The Experimental Study Of Porphyromonas Gingivalis Peptidylarginine Deiminase Exacerbated Ulcerative Colitis

Objective: Periodontitis is an infectious disease caused by dental plaque,which can lead to the destruction of dental support tissues,periodontal pocket formation,progressive adhesion loss and alveolar bone resorption,and eventually lead to tooth loss,which is a common disease affecting people's health.Porphyromonas gingivalis(P.gingivalis)is a spore-free Gram-negative melanin-specific anaerobic bacillus.It is one of the most important periodontal pathogens in the lesion or active area in chronic periodontitis.P.gingivalis is capable of inducing an abnormal immune response in the host cell,disrupting the balance between the host and the microorganism,and causing an ecological imbalance in the microbial group in which the host is harmoniously symbiotic.P.gingivalis has a variety of virulence and potential pathogenic effects.The bacteria itself and its metabolites can reach different organs of the body through the blood,after that induce and exacerbate a variety of systemic diseases.Under physiological conditions,the maintenance of intestinal homeostasis depends on the interaction between gut microbiota,intestinal epithelial system and immune system.Intestinal flora imbalance can cause activation of the host immune response,breaking the immune tolerance of susceptible hosts,which in turn triggers a systemic immune imbalance,eventually leading to a pro-inflammatory response leading to destruction of peripheral tissues.Inflammatory bowel disease(IBD)is a group of chronic non-specific intestinal inflammatory diseases involving the ileum,rectum,colon and even the entire digestive tract.It is one of the most common diseases in intestinal diseases,including ulcerative colitis(UC)and Crohn's disease(CD).The pathogenesis of UC is the result of complex effects of various factors such as environmental factors,intestinal microbes,genetic susceptibility and immune factors.Immunoregulatory disorders are the key factors in their pathogenesis.Recent studies have shown that activation of the intestinal mucosal immune response is the direct cause of UC development,progression and outcome.Human T cells are derived from the thymus and can be divided into two major cell subpopulations,CD4~+ and CD8+,depending on the phenotype.After antigen stimulation,antigen presenting cells and their secreted cytokines can induce differentiation of CD4~+ T cells into Th1,Th2,Th17,regulatory T cells(Tregs)and other cells.Interleukin-17(IL-17)is the main effector molecule secreted by Th17 cells.Th17 cells and autoimmune diseases such as RA,psoriasis and UC are all related.The intestinal mucosa of patients with UC has higher levels of Th17 and IL-17 in the inflammatory state,and mainly expressed in the lamina propria of the intestinal mucosa during the active phase.Th17 and IL-17 may be the important regulators of the pathogenesis of UC.Regulatory T lymphocytes(Tregs)are strongly associated with disease progression and immuno-inflammatory responses reduction by suppressing the proliferation and pro-inflammatory cytokine produced mainly by Th1 and Th17 cells.Th17 cells and Tregs also play crucially important roles during the progression of DSSinduced colon inflammation.Th17 cells,through the IL-17-dependent manner,stimulate activation of inflammatory infiltrations and diffusing hemorrhage of colonic mucosa,accelerating to crypt abscess formation and deterioration of UC.Tregs,owing to promoting the production of anti-inflammatory TGF-? and IL-10,or restraining the expansion of pro-inflammatory Th17 cells to suppress on-going colitis,named as the primary immunosuppressive cells in UC.A large number of oral bacteria of periodontitis patients can flow into the intestine with saliva.It is believed that the microbiota in saliva can affect the balance of the intestinal microbiota to a certain extent.Studies have shown that swallowing P.gingivalis can lead to changes in the intestinal microbiota leading to increased intestinal permeability,endotoxemia and systemic inflammation.Periodontitis contributes to the progression of colitis via a dual microbiome and immune mechanism,which are the ectopic colonization of expanded oral microbiota pathogens in the intestine and induction of abnormal immune responses by migratory Th17 cells.Small intestine structural alterations of epithelial stratification,basal lamina degeneration and neutrophil infiltration are found in the rats with ligature-induced periodontitis.The supplementation of intestinal probiotics may alleviate these defects.Oral infection with P.gingivalis impairs colonic motor functions,implying periodontal pathogens can be defined as a host response modifier in IBD.Local pro-inflammatory cytokines produced and activated at the oral cavity might enter the bloodstream and circulate to the intestines,thus having impacts on IBD.Studies have shown that UC patients have a significant higher incidence of periodontitis,deeper periodontal pocket depth and more tooth loss compared to healthy control group.This evidence indicates that periodontitis may be closely related to UC through a substantially altered microbiome.Peptidylarginine deiminase(PAD)is a protein-modifying enzyme and a critical virulence factor of P.gingivalis,which participates in many vital physiological processes,including cellular differentiation,immune responses and gene transcription.Peptidylarginine deiminase(PAD)can convert host proteins into citrulline by catalyzing the conversion of peptidylarginine residues to citrulline residues.Protein citrullination changes the structure and function of proteins,changes the immunological activity of chemokines,causes abnormalities in the host inflammatory response,and autoimmune reactions occur,resulting in multiple inflammatory diseases,such as systemic lupus erythematosus,rheumatoid arthritis,Alzheimer's disease and UC.Citrullination stimulates the activation of neutrophils and macrophages in the initial stage of the innate immune response to produce new citrullinated peptides.These citrullinated