BCL3 Regulates Macrophage Glycolysis And Polarization By Targeting The NF-κB Pathway

Background Macrophages are highly plastic cells capable of rapidly changing their function through a process defined as polarization.Macrophages can be divided into two major phenotypes: classically activated M1 and alternatively activated M2.M1/M2 macrophage polarization plays an important role in regulating the microenvironment balance within tissues.In addition,the polarization of macrophages also involves the reprogramming of metabolism,such as glycolipid metabolism.Recently,it has been reported that the b-cell chronic lymphoblastic leukemia/lymphoma factor 3（b-cell CLL/lymphoma3,bcl3）is known to regulate inflammatory factor expression in macrophages.And our previous study also found that bcl3 mRNA expression was higher in the adipose tissue of obese mice raised in HFD than in normal mice.It suggests that there should be a link between our bcl3 gene expression and the body’s lipid metabolism,however,it is unclear whether BCL3 is involved in the regulation of macrophage polarization and metabolism.Objective In this study,we should not only explore the role of BCL3 in regulating macrophage polarization and its potential mechanisms,but also investigate how BCL3 regulates macrophage polarization through internal macrophage metabolic reprogramming.Methods 1.Establishment of the mouse model: bcl3-ko mice（presented by Professor Liang Yinming）constructed by CRISPR / Cas9 technology and control WT mice were kept on a normal diet.2.The effect of bcl3 knockdown on macrophage polarization was examined:（1）Extraction of mouse bone marrow macrophages: Two hind limbs of mice were taken,the bone marrow was flushed out,and then induced for seven days to maturity using dedicated medium.（2）Induction of M1 and M2-type macrophages: BMDM 24 h induction by using LPS,IFN-,and IL-4,respectively.（3）Detection of related indicators: The surface markers of M1 and M2 type macrophages and the levels of secreted inflammatory factors were detected by qRTPCR.3.The effect of bcl3 knockdown on glucose in macrophage metabolism was examined:（1）The RNA expression levels of the key enzymes playing important roles in macrophage glucose metabolism were measured by qRT-PCR.（2）Protein expression levels of key enzymes playing an important role in macrophage glucose metabolism were measured by Western blot.（3）Macrophage external acidification rate（ECAR）was determined by seahorse experiment.（4）Bcl3 was overexpressed in the RAW264.7 macrophage line,followed by detection of the protein expression levels of key enzymes playing an important role in macrophage glucose metabolism by Western blot.4.To determine whether the effect on macrophage glucose metabolism after bcl3 knockdown was related to the activation of the NF-κB pathway:（1）The expression levels of the P65 protein associated with the activation of the NF-κB pathway were determined by Western blot and by the immunofluorescence assay（Confocal）.（2）The regulatory effect of BCL3 on the NF-κB pathway was determined by the double-luciferase reporter assay.（3）Macrophages were treated with TAK-242,an inhibitor of the NF-κB pathway,and the protein expression levels of key enzymes that play an important role in the glucose metabolism of macrophages were then measured by Western blot.5.To explore the relationship between BCL3 influence on macrophage polarization and glucose metabolism: After treatment of macrophages were treated with the glycolysis inhibitor 2-DG,the effect of BCL3 on macrophage polarization was detected by qRT-PCR and flow experiments.6.To investigate the effect of bcl3 knockdown on macrophage function:（1）The effect of bcl3 knockdown on macrophage migration was examined by cell scratches as well as by the transwell assay.（2）The effect of bcl3 knockdown on value macrophages was determined by CCK8 assay.（3）The effect of bcl3 on the phagocytic function of macrophages was examined by flow experiments.Results 1.The qRT-PCR results showed that mRNA expression levels of M1 macrophage markers（IL-6,IL-1,MCP-1,CPR132,iNOS）in bcl3 knockout macrophages were upregulated,while mRNA expression levels of M2 macrophage markers（Arg1,CD206）were unchanged.We concluded that compared with the mouse macrophages in the normal group,bcl3 knockout macrophages had a stronger ability to polarize to M1 type,while there was no difference in the ability to polarize to type M2.2.Western blot,Seahorse,and qRT-PCR results showed that the expression level of glycolysis-related gluconolyticases in bcl3 knockout macrophages was upregulated,ECAR levels were elevated,and glycolytic ability was significantly weakened after bcl3 overexpression to macrophage lines,indicating that BCL3 was able to inhibit glycolysis in macrophages.3.Dual-luciferase reporter gene,immunofluorescence confocal,as well as the Westernblot results showed that the knockdown of bcl3 in macrophages was able to promote the NF-κB pathway activation.4.The flow analysis and co-immunoprecipitation experiments showed that BCL3 could interact with nitric oxide synthase（iNOS）to inhibit the expression of nitric oxide synthase.5.The qRT-PCR and Westernblot experiments showed no difference in the ability of the normal group of macrophages and the bcl3 knockdown macrophage cells to undergo polarization to type M1 after the addition of the glycolytic inhibitor 2-DG.6.The scratch assay,Trans-well experiments indicated enhanced migration of macrophages after bcl3 knockdown,and CCK8 experiments indicated enhanced proliferation of macrophages after bcl3 knockdown.Conclusion This study concluded that BCL3 is able to inhibit the glycolytic capacity of macrophages by inhibiting the activation of NF-κB pathway,ultimately leading to a decrease in the ability of macrophages to polarization to M1 type.