Exosomal CagA Derived From Helicobacter Pylori-infected Gastric Epithelial Cells Induces Macrophage Foam Cell Formation And Promote Atherosclerosis

Background and hypothesis:Atherosclerotic cardiovascular disease（CVD）and death remain the leading cause of morbidity and mortality around the world.Due to the high burden of these diseases,massive studies have been conducted to clarify the risk factors of CVD.Despite the developments in the prevention of CVD,there is still a gap between the prevalence of CVD and the risk factor distribution.The classic risk factors for atherosclerosis,namely,cigarette smoking,hypercholesterolemia,hypertension,and diabetes,account for only50%of its incidence.As atherosclerosis is a multi-step process that inflammation takes place in all steps,it has been suggested that chronic infection can contribute to this process.Current epidemiological data and clinical studies suggest that Cytotoxic associated gene-A（CagA）positive H.pylori infection may increase the risk of atherosclerosis and related events.To seek direct evidence for a role of H pylori infection in atherogenesis,we examined the effect of this infection in atherosclerosis-prone mice.Exosomes（EVs）have important functions in intercellular communication and regulation through the transmission from cell to cell or organism to organism.Emerging evidence suggests that in settings of infection,EVs released from host cells can convey pathogen-derived components that serve as important regulators of cellular functions and disease mechanisms.Recent studies shown that EVs may be transported to other organs and tissues to participate in the pathogenesis of H.pylori-induced extra-gastric diseases.Therefore,we determine whether EVs released from the H.pylori-infected host gastric epithelial cells（GECs）enter into systemic circulation and aortic plaque,which thereby play a pro-atherogenic role.Epidemiological and clinical research have provided evidences that CagA-positive H.pylori infection is correlated with high incidence of atherosclerosis and the plaque instability compared with CagA-negative H.pylori.It suggested that CagA,a virulence factor for H.pylori,play an important role in this relationship.A recentstudy showed that serum-derived EVs in patients infected with CagA-positive H.pylori could carry the CagA and enter into the blood circulation.Therefore,we further hypothesized that H.pylori-infected GECs-released EVs mediate CagA delivery into circulation and exosomal CagA promotes atherogenic plaque progression.Aims:（1）To determine if H.pylori infection might influence atherosclerosis in vivo inmice;（2）To determine if EVs released from the H.pylori-infected host gastric epithelialcells promote macrophages-derived foam cell formation in vitro and in vivo;（3）To determine if exosomal CagA promote macrophages-derived foam cellformation and determine the mechanisms involved.Methods and Results:Part I:1.H.pylori infection plays a proatherogenic role in C57BL/6 mice fed a high-fat/cholesterol dietWe developed H.pylori infection in a mouse model using wild-type C57BL/6mice.To this end,C57BL/6 mice fed a normal chow diet（NCD）were treated with H.pylori（strain PMSS1）or vehicle（PBS）by oral gavage three times a week for 4 weeks.After 2 weeks of recovery,uninfected and H.pylori-infected mice then divided into 4groups and were fed either with NCD or a high-fat/cholesterol diet（HFCD）.To confirm that H pylori infection did indeed occur,immunofluorescence staining showed the presence of H.pylori in stomach tissue,which may be required for establishment of subsequent persistent infection.After 16 weeks on NCD,uninfected and H.pylori-infected mice did not show any atherosclerotic lesions in aorta arches and aortic root.In the HFCD-fed groups,we also detected no signs of atherosclerotic plaques in aorta arches from either group,similar to previous studies.However,detailed analysis in cross-sections of aortic roots stained with Hematoxylin-Eosin（HE）and Oil Red O staining found an unusual observation:distinctive foam cell lesions（initial stages）were present adjacent to the aortic valve attachment in H.pylori-infected mice but this type of lesions was not found in the uninfected mice.We observed no differences in body weight and plasma lipid profiles in the NCD-fed groups and HCD-fed groups,indicating that H.pylori infection does not have a major impact on weight gain or lipid metabolism.These results suggest that H.pylori infection does not directedly induce atherosclerosis in wild-type mice but may contribute to the initial stages of pathogenesis of atherosclerosis in high-fat/cholesterol diet-fed atherosclerotic mode.2.H.pylori infection augments atherosclerotic plaque growth and instability in ApoE^-/-miceNext,we studied the effect of H.pylori infection on atherosclerosis in ApoE^-/-mice.After inoculation,uninfected and H.pylori-infected ApoE^-/-mice were given 2-week recovery,put on a high-fat diet（HFD）ad libitum,and then sacrificed at different time points to monitor the kinetics of development of the atherosclerotic plaque.H.pylori-infected ApoE^-/-mice showed more extensive atherosclerotic lesions in the aortic arch and aortic root compared with uninfected ApoE^-/-mice at two time points.Body weights and plasma cholesterol