The Role And Mechanism Of Stat2-Drp1 Mediated Mitochondrial Homeostatic Remodeling In Modulating The Pro-inflammatory Response Of M1 Macrophages

Background:Sepsis is a systemic inflammatory response syndrome caused by biochemical factors,which is also a major public health challenge worldwide.Infection and bacterial endotoxin（Lipopolysaccharide,LPS）stimulate macrophages M1 polarization,which may cause sepsis and death.M1 macrophages are also associated with the development of various diseases,such as obesity and diabetes.Hence,regulation of macrophages pro-inflammatory differentiation is critical for developing new strategy for treatment of various diseases,such as sepsis.Mitochondria,known as the energy production center in eukaryotes,can reshape their own homeostasis in terms of number,morphology,size and structure,which also participate in regulating cell proliferation,apoptosis and differentiation.In recent years,a growing body of literature has emphasized the key role of mitochondria in modulating innate immune and inflammatory processes.Abnormal mitochondria are also observed in macrophages from patients under pathologic conditions,including infection and sepsis.M1macrophage releases large amounts of reactive oxygen species（ROS）,which is necessary for killing pathogenic bacteria,as well as cause mitochondrial damage and membrane potential（Δψm）collapse.However,flow cytometry results reveal a simultaneous increase in ROS andΔψm in LPS-stimulated macrophage,and this is contrary to common sense.Considering that flow cytometry detecting fluorescent signals on a cell-by-cell basis,we speculate that mitochondrial homeostasis in the resting and M1 macrophage are different,which may explain the above paradoxical phenomenon.However,the role and mechanism of mitochondrial homeostasis in macrophage pro-inflammatory differentiation are still unclear.These works aim to provide novel insights into the mechanism of macrophage pro-inflammatory differentiation,and contribute to treatment of various inflammatory diseases,such as sepsis.Aims:1.To explore the changes of mitochondrial homeostasis during the macrophages pro-inflammatory differentiation.2.To investigate the role and mechanism of mitochondrial homeostasis change in regulating the pro-inflammatory response of M1 macrophage.3.To study the molecular mechanism of mitochondrial homeostasis regulation during the process of macrophage pro-inflammatory differentiation.Methods:1.LPS-stimulated RAW264.7,THP-1,BV2 cell lines,as well as mouse peritoneal and bone marrow primary macrophages,CD11c and pro-inflammatory cytokines were detected to evaluate whether macrophage underwent M1-type polarization.In order to clarify the mitochondrial homeostasis alternation in M1-type macrophage,fluorescence staining,transmission electron microscopy and mt DNA copy were conducted to determine the mitochondrial number,morphology,structure andΔψm.2.Relationship between mitochondrial homeostatic remodeling and pro-inflammatory response in M1 macrophage was explored by correlation analysis,and construction of mitochondria-deficient macrophage RAW-ρ⁰.RNA-seq assay was used to analyze the deferentially expressed genes,and screen the key regulators for mitochondrial homeostatic remodeling in LPS-stimulated M1 macrophage.3.Knockdown or inhibition of Drp1 to evaluate whether Drp1 regulates mitochondrial homeostasis and pro-inflammatory response in LPS-stimulated macrophages.Construction of wild-type Drp1(Flag-Drp1^WT),non-phosphorylatable mutant Drp1(Flag-Drp1^S616A)and constitutively active phosphomimetic mutant Drp1^S616E to evaluate the role of Drp1 S616phosphorylation in LPS-stimulated macrophage.Furthermore,inhibition of Drp1 with Mdivi-1 to analyze the function of Drp1 in peritoneal macrophages from septic mice.4.TAK-242,an inhibitor of TLR4,was used to determine the role of LPS-TLR4 signal in regulating Stat2 phosphorylation in LPS-stimulated macrophages.In order to verify whether Stat2 regulated mitochondrial homeostatic remodeling and pro-inflammatory response by promoting Drp1^S616 phosphorylation,macrophages were knockdown Stat2 and then over-expressed with either Drp1^WT or Drp1^S616E5.We analyzed the mitochondrial function alternation in M1 macrophages,and explored the relationship between mitochondrial homeostatic remodeling and mt ROS production.In order to investigate the role of mt ROS in Stat2-Drp1 regulation of macrophages pro-inflammatory differentiation,Stat2 and Drp1 knockdown cells,as well as a mt ROS scavenger Mito Q were used.Results:1.LPS stimulated macrophages M1 phenotype differentiation,indicated by increasing CD11c expression and pro-inflammatory cytokines,including TNF-α,IL-6 and IL-1β.LPS-stimulated macrophages showed higher FSC and SSC signals,which suggested larger cell size and more organelles.Using various cell models and technical means,we found that that mitochondria in LPS-stimulated macrophage underwent profound changes,including increased mass,fragmented morphology and loose cristae.Moreover,M1 macrophages exhibited reducedΔψm,and flow cytometry results ofΔψm might be influenced by mitochondrial mass.2.We found that LPS robustly boosted the production of pro-inflammatory cytokines,including TNF-αand IL-6.Spearman