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Characterization of inflammatory and immune processes

Scientific coordinator of the project: Anne Gonzalez de Peredo

Taking advantage of the great improvements in large-scale proteomic analysis of complex samples, we use state-of-the-art mass spectrometry techniques to study the molecular mechanisms of the inflammatory and immune responses in specific cellular systems, such as endothelial cells and different types of immune cells.
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. 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.

Recently we used such a global proteomic approach 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 in primary 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.

 

References

  • Bouyssié, Gonzalez de Peredo et al (2007) 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 : MCP 6, 1621-1637.
  • Gautier et al (2012). 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 : MCP 11, 527-539.
  • Gautier et al (2016) Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells. Sci Rep 6, 34255.

 

 

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

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), mast cells, and certain subsets of regulatory T cells. IL-33 plays a role in many important diseases. Particularly, several genome-wide association identified the genes of IL-33 and ST2 as major susceptibility loci for human asthma.

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

 

Mechanisms of IL-33 activation by proteolytic maturation

IL-33 contains a N-terminal chromatin-binding domain: it is mainly sequestered in the nucleus of epithelial end endothelial cells from barrier tissues, and is released upon cellular damage or necrose. On the other hand, its C-terminal part is composed of a bioactive IL1-like cytokine domain. The group of J-P Girard  demonstrated that IL-33 is not regulated as IL-1b through maturation by caspase-1 (which rather inactivates IL-33 by cleaving inside the IL1-like domain), but through proteolytic maturation by inflammatory proteases.

We use mass spectrometry analysis to identify highly active mature forms of the protein resulting from processing by activator proteases, and map the cleavage sites in the central domain of IL-33.

 

Detection of Neo-Nterminal peptides in maturated forms of IL-33 through quantitative comparison with peptides from full-length proteinil33_figure-1.png

 

 

Unbiased global proteomic studies to characterize IL-33 action

 

Extracellular IL-33 is known to activate a growing number of target cells (including group 2 innate lymphoid cells, mast cells, regulatory T cells, and endothelial cells). We use large-scale proteomics to characterize the extracellular and intracellular role of IL-33 on these cells.

 

 

 

References

 

  • Lefrancais, E., Roga, S., Gautier, V., Gonzalez-de-Peredo, A., Monsarrat, B., Girard, J. P., and Cayrol, C. (2012) IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G. PNAS 109, 1673-1678.
  • Gautier et al (2016) Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells. Sci Rep 6, 34255.

 

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Proteomic analysis of primary endothelial cells response to the stimulation with extracellular IL-33il33_figure-2.png

 

 

 

 

 

 

 

 

 

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), 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.

 

Characterization of the TCR signalosome using transgenic mouse models

 

In collaboration with R. Roncagalli and B. Malissen (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.

 

ap-ms workflow

 

Using this methodology, we characterized the interactome of PAG (Phosphoprotein associated with glycosphingolipid-enriched microdomains), a ubiquitously expressed transmembrane adaptor protein present in lipid rafts and involved in the regulation of TCR signaling.

Conversely to the previously proposed model, we showed that PAG is quickly phosphorylated and binds CSK and other inhibitory molecules upon T cell activation.

 

Analysis of PAG interactome

 

 

Identification of new interactorsanalysis of pag interactome 1

 

 
 
Binding kinetics of proteins interacting with PAGanalysis_of_pag_interactome_figure-2.png

 

 

Fine characterization of phosphorylation kinetics of PAG tyrosinesanalysis_of_pag_interactome_figure-3.png

 

 

 

We also applied a similar approach to analyze the differences between the molecular networks formed around the E3 ubiquitin–protein ligases CBL and CBLB, two negative regulators of the TCR.

We monitored the dynamics of the CBL and CBLB signaling complexes that assemble in T cells following TCR stimulation, and exploit correlations in protein association with CBL and CBLB as a function of time for predicting the occurrence of direct physical association between them.

By combining co-recruitment analysis with biochemical analysis, we demonstrated that the CD5 transmembrane receptor constitutes a key scaffold for CBL- and CBLBmediated ubiquitylation following TCR engagement.

Analysis of Cbl and Cbl interactomes

 

analysis_of_cbl_and_cbl-b_interactomes_figure-1.png

analysis_of_cbl_and_cbl-b_interactomes_figure-2.png

Characterization of the Themis1 interactome

In collaboration with R. Lesourne (CPTP, Toulouse), we characterized the interactome of Themis1 in primary mouse thymocytes, through immunopurification of endogenous Themis1. Quantitative proteomics identified SHP-1/PTN6 and Vav1 as predominant interacting partners of Themis1. Further biological studies identified a primary role for Themis1 in regulating Grb2 stability and Vav1 effector function. Conversely to the previously proposed model of Themis1 acting as an attenuator of TCR signaling, these studies suggested that Themis1 might rather activate TCR signaling.
 
Analysis of Themis1 interactome

 

 

MS-based analysis of Themis1 interactome in thymocytes

  • Immunopurification of endogenous Themis1
  • Control: same immunoprecipitation in Themis KO thymocytes
  • Label-free quantitative comparison of immunopurified samples from WT and Themis1-/- miceanalysis_of_themis1_interactome_figure-1.png


 

Evaluation of absolute protein partners abundanceanalysis_of_themis1_interactome_figure-2.png

 

References

  • Reginald et al (2015) Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells. Journal of immunology 195, 5472-5481.
  • Voisinne et al (2016) 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 12, 876.
  • Zvezdova et al (2016) Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability. Sci Signal 9, ra51.

 

 

 

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In-depth proteomic analysis of regulatory T cells (Tregs)

Regulatory T cells (Treg) represent a minor sub-population of T lymphocytes which is crucial for the maintenance of immune homeostasis. In collaboration with A. Saoudi (CPTP, Toulouse), we used large-scale quantitative mass spectrometry to define a specific proteomic “signature” of Treg.

Treg and conventional T lymphocyte (Tconv) sub-populations were sorted by flow cytometry and subjected to global proteomic analysis by single-run nanoLC-MS/MS on a fast-sequencing Q-Exactive mass spectrometer. Besides “historical” proteins that characterize Treg, our study identified numerous new proteins that are up- or down-regulated in Treg versus Tconv.

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, in an animal model of inflammatory bowel disease and in co-culture assays.


tregs_figure-1.png

 

 

 


tregs_figure-2.png
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References

  • Duguet F, Locard-Paulet M et al (2017) Proteomic analysis of regulatory T cells reveals the importance of Themis1 in the control of their suppressive function. Mol Cell Proteomics. 2017

 

 

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