Characterization of inflammatory and immune processes

Scientific coordinator: Anne Gonzalez de Peredo (gonzalez@ipbs.fr)

 

Major research axis:

 


 

Proteomic analysis of the inflammatory response of endothelial cells

In collaboration with the team of J-P Girard at IPBS, we use large-scale proteomic analysis associated with label-free quantification to study the pathways and processes activated in endothelial cells (ECs) under inflammatory conditions.

 

More than 10 years ago, we developed original bioinformatics tools to extract peptide signals from raw MS files, measure their intensity, and perform quantitative analysis of protein expression in large-scale studies. Using such methods, we could extensively map the ECs proteome, and provided the most complete proteomic characterization to date of the human EC response to several inflammatory cytokines, such as TNFα, IFNg and IL1β (1,2).

 

We keep on applying such a global proteomic approaches, for example to analyze IL-33 function in endothelial cells. IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. We compared the extracellular and intracellular roles of IL-33 inprimary human endothelial cells, and found that exogenous extracellular IL-33 acts as a cytokine inducing expression of a distinct set of proteins associated with inflammatory responses in endothelial cells, but not as a nuclear factor (3).

 

Selected publications

1- Bouyssie, Gonzalez de Peredo et al. Mascot file parsing and quantification (MFPaQ), a new software to parse, validate, and quantify proteomics data generated by ICAT and SILAC mass spectrometric analyses: application to the proteomics study of membrane proteins from primary human endothelial cells. Molecular & cellular proteomics, 2007.
https://doi.org/10.1074/mcp.t600069-mcp200

 

2-Gautier et al. Label-free quantification and shotgun analysis of complex proteomes by one-dimensional SDS-PAGE/NanoLC-MS: evaluation for the large scale analysis of inflammatory human endothelial cells. Molecular & cellular proteomics, 2012.
https://doi.org/10.1074/mcp.m111.015230

 

3-Gautier et al. Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells. Sci Rep, 2016.
https://doi.org/10.1038/srep34255

Proteomic analysis of the inflammatory response of
endothelial cells upon TNFa/IFNg stimulation

 

Functional mechanisms of interleukin-33, a cytokine playing a major role in innate immunity and allergy

Interleukin-33 (IL-33) is a nuclear cytokine from the IL-1 family with critical roles in tissue homeostasis and repair, type 2 immunity, viral infection, inflammation and allergy. It binds to the ST2 receptor expressed on cells of the innate and adaptive immune system. Major target cells of IL-33 include group 2 innate lymphoid cells (ILC2s), which play an important role in asthma.

In collaboration with the team of J-P Girard at IPBS, we apply mass spectrometry techniques to better characterize the mechanisms of IL-33 action.

Il-33 is activated by proteolytic maturation. We used sensitive mass spectrometry to analyze highly active mature forms of the protein resulting from proteolytic cleavage by activator inflammatory proteases, and map the cleavage sites in the central domain of IL-33 (1). We used similar approaches to finely dissect the mechanisms of IL-33 activation by a large range of environmental allergens (2).

In addition, using large-scale, unbiased proteomic methods, we study the response of target cells upon stimulation with IL-33 and co-stimulatory cytokines.

 

Selected publications

1- Lefrancais et al. IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G. Proc Natl Acad Sci U S A 2012.
https://doi.org/10.1073/pnas.1115884109

 

2-Cayrol et al. Environmental allergens induce allergic inflammation through proteolytic maturation of IL-33. Nat. Immunol. 2018.
https://doi.org/10.1038/s41590-018-0067-5

Mass spectrometry workflow for identification of cleavages sites. Using highly sensitive mass spectrometry, we identified the N-terminal peptides characteristic of the cleavages generated on IL-33 by a whole panel of allergens. These cleavage events induce activation of the cytokine, finally triggering asthma.

 


 

Characterization of the T Cell Receptor (TCR) pathway in primary T lymphocytes

One of our research areas is to decipher, at the cellular and molecular levels, the functional mechanisms of T lymphocytes using proteomic approaches. In collaboration with teams bringing complementary expertise in immunopathology and T cell signaling (B. Malissen, CIML, Marseille; R. Lesourne, CPTP, Toulouse; A. saoudi, CPTP, Toulouse), we are involved in the detailed characterization of the T Cell Receptor (TCR) pathway, by analyzing the signalosome of several key components of this complex system using AP-MS strategies, or the signaling pathways engaged upon activation of T-cells using phosphoproteomics.

In collaboration with B. Malissen and R. Roncagalli (CIML, Marseille), we use KI mouse model expressing different bait proteins of the TCR pathway with a C-terminal One-Strep-tag to enrich endogenous signaling complexes at different time points following TCR activation. Time resolved proteomics allows to obtain dynamics information and to gain insight into the mechanisms of action of several proteins and the internal organization of the interactomes (1-4).

 

Selected publications

1-Reginald et al. Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells. J Immunol 2015.
https://doi.org/10.4049/jimmunol.1501300

 

2-Voisinne et al. Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation. Mol Syst Biol 2016.
https://doi.org/10.15252/msb.20166837

 

3. Gaud et al. The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+T cells. Sci. Signal. 2018.
https://doi.org/10.1126/scisignal.aar3083

 

4. Voisinne et al. Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics. Nat. Immunol, in press.

AP-MS workflow for analysis of the TCR signalosome. We analyze by mass spectrometry signalling complexes that form around canonical proteins used by the proximal TCR signal transduction pathway, and monitor their dynamic of assembly in the first minutes following engagement of the TCR. For that, we use genetically engineered mice models, which express at endogenous level a bait protein with a OneStreptag at their C-terminus, allowing the purification of physiological complexes, directly from primary cells, in a very efficient way.

We could identify a global interaction network involving more than 200 unique proteins and more than 300 high-confidence protein-protein interactions.


 

Proteomic analysis of regulatory T cells

Regulatory T cells (Treg) represent a minor sub-population of T lymphocytes which is crucial for the maintenance of immune homeostasis. Using large-scale quantitative mass spectrometry, we performed an in-depth comparison of the proteomic profile of Treg versus conventional T cells, and defined a specific proteomic “signature” of Treg (1).

We focused on Themis1, a protein particularly under-represented in Treg, and recently described as being involved in the pathogenesis of immune diseases. Using a transgenic mouse model over-expressing Themis1, we provided in vivo and in vitro evidence of its importance for Treg suppressive functions. We also characterized the interactome of Themis1 in primary mouse thymocytes, through immunopurification of endogenous Themis1 (2).

 

Selected publications

1- Duguet F, Locard-Paulet M et al. Proteomic analysis of regulatory T cells reveals the importance of Themis1 in the control of their suppressive function. Mol Cell Proteomics. 2017.
https://doi.org/10.1074/mcp.m116.062745

 

2- Zvezdova et al (2016) Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability. Sci Signal, 2016.
https://doi.org/10.1126/scisignal.aad1576

Proteomic signature of regulatory T cells. Large-scale proteomics allows measuring cellular protein abundaces at high-throughput. By comparing Treg with conventional T cells, a panel of marker proteins specifically up-or down-regulated in this population were identified.