The Mechanism Of Heme Oxygenase-1 Regulating Ferroptosis In Bacillus Calmette-Guerin-infected Macrophages

Tuberculosis(TB)is a zoonotic infectious disease caused by Mycobacterium tuberculosis(M.tb)infection.Macrophages are important intrinsic immune cells;they are also the most important and abundant target and host cells of M.tb infection.A consequence of infection of M.tb is to induce oxidative stress and cellular necrosis in macrophages,which in turn increases the growth and spread of intracellular bacteria,leading infection and injury of more host cells.Therefore,uncovering the molecular regulatory mechanism of macrophage death induced by M.tb has been the priority of research on the pathogenesis of tuberculosis.The major modes of macrophage death caused by M.tb infection that have been identified are apoptosis,necroptosis and pyroptosis.Ferroptosis,a recent discovered cellular necrosis caused by iron-dependent lipid peroxidation,could facilitate the infection and spread of M.tb.Our understanding in the role and mechanism of ferroptosis in TB pathogenesis remains preliminary.Heme oxygenase 1(in human.the abbreviation is HMOX1;in mouse,the abbreviation is Hmoxl),a biomarker of M.tb infection and tuberculosis,plays a role in regulating macrophage iron metabolism and reactive oxygen species homeostasis and is a potential regulator of ferroptosis.However,the role and mechanism of heme oxygenase 1 in regulating ferroptosis in macrophages induced by M.tb infection,and its significance for the proliferation and spread of intracellular bacteria are not clear.Therefore,the following studies were conducted in this paper.1.Investigated the presence of ferroptosis in tuberculosis by analyzing peripheral blood transcriptome data from clinically active TB patients in the GEO database,and studied the relevance of HMOX1 to ferroptosis caused by M.tb infection.2.Establishing macrophage ferroptosis cell model by infection of RAW264.7 cells with different multiplicity of infection(MOI)of BCG for varied time periods.The role of BCG-induced macrophage ferroptosis was clarified.Two ferroptosis agonists,H2O2 and RSL3,were further used to explore the pathway of BCG infection-induced macrophage ferroptosis from two different perspectives:promotion of lipid peroxidation reaction and anti-lipid peroxidation function.3.BCG-infected macrophages were pretreated with NAC to investigate whether there is a negative feedback regulation mechanism between Hmoxl expression and ferroptosis levels.Then,siRNA interfering technique was used to investigate the regulatory role and molecular mechanism of Hmoxl on BCG infection-induced ferroptosis in macrophages,and its effect on intracellular bacterial proliferation and diffusion.The main results of the study were as follows:1.The results of peripheral blood transcriptome analysis of tuberculosis uncovered that ferroptosis was occurred in active TB.HMOX1 was significantly upregulated(p<0.001)and the expression level of GPX4,a marker of ferroptosis,was reduced(p<0.001).Moreover,the expression levels of pro-lipid peroxidation factors ACSL4,ALOX5,NOX4 and VDAC2 were significantly increased(p<0.001,p<0.001,p<0.001,p<0.01).HMOX1 showed a correlation with the expression of a number of important regulators of ferroptosis.2.The macrophage ferroptosis cell model was established by infection of RAW264.7 cells with BCG.BCG at MOI of 5 significantly promoted macrophage ferroptosis.Hmoxl was significantly upregulated only in BCG-infected macrophages(p<0.001).Levels of macrophage ferroptosis induced by BCG infection were similar to the effect of H2O2(p=ns),but the cell viability in the BCG-infected group was extremely significantly higher than that in the H2O2 control group(p<0.001).The RSL3 control group presented a highly significant higher intracellular Fe2+ content,ROS level and lipid peroxidation than the BCG-infected group(p<0.001,p<0.001,p<0.001).The anti-lipid peroxidation protein Gpx4 expression was not significantly different between the RSL3 control group and the BCG-infected group(p=ns).Fspl and cell survival were very significantly reduced in the RSL3 control group(p<0.001,p<0.001).Suggesting that Hmoxl suppressed BCG infection-induced ferroptosis by controlling the level of Fenton-responsive substances and protecting the anti-lipid peroxidation pathway to reduce lipid peroxidation.3.NAC remarkably suppressed BCG infection-induced macrophage ferroptosis and reduced intracellular bacterial load(p<0.001)by scavenging intracellular ROS(p<0.01)and attenuating lipid peroxidation(p<0.01).The knockdown of Hmoxl resulted in intracellular Fe2+content(p<0.001)by upregulating Ncoa4 expression(p<0.001),which promoted the Fenton reaction and lipid peroxidation.Meanwhile,downregulation of Hmox1 led to a continued decrease in the expression of the cysteine uptake channel protein xCT(p<0.001)and cysteine-dependent Gpx4(p<0.001),which further reduced the antilipid peroxidation capacity of macrophages.Downregulation of Hmox1 significantly reduced macrophage viability(p<0.001)and increased the number of intracellular BCG(p<0.001).From above findings,several conclusions were drawn as follows:(1)Transcripts related to cell ferroptosis were identified in peripheral blood of TB patients,among them the upregulation of HMOX1 was strongly correlated with ferroptosis-related transcripts.It was suggested that HMOX1 may influence the infection and spread of M.tb by regulating ferroptosis.(2)At MOI of 5,BCG infection induced macrophage ferroptosis,and Hmoxl expression increased with enhanced ferroptosis.(3)Hmoxl reduced the accumulation of toxic lipid peroxides by curbing the Fenton reaction in BCG infected macrophages.Hmoxl protected the macrophage "xCT-Gpx4" signaling axis and maintained the anti-lipid peroxidation capacity of macrophages.Hmoxl inhibited lipid peroxidation and ferroptosis in macrophages induced by BCG infection,reduced intracellular bacterial load,and had an anti-BCG infection effect.