Periodic Reporting for period 1 - VIBRiOD (VIrus and BacteRia In Oral Disease: the EBV model in periodontitis)
Berichtszeitraum: 2020-05-01 bis 2022-04-30
• Periodontitis is an infectious/inflammatory disease that destroys the tooth-supporting apparatus leading to teeth loss. It is also associated with 50 systemic diseases & cancers. Thus, the development of effective therapies requires deeper insights into the etiopathogenesis of periodontitis.
• Periodontitis is 1 of the most prevalent human diseases, severe periodontitis being the 6th most common disease, with an overall prevalence of 11.2% & 743 million people affected. The total economic impact represents 442 billion USD. Obviously, periodontitis represents a major global health problem with significant social & economic impact. The results of the VIBRiOD have the potential to contribute to the advancement of periodontitis research & its ultimate control.
• VIBRiOD was focused on the etiopathogenesis of periodontitis based on synergies between Epstein-Barr virus (EBV), pathogenic bacteria & human immune response. The objectives were:
1. Detect the EBV infection during periodontitis & establish the relationship to dysbiosis;
2. Understand the relationship between EBV, immune responses & dysbiosis;
3. Characterize the phenotypic & immune properties of infiltrated EBV+ cells;
4. Study the effects of healthy & pathogenic biofilms on EBV viral replication.
• Periodontitis is 1 of the most prevalent human diseases, severe periodontitis being the 6th most common disease, with an overall prevalence of 11.2% & 743 million people affected. The total economic impact represents 442 billion USD. Obviously, periodontitis represents a major global health problem with significant social & economic impact. The results of the VIBRiOD have the potential to contribute to the advancement of periodontitis research & its ultimate control.
• VIBRiOD was focused on the etiopathogenesis of periodontitis based on synergies between Epstein-Barr virus (EBV), pathogenic bacteria & human immune response. The objectives were:
1. Detect the EBV infection during periodontitis & establish the relationship to dysbiosis;
2. Understand the relationship between EBV, immune responses & dysbiosis;
3. Characterize the phenotypic & immune properties of infiltrated EBV+ cells;
4. Study the effects of healthy & pathogenic biofilms on EBV viral replication.
• O1: A total of 54 participants were recruited including 27 healthy (H) volunteers & 27 patients with active periodontitis (PA). To test for the presence of EBV, the EBV R-gene kit was used to detect EBV DNA in the gingival tissues of participants. Following the EBV detection, all the 54 samples were categorized as EBV- or EBV+. EBV was prevalent in 75.9% of the total. Meanwhile, EBV was detected in PA at a higher frequency than in H (81.5% vs 70.4%). The high prevalence of EBV in both H & PA tissues may suggest that EBV spreading is common in gingival tissues even in the absence of inflammatory lesions. To establish the relationship between EBV infection & dysbiosis, several pathogenic bacterial species were detected in EBV- & EBV+ gingival tissues using RT-qPCR. The results were inconclusive to delineate the association between EBV & pathogenic bacteria & further experiments will be required.
• O2: Microarray transcriptional profiling was used to provide a comprehensive description of differential gene expression signatures & biological processes in H & PA-affected tissues that were modulated by EBV infection. 3 scenarios were observed: comparison of the states of H & PA disease, EBV effect in H & PA-affected tissues. In summary, in periodontally-diseased tissues, the host bioprocesses linked to excessive immune/inflammatory responses & tissue degradation were documented. While in the presence of EBV in periodontally-diseased tissues these processes were affected dramatically as EBV augmented the deleterious state of the disease by worsening the inflammatory condition, promoting the B (BC) & plasma (PC) cell infiltration into the lesion & advancing the epithelial tissue degradation. In contrast, in H tissues EBV provoked typical host immune responses against the viral challenge. Overall, this study provides the first evidence to support EBV as a significant pathogenic contributor to periodontitis. These results were presented at the EMBO Workshop as a poster presentation. Also in preparation for submission to the JCI.