level before and after being fed an HFD were also not significantly different between both infected and uninfected groups.Despite similar cholesterol profiles,analysis of Oil Red O-stained atherosclerotic lesion area revealed a significant increase of lipid accumulation in H.pylori-infected ApoE^-/-mice,implying that the pro-atherogenic role of H.pylori may attribute to increased lipid deposition.These results suggest that H.pylori infection promotes foam cell formation and accelerates atherosclerotic plaque growth.We then conducted a more detailed analysis of aortic root plaque composition.Immunohistochemical analysis showed that increases in multiple indices of plaque instability including increased macrophages infiltration,decreased a-smooth muscle actin（a-SMA）positive structures and decreased collagen I⁺content.In light of these changes,atherosclerotic plaques in the H.pylori-infected ApoE^-/-mice contained large necrotic core areas,which were progressively enlarged during 8 and 16 weeks of WD feeding.Further,the thickness of the fibrous cap in plaques exposed to H.pylori infection was significantly decreased relative to uninfected groups.Both increases of necrotic areas and decreases of the fibrous cap thickness are important features of vulnerable plaques.Above data indicate that H.pylori-infection augments atherosclerotic plaque growth and increases plaque instability and potential plaque disruption.3.H.pylori infection promotes macrophage-derived foam cell formation in vivoAccumulation of cholesterol in macrophages during early stages of the atherosclerotic plaque formation is a critical step during the development of this disease.Once the excessive accumulation of lipid would progress toward a more advanced stage of atherosclerosis.Above findings having identified a link between H.pylori infection,lesional lipid deposition and atherosclerosis.Therefore,we first analyzed whether H.pylori infection contribute to macrophages-derived foam cell formation.Immunostaining Bodipy493/503 and CD68 showed that H.pylori infection significantly promote neutral lipid accumulation in the lesional macrophages.These findings suggest that H.pylori infection causes a marked acceleration and increases in atherosclerosis that is dependent on macrophage-derived foam cell formation.Part II:1.Exosomes released from H.pylori-infected gastric epithelial cells enhance ox-LDL-induced foam cell formation in vitroTo characterize EVs from H.pylori-infected GECs,we first established a novel in vitro infection model.We infected human normal GECs lines（GES-1）with H.pylori12 hours and washed 4X with PBS to remove unattached bacteria and then added gentamicin to prevent extracellular bacterial growth.Conditioned supernatant was then collected after 48?hours of culture and EVs were purified by standard differential centrifugation.Utilizing transmission electron microscopy（TEM）and nanoparticle tracking analysis（NTA）,we observed that uninfected（GES-EVs）and H.pylori-infected GES-1-derived EVs（Hp-GES-EVs）contained primarily double-layered vesicles with diameter distribution between 110-150 nm,which correspond to the expected size of EVs.We further confirmed the presence of three exosome-specific markers CD63,CD9 and TSG101 in GES-EVs and Hp-GES-EVs.To demonstrate the exosomal transfer between GECs and macrophages,we labeled GES-EVs and Hp-GES-EVs with PKH26 dye and incubated them with Raw264.7 cells and found that these GES-derived EVs rapidly enter into the recipient cells.Next,we evaluated the role of Hp-GES-EVs in promoting the formation of macrophage-derived foam cell.Treatment with GES-EVs did not affect the percentage of oxLDL-induced foam cells compared to the untreated controls.However,exposure of Raw264.7 cells to Hp-GES-EVs led to an increase in the percentage of foam cells.Cellular lipid content analysis also revealed that Hp-GES-EVs increased the total cholesterol content in ox-LDL-treated Raw264.7 cells.These data indicate that H.pylori-infected GECs-released EVs are sufficient to enhance macrophages-derived foam cell formation.2.Exosomes released from H.pylori-infected gastric epithelial cells enhance ox-LDL-induced foam cell formation in vitroTo validate our finding in vivo,we first assessed the biodistribution of Hp-GES-EVs and whether circulating Hp-GES-EVs could enter into the aortic plaques.Dil-labelled Hp-GES-EVs were intravenously injected into ApoE^-/-mice fed an HFD for16 weeks and the live mice were imaged using an in vivo imaging system（IVIS）at 24hours after injection.From the representative images,the IVIS analysis showed a clear Dil fluorescence signal in the thoracic areas of Dil-labelled Hp-GES-EVs treated ApoE^-/-mice but no thoracic Dil fluorescence was observed in PBS-treated ApoE^-/-mice.To confirm that the signal originated from the atherosclerotic plaques,we examined plaque lesion of aortic root tissues and observed that Dil-labelled Hp-GES-EVs were up-taken in macrophage-rich lipid core.These finding suggest that circulating Hp-GES-EVs can be up-taken in the plaque and may play a pro-atherogenic role.To determine whether Hp-GES-EVs are responsible for H.pylori infection-induced acceleration of atherosclerosis,we periodically injected PBS,GES-EVs and Hp-GES-EVs to ApoE^-/-mice.Administration