peptides stimulate as well as the formation of autoantibodies and promote the perpetuation and progression of intestinal inflammation.The expression of citrullinated peptides in colonic biopsy of IBD patients was higher than that of the control group.Immunohistochemistry showed that PAD staining was strong in the intestinal lamina propria cells of DSS-induced acute colitis mouse models and UC patients,confirming that citrullination can promote inflammation.Porphyromonas gingivalis peptidylarginine deiminase(PPAD)is synthesized and released by P.gingivalis in membrane vesicles,which is able to accelerate protein citrullination in gingival tissues and encoded by the PG1424 gene.PPAD may be an important crosstalk factor in studying the underlying mechanisms of P.gingivalis and even the relationship between periodontitis and autoimmune diseases.Whether PPAD contributes to UC has not been investigated.In our study,?PG1424 mutant strain was constructed by gene knockout technology and identified to provide a theoretical basis for studying the function of PPAD.The UC mice model was established by dextran sodium sulfate to study the role of PPAD in immune response in intestinal diseases,and to explore whether PPAD will affect the severity of UC.At the same time,P.gingivalis W83 and ?PG1424 were co-cultured with CD4~+ T cells respectively,and the percentage of Th17 cell and Tregs,expression levels of IL-17 and IL-10 in serum were detected whether P.gingivalis can promote inflammatory immunity by promoting the transformation of CD4~+ T cells into pro-inflammatory Th17 cells and exacerbates the development of UC.Methods: 1.The homologous recombination vector of PG1424 gene was constructed and electroporated into P.gingivalis W83 strain to inactivate the PG1424 gene in the genome.The exogenous insertion gene is an erythromycin resistance gene with erythromycin resistance,and the positive strain can be screened by the erythromycin resistance medium.It was identified by methods such as realtime PCR to provide a bacterial model for studying the function of PG1424 gene.2.30 mice(C57BL/6)were divided into 5 groups equally and randomly.Group 1(the control group): normal diet and water for 40 days.Group 2(P.gingivalis group): normal diet and water for 40 days,every other day was fed with l × l09 CFU/m L P.gingivalis W83 to the end of the 40 th day.Group 3(UC group): normal diet and water for 40 days,the UC model was constructed with 3% DSS solution for 10 days from the 31 st day to 40 th day.Group 4(UC wild strain group): normal diet and water for 40 days,every other day was fed with l×l09 CFU/m L P.gingivalis W83 from 1st day to the 40 th day,from the 31 st day 3% DSS solution was constructed for 10 days.Group 5(UC mutant strain group): the same to the Group 4,except feeding with ?PG1424 instead of P.gingivalis W83.During the experiment,the general condition of the mice was observed daily,the body weight was weighed,the disease activity index and the tissue damage index were calculated,and the severity of colitis was evaluated.On the 40 th day of the experiment,the mice in the five groups were removed from the eyeballs for blood collection,and the serum was separated and sacrificed by cervical dislocation.The colon was isolated to measure the length of the colon.HE of the colon tissue was performed for histopathological biopsy.Flow cytometry and ELISA kit were used to detect the percentage of Th17 cell and Tregs,expression levels of IL-17 and IL-10 in peripheral venous blood.3.In vitro study,P.gingivalis W83 and ?PG1424 were co-cultured with CD4~+ T cells for 72 h,respectively.The percentage of Th17 cell and Tregs,expression levels of IL-17 and IL-10 were detected to investigate whether PPAD can exacerbate the occurrence and development of UC by promoting inflammatory immune response by means of boosting the transformation of CD4~+ T cells to pro-inflammatory Th17.Results: 1.The homologous recombinant vector of PG1424 gene was constructed and electroporated into P.gingivalis W83 strain,and the foreign gene was inserted to inactivate PG1424 gene.The positive strains were screened by erythromycin resistant medium,and the PG1424 gene-inactivated strains were successfully constructed by realtime PCR.2.The three groups of mice drinking DSS solution showed significant weight loss,shortened colon length,and a large amount of blood in the intestinal lumen.DAI index,TDI index and histopathological results showed that the inflammatory changes were heavier,the ratio of Th17 cells and serum IL-17 levels were significantly higher,the ratio of Tregs and IL-10 were significantly lower,and the results of UC wild strain group were significant different from UC group and UC mutant strain group.There were no significant differences between the UC group and UC mutant strain group.Compared with the control group,the P.gingivalis group had slightly higher results than the control group,but no significant difference was observed.3.In vitro cytology experiments showed that both P.gingivalis W83 and ?PG1424 promoted the transformation of CD4~+ T cells to pro-inflammatory Th17 and the upregulation of Th17 in the supernatant,the major effector cytokine.The proportion of Tregs and the expression level of IL-10 decreased.Compared with ?PG1424,P.gingivalis W83 strain promoted the effect of CD4~+ T cells on pro-inflammatory Th17 transformation and IL-17 expression up-regulation obviously.Conclusions: 1.P.gingivalis W83 could exacerbate the severity of UC and might be one of the risk factors for intestinal disease.2.Compared with ?PG1424,mice infected with P.gingivalis W83 strain were more severe in UC,suggesting that PPAD might be the underlying mechanism of P.gingivalis W83 strain inducing CD4~+ T cells to promote proinflammatory Th17 transformation,promoting UC inflammatory response,aggravating the degree of UC.3.PPAD played an important role in the transformation of CD4~+ T cells into Th17,which exacerbated the severity of UC.The in-depth study of PPAD provided a new research direction for exploring the related factors of UC immune abnormal response.