Microbial products are sensed through Toll-like receptors (TLRs) and trigger a program of dendritic cell (DC) maturation that enables DCs to activate T cells. Although an accepted hallmark of this ...response is eventual down-regulation of DC endocytic capacity, we show that TLR ligands first acutely stimulate antigen macropinocytosis, leading to enhanced presentation on class I and class II major histocompatibility complex molecules. Simultaneously, actin-rich podosomes disappear, which suggests a coordinated redeployment of actin to fuel endocytosis. These reciprocal changes are transient and require p38 and extracellular signal-regulated kinase activation. Thus, the DC actin cytoskeleton can be rapidly mobilized in response to innate immune stimuli to enhance antigen capture and presentation.
Rsk kinases play important roles in several cellular processes such as proliferation, metabolism, and migration. Until recently, Rsk activation was thought to be exclusively initiated by Erk1/2, but ...in dendritic cells (DC) Rsk is also activated by p38 mitogen-activated protein (MAP) kinase via its downstream substrates, MK2/3. How and why this noncanonical configuration of the MAP kinase pathway is adopted by these key immune cells are not known. We demonstrate that the Erk1/2-activated C-terminal kinase domain of Rsk is dispensable for p38-MK2/3 activation and show that compared with fibroblasts, a greater fraction of p38 and MK2/3 is located in the cytosol of DC prior to stimulation, suggesting a partial explanation for the operation of the noncanonical pathway of Rsk activation in these cells. p38/MK2/3-activated Rsk phosphorylated downstream targets and is physiologically important because in plasmacytoid DC (pDC) stimulated with Toll-like receptor 7 (TLR7) agonists, Erk1/2 activation is very weak relative to p38. As a result, Rsk activation is entirely p38 dependent. We show that this unusual configuration of MAP kinase signaling contributes substantially to production of type I interferons, a hallmark of pDC activation.
Langerhans cells (LC), the dendritic cells of the epidermis, are distributed in a distinctive regularly spaced array. In the mouse, the LC array is established in the first few days of life from ...proliferating local precursors, but the regulating signaling pathways are not fully understood. We found that mice lacking the kinase phosphoinositide-dependent kinase 1 selectively lack LC. Deletion of the phosphoinositide-dependent kinase 1 target kinases, ribosomal S6 kinase 1 (Rsk1) and Rsk2, produced a striking perturbation in the LC network: LC density was reduced 2-fold, but LC size was increased by the same magnitude. Reduced LC numbers in Rsk1/2(-/-) mice was not due to accelerated emigration from the skin but rather to reduced proliferation at least in adults. Rsk1/2 were required for normal LC patterning in neonates, but not when LC were ablated in adults and replaced by bone marrow-derived cells. Increased LC size was an intrinsic response to reduced LC numbers, reversible on LC emigration, and could be observed in wild type epidermis where LC size also correlated inversely with LC density. Our results identify a key signaling pathway needed to establish a normal LC network and suggest that LC might maintain epidermal surveillance by increasing their "footprint" when their numbers are limited.
Protein kinase inhibitors frequently have interesting effects that cannot be fully ascribed to the intended target kinase(s) but identifying additional targets that might explain the effects is not ...straightforward. By comparing two different inhibitors of the Rsk (p90 ribosomal S6 kinase) kinases, we found that the increasingly used compound BI-D1870 had biological effects in murine DCs (dendritic cells) that could not be solely ascribed to Rsk or other documented targets. We assessed the ability of BI-D1870 and a second Rsk inhibitor, BIX 02565 to protect enzyme active sites from reaction with biotinylated nucleotide acyl phosphates. Using SILAC (stable isotope labelling by amino acids in cell culture)-labelled DC lysates as a source of enzyme targets, we identify several kinases that interact with BI-D1870 but not with BIX 02565. We confirmed that these kinases, including Slk, Lok and Mst1, are inhibited by BI-D1870 but to a much lesser extent by BIX 02565 and that phosphorylation of some of their substrates is blocked by BI-D1870 in living cells. Our results suggest that the BI-D1870 inhibitor should be used with caution. The SILAC-based methodology we used should be useful for further comparative unbiased profiling of the target spectrum of kinase inhibitors with interesting biological effects under conditions that closely mimic those found in cells.
