Characterisation of the citrullinated proteome targeted by autoantibodies in rheumatoid arthritis
Prosjekt
- Prosjektnummer
- 2009067
- Ansvarlig person
- Gustavo de Souza
- Institusjon
- Oslo universitetssykehus HF
- Prosjektkategori
- Doktorgradsstipend
- Helsekategori
- Inflammatory and Immune System
- Forskningsaktivitet
- 1. Underpinning
Rapporter
As previously agreed with HSØ and the current project leader, the project in question was redesigned to improve its chance for a successful completion. This has to be done due to the fact that the original project leader left his position at the UiO and moved back to his home country in the end of 2010, as documented in the report of the year 2011.
As previously reported, the PhD candidate collected all data required for completion of her PhD by the end of 2013 and started working in in the drafting of the articles required for the thesis submission. As stated before, the PhD candidate received no salaries from this project since 2014, neither other expenses were performed. The progress has been slow, but progress was achieved: the main data collected was finally accepted for publication in the strongest mass spectrometry journal available, The Molecular and Cellular Proteomics, in November 2015.
We are now in the process of writing the final articles and hopefully the PhD defense will be achieved during the current year of 2016.
Not applicable.
As previously agreed with HSØ and the current project leader, the project in question was redesigned to improve its chance for a successful completion. This has to be done due to the fact that the original project leader left his position at the UiO and moved back to his home country in the end of 2010, as documented in the report of the year 2011.As previously reported, the PhD candidate collected all data required for completion of her PhD by the end of 2013 and started working in in the drafting of the articles required for the thesis submission. While the progress had been slow, I am confident the thesis will be completed in due time. It mut be made clear thought that the PhD candidate received no salaries from this project in 2014, neither other expenses were performed.
In summary, we compared the proteome of primary Th1 and Th17 cell clones to characterize proteins relevant to T-helper cell differentiation, using a “label-free” mass spectrometry-based proteomic approach. This data was collected at the Proteomics Core Facility at Rikshospitalet. The candidate characterized and quantified over 7500 proteins from those cells, and biological replicates were fairly stable, demonstrating little perturbation from in vitro conditions. Transcriptomics data was also collected with the support of the Genomics Core Facility at Radiumhospitalet.
The candidate also collected proteomic data of the same cells in resting and activated states, aiming to identify molecules related to T cell activation. The expertise brought through the project above allowed the candidate to employ a similar proteomic approach to test the effect of methotrexate in a T-cell model (Jurkat). This drug is largely used in autoimmune disorders, including rheumatoid arthritis. In this case, due to the ease to grow the model in culture, the candidate used an elegant stable (non-radioactive) isotopic labeling of the cells to perform quantitative proteomics. Cytosolic analysis revealed the identification of more than 6000 proteins. As I reported previously I am satisfied that the candidate received excellent training in both quantitative proteomics and immunology.
One of the many interesting observations achieved in this project was the discovery of a specific population of the Th1 CD4+ T cells expressing proteins suggesting that some of Th1 cells could have certain phenotypic features very rarely observed in CD4+ cells (only reported once in Th17 cells expressing very high levels of NKG2D) and most commonly seen in CD8+ cells. Using flow cytometry we confirmed that our observations were not restricted to the two Th1 cells used in the proteomic experiments, since 7 out of 20 other different Th1 clones also expressed the molecules identified by proteomics (and most importantly, were also CD8-). We performed several validation experiments to demonstrate that such cells not only exist in vitro, but they also present in vivo and possess some clinical relevance. The analysis of this data is under way and is the missing part to finish the 1st publication. We are confident that with these final results, the candidate 1st article for her PhD thesis can be of potential high impact factor.
As previously agreed with HSØ and the current project leader, the project in question was redesigned to improve its chance for a successful completion. This has to be done due to the fact that the original project leader left his position at the UiO and moved back to his home country in the end of 2010, as documented in the report of the year 2011.The PhD candidate collected all data required for completion of her PhD validated the data with help of collaborators from the group. We are currently working in the drafting of the 1st article to be soon submitted. The candidate had been also working in the data analysis and planning of at least another two articles, possibly three.
As described previously, our main aim was to compare the proteome of primary Th1 and Th17 cell clones. Initially, the candidate selected 4 distinct T cell lines previously isolated from biopsies of patients with Crohn’s disease (two cells for each phenotype). In addition, one cell for each phenotype was also grown twice in different batches, as well as using different stimuli methods, resulting a total of 5 biological replicates for each Th phenotype. This was done to assess any in vitro perturbation which could probably occur in individual replicates. The candidate characterized and quantified over 7500 proteins from those cells, and biological replicates were fairly stable, demonstrating little perturbation from in vitro conditions. Transcriptomics data was also collected with the support of the Genomics Core Facility at Radiumhospitalet.
The candidate also collected proteomic data of the same cells in resting and activated states, aiming to identify molecules related to T cell activation. Candidate also performed experiments comparing gluten-specific T cell clones isolated from Celiac disease. The expertise brought through the project above allowed the candidate to employ a similar proteomic approach to test the effect of methotrexate in a T-cell model (Jurkat). Cytosolic analysis revealed the identification of more than 6000 proteins. Overall I am satisfied that the candidate received excellent training in both quantitative proteomics and immunology.
