared to WT.2.（1）Compared with WT,BMDMs from Htr A2^{mnd2（+/-）}mice showed decreased expression of DRP1 and OMA1,increased expression of MFN1,and no significant difference in OPA1 expression;BMDMs from Htr A2^{mnd2（-/-）}mice showed no significant difference in DRP1 expression and increased expression of MFN1 and OMA1,and at the same time,expression levels of hydrolytically truncated OPA1 protein were correspondingly increased.After LPS stimulation,compared with WT,BMDMs from Htr A2^{mnd2（+/-）}mice showed significantly lower expression of DRP1 and OMA1,higher expression of MFN1,and significantly higher expression level of OPA1;BMDMs from Htr A2^{mnd2（-/-）}mice showed lower expression of DRP1 and OPA1,higher expression of MFN1,and significantly higher expression of OMA1.After IL-4 stimulation,compared with WT,BMDMs from Htr A2^{mnd2（+/-）}mice showed significantly lower expression of DRP1,significantly higher expression of MFN1,lower expression of OMA1,and no significant difference in OPA1 protein expression;BMDMs from Htr A2^{mnd2（-/-）}mice showed lower expression of DRP1 and MFN1,significantly higher expression of OPA1,and OPA1 expression was significantly reduced.（2）HSP60 and HSP90 protein expression was slightly increased in BMDMs from Htr A2^{mnd2（+/-）}and Htr A2^{mnd2（-/-）}mice compared to WT.After LPS stimulation,there was no significant difference in HSP60 and HSP90 expression in BMDMs from Htr A2^{mnd2（+/-）}mice and a significant decrease in HSP60 and HSP90 expression in BMDMs from Htr A2^{mnd2（-/-）}mice compared with WT.After IL-4 stimulation,both HSP60 and HSP90 expression were decreased in BMDMs from Htr A2^{mnd2（+/-）}mice and significantly decreased in BMDMs from Htr A2^{mnd2（-/-）}mice compared with WT.3.（1）Compared with WT,the M2 polarization marker genes Arg1 and Mrc1transcript levels were decreased and YM1 transcript levels were increased in BMDMs from Htr A2^{mnd2（+/-）}mice;Arg1 and Mrc1 transcript levels were significantly decreased and YM1 transcript levels were increased in BMDMs from Htr A2^{mnd2（-/-）}mice.After IL-4 stimulation,Arg1,Mrc1,and YM1 transcript levels were increased in BMDMs from Htr A2^{mnd2（+/-）}mice,and Arg1,Mrc1,and YM1 transcript levels were significantly reduced in BMDMs from Htr A2^{mnd2（-/-）}mice.M1 polarization marker genes in BMDMs from Htr A2^{mnd2（+/-）}mice IL-1α,IL-1β,and IFN-γwere all significantly reduced in transcript levels,whereas BMDMs from Htr A2^{mnd2（-/-）}mice had increased transcript levels of IL-1αand IL-1βand decreased transcript levels of IFN-γ.（2）ATP production was reduced and glucose uptake and lactate production were significantly increased in BMDMs from Htr A2^{mnd2（+/-）}mice compared with WT;ATP production was reduced and glucose uptake and lactate production were increased in BMDMs from Htr A2^{mnd2（-/-）}mice.After IL-4 stimulation,ATP production and glucose uptake as well as lactate production were increased in BMDMs from Htr A2^{mnd2（+/-）}mice,and ATP production and glucose uptake were decreased and lactate production was increased in BMDMs from Htr A2^{mnd2（-/-）}mice.（3）OCR and ECAR were elevated in Htr A2^{mnd2（+/-）}and Htr A2^{mnd2（-/-）}mice compared to WT and remained elevated in Htr A2^{mnd2（+/-）}and Htr A2^{mnd2（-/-）}mice after stimulation with IL-4.4.Compared with WT,Htr A2^{mnd2（+/-）}and Htr A2^{mnd2（-/-）}mice had a higher number of mitochondria in BMDMs.After IL-4 stimulation,the number of mitochondria was further increased in BMDMs from Htr A2^{mnd2（+/-）}mice and decreased in BMDMs from Htr A2^{mnd2（-/-）}mice.5.（1）Mitochondrial ROS levels were elevated in BMDMs from Htr A2^{mnd2（+/-）}mice compared with WT and significantly elevated in BMDMs from Htr A2^{mnd2（-/-）}mice.After IL-4 stimulation,there was no significant difference in mitochondrial ROS levels in BMDMs from Htr A2^{mnd2（+/-）}mice,and mitochondrial ROS levels were elevated in BMDMs from Htr A2^{mnd2（-/-）}mice compared with WT.（2）8-OHDG levels were elevated in BMDMs from Htr A2^{mnd2（+/-）}mice compared with WT,and 8-OHDG levels were significantly elevated in BMDMs from Htr A2^{mnd2（-/-）}mice.After IL-4 stimulation,8-OHDG levels were elevated in BMDMs from Htr A2^{mnd2（+/-）}mice compared with WT,and 8-OHDG levels were significantly elevated in BMDMs from Htr A2^{mnd2（-/-）}mice.8-OHDG levels were significantly elevated in BMDM.（3）Membrane potentials of BMDMs from Htr A2^{mnd2（+/-）}and Htr A2^{mnd2（-/-）}mice were elevated compared to WT,and after IL-4 stimulation,membrane potentials of BMDMs from Htr A2^{mnd2（+/-）}mice were still elevated and membrane potentials of BMDMs from Htr A2^{mnd2（-/-）}mice were significantly reduced.Conclusion:Dysregulation of mitochondrial quality control due to Htr A2/Omi protease deficiency inhibits IL-4-induced macrophage M2 polarization shifts with oxidative phosphorylation as the primary metabolic pathway and collectively leads to a pro-and anti-inflammatory imbalance through increased levels of oxidative stress,which in turn leads to sustained activation of chronic inflammation.Regulation of inflammatory response homeostasis by restoring impaired mitochondrial quality control may be a potential direction to attenuate chronic inflammatory activation.