A crucial role for ß2 integrins in podosome formation, dynamics and Toll-like-receptor-signaled disassembly in dendritic cells.

The dynamic properties of podosomes, their ability to degrade the underlying matrix and their modulation by Toll-like receptor (TLR) signaling in dendritic cells (DCs) suggests they have an important role in migration. Integrins are thought to participate in formation and dynamics of podosomes but the multiplicity of integrins in podosomes has made this difficult to assess. We report that murine DCs that lack ß2 integrins fail to form podosomes. Re-expression of ß2 integrins restored podosomes but not when the membrane proximal or distal NPxF motifs, or when an intervening triplet of threonine residues were mutated. We show that ß2 integrins are remarkably long-lived in podosome clusters and form a persistent framework that hosts multiple actin-core-formation events at the same or adjacent sites. When ß2 integrin amino acid residues 745 or 756 were mutated from Ser to Ala, podosomes became resistant to dissolution mediated through TLR signaling. TLR signaling did not detectably modulate phosphorylation at these sites but mutation of either residue to phospho-mimetic Asp increased ß2 integrin turnover in podosomes, indicating that phosphorylation at one or both sites establishes permissive conditions for TLR-signaled podosome disassembly.

Proximal effects of Toll-like receptor activation in dendritic cells.

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.

TLR signalling regulated antigen presentation in dendritic cells.

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.

A role for ARF6 in dendritic cell podosome formation and migration.

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.

TLR ligand-induced podosome disassembly in dendritic cells is ADAM17 dependent.

Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin-rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling-induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor alpha-converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this "sheddase" in regulating an actin-based structure.

Enhanced dendritic cell antigen capture via toll-like receptor-induced actin remodeling.

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.