One of the many interesting observations achieved was the discovery of a specific population of the Th1 CD4+ T cells expressing proteins suggesting that some of Th1 cells could have certain phenotypic features very rarely observed in CD4+ cells (only reported once in Th17 cells expressing very high levels of NKG2D) and most commonly seen in CD8+ cells. We confirmed that our observations were not restricted to the two Th1 cells used in the proteomic experiments, since 7 out of 20 other different Th1 clones also expressed the molecules identified by proteomics (and most importantly, were also CD8-). We confirmed that the phenotypic feature could be achieved in vitro when the cells were stimulated. We were then concerned that our observation could be an artifact resultant from cells growing in artificial conditions. However, once again using flow cytometry in cells freshly isolated from gut biopsy of one patient, we confirmed that these cells with unique phenotype do exist in vivo and represent close to 17% of the Th1 CD4+ cells in the donor. We are now testing further donors to check how often these cells are present per patient, and also in control donors (hospital patients with biopsies collected but without inflammation disorders such as Chron’s or celiac disease). We hope to conclude that such cells not only exist in vivo, but they also possess some clinical relevance since they are often observed in patients with gut inflammation but not in controls. We are confident that with these final results, the candidate 1st article for her PhD thesis can be of potential high impact factor.
Finally, the candidate disputation is planned for 2nd half of 2014.
As previously agreed with HSØ and the current project leader, the project was redesigned to improve its chance for a successful completion. This has to be done due to the fact that the original project leader left his position at the UiO and moved back to his home country in the end of 2010, as documented in the report of the year 2011.The progress of the project in the year of 2012 has been extremely satisfactory. The PhD candidate managed to collect 90% of all data required for completion. As is now, the candidate is finishing the last couple of bench experiments, performing heavy data analysis and writing the drafts of three articles. While there has been a short delay in the planned schedule, it mostly happened due to the fact that the results obtained were of quality above the expected (as described below) and this required additional efforts to process them. Therefore, in late 2012 we requested an extension of the project to August 2013 with current funds, which was approved by HSØ research administration.
As described previously, the main aim is to compare the proteome of primary Th1 and Th17 cells, in order to observe molecules relevant to T-helper cell differentiation. For this the candidate used a “label-free” proteomic approach. Initially, the candidate selected 4 distinct T cell lines previously isolated from biopsies of patients with Crohn’s disease (two cells for each phenotype). In addition, one cell for each phenotype was also grown twice in different batches, as well as using different stimuli methods (soluble cytokine vs. bead-immobilized cytokines), resulting a total of 5 biological replicates for each Th phenotype. This was done to assess any in vitro perturbation which could probably occur in individual replicates. Through using high-end instrumentation that became available to the lab in 2012, the candidate characterized and quantified over 7500 proteins from those cells. As is, candidate is now focusing in understanding the participation of transcription factors that are differentially expressed between the Th1 and Th17 cells. The candidate also demonstrates that the biological replicates are very stable in vitro, regardless if cells had been grown in different periods or with different cytokine feeding methods. This is an interesting observation that will possibly grant the candidate a publication in itself.
The expertise brought through the project above allowed the candidate to employ a similar proteomic approach to test the effect of methotrexate in a T-cell model (Jurkat). This drug is largely used in autoimmune disorders, including rheumatoid arthritis. In this case, due to the ease to grow the model in culture, the candidate used an elegant stable (non-radioactive) isotopic labeling of the cells to perform quantitative proteomics. For similar reasons, the candidate was also able to collect both cytosolic and nucleic fractions of the cells. Cytosolic analysis revealed the identification of more than 6000 proteins, and the analysis of nucleic fraction is currently under way. With the expertise acquired for this experiment, the candidate became a capacitated proteomics researcher in two of the most used quantitative approaches available.
As previously agreed with HSØ and the current project leader, the project in question was redesigned to improve its chance for a successful completion. This has to be done due to the fact that the original project leader left his position at the UiO and moved back to his home country in the end of 2010, as documented in last year report.This realistic change in project direction was critical for the proper development of the PhD candidate. Her performance in 2011 had been satisfactory, and is my belief that the minimal requirements for obtaining the PhD title will be completed by the end of 2012. The PhD candidate is very close to submit her first article to an indexed journal, and data for another two articles is already being collected. In addition, to perform this tasks, the PhD candidate had been developing mass spectrometry/data analysis skills which are limited in the Norwegian research community and are in great demand, which improves her competitive edge to follow up her career. For example, in the summer of 2011 the candidate received training in a proteomics course offered at the top proteomics group in the field, at the Dept. of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry in Martinried, Germany, under the direction of Dr. Matthias Mann.
