Role Of IL-33 On The Polarization And Metabolism Of THP1

Macrophages are an important part of innate immunity,which is the first line of defense of the human immune system against infection and pathogen invasion.Macrophages originate from monocytes and are stimulated by the surrounding environment to differentiate into classically activated(M1)macrophages and substitutively activated(M2)macrophages.M1-type macrophages are characterized by the secretion of pro-inflammatory cytokines,including TNF-α and IL-1β,which promote the type Ⅰ T cell response of antigen-activated T cells and support the body’s intracellular anti-infection and anti-tumor immune responses.M1 macrophages enhance antibacterial activity by up-regulating superoxides(ROS),generating active nitrogen intermediates(NO)and increasing antimicrobial peptides.M2-type macrophages produce anti-inflammatory cytokines,such as TGF-β,IL-10,and induce type Ⅱ immune response,which mainly plays a role in promoting tissue repair,fibrosis and tumor.IL-33,a member of the IL-1 family,is a pleiotropic cytokine involved in all stages of the macrophage-related immune response,from initiation to resolution.It plays an immunomodulatory role in autoimmune diseases,infectious diseases,cancer and other diseases by regulating the phenotype polarization of macrophages and the release of different types of cytokines to promote or prevent the progression of diseases.Studies have shown that IL-33 up-regulates the ability of M1 macrophages to secrete pro-inflammatory factors and some chemokines after LPS stimulation.However,other studies have shown that IL-33 leads to the release of anti-inflammatory cytokines and promotes the activation of M2 macrophages.Although IL-4 and IL-13 have direct effects on the induction of M2 macrophages,IL-33 can also induce M2 macrophages through the production of Th2 cytokines.Many studies have shown that IL-33 regulates the polarization of macrophages through the regulation of cell metabolism.IL-33 localized in the nucleus has transcriptional inhibition properties such as inhibition of the downstream genes of NF-κB via directly binding to NF-κB.Full length IL-33(flIL-33)stored in nuclear of cells in physiological conditions,and released upon cell damage or cell death.Once released into extracellular space,flIL-33 could be cleaved into mature form of IL-33(cIL-33)which is much more bioactive.Cytokine IL-33 could bind to its receptor ST2 and activate the downstream pathways.Different domains of IL-33 have different or even opposite functions,which may be the main reason for the multiple roles of IL-33 during immune responses.Thus,this experiment aims to investigate the effect of IL-33 on the polarization of macrophages,and what metabolic pathways affect the polarization of macrophages during the polarization process,and analyze the possible pathways causing this mechanism.Specific research contents are as follows:1.Establishment of different fragments of IL-33 transduced THP1 stable transplantersLentivirus vectors encoding full-length(fl)IL-33,nuclear(n)IL-33 and C-terminal(C)IL-33 were transfected into THP1 cell lines,respectively.After purinomycin screening,monoclonal proliferation was performed to obtain THP1 stable transmutation strains with different vectors.DNA electrophoresis,qPCR,Western blot and immunofluorescence were used to detect the transfection effect.The results showed that each IL-33 lentiviral vector was successfully transferred to THP1,and ctrl,THP1/flIL-33,THP1/nIL-33 and THP1/cIL-33 stable cell lines were successfully constructed.2.The effect of different fragments of IL-33 on THP1 cell polarization was detectedAfter M0 stimulation with PMA,ctrl,THP1/flIL-33,THP1/nIL-33 and THP1/cIL-33 were stimulated with LPS+IFN-γ or IL-4,respectively.PBS was used as the control group.The differentiation of monocytes into M1-type or M2-type macrophages was verified by qPCR and Western blot,and the effects of different fragments of IL-33 on the polarization of THP1 cells were analyzed.The results showed that all IL-33 fragments inhibited the polarization of monocyte THP1 to M1 macrophages and promoted the polarization of THP1 to M2 macrophages.The role of cIL-33 is more obvious.3.The effect of different fragments of IL-33 on THP1 cell metabolism was detected3.1 Effects of different fragments of IL-33 on metabolism of Ml-type macrophagesIn order to study the effect of IL-33 on the metabolism of M1-type macrophages,ctrl,THP1/flIL-33,THP1/nIL-33 and THP1/cIL-33 cells were induced