The adaptive immune response depends on dendritic cell (DC) activation by microbial products that signal via pattern recognition receptors and activate mitogen-activated protein kinases, NFκB and ...PI3K. The contribution of the AGC kinase family, including protein kinase B, protein kinase C, p90kDa ribosomal S6 kinase, and S6 kinase, has been little investigated because the probable redundancy among their isoforms makes their study difficult. We took advantage of the fact that all these kinases are regulated by the upstream master kinase 3-phosphoinositide-dependent kinase 1 (PDK1). Here we analyze various properties of DC from mice expressing ∼10% of normal PDK1 (PDK1fl/–). DC populations in lymphoid and nonlymphoid tissues appeared normal in PDK1fl/– mice, and some in vitro responses to lipopolysaccharide (LPS) such as cytokine production were normal in cultured bone marrow DC. However, LPS-induced expression of class II major histocompatibility complex and CD86 were elevated in PDK1fl/– BMDC and PDK1fl/– spleen DC produced more interleukin-10 and -12, implying an attenuating role for PDK1. Unexpectedly, PDK1fl/– DC had a significantly reduced capacity for LPS-stimulated macropinocytosis and phagocytosis that correlated with a lowered F-actin/G-actin ratio, apparently because of increased actin depolymerization. Several PDK1-regulated kinases, some of which feed into actin regulators, showed reduced activation in PDK1fl/– DC. Reintroduction of PDK1 restored S6 kinase activity, increased levels of F-actin, and boosted macropinocytosis thus linking PDK1 and its downstream effectors to the unusual phenotype of PDK1fl/– DC.
ADP-ribosylation factor 6 (ARF6) is a widely expressed GTPase that influences both membrane traffic and actin cytoskeleton function. Its role in dendritic cells (DC) has not previously been ...investigated. We analysed the effect of retroviral expression of ARF6 GDP/GTP binding and other functional mutants in primary murine DC. Maturation in response to lipopolysaccharide (LPS) proceeded normally in DC expressing ARF6 mutants and production of inflammatory cytokines was similarly unaffected. Although LPS-stimulated macropinocytosis was suppressed by expression of the GTP-binding Q67L ARF6 mutant we detected no overall activation of ARF6 by LPS. The ability of immature DC to migrate towards CCL3 and to a lesser extent, of mature DC to migrate towards CCL19, was compromised by expression of either the Q67L or the GDP-binding T44N mutant. Examination of the actin cytoskeleton in these cells revealed that both mutants strongly inhibited the formation of F-actin-rich podosomes, providing a possible explanation for the effects of ARF6 mutants on DC migration. Thus, these studies identify responses in DC that require normal ARF6 function, though not necessarily further ARF6 activation. They reveal for the first time a role for ARF6 in podosome formation and demonstrate functional effects of the T44N ARF6 mutant.
Toll-like receptor (TLR) signals induce dendritic cell (DC) differentiation and influence the immunological outcome of their interactions with T cells. Recent in vitro studies demonstrate that TLR ...signals also trigger striking reorganisation of the DC vacuolar compartments, the cytoskeleton and the machinery of protein translation and turnover. Moreover, TLR ligation within endosomes and phagosomes appears to establish organelle autonomous signals. These changes, which mostly occur within minutes to a few hours after TLR engagement, are adaptations relevant to the antigen capture, processing and migratory phases of the DC life history.
Upon conjugation with cognate antigen-presenting cells (APCs), T lymphocytes undergo a sustained Ca2+i increase resulting from the engagement of TCR and of accessory molecules with ligands expressed ...on the surface of APCs. We investigated the contribution of the accessory molecule CD2 to the activation of phospholipase Cγ1 (PLCγ1)/calcium pathway in antigen-stimulated T cells. We show that CD2 binding with its ligand CD58 expressed on the surface of APCs augments and sustains antigen-induced Ca2+i increase in individual T cells interacting with APCs. We also show that in conditions in which CD2–CD58 interaction is impeded, the recruitment of PLCγ1 to the immunological synapse (IS) is reduced. Interestingly, in these conditions PLCγ1 phosphorylation in the regulatory tyrosine 783 is also defective. Our results indicate that TCR- and CD2-derived signals converge for the recruitment and activation of PLCγ1 at the IS and shed new light on the accessory function of CD2 in T cell activation by specific antigen.
Recent evidence suggests that TLR signalling in dendritic cells (DCs) transiently enhances antigen endocytosis and autophagy, augments the assembly of key antigen transport and processing systems, ...qualitatively modulates protein translation and induces a temporary cessation of DC motility. These rapid changes require activation of the MAP kinases, PI3-kinase and downstream signalling pathways and are observed in both myeloid DC and, with variations on the theme, in plasmacytoid DC.
PDF