of Hp-GES-EVs significantly augmented areas of atherosclerotic lesion and lipid deposition compared with GES-EVs-injected or PBS-injected ApoE^-/-mice.Consistent with the result of H.pylori infection in vivo and in vitro experiment,Hp-GES-EVs treatment significantly increased macrophage-derived foam cell accumulation in the plaque.There was not statistically significant difference of plaque size,and the percentage of macrophage-derived foam cell between GES-EVs-injected and PBS-injected ApoE^-/-mice.Together,the above data support a key role of Hp-GES-EVs in H.pylori infection-induced acceleration of atherosclerosis through contributing to macrophage-derived foam cell formation in vitro and in vivo.Part III:1.H.pylori CagA is secreted in exosomes and promotes ox-LDL-induced foam cell formationTo explore whether Hp-GES-EVs contained CagA,we first detected CagA expression in cell lysates and EVs prepared from H.pylori-infected and uninfected cells.As expected,CagA was specifically detected in cell lysates of H.pylori-infected GES-1 and Hp-GES-EVs.In addition,the presence of CagA in Hp-GES-EVs from gentamicin-treated cells suggests that the observed CagA expression was not due to free extracellular CagA and CagA contained in bacterially secreted vesicles.To determine whether EVs-associated CagA could enhance macrophages-derived foam cell formation and the underlying mechanism following H.pylori infection,we directly treated Raw264.7 cells with endotoxin-free recombinant CagA（rCagA）,and found that rCagA recapitulated the pro-atherogenic effect of Hp-GES-EVs on macrophages.Treatment of Raw264.7 cells with rCagA significantly promoted ox-LDL-induce foam cell formation and increased total cholesterol content compared with untreated controls.Macrophage-derived foam cells are generated by uncontrolled uptake of modified lipoproteins such as ox-LDL and/or impaired cholesterol efflux.Therefore,we next investigated whether the increased uptake of ox-LDL and/or impaired cholesterol efflux could account for the increased foam cell formation in rCagA-treated macrophages.To this end,we treated Raw264.7 cells with the fluorescently labelled ox-LDL（Dil-oxLDL）and found that rCagA did not affect cholesterol uptake by detecting the fluorescence intensity.However,rCagA treatment significantly decreased cholesterol efflux to ApoA1 and HDL by performing the cholesterol efflux assay.To delineate the mechanism by which rCagA promoted foam cell formation,we next examined the expression of SRs and RCTs in rCagA-treated macrophages.Consistently,rCagA treatment decreased the expression of ABCA1,ABCG1 and SR-BI at both mRNA and protein levels in a dose-dependent manner,but did not affect the expression of SR-A,CD36 and LOX-1.Taken together,these results demonstrate that CagA enhances foam cell formation by decreasing cholesterol efflux capacity.These findings also highlight a novel mechanism by which H.pylori-infected host GECs release CagA-expressing EVs in a way that promotes macrophages-derived foam cell formation.2.Systemic administration of rCagA exacerbates atherosclerosis in ApoE^-/-miceTo further confirm the functional role of CagA in vivo,we evaluated whether systemically administrated of rCagA to HFD-fed ApoE^-/-mice for 12 weeks were sufficient to accelerate the growth and instability of atherosclerotic plaque.As expected,treatment of rCagA significantly increased atherosclerotic plaque burden in the aorta and aortic sinus compared with control groups.En face analysis of Oil Red O and Bodipy493/503-staing revealed that lipid accumulation in the lesional macrophages were also increased in the rCagA-injected groups.Analysis of plaque morphology showed that increased macrophages infiltration,decreased collagen I⁺content and decreased a-SMA positive structures.Moreover,necrotic core area in the plaques was significantly bigger in the rCagA-injected ApoE^-/-mice.There was no significant difference among these groups in body weight,plasma cholesterol,and triglyceride levels.Collectively,these results similar to that observed in Hp-GES-EVs treated mice and suggest that elevated circulating CagA are necessary and sufficient to accelerate the growth and instability of atherosclerotic plaque.3.rCagA-induced ABCA1/ABCG1/SR-BI downregulation depends on PPARγ-LXRαsignalingTo identify the molecular mechanism involved in the transcription downregulation of cholesterol efflux transporters,we examined the status of key transcription factors regulating cholesterol efflux in macrophages,which is known as the members of nuclear receptors family including PPARγand LXRα.rCagA treatment dose-dependently decreased PPARγand LXRαexpression at both mRNA and protein levels.These studies suggest that PPARγand LXRα,two key upstream transcription factors,are involve in rCagA-induced the depression of cholesterol efflux and the downregulation of transporters expression.Conclusions:These results provide evidences that long-term H.pylori infection can accelerate atherogenic plaque progression and exosomal CagA derived from Helicobacter pylori-infected gastric epithelial cells induces macrophage foam cell formation and promote atherosclerosis.These findings provide mechanistic insights on H.pylori infection-associated cardiovascular risks.