IRAP+ endosomes restrict TLR9 activation and signaling.

The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP+ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP+ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.

Cytoskeletal tension actively sustains the migratory T-cell synaptic contact.

When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott-Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact.

TLR7 trafficking and signaling in B cells is regulated by the MHCII-associated invariant chain.

Toll-like receptor 7 (TLR7) is an endosomal receptor that recognizes single-stranded RNA from viruses. Its trafficking and activation is regulated by the endoplasmic reticulum (ER) chaperone UNC93B1 and lysosomal proteases. UNC93B1 also modulates major histocompatibility complex class II (MHCII) antigen presentation, and deficiency in MHCII protein diminishes TLR9 signaling. These results indicate a link between proteins that regulate both innate and adaptive responses. Here, we report that TLR7 resides in lysosomes and interacts with the MHCII-chaperone molecule, the invariant chain (Ii) or CD74, in B cells. In the absence of CD74, TLR7 displays both ER and lysosomal localization, leading to an increase in pro-inflammatory cytokine production. Furthermore, stimulation with TLR7 but not TLR9, is inefficient in boosting antigen presentation in Ii-deficient cells. In contrast, in B cells lacking TLR7 or mutated for UNC93B1, which are able to trigger TLR7 activation, antigen presentation is enhanced. This suggests that TLR7 signaling in B cells is controlled by the Ii chain.

Toll-like receptor 5 (TLR5), IL-1ß secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing.

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-1ß release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5(-/-) AMs, demonstrating the dependence of IL-1ß production on TLR5. We showed that this IL-1ß 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-1ß formation.

Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation.

Intracellular Toll-like receptors (TLRs) expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV), a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP) deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8(+) T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases.

DOCK8 is essential for LFA-1-dependent positioning of T follicular helper cells in germinal centers.

T follicular helper (Tfh) cell migration into germinal centers (GCs) is essential for the generation of GC B cells and antibody responses to T cell-dependent (TD) antigens. This process requires interactions between lymphocyte function-associated antigen 1 (LFA-1) on Tfh cells and ICAMs on B cells. The mechanisms underlying defective antibody responses to TD antigens in DOCK8 deficiency are incompletely understood. We show that mice selectively lacking DOCK8 in T cells had impaired IgG antibody responses to TD antigens, decreased GC size, and reduced numbers of GC B cells. However, they developed normal numbers of Tfh cells with intact capacity for driving B cell differentiation into a GC phenotype in vitro. Notably, migration of DOCK8-deficient T cells into GCs was defective. Following T cell receptor (TCR)/CD3 ligation, DOCK8-deficient T cells had impaired LFA-1 activation and reduced binding to ICAM-1. Our results therefore indicate that DOCK8 is important for LFA-1-dependent positioning of Tfh cells in GCs, and thereby the generation of GC B cells and IgG antibody responses to TD antigen.

In Vitro Digestion with Proteases Producing MHC Class II Ligands.

Proteases generate peptides that bind to MHC class II molecules to interact with a wide diversity of CD4+ T cells. They are expressed in dedicated organelles: endosomes and lysosomes of professional antigen-presenting cells (pAPCs) such as B cells, macrophages, and dendritic cells. The identification of endosomal proteases which produce antigenic peptides is important for example for better vaccination and to prevent autoimmune diseases. Here, we describe a panel of techniques (in vitro digestion assays of protein with recombinant proteases or purified endosomes/lysosomes, T cell stimulation) to monitor the production of MHC class II ligands.

Author Correction: TLR9 activation via microglial glucocorticoid receptors contributes to degeneration of midbrain dopamine neurons.

The originally published version of this Article contained an error in the subheading "Microglial GR does not affect DN loss triggered by TLR4 and TLR7," which was incorrectly given as "Microglial GR does affect DN loss triggered by TLR2 and TLR4". This has now been corrected in both the PDF and HTML versions of the Article.

TLR9 activation via microglial glucocorticoid receptors contributes to degeneration of midbrain dopamine neurons.

Inflammation is a characteristic feature of Parkinson's disease (PD). We examined the role of TLR9 and its regulation by glucocorticoid receptors (GRs) in degeneration of substantia nigra dopamine neurons (DNs). TLR9 agonist, CpG-ODN, induced DN degeneration in mice lacking GR in microglia but not in controls. TLR9 deletion reduced DN loss in neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. GR regulates TLR9 activation during MPTP neurotoxicity as TLR9 antagonist suppressed increased DN loss in microglia/macrophage GR mutant mice. GR absence in microglia enhanced TLR9 translocation to endolysosomes and facilitated its cleavage leading to pro-inflammatory gene expression. GR-dependent TLR9 activation also triggered DN loss following intranigral injection of mitochondrial DNA. Finally, microglial GR sensitivity to A53T-alpha-synuclein induced DN degeneration as well as decreased microglial GR expression observed in SN of PD brain samples, all suggest that reduced microglial GR activity in SN can stimulate TLR9 activation and DN loss in PD pathology.

DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs.

Patients deficient in the guanine nucleotide exchange factor DOCK8 have decreased numbers and impaired in vitro function of Tregs and make autoantibodies, but they seldom develop autoimmunity. We show that, similarly, Dock8-/- mice have decreased numbers and impaired in vitro function of Tregs but do not develop autoimmunity. In contrast, mice with selective DOCK8 deficiency in Tregs develop lymphoproliferation, autoantibodies, and gastrointestinal inflammation, despite a normal percentage and in vitro function of Tregs, suggesting that deficient T effector cell function might protect DOCK8-deficient patients from autoimmunity. We demonstrate that DOCK8 associates with STAT5 and is important for IL-2-driven STAT5 phosphorylation in Tregs. DOCK8 localizes within the lamellar actin ring of the Treg immune synapse (IS). Dock8-/- Tregs have abnormal TCR-driven actin dynamics, decreased adhesiveness, an altered gene expression profile, an unstable IS with decreased recruitment of signaling molecules, and impaired transendocytosis of the costimulatory molecule CD86. These data suggest that DOCK8 enforces immunological tolerance by promoting IL-2 signaling, TCR-driven actin dynamics, and the IS in Tregs.

A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton.

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.

Invariant chain is a new chaperone for TLR7 in B cells.

The innate immune system provides the first barrier against pathogens. Intracellular Toll-like receptors (TLR3, 7 and 9) localise in endosomes and sense nucleotides from viruses and bacteria. This recognition induces their conformational changes resulting in the production of proinflammatory cytokines and MHC class II (MHCII) antigenic presentation. In the absence of stimulation, TLRs are retained in the endoplasmic reticulum. Upon stimulation, they relocate to the endo-lysosomal compartment, allowing the recruitment of the adaptor molecules, MyD88 or TRIF. Increasing evidences describe a cross talk between proteins that regulate both innate and adaptive immune responses. For example, proteolytic enzymes which are required for breaking down exogenous antigen to generate suitable peptides for MHCII molecules are also essential to activate endosomal TLRs and MHCII molecules were recently described to regulate TLR signalling. But other proteins are possibly involved and regulated differentially between cell types. We have observed that intracellular TLR trafficking and signalling in B cells are different from dendritic cells and macrophages and involved the MHCII chaperone molecule, the invariant chain (Ii).

Intracellular Toll-like receptor recruitment and cleavage in endosomal/lysosomal organelles.

Microbial pathogens are recognized through multiple, distinct receptors such as intracellular Toll-like receptors (TLRs 3, 7, 8, 9, and 13) which reside in the endosomes and lysosomes. TLRs are sensitive to chloroquine, a lysomotropic agent that neutralizes acidic compartments indicating a role for endo/lysosomal proteases for their signaling. Indeed, upon stimulation, full-length TLR7 and 9 are cleaved into a C-terminal fragment and this processing is highly dependent on a cysteine protease named asparagine endopeptidase (AEP) in dendritic cells. A recruitment and a boost in AEP activity, which was induced shortly after TLR7 and 9 stimulation, are shown to promote TLR7 and 9 cleavage and correlate with an increased acidification in endosomes and lysosomes. Moreover, mutating a putative AEP cleavage site in TLR7 or 9 strongly decreases their signaling in DCs, suggesting perhaps a direct cleavage of TLR7 and 9 by AEP. These results demonstrate that TLR7 and 9 require a proteolytic cleavage for their signaling and identified a key endocytic protease playing a critical role in this process.

In vitro digestion with proteases producing MHC class II ligands.

Proteases generate peptides that bind to MHC class II molecules to interact with a wide diversity of CD4(+) T cells. They are expressed in dedicated organelles: endosomes and lysosomes of professional antigen presenting cells (pAPCs) such as B cells, macrophages, and dendritic cells. The identification of endosomal proteases which produce antigenic peptides is important, for example, for better vaccination and to prevent autoimmune diseases. Here, we describe a panel of technics (in vitro digestion assays of protein with recombinant proteases or purified endosomes/lysosomes, T cell stimulation) to monitor the production of MHC class II ligands.