Cellular Energy Metabolism Regulates Macrophage CBM Complex And Inflammatory NF-κB Signaling

Background and Significance:A large number of experimental and clinical studies have found that high glucose is often accompanied by inflammation,for example,hyperglycemia/diabetes patients are often accompanied by chronic inflammation,which is an important clinical manifestation of chronic metabolic syndrome;Another example is that many malignant tumor cells need to consume a lot of glucose（Warburg effect）,but they are often in the microenvironment of chronic inflammation.In experimental studies of infectious diseases,glucose is also an important driver of lethal inflammatory responses（known as cytokine storms）^[1].Unfortunately,little is known about how glucose regulates inflammatory signals.Our previous studies have shown that glucose can regulate the signaling axis of LKB1-AMPK protein kinase in T cells through the level of cellular energy metabolism,and then regulate the formation of CARD-BCL10-MALT1（CBM complex）through phosphorylation of BCL10,thus regulating the inflammatory signaling pathway of NF-κB.Since macrophages are central cells mediating inflammatory responses,it remains unclear whether glucose metabolism/cellular energy metabolism can also regulate NF-κB inflammatory signaling through this signaling pathway.This study focused on the regulation of glucose metabolism on NF-κB signaling pathway in macrophages,and explored the direct influence of energy stress signals on classical NF-κB pathway in macrophages.That is,glucose starvation-LKB1-AMPK-CARD-BCL10-MALT1（CBM complex）-NF-κB signaling axis can affect the inflammatory signal in macrophages,and reveal the molecular mechanism of glucose energy metabolism regulating the inflammatory signal in macrophages.This study not only provides new experimental evidence to clarify the inflammatory effect of high glucose,but also provides a theoretical explanation for the anti-inflammatory effect of clinical hypoglycemic drugs.Methods:（1）Macrophages cell line RAW264.7 cultured in vitro was stimulated by glucose starvation,metformin or 2-deoxyglucose（2DG）,and the protein expression level of IκBα,an inhibitory molecule of NF-κB,was detected by Western blot.The transcription level of the downstream cytokine TNF-αof NF-κB was detected by q PCR.（2）RAW264.7 cells were treated with different glucose concentrations,the expression level of IκBαprotein was detected by Western blot,and the transcription level of TNF-αwas detected by q PCR.（3）Primary peritoneal macrophages of mice were extracted,treated with metformin or 2DG,m RNA was extracted for reverse transcription,and the expression of TNF-αwas detected by q PCR.（4）Lentivirus-mediated short hairpin（sh）RNA was used to establish the RAW264.7 cell line with LKB1 stable silence or Scramble,and Scramble control cells and LKB1 silent cells were treated with glucose starvation.Western blot was used to compare the activation of NF-κB between the two groups.（5）RAW264.7 cells were treated with glucose starvation and 2DG with or without AMPK inhibitor,Western blot was used to detect the difference in NF-κB signal activation between the two groups.（6）Lentivirus-mediated short hairpin（sh）RNA was used to establish the RAW264.7 cell line with BCL10/MALT1 stable silence or Scramble,Scramble cells and MALT1 or BCL10silent cells were treated with glucose starvation,respectively.Western blot was used to detect the difference of NF-κB signal activation between the two groups.（7）Transient overexpression of HA-MALT1 and Myc-BCL10 was observed in 293T cells,and the formation of BCL10 and MALT1 complex was detected by co-immunoprecipitation under high and no glucose culture environment.（8）The binding status of endogenous BCL10 and MALT1 complex in RAW264.7 cells treated with 2DG was detected by immunoprecipitation.（9）Western blot was used to detect the activation differences of NF-κB signal in Scramble and BCL10 stable silenced RAW264.7 stimulated by LPA,LPS and TNF-α,and to explore the activation of NF-κB mediated by CBM complex under different stimulation conditions.（10）RAW264.7 cells cultured with high or no glucose were stimulated with LPA,LPS or TNF-α.Western blot was used to detect the effect of high or no glucose culture on the activation of NF-κB activated by LPA,LPS or TNF-α,and q PCR was used to detect the difference in expression of TNF-α.Results:1.Energy stress can inhibit basal NF-κB activity in macrophages（1）Glucose starvation,metformin and 2-deoxyglucose（2DG）inhibited NF-κB signaling in RAW264.7 and mouse primary peritoneal macrophages,manifested by the accumulation of IκBαprotein and the decrease of TNF-αm RNA level;（2）Low glucose inhibited the expression level of RAW264.7 IκBαprotein and TNF-αm RNA;Metformin and 2-deoxyglucose（2-DG）can inhibit the expression of the downstream cytokine TNF-αof NF-κB in primary peritoneal macrophages of mice.2.Energy stress regulates NF-κB activity in macrophages through the LKB1-AMPK signaling axis（1）Western blot showed that compared with control RAW264.7 cells,LKB1 knockdown blocked glucose starvation-induced inhibition of NF-κB;（2）Western blot showed that compared with RAW264.7 cells in the control group,AMPK inhibitor treatment blocked glucose starvation and 2DG induced accumulation of IκBαprotein.3.Energy stress regulating NF-κB activity in macrophages is realized by regulating the CBM complex（1）Western blot showed that compared with the Scramble control group,the knockdown of BCL10 and MALT1 in macrophages significantly blocked the accumulation of glucose starvation induced IκBαprotein;（2）Exogenous co-immunoprecipitation showed that the complex of BCL10 and MALT1disappeared under the treatment of glucose starvation;（3）Endogenous co-immunoprecipitation showed that the complex of BCL10 and MALT1disappeared under 2DG treatment.4.Energy stress had a strong inhibitory effect on LPA-stimulated NF-κB signaling,but had no effect on LPS and TNF-αinduced NF-κB signaling.（1）Western blot showed that the knockdown of BCL10 in RAW264.7 cells inhibited the activation of NF-κB induced by LPA,but had no effect on the activation of NF-κB induced by LPS and TNF-α;（2）Western blot and q PCR showed that in RAW264.7 cells glucose starvation inhibited the activation of NF-κB induced by LPA,but did not affect the activation of NF-κB induced by LPS and TNF-α.Conclusion:In macrophages,CARD9-BCL10-MALT1（CBM）complex can be mediated by the LKB1-AMPK signaling axis under energy stress,thereby affecting the basal condition and LPA-induced classical NF-κB inflammatory signaling pathway in macrophages.Thus,the expression of TNF-α,a downstream inflammatory factor,is affected.