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Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E(2) (PGE(2)). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE(2) causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE(2)-mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D and 3-D geometric cues control the spatial organization of podosomes. More importantly, our studies demonstrate the importance of substrate dimensionality in regulating podosome dissolution and suggest that substrate dimensionality plays an important role in controlling DC activation, a key process in initiating immune responses.

Dendritic cells (DC) take up pathogens through phagocytosis and process them into protein and lipid fragments for presentation to T cells. So far, the proteome of the human DC phagosome, a detrimental compartment for antigen processing and presentation as well as for DC activation, remains largely uncharacterized. Here we have analyzed the protein composition of phagosomes from human monocyte-derived DC. For LC-MS/MS analysis we purified phagosomes from DC using latex beads targeted to DC-SIGN, and quantified proteins using a label-free method. We used organellar enrichment ranking (OER) to select proteins with a high potential to be relevant for phagosome function. The method compares phagosome protein abundance with protein abundance in whole DC. Phagosome enrichment indicates specific recruitment to the phagosome rather than co-purification or passive incorporation. Using OER we extracted the most enriched proteins that we further complemented with functionally associated proteins to define a set of 90 phagosomal proteins that included many proteins with established relevance on DC phagosomes as well as high potential novel candidates. We already experimentally confirmed phagosomal recruitment of Galectin-9, which has not been previously associated with phagocytosis, to both bead and pathogen containing phagosomes, suggesting a role for Galectin-9 in DC phagocytosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Gene expression is commonly used to study the activation of dendritic cells (DCs) to identify proteins that determine whether these cells induce an immunostimulatory or tolerogenic immune response. RNA expression, however, does not necessarily predict protein abundance and often requires large numbers of experiments for statistical significance. Proteomics provides a direct view on protein expression but is costly and time consuming. Here, we combined a comprehensive quantitative proteome and transcriptome analysis on a single batch of immature and cytokine cocktail matured human DCs and integrated resulting data sets at the pathway level. Although overall correlation between differential mRNA and protein expression was low, correlation between components of DC relevant pathways was significantly higher. Differentially expressed proteins and genes partly mapped to identical but also to different pathway components demonstrating that RNA and protein data not only supported but also complemented each other. We identified 5 dominant pathways, which confirmed the importance of cytokines, cell adhesion, and migration in DC maturation and also indicated a fundamental role for lipid metabolism. From these pathways we extracted novel maturation markers that might improve DC vaccine design. For several of the candidate markers we confirmed widespread significance examining DCs from multiple individuals, underscoring the validity of our approach. We conclude that integration of different but related data sets at the pathway level can significantly increase the predictive power of multi "omics" analyses.

Chronic infections are caused by microorganisms that display effective immune evasion mechanisms. Dendritic cell (DC)-dependent T cell-mediated adaptive immunity is one of the mechanisms that have evolved to prevent the occurrence of chronic bacterial infections. In turn, bacterial pathogens have developed strategies to evade immune recognition. In this study, we show that gram-negative and gram-positive bacteria differ in their ability to activate DCs and that gram-negative bacteria are far more effective inducers of DC maturation. Moreover, we observed that only gram-negative bacteria can induce loss of adhesive podosome structures in DCs, a response necessary for the induction of effective DC migration. We demonstrate that the ability of gram-negative bacteria to trigger podosome turnover and induce DC migration reflects their capacity to selectively activate TLR4. Examining mice defective in TLR4 signaling, we show that this DC maturation and migration are mainly Toll/IL-1 receptor domain-containing adaptor-inducing IFNbeta-dependent. Furthermore, we show that these processes depend on the production of PGs by these DCs, suggesting a direct link between TLR4-mediated signaling and arachidonic metabolism. These findings demonstrate that gram-positive and gram-negative bacteria profoundly differ in their capacity to activate DCs. We propose that this inability of gram-positive bacteria to induce DC maturation and migration is part of the armamentarium necessary for avoiding the induction of an effective cellular immune response and may explain the frequent involvement of these pathogens in chronic infections.

Podosomes are dynamic adhesion structures found in dendritic cells (DCs) and other cells of the myeloid lineage. We previously showed that prostaglandin E2 (PGE2), an important proinflammatory mediator produced during DC maturation, induces podosome disassembly within minutes after stimulation. Here, we demonstrate that this response is mediated by cAMP elevation, occurs downstream of Rho kinase and is dependent on myosin II. Whereas PGE2 stimulation leads to activation of the small GTPase RhoA, decreased levels of Rac1-GTP and Cdc42-GTP are observed. These results show that PGE2 stimulation leads to activation of the RhoA Rho-kinase axis to promote actomyosin-based contraction and subsequent podosome dissolution. Because podosome disassembly is accompanied by de novo formation of focal adhesions, we propose that the disassembly/formation of these two different adhesion structures is oppositely regulated by actomyosin contractility and relative activities of RhoA, Rac1 and Cdc42. [ABSTRACT FROM AUTHOR]