The Immunoregulation Of DUSP5-Mediated Fatty Acid Metabolism In BCG-infected Macrophages

The widespread epidemic of tuberculosis poses a serious threat to human health.Fatty acid is the main energy source of Mycobacterium tuberculosis(Mtb).Host-derived fatty acid is utilized by Mtb to maintain its reproduction,which reprogram host cells fatty acid metabolism ultimately.As an significant part of energy metabolism,fatty acid metabolism not only provides energy support for host cells,variety of metabolic intermediates can be used as signal molecules to take part in host immune responses.Dual specificity phosphatase 5(DUSP5)modulate MAPK signaling activation via dephosphorylation and plays role in host cell immune response.Studies have confirmed lipometabolic disturbance including fatty liver are closely related to DUSPs.However,the regulatory effect of DUSP5 during Mtb infection is still unclear.Bacillus Calmette-Guerin(BCG)was taken as the research object in this study,besides,RAW264.7 macrophages and C57BL/6J mouse lung BCG infection models were established.Small interfering RNA(siRNA)knockdown RAW264.7 macrophages DUSP5 expression,the effect of DUSP5 on autophagy,fatty acid metabolism and inflammatory response were syudied in vitro.DUSP5 interfered adeno-associated virus(AAV)was used to surpressed the expression of DUSP5 in C57BL/6J mice lungs,the effect of DUSP5 on the bacterial load and lung inflammation after BCG infection was explored in vivo.To investigate the effects of fatty acid oxidation on autophagy and inflammatory response of RAW264.7 macrophages infected with BCG,the fatty acid oxidation pathway was inhibited by etomoxir(Eto).The main findings are as follows:1.DUSP5 expression and reactive oxygen species(ROS)production were induced by BCG infection,and their level were decreased significantly after C29(TLR2 inhibitor),si-TLR2 and TLR2 neutralized antibody treatment(P<0.01).The expression of p-ERK1/2,p-p38,p-JNK and DUSP5 were decreased in N-Acetyl-L-cysteine(NAC)treated cells.Further studies showed that BCG infection activated ERK1/2?p38 and JNK which promote Nrf2 expression.ML385,a inhibitor for Nrf2,surpressed DUSP5 protein expression(P<0.01).These results indicated that BCG induced the expression of DUSP5 through TLR2/ROS/MAPK/Nrf2 signaling pathway in macrophages.2.In vivo studies showed that DUSPS knockdown decreased the BCG load in mice lungs.BCG infection indeced RAW624.7 macrophages autophagy.Si-DUSP5 transfection promoted expression of Beclinl,Atg5,Atg7 and LC3-?(P<0.01),and autophagosome accumulation was observed inside RAW264.7 cells.Besides,autophagy flux was promoted in DUSP5 knockdown cells.Further study showed si-DUSP5 elevated the expression of p-ERK1/2 and p-P90RSK(P<0.01).PD98059 treatment decreased the expressions of ATG5 and LC3-?(P<0.01)which accompanied with autophagosomes decreasement.Above suggested DUSP5 knockdown activated ERK1/2 signaling which promotes macrophage autophagy during BCG infection.3.DUSP5 knockdown inhibits IL-1? and IL-6 expression,and the phosphorylation level of NF-?B was decreased in BCG infected RAW264.7 macrophages.In vivo studies confirmed that DUSP5 knockdown alleviated lung tissue inflammation and promoted the expression of anti-inflammatory cytokines IL-10 and IL-13.These results indicate that DUSP5 plays a pro-inflammatory role during BCG infection.4.BCG infection promoted fatty acid oxidation of RAW264.7 macrophages.Intracellular lipid droplets,free fatty acids and triglycerides were all elevated after DUSP5 siRNA transfection.Meanwhile,DUSP5 knockdown inhibited the expression of CPT-1A and PPARa which participated in fatty acid oxidation,and the expression of fatty acid synthesis proteins SCD1,FASN and PPARy were up-regulated.These results indicated that DUSP5 mediated fatty acid oxidation induced by BCG infection.5.During BCG infection,attenuated lungs inflammation and reduced BCG load were observed in C57BL/6J mice injected with Eto.Eto pretreatment promoted the the expression of Beclinl,LC3-? and Rab7(P<0.01),and acummulated autophagosomes and promoted autophagy flux were detected in BCG-infected RAW264.7 macrophages.However,the expression of IL-1?,IL-6 and TNF-? were inhibited by Eto.Further research found that Eto promoted ROS production in RAW264.7 macrophages,and NAC significantly down-regulated the expression of LC3-?(P<0.05).It is suggested that during BCG infection,DUSP5-mediated fatty acid oxidation inhibits the production of ROS,thereby inhibiting the autophagy of macrophages,and it play negtive role in BCG induced inflammation.In summary,BCG infection induces DUSP5 expression,thereby promoting fatty acid oxidation in macrophages.In this process,DUSP5-mediated fatty acid oxidation inhibits ROS production and surpressed ERK1/2 activity,thus inhibits the BCG induced macrophage autophagy,which will provid a safe intracellular environment for intracellular bacteria survival in macrophages.In addition,DUSP5-mediated fatty acid oxidation also promoted the expression of pro-inflammatory factors such as IL-1?,IL-6 and TNF-?.The above indecates that that during BCG infection,the fatty acid oxidation pathway mediated by DUSP5 plays a regulatory role in promoting inflammation and suppressing autophagy.DUSP5 may serve as novel target for host-directed therapy against Mtb and may provide a new direction for the immunometabolism of tuberculosis.