Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.

A deficit in early clearance of Pseudomonas aeruginosa (P. aeruginosa) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated P. aeruginosa or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type P. aeruginosa strain) by primary AMs was causally associated with increased IL-1β release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5−/− AMs, demonstrating the dependence of IL-1β production on TLR5. We showed that this IL-1β production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1 −/− and AEP −/− mice were unable to kill P. aeruginosa. Altogether, these findings demonstrate that TLR5 engagement plays a major role in P. aeruginosa internalization and in triggering IL-1β formation.flagellin | interleukin-1 | lysosomal protease T he opportunist Gram-negative bacterium, Pseudomonas aeruginosa, is particularly important in nosocomial pneumonia and in chronic lung diseases such as cystic fibrosis (1). Alveolar macrophages (AMs) lie at the forefront of lung defense against pathogens such as P. aeruginosa. The main function of AMs is to clear pathogens (2), and a deficiency in early recognition of P. aeruginosa by AMs has been suspected in these pathologies (3,4). Research has shown that pathogen-associated molecular patterns (PAMPs) are recognized by specific Toll-like receptors (TLRs) at the surface of phagocytes and mucosal epithelial cells. Surprisingly, although numerous studies have associated the ligand-induced TLR engagement to cytokine and chemokine production from phagocytes (5, 6), comparatively fewer studies have investigated the importance of TLRs in pathogen phagocytosis and killing. Furthermore, these studies have mostly used macrophages from bone marrow-differentiated cells (BMDMs), few have used live bacteria, and even fewer have used flagellated bacteria such as P. aeruginosa. Moreover most studies have used primed phagocytes (with LPS, zymosan) to boost pathogen uptake. Despite these caveats, the recruitment of membrane TLRs to phagosomes upon phagocytosis has been demonstrated (7-10), except for TLR5. TLR2, TLR4, and the adaptor molecule MyD88 have been shown to be important molecules in processing of heat-killed Escherichia coli and Staphylococcus aureus by BMDMs in late endosomes and lysosomes (9-11), suggesting that a blockade in phagosome maturation was occurring in phagocytes deleted for these molecules.TLR5 is thought to be one of the key receptors implicated in the recognition of P. aeruginosa (5, 8), bu...