correlation analysis showed that both TNF-αand IL-6 in supernatants positively correlated with mitochondrial mass in BMDMs.Et Br exposure to develop RAW-ρ⁰ cells that lack mt DNA,TEM and MTG staining results indicated that RAW-ρ⁰ cells have lower mitochondrial mass than wild-type RAW cells in the presence,or absence,of LPS.Importantly,LPS-challenged RAW-ρ⁰ showed significantly lower TNF-αand IL-6 expression and production compared to wild-type RAW.Hence,mitochondrial mass increase was necessary for initiating the pro-inflammatory response of macrophages.In general,excessive mitochondrial fission usually resulted in increasing number and decreasing length,which suggested that abnormal mitochondrial dynamics might be the key mechanism for mitochondrial homeostatic remodeling in M1 macrophages.RNAseq analysis revealed that Stat2 and Drp1 may be the core regulatory genes for mitochondrial homeostatic remodeling in M1 macrophages.3.Drp1 is known as a key regulator of mitochondrial fission,and we found that LPS-stimulated the increase of Drp1 expression and S616 phosphorylation in macrophages.Knockdown or inhibition of Drp1 attenuated LPS-induced mitochondrial homeostatic remodeling and macrophages pro-inflammatory differentiation.Re-expressed wild-type Drp1(Flag-Drp1^WT)and non-phosphorylatable mutant Drp1(Flag-Drp1^S616A)in Drp1knockdown cells,we found that Drp1 S616 inactivating mutation inhibited LPS-induced mitochondrial homeostatic remodeling and pro-inflammatory response.In turn,RAW cells over-expressing a constitutively active phosphomimetic mutant of Drp1(Drp1^S616E)showed higher mitochondrial mass and pro-inflammatory cytokines,even without LPS stimulation.Moreover,in vivo assays proved that Drp1 boosted mitochondrial homeostatic remodeling and pro-inflammatory differentiation in peritoneal macrophages of septic mice.4.Stat2 is a key intracellular signal transduction factor,RNAseq analysis suggested that Stat2 might regulate immune response,mitochondria,biomacromolecule synthesis and protein phosphorylation.Here,we found that LPS-stimulated macrophages showed increased expression of Stat2 and p-Stat2,while TLR4 inhibitor TAK-242 blunted LPS-induced Stat2 phosphorylation.In the early stage of LPS stimulation,macrophages exhibited increased expression of mitochondrial biosynthesis genes,including PGC-1α,Nrf1 and TFAM,while knockdown Stat2 inhibited mitochondrial biosynthesis.Furthermore,knockdown of Stat2 remarkably attenuated LPS-mediated Drp1^S616phosphorylation,mitochondrial mass increase and pro-inflammatory cytokine expression.RAW cells with Stat2 knockdown were overexpressed with either Drp1^WT or Drp1^S616E.The results showed that transfection with Drp1^WT failed to increase mitochondrial fragmentation and inflammatory cytokines,while overexpression of Drp1^WT showed obvious enhanced effect.We therefore conclude that Stat2 promotes the mitochondrial remodeling and pro-inflammatory response of macrophages by regulating Drp1^S616 phosphorylation.5.As a key organelle in eukaryotic cells,the major function of mitochondria is to synthesize ATP and produce ROS.We demonstrated that LPS-stimulated macrophages underwent metabolism reprogramming from mitochondrial oxidative phosphorylation to aerobic glycolysis.In LPS-stimulated macrophages,ATP levels was decreased and mt ROS content increased,and mt ROS and mitochondrial number showed a positive correlationship.In response to LPS,mitochondria-deficient RAW-ρ⁰ cells exhibited deficiency for mt ROS production.Therefore,mitochondrial function M1 macrophages shifted from ATP synthesis to ROS production,which was the main source of intracellular ROS.Knockdown of Stat2and Drp1 attenuated LPS-induced metabolism reprogramming and ROS production.Mito Q,an mt ROS-specific scavenger,inhibited LPS-induced NFκB phosphorylation and nuclear translocation,as well as the transcriptional expression of TNF-αand IL-6.These results suggested that mt ROS is necessary for Stat2-Drp1 in regulating macrophages pro-inflammatory responses.Conclusions:1.Mitochondria in M1 macrophages undergo enhanced fission and homeostatic remodeling,indicating by increased mass,shorter length and reduced cristae structure.Notably,the increase of mitochondrial mass has not been reported before,and may be used as a novel marker of M1 macrophages.2.M1 macrophages undergo metabolism reprogramming,and switch their core metabolism mitochondrial oxidative phosphorylation to aerobic glycolysis.Meanwhile,the mitochondria function shifts from ATP synthesis to ROS production.3.Mitochondrial homeostatic remodeling is necessary for initiating pro-inflammatory response in LPS-treated macrophage.Stat2-Drp1 promotes macrophage pro-inflammatory differentiation by remodeling mitochondrial homeostasis,and ROS may play a critical role during this procession.4.Drp1 and its S616 site phosphorylation play a key role in regulating mitochondrial homeostatic remodeling and macrophages pro-inflammatory response.Inhibition of Drp1alleviates LPS-induced sepsis in mice.5.Stat2 promotes Drp1^S616phosphorylation and up-regulates mitochondrial biosynthesis,which contributes to remodeling of mitochondrial homeostasis.Stat2 is a novel identified regulator for pro-inflammatory response of macrophages.