The Role And Mechanism Of Piezo1 In Remodeling The Involvement Of Macrophages In Inflammation

Part 1.Piezo 1 regulates macrophage glycolysis and development of DSS-induced colitis in mice through the Ca2+/CaMKⅡ/Hif1α axis.Background:Macrophages are classical innate immune cells that are the major component of the body’s first line of defense.Macrophages can carry out immune defense and immune self-stabilization through phagocytosis,antigen presentation and immune regulation,so as to intensively regulate the change process of host inflammatory response.Macrophages are also highly plasticized innate immune cells whose phenotype and function are regulated by a variety of environmental factors,such as temperature,pH and oxygen concentration.In recent years,various types of mechanical stress have been reported to be closely related to the inflammatory response of macrophages.For example,cyclical hydrostatic pressure and physical properties of biomaterials can be sensed by macrophages and promote their polarization towards to pro-inflammatory phenotypes.These environmental factors can fine-tune the immune inflammatory response of macrophages,but their underlying mechanisms have not been thoroughly studied.Metabolic reprogramming is critical to the functional state of macrophages.Activation of proinflammatory macrophages results in a metabolic switch from oxidative phosphorylation(OXPHOS)to aerobic glycolysis,similar to the Warburg effect in tumors.Aerobic glycolysis supports the energy and nutrition demand of activated macrophages by quickly providing metabolic intermediates for molecule synthesis,although it is an inefficient pathway in terms of ATP production.The dysregulation of immune metabolism is closely related to the occurrence of inflammation.Correcting the abnormal metabolic state of immune cells may also be a promising antiinflammatory strategy in the future.However,how the changes of physical signals affect the metabolic state of macrophages remains unclear.The mechanotransduction signal pathway of macrophages is very complex,and how cells convert mechanical signals into biochemical signals needs further study.The important role of Piezo 1,the mechanically sensitive ion channel,in macrophages has been reported several times in recent years.Piezol,a mechanically sensitive,nonselective cationic channel that can be activated by pressure,shear forces,cyclical hydrostatic pressure and the chemical agonist Yoda1,plays the crucial role in vascular development,erythrocyte homeostasis,bone remodeling and cardiac signal transduction.It has also been reported that TLR4 may form a complex with Piezo 1,which promotes the remodeling of the cytoskeleton and thus activates the natural immune response of macrophages.Given the close connection between the metabolic state and the immune response,we suspect that the activation of Piezo 1 channel may help the metabolic reprogramming of macrophages,but this hypothesis has not been investigated.Inflammatory bowel disease(IBD)is a type of acute inflammation initiated and regulated by macrophages with extensive mechanical changes.Increased blood flow in the intestine aggravates capillary and interstitial pressure.Long-term inflammation causes fibrosis in the intestinal wall and increases matrix hardness.These mechanical properties are closely related to the severity of inflammation.Therefore,we constructed Piezo 1 specific deficiency mouse model and induce colitis by DSS to investigate whether Piezol expression may influence the development of colitis by influencing the metabolic state of macrophages.Object:Mechanical stress can intensively regulate the phenotype and function of macrophages,but how the mechanosensitive signals reshape macrophage function remains unclear.The purpose of this study is to investigate the regulatory effect of Piezol,the mechanically sensitive ion channel protein,on the glucose metabolism and immune response of macrophages.Methods:1.Construct the myeloid specific Piezo1 deficiency mice model by hybridize Lyz2cre/+and Piezolflox/flox.2.After the mice were euthanized,the primary bone marrow derived macrophages(BMDMs)were cultured with 100ng/ml M-CSF for 5-7 days,and the cells were scraped,counted and planted on plates for the following experiments.3.Cells were treated with specific chemical reagents(Yodal/KN93/LPS/IL4)or transfected with siRNA,and the time of cell collection was determined according to different time requirements.4.Real-time quantitative PCR(RT-qPCR):Total RNA was extracted and reversetranscribed into cDNA.Roche real-time quantitative PCR instrument was used for RNA quantification.5.Western blot:Extract the total protein,measure the concentration,add the loading buffer and heating and calculate the sample