Functional genomics to dissect complex human lupus phenotype – LEDsGO

We have identified an overexpression of Trib1 and Fkbp11 genes in B cells from lupus patients in remission phases. In order to better understand the consequences of this overexpression on B cell phenotype and to identify the mechanisms of the maintenance of the clinically silent phases, we have developed transgenic mice overexpressing Fkbp11 or Trib1. These mice have an interesting phenotype that we propose to study in this project. Interestingly, Fkbp11 overexpressing mice produce some autoantibodies. In order to understand the mechanisms underlying this autoantibody production, we crossed these mice with other transgenic mice allowing for a fine analysis of autoreactive B cell activation and autoantibody production, and also with mice with a strong genetic background for the development of autoimmunity, in order to see if Fkbp11 overexpression exacerbates the symptoms of the disease.
Concerning Trib1, we have produced B cell specific Trib1 overexpressing mice. These mice have a decreased antibody production, which is very interesting to explain the settings and maintenance of remission phases in lupus patients. This project proposes to study in detail the mechanisms of this decrease of antibody production, in the transgenic mice at a basal level, and after immunization with several antigens (to mimic a vaccination) or viral or bacterial infection.
We also propose to search for Trib1 partners in B cells, via mass spectrometry, in order to dissect the molecular pathways underlying this phenotype. The identified partners will be validated by complementary techniques. Then mutants of these partners could be generated to test their effect on B cell phenotype.

Fkbp11 overexpressing mice develop a phenotype of B cell hyperactivation, in addition to a B cell tolerance breakdown (increased production of autoantibodies). We have shown that Fkbp11 overexpression exacerbated some features of autoimmunity in the B6lpr/lpr murine model of lupus. We also used the well-documented 56R anti-DNA murine model to study in more detail the mechanism of autoantibody production. Our results on 56R mice demonstrate that the production of autoantibodies is increased when Fkbp11 is overexpressed. This phenotype was not readily explained by detectable abnormalities in central tolerance (mechanisms allowing for the elimination of autoreactive B cell in normal individuals). Secondly, our data showed that overexpression of Fkbp11 can promote the initiation of plasma cell differenciation (the plasma cells being the antibody secreting cells), and localize FKBP19 protein (encoded by Fkbp11 gene) upstream of Pax5 (on of the master regulators of plasma cell differentiation) during the plasma cell differentiation pathway.
Concerning Trib1, we have completed the analysis of Trib1 overexpressing mice and have shown that this decrease of antibody production by B cells was noticed in several situations (basal level, vaccination…), and that this phenotype is not the consequence of an increased B cell death, a decreased B cell proliferation, a failure in class switching process (allowing for the production of antibodies of different classes) or in plasma cell differentiation, but seems rather to be linked to a defect in B cell antibody secretion process. Finally, we showed that this defect also concerns autoantibody production. In conclusion, we describe here a new role of Trib1 as a negative regulator of B cells, which could constitute a new mechanism of B cell control during remission phases of lupus.
These results have important consequences in fundamental biology but also in the comprehension of the physiopathological pathways implicated in lupus.

In order to better explain the mechanisms of the different traits of phenotype that we observed in our transgenic murine models, we propose to search for FKBP19 (protein encoded by Fkbp11) and Trib1 partners in B cells. This could help to identify new biological pathways implicated in autoantibody production, in the differentiation of B cells to antibody/autoantibody secreting cells, and in the control of antibody/autoantibody production.
We have begun the experiments for the research of Trib1 partners in B cells in order to identify the biological pathways that are deregulated by Trib1 overexpression, and that could be implicated in the decrease of antibody production in our transgenic model. We have identified very interesting partners, which role in B cells in today unknown. We plan now to complete these experiments, and validate the partners with complementary techniques (immunoprecipation, Western-Blot, production of mutants). In parallel, we have started the analysis of the activation of several signaling pathways important in B cell function, and have shown a hypophosphorylation (i.e. a decrease of activation) of Erk MAPK in Trib1 overexpressing B cells. These results are important because Trib1 is known, in other cells than B cells, to regulate this MAPK pathway. It will be important to see if the immunosuppressive phenotype observed in Trib1 overexpressing cells is reproduced when the MAPK Erk pathway is controlled by the use of inhibitors of Erk. We have also initiated experimental infections to see if the immunosuppressive phenotype of Trib1 overexpressing B cells also concerns humoral responses to pathogens. Finally, we plan to investigate TRIB1 abnormal overexpression in some primary immunodeficiencies with hypogammaglobulinemia, i.e. diseases with abnormalities in antibody production.

Publications :
1. Overexpression of Fkbp11, a feature of lupus B cells, leads to B cell tolerance breakdown and initiates plasma cell differentiation.