• O3: In vitro model was established for BC sequential differentiation into PC as a multi-step 11-day culturing process. At the same time, this BC-to-PC transition was accompanied by the infection of BCs with EBV. Generated various cell populations were further analyzed for the gene expression regarding BC-to-PC development markers, EBV infection & cytokine profile using RT-qPCR. For determination of BC phenotype, the expression of PAX5 & CD21 were examined showing a progressive decrease from D1 to D11 in non-infected and infected cultures. For PC profile, the expression of BLIMP1, CD38 & CD138 were studied. The CD38 was increased from D1 to D11 in non-infected & infected cultures. The fate of BLIMP1 & CD138 were ambiguous in our experiments. As the EBV infection indicator, the expression of 2 latent & 3 lytic genes were monitored. In non-infected cultures there was no expression of all EBV genes, while in infected cultures from iD4 to iD11 the expression of 5 EBV genes dropped down progressively. A broad literature review is required to interpret these observations. For cytokine profile, IL1β, IL6, IL8 & TNFα were monitored. The IL1β increased from D1 to D11 in non-infected & infected cultures, IL6 & TNFα were the highest in D1, while IL8 was similarly high & stayed stable in all the non-infected & infected cultures. For the understanding of cytokine profile, a broad literature search is also needed following which a possible research article is feasible.
• O4 was not achieved because of the complexity & lack of time related to COVID19 restrictions. Instead, an attempt was made to investigate EBV infection of junctional epithelial cells (JECs) isolated from periodontal tissues. JECs & EBV infection of JECs were detected by immunofluorescent staining of cytokeratin 19 & EBNA1, respectively. In addition, EBV gene expression profiles were determined by RT-qPCR for EBNA1, LMP2 & BZLF1. The micrographs from non-infected cells showed no fluorescence signal from EBNA1, while infected JECs were positive for EBNA1. Regarding the EBV gene expression in JECs, there was no expression of EBV genes in non-infected JECs, while all EBV transcripts were detected in EBV-infected JECs in the following decreasing order: LMP1>BZLF1>EBNA1. These promising results that JECs may be a possible target of EBV infection could be an important section of an imminent publication.
• O2: Microarray transcriptional profiling was used to provide a comprehensive description of differential gene expression signatures & biological processes in H & PA-affected tissues that were modulated by EBV infection. 3 scenarios were observed: comparison of the states of H & PA disease, EBV effect in H & PA-affected tissues. In summary, in periodontally-diseased tissues, the host bioprocesses linked to excessive immune/inflammatory responses & tissue degradation were documented. While in the presence of EBV in periodontally-diseased tissues these processes were affected dramatically as EBV augmented the deleterious state of the disease by worsening the inflammatory condition, promoting the B (BC) & plasma (PC) cell infiltration into the lesion & advancing the epithelial tissue degradation. In contrast, in H tissues EBV provoked typical host immune responses against the viral challenge. Overall, this study provides the first evidence to support EBV as a significant pathogenic contributor to periodontitis. These results were presented at the EMBO Workshop as a poster presentation. Also in preparation for submission to the JCI.
• O3: In vitro model was established for BC sequential differentiation into PC as a multi-step 11-day culturing process. At the same time, this BC-to-PC transition was accompanied by the infection of BCs with EBV. Generated various cell populations were further analyzed for the gene expression regarding BC-to-PC development markers, EBV infection & cytokine profile using RT-qPCR. For determination of BC phenotype, the expression of PAX5 & CD21 were examined showing a progressive decrease from D1 to D11 in non-infected and infected cultures. For PC profile, the expression of BLIMP1, CD38 & CD138 were studied. The CD38 was increased from D1 to D11 in non-infected & infected cultures. The fate of BLIMP1 & CD138 were ambiguous in our experiments. As the EBV infection indicator, the expression of 2 latent & 3 lytic genes were monitored. In non-infected cultures there was no expression of all EBV genes, while in infected cultures from iD4 to iD11 the expression of 5 EBV genes dropped down progressively. A broad literature review is required to interpret these observations. For cytokine profile, IL1β, IL6, IL8 & TNFα were monitored. The IL1β increased from D1 to D11 in non-infected & infected cultures, IL6 & TNFα were the highest in D1, while IL8 was similarly high & stayed stable in all the non-infected & infected cultures. For the understanding of cytokine profile, a broad literature search is also needed following which a possible research article is feasible.