amount.Proteins were separated using SDS-PAGE and transferred to the PVDF membrane.Use the primary antibody and secondary antibody to incubate the membrane.After taking photos,use ImageJ software to measure the protein expression level.6.Cytokine detection:Supernatant of cultured cells was collected.Centrifugate the samples and add the corresponding antibody,incubated for 1-2h.Washed the antibody and add streptavidin-HRP for 30min.After washing,add color developing solution,stop the reaction when the color developing degree is appropriate.7.Seahorse Metabolic Assay:Use Agilent glycolysis rate test kit.The plates were planted in advance and given drug stimulation,and the plates were hydrated overnight.On the day of the experiment,the detection solution was configured,and the cells were washed with the detection solution twice.Rot/AA and 2-DG compounds with appropriate concentration were added into the A/B ports.Use the glycolysis rate base experiment template.Data analysis was performed with a glycolysis rate measurement report generator.8.Glucose Uptake Assays:AAT Bioquest Cell Meter 2-NBDG glucose uptake kit was used,2-NBDG staining solution was incubated for 30min and then washed,and fluorescence intensity of cells was detected by fluorescence microscope or flow cytometry.9.Calcium imaging:Use the Thermo Fluo-4 calcium imaging kit.The staining solution was incubated with 5uM for 15-30min.The staining solution was removed and fluorescence intensity of cells was detected by fluorescence microscope or flow cytometry.10.Immunofluorescence and immunohistochemical staining:TritonX-100 was added directly after fixation in frozen sections.The paraffin sections dewaxing hydration and antigen repair.Add the diluted donkey serum for 30min and incubate the primary antibody overnight.After washing,add secondary antibody and incubate at room temperature for 1h.Visualized staining of tissue was performed with a fluorescence microscope.11.Metabolome assay:Metabolite quantification was performed by BioTree Biotech using liquid chromatography-tandem mass spectrometry(LC-MS/MS).The final concentration(CF,nmol·L-1)is equal to the calculated concentration(CC,nmol·L-1)multiplied by the dilution factor(Dil).12.Single cell sequencing analysis:Seurat R package was used to screen cells with high mitochondrial genes and cells with low gene count.Data were normalized and scaled using the LogNormalize method.Variable genes were detected using FindVariable Features.Cells were clustered using the FindClusters method and visualized using t-distributed stochastic neighbor embedding(t-SNE).Differentially expressed genes(DEGs)were determined using the FindAllMarkers function.GO analysis was performed by enrichGO.12.In vivo model:colitis mouse model was constructed by free reference of 3%DSS for 7 days,body weight of mice was monitored,colon length and histological score were measured,and innate immune cells in colon were isolated for RNA determination.13.Flow cytometry:single-cell suspension was prepared,appropriate concentration was adjusted,CD16/32 blocking antibody was added,and corresponding flow cytometry antibody was added to detect cell surface antigens.14.Data analysis:The experiment was repeated at least three times,and the data were expressed as mean ± standard deviation.Shapiro-Wilk test was used for normality.Comparisons between groups were made using one-way analysis of variance or an unpaired two-tailed student t test.P<0.05 was considered statistically significant.Results:1.Piezo 1 activation on macrophages promotes the expression and secretion of LPSinduced inflammatory cytokines.Piezo 1 is highly expressed in myeloid cells,and the deficiency of Piezo 1 reduces the production of inflammatory cytokines at rest and under cyclical hydrostatic pressure after LPS or LPS/nigericin stimulation.In addition,Yodal promotes LPS or LPS/Nigerian-induced secretion of inflammatory cytokines.LPS or LPS/nigeritin induced transcription levels of IL-6,TNF-α,and IL-1β were also significantly reduced in both resting state and cyclical hydrostatic treatment in Piezo 1deficient macrophages.2.Piezo1 promotes glycolysis reprogramming in the pro-inflammatory condition of macrophages.Single-cell sequencing analysis shows that Piezo 1 deficiency regulates the genes involved in the biological process of carbon metabolism.Seahorse XF glycolysis rate results show that the loss of Piezo 1 significantly affected several indicators of the rate of glycolysis.At the same time,LPS is added to induce the transformation of macrophages into a proinflammatory state and Piezo 1 knockout also significantly disrupted the LPS-induced metabolic reprogramming.The 2-DG treatment eliminates the difference in the expression of inflammatory factors