Julie Ruer-Laventie, Lea Simoni, Jean-Nicolas Schickel, Anne Soley, Monique Duval, Anne-Marie Knapp, Luc Marcellin, Delphine Lamon, Anne-Sophie Korganow, Thierry Martin, Jean-Louis Pasquali, & Pauline Soulas-Sprauel.
Immunity, inflammation and Disease, in press

Participation to congress:
Oral communications:
1. Ruer-Laventie J., Schickel J-N, Simoni L.*, Soley A., Marcellin L., Duval M., Knapp A-M, Martin T., Korganow A-S, Pasquali J-L, Soulas-Sprauel P. Overexpression of FKBP11, a feature of lupus B cells, leads to B cell tolerance breakdown and initiates plasma cell differentiation. Autoimmunity 2014 Congress. Nice, France, 26-30 mars 2014.
* Communication orale effectuée par L. Simoni, thesis student.

2. Soulas-Sprauel P. Functional genomics to dissect complex human lupus phenotype. Journée mondiale du lupus Strasbourg 2015. Strasbourg, France. 10-11 mai 2015.

Posters:
1. Ruer-Laventie J., moni L., Schickel J-N, Soley A., Duval M., Knapp A-M, Marcellin L., Korganow A-S, Pasquali J-L, Martin T., Soulas-Sprauel P. Overexpression of Fkbp11, a feature of lupus B cells, leads to B cell tolerance breakdown and initiates plasma cell differentiation. Keystone Congress. The golden anniversary of B cell discovery. Banff, Canada. 22-27 mars 2015.

2. Simoni L., Ruer-Laventie J., Soley A., Duval M., Martin T., Pasquali J-L, Korganow A-S, Soulas-Sprauel P. Immunosuppressive role of Trib1 in B cells. Keystone Congress. The golden anniversary of B cell discovery. Banff, Canada. 22-27 mars 2015.

This project proposes to develop functional genomic studies to dissect complex human lupus phenotype. SLE is a severe and heterogeneous autoimmune disease, characterized by the production of various pathogenic autoantibodies, which participate in immunopathology and end-organ damages. Both genetic (most often in a polygenic manner) and environmental factors contribute to the development of the disease.
The heterogeneity of SLE is evident from different points of views: the clinical phenotype is different from patient to patient, as well as the genetic susceptibility to the disease. We must consider that adult SLE arises from the building up of many subtle gene variations, each one adding a new brick on the SLE susceptibility, and each one contributing to a phenotypic trait to the disease.

Trying to understand the mechanism of the different phenotypic traits of the disease (loss of immune tolerance leading to autoantibody production, defect of apoptotic debris clearance, immune complexes related kidney pathology, diverse skin manifestations, arthritis…) is a huge and essential effort. On a strategic point of view, one can guess at least two different roads to identify such molecular mechanisms of the SLE phenotypic expressions. The first one starts from the genomic variants already identified during genome wide association studies (GWAS). However, these variations usually do not identify the causal gene, and it is likely that more than one gene variant is necessary to induce a phenotypic trait (because odds ratio compared to healthy individuals are weak). The second starts from a detailed wide analysis of gene expression variation in a precise cell type: this analysis should identify some deregulated genes as a resultant of the consequences of the biological effects of the diverse genetic variants present in a patient.

This proposal uses the second road. We already identified a list of deregulated genes in purified B cells of quiescent SLE patients. We started to create new mouse models to reproduce the human SLE gene expression variations and have already shown that this functional genomic approach is successful (1). We now propose to focus on two highly promising genes for which we have preliminary results: 1) FKBP11, encoding an enzyme of peptidyl-prolyl cis/trans isomerase (PPIase) FKBP family, is overexpressed in B cells from quiescent SLE patients. FKBP11 lentigenic overexpressing mice develop some known phenotypic features of human SLE, notably autoantibody production; 2) Trib1, a regulator of MAPK signalling, is also overexpressed in B cells from quiescent SLE patients. B cell specific conditional Trib1 Knock-In mice develop an immunosuppressive phenotype. Therefore, Trib1 overexpression could be implicated in the maintenance of the quiescent phase of SLE.

The aim of this project is to understand the mechanisms of our transgenic mouse model’s phenotype: analysis of B cell tolerance breakdown and plasma cell differentiation in FKBP11 lentigenic mice, and analysis of the mechanisms of immunosuppressive phenotype in B cell specific Trib1 overexpressing mice. In addition, we propose to identify FKBP19 (protein encoded by FKBP11 gene) and Trib1 partners by proteomic analysis (immunoprecipitations and mass spectrometry analysis), in order to better define biological pathways regulated by these 2 proteins. In a last specific aim, we propose to confirm our results in human SLE B cells.

This project will help to better characterize the role of these 2 genes (today mostly unknown) in B cell function (and in immune system in general), and in autoimmunity (for FKBP11) and immunosuppression (for Trib1), with encouraging preliminary results. This could lead to the identification of new biological pathways for SLE development and for remission phase’s establishment in SLE patients, with potential therapeutic applications.

References:
1) Schickel, J.N. (last author: P. Soulas-Sprauel). EMBO Mol Med, 2012, 4(12): p. 1261-75.