Dectin-1 is a Pathogen Recognition Receptor expressed on myeloid immune cells, known to play a major role in the antimicrobial immune response. It belongs to the C-type lectin Receptors (CLR) family and can bind to carbohydrate structures, such as β-glucans present on pathogens. This recognition triggers the phosphorylation of key tyrosine residues in the ITAM domain of Dectin-1, leading to the activation of SYK-dependent and independent signaling pathways, and finally to the secretion of pro-inflammatory cytokines and reactive oxygen species. Even though Dectin-1 is an important CLR known to be involved in many diseases such as fungal infections, inflammation and autoimmune diseases, its signaling mechanisms remain unclear. In this project, we used global phosphoproteomics to obtain a time-resolved characterization of the early signaling events taking place in the first minutes following Dectin-1 engagement, and identify key intracellular signaling proteins involved in that pathway. In addition, we established a high-throughput approach for secretome analysis, to monitor in a fast and global manner the inflammatory mediators secreted by immune cells 24 h after Dectin-1 stimulation.

To that aim, ER-Hoxb8 immortalized murine cell lines from either WT or Dectin-1 KO progenitors were differentiated into dendritic cells and stimulated with depleted Zymosan, a canonical ligand for Dectin-1, during different time length. Phosphorylated peptides were enriched using a two-step protocol based on TiO2 and phospho-Tyrosine antibody affinity purification, and resulting phosphopeptides were analyzed on an Orbitrap-Exploris instrument equipped with a FAIMS interface. To monitor the secretory phenotype of DCs, a high-throughput approach for secretome analysis was developed using the Evosep chromatographic system and DIA-PASEF analysis on a TIMS-TOF SCP mass spectrometer.

Using this analytical pipeline, we could globally detect more than 10,000 class I phosphosites, and identify after statistical analysis more than 300 phosphosites significantly regulated upon Dectin-1 stimulation. These included sites from known proteins of the Dectin-1 signaling pathway, as well as novel targets that were selected for further functional validation by generating the corresponding KO cells. We could also provide a detailed characterization of the DC phenotypic response following Dectin-1 activation, with more than 500 proteins significantly over-represented in the secretome of activated cells, including many canonical inflammatory mediators. The high-throughput secretomic analytical workflow based on the Evosep system and fast DIA-MS runs provides a quick and comprehensive read-out to monitor the phenotypic response of immune cells, allowing the systematic screening of different KO cells in on-going validation studies.