Gingipain Of Porphyromonas Gingivalis Manipulates M1 Macrophage Polarization Through C5a Pathway

Background: Chronic periodontitis is a chronic inflammatory disease,which is characterized by the destruction of periodontal connective tissue and alveolar bone loss.Macrophages can undergo specific differentiation in response to local tissue environment such as microbial products to differentiate into different functional phenotypes.Macrophages can be fully polarized to become two different functional phenotypes: classically activated macrophages(M1 macrophages)and alternatively activated macrophages(M2 macrophages)in response to diverse stimuli.The M1 macrophages express co-stimulatory CD86 and produce high levels of pro-inflammatory cytokines,like IL-12,IL-23,iNOS,TNF-?,IL-1? and IL-6 to drive antigen specific Th1 and Th17 cell inflammatory responses and fight pathogens,which mediates macrophage bactericidal action.Cytokines TNF-?,IL-1? and IL-6 are also bone-resorptive cytokines to mediate destruction of periodontal tissue.Porphyromonas gingivalis(P.gingivalis,Pg),a gram-negative anaerobe with a plethora of virulence factors including lipopolysaccharides(LPS),gingipain and fimbriae,is strongly associated with periodontitis.Gingipains are typsin-like cysteine proteases,and are considered enzymes with complement 5 convertase-like activity that can locally increase concentration of C5 a ligand,These C5 a ligands help P.gingivalis to co-activate complement C5 a receptor and TLR2/4 on immune cells,induce signaling crosstalk in macrophages,and inhibit their M1 macrophage polarization,whereas the same pathogen-instigated C5 a R-TLR2/4 crosstalk upregulates other inflammatory and bone-resorptive cytokines such as TNF-??IL-1? and IL-6.In conclusion,gingipain targets C5 a R to inhibit M1 macrophage killing functions and cause periodontal disease.Methods: 1.Preparation of gingipainThe gingipain was directly extracted from the supernatant of Pg ATCC 33277 as previously descrebed by sheets,then was identified by Mass Spectrography.The activity of the gingipain was determined by chromogenic substrate BAPNA.2.The effect of gingipain on macrophage polarization RAW264.7 cell was co-cultured with various enzymatic activity of gingipain(0.25,1,4,8U/L)and inactive gingipain at 24 h,then q RT-PCR was used to evaluate the related cytokines gene expression of M1.3.The effects of gingipain on the expression of bacteriostatic cytokines in M1 macrophages induced by Ec-LPS and Pg-LPS.Experimental groups were established: the negative control group,LPS group,LPS+gingipain group,LPS+gingipain+PMX-53 group.After 24 hours,q RT-PCR,ELISA and Flow cytometry were used to evaluate the related costimulatory molecules and cytokines expression of M1.Results: 1.The gingipain was characterized as Rgp A,the enzymatic activity of Rgps was 20U/L.2.The gingipain promoted gene expressions of IL-12,IL-23,iNOS,TNF-?,IL-1? and IL-6 with dose increase of gingipain in general,especially at the dose(4 U/L)of gingipain(P<0.05).Compared to the group of inactive gingipain,active gingipain could weakly promote the expression of above cytokines,and PMX-53 could enhance active gingipain stimulation for IL-12,IL-23,and iNOS at gene expression,but not inactive gingipain group(P<0.05).The PMX-53,however,decreased the effects of active gingipain on TNF-?,IL-1?,and IL-6 at gene expression,but not inactive gingipain(P<0.05).3.Gingipain plus Ec-LPS decreased expresstions of IL-12,IL-23,iNOS,TNF-?,IL-1? and IL-6 in which Ec-LPS-induced increase(P<0.01).PMX-53 could enhance gingipain+Ec-LPS group stimulation for IL-12,IL-23,and iNOS at gene and protein expression(P<0.01),but decreased the effects of gingipain+Ec-LPS group on TNF-?,IL-1?,and IL-6 at gene expression(P<0.01).4.For gingipain plus Pg-LPS treated RAW264.7 macrophages,gingipain weakly enhanced expressions of cytokines of IL-12,IL-23 in which Pg-LPS induced increase(P<0.01),but not IL-10 and CD206 while gingipain decreased expressions of CD86 and cytokines of iNOS?TNF-?,IL-1? and IL-6(P<0.05)in which Pg-LPS induced increase.PMX-53 could enhance gingipain+Pg-LPS group stimulation for IL-12,IL-23,and iNOS at gene and protein expression(P<0.05),but decreased the effects of gingipain+Ec-LPS group on TNF-?,IL-1?,and IL-6 at gene expression(P<0.05).Conclusions: 1.M1 Macrophage phenotypes can be weakly induced by gingipain in vitro.2.Gingipain is a critical regulator,more like a balancer to manipulate M1 macrophage polarization in order to benefit P.gingivalis infection through the C5 a pathway.3.The C5 a R antagonist PMX-53 has potential application to prevent bone absorption.