Proteus Mirabilis Targets Atherosclerosis Plaques Of Human Coronary Arteries Via DC-SIGN(CD209)

Background Atherosclerosis(AS)is a chronic inflammatory disease caused by many factors.Bacterial DNAs are constantly detected from the atherosclerotic plaques,and around fifty percent of bacterial species identified in the plaques are members of Gram-negative Proteobacteria family,suggesting that a combination of persistent infection and inflammation may play a role in the formation of plaques.However,the mechanism by which bacterial infection affects the progression of AS is still unclear.A series of previous studies in our laboratory found that the core oligosaccharides of many Gram-negative bacteria were able to interact with C-type lectin receptors—DC-SIGN(CD209)or Langerin(CD207).Studies showed that DC-SIGN was expressed in atherosclerotic plaques,and coronary atherosclerotic plaques could be stimulated to produce anti-outer membrane proteins of Proteus mirabilis(P.mirabilis)cross-reacting antibody.In this study,we explored whether P.mirabilis,a member of Proteobacteria,would interact with coronary atherosclerotic plaques through C-type lectin receptors,thereby affecting the progression of AS.Methods 1 The phenotypic analysis of lipopolysaccharide(LPS)from P.mirabilis 1.1 After LPS was purified,the O-antigen expression of P.mirabilis was detected by SDS-PAGE silver staining.1.2 Since PLA required rough LPS to be active,the plasmid expressing PLA protein was transferred to P.mirabilis,and the function of PLA was measured to further detect the LPS phenotype of P.mirabilis.2 Cell invasion assays 2.1 P.mirabilis was labelled with CFDA-SE,then gentamicin protection assay and flow cytometry were used to detect the ability of the bacteria to invade CD209-expressing mouse peritoneal macrophages and macrophage cell line(RAW264.7)that lacks the expression of CD209 receptors.2.2 Gentamicin protection assay was used to detect the invasion of P.mirabilis with and without O-antigen to stably transfected CHO cell line that expresses h DC-SIGN(CHO-h DC-SIGN)and h Langerin(CHO-h Langerin).2.3 Gentamicin protection assay was used to detect whether the interaction between P.mirabilis and CHO-h DC-SIGN and CHO-h Langerin could be inhibited by the added mannan,anti-CD209 antibody and anti-CD207 antibody.3 CD209 receptor is mainly expressed on the surface of macrophages in coronary atherosclerotic plaques 3.1 A part of the fresh plaques(experimental group)and normal artery(control group)were excised and paraffin-embedded.The expression of CD209 and CD207 receptors in tissues were detected by immunohistochemistry.3.2 Double immunofluorescence staining was used to detect whether CD209 was co-localized with the macrophage marker CD68.4 The adhesion assays of coronary atherosclerotic plaques 4.1 The fresh plaques were cut into 1mm3 and put in the culture medium.Then P.mirabilis with and without O-antigen were added to detect the adhesion ability of plaques.At the same time,the adhesion of bacteria to normal artery was used as a control.4.2 Detecting whether the interaction between P.mirabilis and plaques could be inhibited by mannan and anti-CD209 antibody.5 Sequencing of bacterial 16 S r DNA in coronary atherosclerotic plaques Sequencing library was prepared by using 16 S r DNA product of plaques as template,and the full-length sequence of 16 S r DNA was sequenced to analyze the bacterial composition in plaques.6 In vivo assays Apolipoprotein E knockout(Apo E-/-)AS model mice and wild-type(WT)mice were infected by P.mirabilis via tail vein.Then the mice were killed 2,12,24 and 48 hours later,respectively.The ability of bacteria to spread to the heart was analyzed.Results 1 The P.mirabilis used in this study is rough strain SDS-PAGE silver staining and PLA activation assay showed that the O-antigen chain of LPS of P.mirabilis was reduced,which may allow the exposure of the LPS core.2 CD209 and CD207 are receptors of P.mirabilis 2.1 P.mirabilis invaded primary macrophages much more effectively than that of RAW264.7,indicating that CD209 can interact with P.mirabilis.2.2 P.mirabilis invaded both CHO-h DC-SIGN and CHO-h Langerin cells more effectively than CHO cells.However,P.mirabilis with O-antigen could not promote the invasion,which indicated that the expression of O-antigen can reduce the interaction between bacteria and receptors.In addition,the invasion of receptors-expressing cells by P.mirabilis could be blocked by the inhibitors mannan,anti-CD209 antibody and anti-CD207 antibody.3 CD209 receptor is mainly expressed on the surface of macrophages in coronary atherosclerotic plaques 3.1 Immunohistochemistry showed that the all collected plaques expressed CD209 receptor,but only one specimen expressed CD207 receptor.Both CD209 and CD207 were not expressed in all normal arterial specimens.3.2 Double immunofluorescence staining showed that DC-SIGN was predominantly co-localized with CD68,the marker of macrophages.4 P.mirabilis interacts with coronary atherosclerotic plaques via CD209 receptor P.mirabilis adhered plaques effective than that health arteries did.However,P.mirabilis with O-antigen hardly adhered to plaques,which indicated that the expression of O-antigen reduced the interaction between bacteria and plaques.In addition,the adhesion of P.mirabilis to plaques was inhibited by mannan,anti-CD209 antibody and anti-CD207 antibody.5 In this study,one of the most abundant phylum in coronary atherosclerotic plaques was Proteobacteria The microbiotas from two plaques were analyzed by 16 S r DNA sequencing.One of the most abundant phylum was Proteobacteria and the other was Firmicute,which revealed the complexity of microbial composition during the development of AS.6 P.mirabilis can spread with blood flow to the heart of Apo E-/-AS model mice Apo E-/-and WT mice were infected with P.mirabilis via intravenous injection.Grinding homogenate after 2,12,24,and 48 h revealed that Apo E-/-mice had a large number of bacteria and reached a peak at 24 h.However,the hearts of WT mice had fewer bacteria.Conclusions This study demonstrated that DC-SIGN(CD209)expressed on coronary atherosclerotic plaques can act as a cellular receptor for Proteus mirabilis,which suggests the colonization of bacteria in plaques to cause inflammation.