The actin-nucleator promoting factor WASp regulates endo-lysosomal maturation and Toll-like receptor 9 signaling in Dendritic Cells.

Toll-like receptors (TLRs) are innate immune receptors, which play a key role in both innate and adaptive immune responses, are important to recognize pathogenic components and to trigger inflammatory responses. However, TLRs could be inappropriately activated by self and endogenous nucleic acids leading to unwarranted inflammation with dangerous outcomes, including autoimmune diseases. Intracellular TLRs compartmentalization is essential to regulate initiation and termination of signalling, thereby avoiding excessive inflammation. Therefore, studies of mechanisms and adaptors that limit unrestrained activation of innate immune cells upon TLRs triggering are of increasing interest as their potential in understanding autoimmunity. Wiskott-Aldrich Syndrome (WAS) is a X-linked primary immune deficiency, caused by mutations on WAS-protein (WASp), a hematopoietic specific actin nucleator promoting factor (NPFs). WAS is characterized by recurrent infections, and a marked predisposition to develop autoimmune phenomena. This thesis aimed to dissect the molecular mechanism that leads to excessive production of type-I interferon in the context of WAS, using a model of WASp null dendritic cells (WKO DCs). First, we developed and validated new cellular models to study WASp. We demonstrated that, after TLR9 stimulation, WKO cells produce higher amount of type-I IFN and proinflammatory cytokines, both in BM-DCs and Hoxb8-DCs models. Moreover, our dose-response assay of endogenous ligands showed that WKO display a lower TLR9 activation threshold compared to wild-type (WT) DCs. Intriguingly, our morphological analysis revealed structural alterations in endo-lysosomes of WKO DCs. Using flow cytometry and endocytic probes we observed that the lack of WASp leads to aberration in cargo recycling and degradation. We then investigated whether WKO structural and functional remodeling could influence TLR9-ligand intracellular trafficking. By blocking protein synthesis, we demonstrated that WASp expression is important to control TLR9 degradation rate. In addition, biochemical and immunofluorescence analysis revealed that exogenous and endogenous TLR9 ligands are engulfed in WKO cells. In particular, ligands traffic slowly across the first endocytic organelles and are degraded less efficiently than in WT DCs. Finally, using actin inhibitors, we observed that treated cells display impaired endo-lysosome maturation and reduced TLR9 degradation. Most importantly, TLR9 signalling is enhanced leading to higher production of type-I IFN. Taken together, these results prove that the perturbation of actin dynamics closely resembles WASp deficiency phenotype. In conclusion, our findings shed light on the mechanism underlying excessive TLR9 activation: actin nucleation mediated by WASp is required to maintain a correct endo-lysosomal organization and to control TLR9 threshold, activation and signalling in DCs.