by PMA to be M0,PBS was used as the control group,and LPS+IFN-γ were used to induce the differentiation of M0 into M1 as the experimental group.Differences in energy metabolism(ATP,lactic acid,pyruvate production,glucose uptake,OCR and ECAR)were measured between the control group and the experimental group.The results showed that:Glucose uptake,pyruvate production and ATP production decreased significantly,while lactic acid production showed no significant difference,OCR increased,ECAR decreased,OCR/ECAR increased,and the regulation effect of cIL-33 was more obvious.The results showed that IL-33 promoted the energy metabolism of THP1 cells dependent on oxidative phosphorylation(OXPHOS)under the stimulation of LPS+IFN-γ.3.2 Effects of different fragments of IL-33 on metabolism of M2-type macrophagesIn order to study the effect of IL-33 on the metabolism of M2-type macrophages,Ctrl,THP1/flIL-33,THP1/nIL-33 and THP1/cIL-33 cells were induced by PMA to be M0,PBS was used as the control group,and IL-4 was used to induce M0 to be differentiated into M2 as the experimental group.Differences in energy metabolism(ATP,lactic acid,pyruvate production,glucose uptake,OCR and ECAR)were measured between the control group and the experimental group.The results showed that:Glucose uptake,ATP production and lactic acid production decreased significantly,pyruvate production was basically no difference,OCR increased,ECAR decreased,OCR/ECAR increased,and the regulatory effect of cIL-33 was more obvious.These results indicated that IL-33 promoted the energy metabolism of THP1 cells by oxidative phosphorylation(OXPHOS)under the stimulation of IL-4.3.3 Possible mechanism of the effect of different fragments of IL-33 on macrophage metabolismBy measuring the mitochondrial membrane dynamics of PBS,LPS+IFN-γ,IL-4 groups,the changes of mitochondrial membrane dynamics were observed.The results showed that the mitochondrial membrane dynamics of PBS and IL-4 groups increased,while that of LPS+IFN-γ group decreased.Western blot detected the expression of cell related autophagy proteins.The results showed that the overexpression of IL-33 fragments led to the increase of mitochondrial autophagy protein expression when THP1 was polarized towards M1,and the decrease of mitochondrial autophagy protein expression when ThP1 was polarized towards M2.Immunofluorescence was uesd to observe the changes of autophagy protein LC3 and the number of mitochondria in the PBS and IL-4 groups.The results showed that the number of mitochondria in the PBS and IL-4 groups increased and the expression of autophagy protein decreased,while the number of mitochondria in the LPS+IFN-γ group decreased and the expression of autophagy protein increased.The changes in the number of autolysosomes and mitochondria were analyzed by biological electron microscopy.The results showed that:Mitochondria of cIL-33 increased and autophagosome decreased in the PBS group;mitochondria of cIL-33 decreased and autophagosome increased in the LPS+IFN-γ stimulated groups;mitochondria of cIL-33 increased and autophagosome decreased in the IL-4 stimulated group.These results indicated that IL-33 promoted oxidative phosphorylation by inhibiting mitochondrial autophagy and increasing the number of mitochondria in THP1 cells.4.Possible pathways of different fragments of IL-33 affecting THP1 cell polarization and metabolismTranscriptome sequencing analysis of ctrl,flIL-33,nIL-33 and cIL-33 showed that the ECM-receptor interaction pathway of THP1/cIL-33 and THP1/flIL-33 was significantly enriched.These results indicated that IL-33 acted on ECM receptor in the form of cytokine and activated MAPK pathway.While flIL-33 and cIL-33 mainly activated extracellular receptor ST2 and downstream MyD88,MAPK-ERK and PI3K-AKT pathways,and then activated mTOR and NF-κB;nIL-33 plays an immunomodulatory role mainly through the calcium signaling pathway related gene regulatory network.In summary,this work is to investigate how IL-33 affects macrophages polarization and metabolism.The results indicated that IL-33 decreased the M1-like polarization while increased the M2-like polarization of THP1 cells.IL-33 also increased the OXPHOS of THP1 cells in both polarization conditions.IL-33 play these functions via binding of its receptor as a cytokine and affects cellular Calcium signaling pathway as a nuclear transcription factor.This study provides further understanding of underling mechanisms of macrophage polarization and its immunoregulation roles.