Role of intracellular trafficking in the control of Immunoglobulin Receptor-mediated diseases – IDEA

Cell biology: protein inactivation (lentiviral shRNA or CRISPR/Cas9); protein over-expression, APEX2-mediated in situ biotinylation followed by the isolation of FcR signaling platforms and their identification by mass-spectrometry, flow cytometry, Imaging flow cytometry (AMNIS), improved resolution confocal microscopy and videomicroscopy, murine models of FcR mediated inflammation.

We demonstrated that different FcRs follow distinct endocytic pathways after activation, as following: FcaRI is internalized into lysosomes, Fc?RIIA is retained in early endosomes and the Fc?RIIB is internalized in autophagosomes. The high affinity Fc?RI is directed in endosomal compartments described by the Insulin Responsive AminoPeptidase (IRAP). We found that internalized Fc?RI uses endosomal-signaling platforms, which depend on the presence of IRAP and Rab14. Destabilization of the endosomal signaling platforms compromised the ability of peritoneal macrophages to kill tumor cells by antibody-dependent cell mediated cytotoxicity. These results are available on BioRxiv as a manuscript (https://www.biorxiv.org/content/10.1101/2021.06.24.449774v1).
In parallel, motivated by our in vitro data on FcR endosomal signaling, we investigated in vivo the role of IRAP in FcR induced inflammation. We showed that IRAP-deficient versus wild-type mice exhibit a less severe IgE-dependent passive systemic anaphylaxis, IgG-triggered active systemic anaphylaxis and autoimmune arthritis. Mechanistic analysis in mast cells revealed that IRAP intracellular vesicles rapidly recruit to the plasma membrane upon FceRI stimulation with IRAP-deficient cells exhibiting a reduced calcium signal and reduced secretory responses. Active SykY524/525 was decreased and less prominent at the plasma membrane in IRAP-deficient cells. Syk inhibition did not depend on early signaling events mediated by Lyn kinase but relied on SHP1 phosphatase activity as in IRAP-deficient mast cells the inactivation on Ser591 was decreased, thereby enhancing FcR-induced inflammatory response. Ex vivo profiling after IgG-triggered anaphylaxis confirmed decreased phosphorylation of Syk and SHP-1S591 in isolated neutrophils and monocytes.
In summary, our findings demonstrate that IRAP endosomes are required for signal amplification for 3 major immune immunoglobulin receptors: Fc?RIIA, Fc?RI and FceRI.

To understand the molecular mechanism by which IRAP amplifies FcR signaling we set up an in situ biotinylation assay by fusing the human Fc?RIIA to the peroxidase APEX2. We verified the expression and correct localization of Fc?RIIA-APEX2 fusion protein and set up the biotinylation protocol. The DC2.4 cells expressing Fc?RIIA-APEX2 were activated with anti- Fc?RIIA F(ab)2 antibodies cross-linked with secondary antibodies and the receptor internalization was verified by confocal microscopy. The proteins biotinylated in situ were purified and sent to mass-spectrometry platform of Pasteur Institute. When the results will be available, we will validate the most interesting candidates by protein deletion, protein over-expression and functional assays.

1. The high affinity IgE receptor: a signaling update. Blank U, Huang H, Kawakami T. Curr Opin Immunol. 2021 Apr 7;72:51-58. doi: 10.1016/j.coi.2021.03.015. Online ahead of print. PMID: 33838574 Review.
2. Cytoskeletal Transport, Reorganization, and Fusion Regulation in Mast Cell-Stimulus Secretion Coupling. Ménasché G, Longé C, Bratti M, Blank U. Front Cell Dev Biol. 2021 Mar 16;9:652077. doi: 10.3389/fcell.2021.652077. eCollection 2021. PMID: 33796537 Free PMC article. Review
3. High affinity Fc?R activating function depends on IRAP+ endosomal-signaling platforms. Samira Benadda, Mathilde Nugues, Marcelle Bens, Mariacristina De Luca, Olivier Pellé, Renato C. Monteiro, Irini Evnouchidou, Loredana Saveanu
doi: doi.org/10.1101/2021.06.24.449774

Activated cell surface receptors are rapidly endocytosed. It has been established that endocytosis of growth factor receptors (RTKs) and G-proteins coupled receptors (GPCRs ) can either lead to abolition or to amplification of signalling, depending on the strength of receptor activation and/or on the endocytosis pathways involved in receptor internalisation. Key immune receptors, such as the activating Fc immunoglobulin Receptors (FcRs) are also internalised after ligand binding, but it is not clear if their endocytosis terminates or sustains signalling. This question is particularly pertinent in the case of FcRs, as they can mediate either activating or inhibitory signalling depending on the valency of their engagement. While immune complexes (IC) triggers an inflammatory response, the binding of soluble IgG (such as IVIg) inhibits the inflammatory reaction. The mechanisms by which the same receptor is able to trigger either pro-, or anti-inflammatory responses according to the ligand valency are not clear. Yet, they may be crucial to understand the pathogenic processes underlying FcR-mediated diseases, including glomerulonephritis with the deposition of immune complexes, arthritis, allergy and asthma.
The IDEA project is based on preliminary data suggesting that FcRs internalisation in cell-specific storage endosomes described by the Insulin Responsive AminoPeptidase (IRAP) dictates the pro- or anti-inflammatory outcome of FcR activation. We will investigate the molecular mechanisms that regulate the internalisation of activated FcRs and the trafficking steps that regulate their signalling both in vitro and in vivo in 4 working packages as follows:
In the WP1, by a combination of state of the art methods such as, in vivo biotinylation via the new peroxidase APEX2, CrispR/Cas9 gene inactivation, improved resolution microscopy, we plan to identify from which intracellular compartment the activating Fc?Rs can trigger the inflammatory response. This fundamental work will therefore provide a comprehensive map of Fc?R cellular signalling platforms opening new perspectives in the study of molecular mechanisms leading to Fc?R-mediated inflammation.
In WP2, we will investigate the role of the endocytic signalling platform under conditions of activating and inhibitory conditions of triggering. We will further validate our results in vivo using murine models of Fc?R-mediated diseases, such as glomerulonephritis and arthritis. Finally, we will also study the endocytic trafficking and signalling of FcRs in human peripheral blood monocytes obtained from healthy donors and patients suffering from Fc?R-mediated inflammatory diseases, such as lupus and arthritis. The results obtained on human samples might reveal new biomarkers useful for the diagnosis and prognosis of Fc?R-mediated inflammatory diseases.
In the WP3, we will investigate if, as suggested by our preliminary data, the signalling of FceRI, the major receptor involved in allergy is also controlled by the endocytic system. We will analyse in vitro the FceRI trafficking and signalling in mast cells lacking components of the newly identified endocytic platform as defined in WP1. Using different allergy models we will validate in vivo the results obtained on FceR signalling.
Finally, in WP4, we will use murine models of FcR-mediated diseases to test the impact of new small molecules targeting the endocytic signalling and which in preliminary experiments have been demonstrated to alleviate FcR-initiated inflammation.