Studies On The Role And Mechanism Of ATP-citrate Lyase In ITreg Differentiation

As a critical subset of immunosuppressive cells,inducible regulatory T cells(iTreg)differentiated from activated CD4⁺T cells(Th0)in the presence of TGF?1play a central role in the maintenance of immune homeostasis.Deregulation of Treg number and(or)functions are frequently associated with distinct inflammatory disorders,such as inflammatory bowel disease(IBD).Although iTreg are derived from Th0,they are characterized with distinct metabolic patterns.As reported,Th0rely on fatty acid synthesis(FAS),whereas iTreg are dependent on fatty acid oxidation(FAO).Because FAS and FAO are mutually exclusive,only one of them can be dominant in a specific type of cells.A shift from FAS to FAO has to take place during iTreg differentiation.Although the downregulation of FAS by inhibitor is important for iTreg differentiation,the mechanism regulating FAS during this process is still unclear.Considering that FAS is controlled by a set of key enzymes,we started with assessing their enzymatic activities during iTreg differentiation.Next,we evaluated changes in cell metabolism and iTreg differentiation induced by altered activity of the target enzyme.Thirdly,we explored the underlying mechanism regulating iTreg differentiation.Fourthly,we successfully alleviated inflammatory diseases in mice by manipulating the activity of the target enzyme.In our study,we firstly analyzed enzymatic activities of the key enzymes in FAS pathway,including ATP citrate lyase(ACLY),acetyl-Co A carboxylase(ACC)and fatty acid synthetase(FASN).Only the enzymatic activity of ACLY declined dramatically during iTreg differentiation from Th0.Enhancing ACLY activity by ACLY overexpression inhibited iTreg differentiation,while repressing ACLY activity by targeting si RNAs or specific inhibitor promoted iTreg differentiation.This suggests that the decline of ACLY activity is important for iTreg differentiation.Secondly,we explored the mechanism for iTreg differentiation mediated by ACLY.[U-¹³C]-glucose tracing flux analysis revealed a decline in FAS upon ACLY inhibition.Importantly,our rescue experiments showed that the addition of malonyl-Co A,an essential metabolite in FAS pathway,abolished ACLY-inhibition-induced iTreg differentiation.Notably,malonyl-Co A is a well-known inhibitor for carnitine palmitoyltransferase 1(CPT1),which is the rate-limiting enzyme in FAO pathway.Moreover,we found that ACLY inhibition resulted in a decrease in malonyl-Co A,but an increase in CPT1 activity and FAO.When CPT1 was inhibited or knocked down,ACLY-inhibition-induced iTreg differentiation was compromised.These data suggest that ACLY inhibition may promote iTreg differentiation via malonyl-Co A-CPT1-FAO axis.Thirdly,we explored the mechanism responsible for ACLY reduction during iTreg differentiation.Instead of transcription,declined ACLY activity during iTreg differentiation was actually due to a reduction at protein level.Further analysis on protein synthesis and degradation showed that TGF?1 enhanced the interaction of CUL3-KLHL25,an E3 ubiquitination ligase,with ACLY and in turn increased ACLY ubiquitination and degradation.When we removed CUL3 to block ACLY ubiquitination,iTreg differentiation were compromised.Importantly,simultaneous depletion of ACLY partially rescued CUL3-deficiency-induced defects in iTreg differentiation.In parallel,we also confirmed the important role of CUL3-KLHL25-mediated ACLY ubiquitination in human iTreg differentiation.These results suggest that ACLY ubiquitination mediated by CUL3-KLHL25 is required for TGF?1-induced iTreg differentiation.Ultimately,we found that inflammatory diseases in mice were alleviated upon ACLY repression.Using established mouse models with IBD(Inflammatory bowel disease)or allergic diarrhea,we successfully demonstrated that repressing ACLY led to improved iTreg differentiation and thereby alleviation of inflammation.Taken together,our data pinpoint a novel mechanism for TGF?1-induced iTreg differentiation.TGF?1 stimulation causes CUL3-KLHL25-mediated ACLY ubiquitination and degradation,which facilitate metabolic reprogramming,in particular a shift from FAS to FAO,to promote iTreg differentiation.Together,our study not only uncovers a novel mechanism for iTreg differentiation,but also provides a potential target for iTreg-manipulation-based immunotherapy.