Briefly, the candidate scientific achievements so far are as follow: as described in the previous report, we intended to compare the proteome of Th1 and Th17 cells, in order to observe molecules relevant to T-helper cell differentiation. The candidate characterized and quantified over 2600 proteins from two of such cells, and initially selected a few transcription factors which were differentially expressed between the Th1 and Th17 cells. Further validation using antibodies and different biological replicates (different cells with similar cytokine profiles) had show that at least one of the transcription factors is constantly observed as a highly expressed protein in interferon-gamma (Th1) producing cells. The function of this factor is poorly characterized in the literature, and we believed we uncovered an important molecular feature of T-helper cells differentiation.
Finally, the characterization of these cells also served us as a pilot project, to estimate the level of proteomic coverage (i.e. minimal number of protein identification) we might achieve using them. The candidate is now currently testing the effect of methotrexate in T-cells. This drug is largely used in autoimmune disorders, including rheumatoid arthritis. As described above, the data for this experiment is already being collected, and utilizes elegant stable (non-radioactive) isotopic labeling of the cells.
In October 2010, the leader of the Proteomic Core Facility (PCF) at Rikshospitalet and leader of this project, Burkhard Fleckenstein, had left his position to follow his career in Germany. For his position, the Institute of Immunology hired Gustavo de Souza, a researcher with formation in Cell Biology and quantitative Mass Spectrometry-based Proteomics. Dr. de Souza assumed all tasks from Burkhard Fleckenstein including PCF service management and leadership of the research group.Without question, this will have a big impact in the project development and in the development of Ms. Tahira Riaz as a scientist, if project aims are not realistically modified. Understandably, project progression for the year of 2010 was very limited, given the fact that project leader had left the group. As a consequence, the project is now behind schedule. Furthermore, it is reasonable to argue that the project need to have further polishing in order to the PhD candidate to achieve the minimal criteria for a successful program completion. This will have to take in consideration not only the aims initially proposed, but also the different level of expertise in Cell Biology from the new group leader.
In rheumatoid arthritis (RA), T cells play a key role in the autoimmune response, specially Th17 T-cells. We plan to initially characterize the proteome of Th1 and Th17 cells, in order to observe molecules relevant to Th17 differentiation and is possible role in RA pathogenesis. In addition, by using cell culture T-cell models, we will investigate the role of TNF-alpha blockers in this cell biology. This compound is now largely used in RA treatment, yet its molecular basis is poorly understood. If time allows, Ms Tahira Riaz will use her currently level of progression with the ETD instrumentation at PCF to investigate relevant post-translational modifications as phosphorylation and citrullination. Altogether, I am confident that with such project modifications Ms Tahira Riaz will fulfill any requirements for a successful PhD within the remaining period for this grant.
The feasibility of such changes is extremely straightforward, as I have been showing in previous orientation work with primary human macrophages (Tomazella GG et al., Proteome Science v.7 p.32, 2009; Tomazella GG et al., Journal of Proteome Research v.9 p.2030, 2010) and primary human monocyte-derived dendritic cells (Tomazela GG, Hovden AO, Thiede B, Wiker HG, Appel S, de Souza GA. Article in preparation).
Ms Tahira Riaz has been selected as a PhD student and started her work in August 2009. She is using proteomics to study the role of protein citrullination in rheumatoid arthritis. In the beginning she focused on the required mass spectrometric techniques.The application for funding for a PhD student was given without a name of a candidate. The position was announced in early 2009, but an appropriate candidate was not identified before May 2009. In August 2009, the selected candidate, Ms Tahira Riaz, has started to work as a PhD student in our group (3-years contract).
Ms Riaz has done her Master thesis in the proteomics laboratory of Bernd Thiede (Biotechnology Centre, Oslo) and has worked as an engineer at the FUGE/NFR proteomic node at UMB in Ås. Thus, Ms Riaz has a profound background in mass spectrometry based proteomics and is nicely qualified for her PhD project.
The project aims to study protein citrullination in rheumatoid arthritis (RA). Proteins with this posttranslational modification represent targets recognized by anti-citrullinated protein antibodies (ACPA) which are diagnostic markers of RA. We will specifically enrich citrullinated proteins and peptides from synovial fluid of RA patients and identify them by mass spectrometric analysis. Since Ms Riaz started in August 2009, she has focused on the setup of LC-MS/MS analysis on our new ion trap mass spectrometer which is equipped with electron transfer dissociation (ETD) technology for a better identification of these proteins. This instrumentation was purchased in September 2009 and is essential for Ms Riaz’ future work in the project. In addition, Ms Riaz has performed experiments using capillary electrophoresis as another very sensitive analytical method. Altogether, Ms Riaz’ project has had a good start and we expect interesting results to be published in the future.
Vitenskapelige artikler
Riaz T, Sollid LM, Olsen I, de Souza GA
Quantitative Proteomics of Gut-Derived Th1 and Th1/Th17 Clones Reveal the Presence of CD28+ NKG2D- Th1 Cytotoxic CD4+ T cells.
Mol Cell Proteomics 2016 Mar;15(3):1007-16. Epub 2015 des 4
PMID: 26637539 - Inngår i doktorgradsavhandlingen
Deltagere
- Tahira Ahmad Riaz Doktorgradsstipendiat
- Gustavo Antonio De Souza Prosjektleder
- Tahira Riaz Doktorgradsstipendiat
- Ludvig Magne Sollid Biveileder
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