caused by the Piezo 1 deficiency.The metabolome shows that Piezo 1 promotes the accumulation of glycolysis products after cyclic hydrostatic pressure treatment.3.Yoda1 stimulated and induced aerobic glycolysis of macrophages.Yoda 1 treatment increases the glycolysis levels in resting and LPS-stimulated BMDMs.Moreover,the knockout of Piezo 1 reduces the promoting effect of Yoda1 on the glycolysis of BMDMs,suggesting that Yodal-mediated metabolic regulation is dependent on Piezol.4.Mechanically,calcium ion influx mediated by Piezo 1 enhances the activation of calcium-dependent signals,thereby activating CaMKII phosphorylation.CaMKII regulates the protein expression level of Hif1α and its coactivator P300 and the nuclear translocation of Hif1α.Just as the blocking of Hif1α eliminates the difference in glycolysis between the normal and Piezol deficiency cells,the intervention of KN93 lessens the metabolic difference between the two.The Ca2+-CaMKII-Hifla signaling pathway participates in the metabolic reprogramming induced by Piezo1.5.Piezol deficiency in macrophages improves the process of ulcerative colitis in mice and is associated with decreased Hifla expression and glycolysis levels.In addition,exogenous Yodal significantly increased the infiltration of immune cells and the progression of DSS induced colitis.Conclusion:The mechanically-sensitive ion channel protein Piezol regulates aerobic glycolysis of macrophages through the Ca2+/CaMKII/HIF1α axis and promotes the LPS-induced immune response.Part 2.The mechanical activation of adipose tissue macrophage mediated by Piezo1 protects against dietinduced obesity by regulating sympathetic activity.Background:The global obesity epidemic is a public health concern.It causes more than two million deaths each year and is the second leading risk factor after smoking.Obesity significantly increases the incidence of diseases including type 2 diabetes,cardiovascular disease and cancer.The main cause of obesity is that the body consumes more calories than it consumes,which leads to excessive accumulation of fat in the adipose tissue and causes chronic metabolic inflammation throughout the body.In order to balance the energy of the whole body,adipose tissue can be roughly divided into two different types.White adipose tissue(WAT)is composed of monovesicular adipose cells,which store excess energy,while brown adipose tissue(BAT)is mainly composed of polyvesicular adipose tissue,which is abundant in early childhood and gradually decreases after adulthood.It can burn energy and generate heat.It plays an important role in temperature regulation and food induced heat production at low temperature.In addition,the researchers found that brown-like cells,known as beige adipose tissue,appeared scattered in white adipose tissue under the skin of mice when exposed to persistent external stimuli,such as cold stimulation or beta-adrenergic agonists.Beige adipose tissue has thermogenic properties that promote energy expenditure.Increasing the transformation of white fat into brown adipose tissue and improving the function of brown fat provide a new target for the prevention and treatment of metabolic diseases such as obesity.Adaptive thermogenesis,based on the anatomy of brown adipose tissue and beige adipose cells,activates non-shivering thermogenesis in response to constant external stimuli such as low temperature exposure and nutrition.Adaptive thermogenesis is initiated by the release of catecholamine by sympathetic neurons in response to cold stimulation.Catecholamine acts on β3-adrenergic receptors on adipocytes,activating adrenal signaling pathways and promoting adipocyte thermogenesis programs.Due to its role in increasing energy expenditure,improving the body’s adaptive thermogenesis has become an important strategy to combat obesity and related complications such as type 2 diabetes and insulin resistance.Adipose inflammation in obesity activates recruitment of different types of immune cells,such as T cells,B cells,mast cells,and macrophages,in which adipose tissue macrophages(ATMs)play a crucial regulatory role.It has been reported that macrophages in adipose tissue account for about 5%of the vascular matrix in lean body,while up to 20%-50%in obese body.Infiltrating macrophages continue to aggravate systemic inflammation by secreting inflammatory factors,which is the main contributor to obesity inflammation.Macrophages are usually divided into pro-inflammatory cells(M1)and anti-inflammatory cells(M2).With the occurrence of obesity,proinflammatory macrophages are over-activated,and increase the progression of adipose inflammation and systemic insulin resistance by secreting inflammatory factors such as IL-1β,IL-6 and TNF-α.However,reversing the