• O4 was not achieved because of the complexity & lack of time related to COVID19 restrictions. Instead, an attempt was made to investigate EBV infection of junctional epithelial cells (JECs) isolated from periodontal tissues. JECs & EBV infection of JECs were detected by immunofluorescent staining of cytokeratin 19 & EBNA1, respectively. In addition, EBV gene expression profiles were determined by RT-qPCR for EBNA1, LMP2 & BZLF1. The micrographs from non-infected cells showed no fluorescence signal from EBNA1, while infected JECs were positive for EBNA1. Regarding the EBV gene expression in JECs, there was no expression of EBV genes in non-infected JECs, while all EBV transcripts were detected in EBV-infected JECs in the following decreasing order: LMP1>BZLF1>EBNA1. These promising results that JECs may be a possible target of EBV infection could be an important section of an imminent publication.
Researcher’s training
Autoclave training on the proper & safe use of the autoclaves; Statistical training “R software”; Course “French foreign language”; Statistics & bioinformatics “Ingenuity Pathway Analysis”.
Transfer of knowledge
The project ensured a 2-way transfer of knowledge between the researcher & the host. The researcher expanded her knowledge; gained experience in new techniques. She participated in the training & up-skilling of PhD students, supported the host lab in the development of new methods, helped solve the problems raised in the lab, etc.
Dissemination of project results
The results were presented at EMBO Workshop Pathogen Immunity & Signaling.
The output of the research may be of interest to dental practitioners. The knowledge generated during the project can be implemented into everyday clinical care.
The fellow is a member of the collaborative Detect EBV group. An interview was given by the fellow for #detectEBV.
A presentation on World Oral Health Day was given by the fellow for a “French foreign language” course to reach UCA’s personnel to advance oral health.
Impact on researcher’s career
The project enhanced the future career prospects of the researcher by granting competencies in new fields; added a range of additional skills; added valuable scientific publications to her CV (first author: 1 published, 1 in preparation, 2 anticipated; co-author: 1 published, 2 anticipated); provided collaborations with highly qualified partners.
The next career step is the 1-year contract with the CHU Nice & the host lab (PHRC HERPARO). Also, the researcher submitted a proposal to obtain funding from Eklund Foundation for 18-month research.
Autoclave training on the proper & safe use of the autoclaves; Statistical training “R software”; Course “French foreign language”; Statistics & bioinformatics “Ingenuity Pathway Analysis”.
Transfer of knowledge
The project ensured a 2-way transfer of knowledge between the researcher & the host. The researcher expanded her knowledge; gained experience in new techniques. She participated in the training & up-skilling of PhD students, supported the host lab in the development of new methods, helped solve the problems raised in the lab, etc.
Dissemination of project results
The results were presented at EMBO Workshop Pathogen Immunity & Signaling.
The output of the research may be of interest to dental practitioners. The knowledge generated during the project can be implemented into everyday clinical care.
The fellow is a member of the collaborative Detect EBV group. An interview was given by the fellow for #detectEBV.
A presentation on World Oral Health Day was given by the fellow for a “French foreign language” course to reach UCA’s personnel to advance oral health.
Impact on researcher’s career
The project enhanced the future career prospects of the researcher by granting competencies in new fields; added a range of additional skills; added valuable scientific publications to her CV (first author: 1 published, 1 in preparation, 2 anticipated; co-author: 1 published, 2 anticipated); provided collaborations with highly qualified partners.
The next career step is the 1-year contract with the CHU Nice & the host lab (PHRC HERPARO). Also, the researcher submitted a proposal to obtain funding from Eklund Foundation for 18-month research.