polarization of M1 to M2 can improve inflammation and increase insulin sensitivity by increasing the synthesis of anti-inflammatory cytokine IL-10.Although adipose tissue macrophages in obesity share immune characteristics with macrophages in other typical inflammatory responses,they also exhibit a unique phenotype regulated by environmental factors.Therefore,a simple dichotomy to define adipose tissue macrophages phenotype in terms of M1/M2 is incomplete.In recent years,two new types of macrophages have been gradually recognized,namely metabolically activated macrophages and oxidized macrophages.Metabolically activated macrophages are induced by saturated fatty acids,possess both M1 and M2 characteristics,and buffer the excess lipids in the environment appropriately.Mox is induced by Oxidized phospholipids(OxPLs),and oxidized tissue damage mediates overexpression of Nrf2 with decreased phagocytosis and chemotaxis.These studies demonstrate the dynamic plasticity of macrophages in response to signal changes in tissue niches.In addition to the inflammatory function,the mechanism of the association between macrophages and norepinephrine has been widely reported,but the specific mechanism is still controversial.Some articles have proposed that M2 macrophages in white adipose tissue can produce NE to maintain thermogenesis,but other studies refuted this theory and confirmed that macrophages cannot regulate thermogenesis by producing NE through the construction of conditional knockout mice.Since then,many articles have demonstrated that macrophages can regulate local sympathetic activity without directly secreting NE,such as overexpression of SLC6a2 and MAOA,uptake and degradation of NE,thereby reducing local levels of NE,or secretion of SLIT3 to promote sympathetic activity and improve obesity.These studies have identified an unpredictably complex role of macrophages in the regulation of sympathetic homeostasis,with potential to treat obesity.Objectives:The present study aims to explore whether Piezo1 of macrophages is activated by the changes in the microenvironment in adipose tissue during obesity,and how Piezo1 of the activation affects the energy balance and glucose homeostasis throughout the body.Methods:1.The mice knocked out by myeloid Piezo1 are constructed,and both the experimental group Lyz2crePiezo1flox/flox mice(KO)and the control group Piezo1flox/flox mice(WT)are selected for each experiment.2.A high-fat diet obesity model fed with 60%diet was established for 12 weeks,and body weight was monitored.3.Glucose homeostasis was detected by glucose tolerance test and insulin resistance test.4.ATMs transplantation experiment:WT mice were used as recipient mice.Whole bone marrow cells of WT or KO mice were transplanted respectively and fed low-fat or highfat diet.5.Metabolic cage detection:OXYMAX-CLAMS system was used to automatically record the physiological parameters of experimental mice within 72 hours,including food intake,activity,oxygen consumption VO2(mL/kg/h),energy consumption(heat production rate,kcal/h),respiratory exchange rate,etc.6.Serological experiment:Test kit to detect cholesterol,triglyceride,low density lipoprotein and norepinephrine,etc7.ATMs sorting:The adipose tissue was dissociated by collagen enzyme,the vascular matrix was obtained by stratification,and the F4/80 positive cells were separated by kit for follow-up experiments.8.Real-time quantitative PCR(RT-qPCR):Cell RNA was extracted and reversetranscribed into cDNA.Roche PCR real-time quantitative PCR instrument was used for RNA quantification.9.Western blot:Extract the total protein of cells,measure the concentration,add the sample buffer for heating,calculate the sample amount,and carry out protein electrophoresis transfer,sealing,primary antibody incubation and secondary antibody incubation.After taking photos,use ImageJ software to measure the protein expression level.10.Immunofluorescence and immunohistochemical staining:TritonX-100 holes were drilled directly after fixation in frozen sections,and holes were drilled after fixation,dewaxing hydration and antigen repair in paraffin sections.Diluted donkey serum was sealed for 30min and incubated overnight at 4 degrees of primary antibody.After washing,add secondary antibody and incubate at room temperature for 1h.After the sealing tablets were added,the fluorescence section could select the anti-fluorescence quenching sealing tablets containing DAPI,and the edge was sealed for microscopic observation.11.RNA-seq data analysis:The raw data was filtered,and the clean data was compared to the reference genome and the reference gene set.RSEM was used for gene expression quantification,pheatmap was used for expression clustering heatmap,DEGseq was used for differential gene detection,and differences with Q value<0.05 were selected for enrichment analysis.The first 1000 differentially ranked genes were selected,and Phyper was used for enrichment analysis based on hypergeometric test.12.Cell culture:Mouse whole bone marrow cells were induced by 100ng/ml M-CSF for 5-7 days,and the scraped cells were counted as BMDMs.3T3-L1 was cultured with induction medium for 72-96h and then replaced with lipid maintenance medium for 35 days until mature adipocytes were differentiated.PC 12 was induced into mature sympathetic cells using β-NGF.13.Data analysis:The experiment was repeated at least three times,and the data were expressed as mean ±standard deviation.Shapiro-Wilk test was used for normality.Comparisons between groups were made using one-way analysis of variance or an unpaired two-tailed student t test.P<0.05 was considered statistically significant.Results1.The loss of Piezo 1 on macrophages exacerbates the development of obesity in mice.The construction of a transgenic mouse model in which Piezol is specifically knocked out of the myeloid cells found that,unlike other models of inflammation,the absence of Piezol did not relieve the inflammation.Instead,it exacerbated the development of obesity,glucose intolerance and insulin resistance in the mice.Immunohistochemistry showed increased hepatic lipid deposition and compensatory islet hypertrophy in KO mice.2.Myelopiezol regulates body weight gain and glucose homeostasis in obese mice by a nonclassical polarized pathway.Using flow cytometry to analyze the polarization of infiltrating macrophages in adipose tissue,we found that KO mice were more obese,but their adipose tissue macrophages showed no significant M1-polarization trend.The F4/80 positive adipose tissue macrophages were sorted by magnetic beads and RNA sequencing was performed.The discovery that genes down-regulated by Piezo1 loss are rich in extracellular matrix remodeling pathways,such as ECM receptor interactions and local adhesive plaques,may hint at the reason for the activation of Piezo1 in ATMs.Since KEGG did not show any inflammatory pathways,we performed GSEA enrichment analysis on related genes,and the results showed that the activation degree of inflammatory pathways in adipose tissue macrophages in WT was higher than that in adipose tissue macrophages in KO.Meanwhile,M1-related genes were highly expressed in adipose tissue macrophages of WT,such as IL-1β and IL-6,while M2related genes were highly expressed in adipose tissue macrophages of KO,such as IL10.Thus,although WT mice are less obese,the expression of the inflammatory gene is higher in adipose tissue macrophages in WT mice,leading us to speculate that Piezo 1 in ATMs regulates body weight and glucose tolerance through an unconventional polarization pathway.3.Myelopiezol expression enhances energy expenditure and adaptive thermogenesis in mice exposed to the high-fat diet.Metabolic cage tests showed that O2 consumption and heat production were significantly reduced in KO mice fed a high fat diet.WB,PCR and RNA-seq all suggested that fat metabolism was inhibited in KO mice.By monitoring body temperature under cold stimulation,adaptive thermogenesis was significantly reduced in KO mice regardless of high fat diet or low fat diet.We therefore speculate that the loss of Piezo1 in macrophages regulates obesity by affecting sympathetic nerve activity.4.Myeloid Piezo1 enhances sympathetic TH expression.Through in vitro co-culture experiments,it was found that the BMDMs knocked out by Piezo1 do not affect the function of fat or the TH expression of sympathetic nerves.However,the Yodalactivated BMDMs significantly increase the TH signaling pathway of sympathetic nerves,but do not affect the lipid metabolism of adipocytes.Therefore,the BMDMs function through their interaction with the sympathetic nerves.5.The activation of myeloPIEZO1 improves sympathetic activity by increasing the expression of Slit3.After Piezo1 knockout,neither of the genes involved in NE degradation in adipose tissue macrophages(SLC6a2 and MAOA)were significantly changed,but axon-guided pathways were present in KEGG analysis.In cluster heat maps,we found decreased Slit3 expression in KO mice,which has been reported to bind with ROBO1 on the sympathetic nerve to increase sympathetic activity.Thus,the activation of Piezol in macrophages increases the expression of Slit3,which activates the sympathetic nerve and promotes fat thermogenesis.Conclusion:The mechanical activation of adipose tissue macrophages mediated by Piezo1 regulates sympathetic nerve activity through a nonclassical inflammatory pathway,promotes adaptive thermogenesis and regulates systemic glucose homeostasis and insulin sensitivity.