Metelo, J; (2013) Contribution of Integrins and Actin Regulators to Human Dendritic Cell Podosome Biology. Doctoral thesis , UCL (University College London).

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Abstract

Dendritic cells (DC) are key cells of the innate immune system required to prime adaptive immunity. Migration is central to their function to enable immune surveillance of the whole body and for prompt activation of the adaptive immune system. Immature DC assemble specialised actin structures called podosomes which are thought to be critical for efficient adhesion-mediated migration. Podosomes are, therefore, considered to be essential for DC function. Despite the great increase in literature regarding podosomes and related structures over recent years, still much is unknown about critical components, regulation and function of these structures in DC. Cytoskeletal studies of DC have been complicated by the fact that tools commonly employed for biological manipulation may constitute activation stimuli for DC and dramatically alter the DC cytoarchitecture. A panel of human THP1DC knock-down cell lines was generated using RNAi technology targeting factors suspected or known to be important for podosome formation and/or function such as the integrins CD18 and CD29 and the actin regulators HS1, WASp and WIP. Results obtained from functional analysis of the knock-down cell lines confirm CD18 to be specifically recruited to the DC podosomes and to be essential for their assembly. On the contrary, CD29 knock-down did not attenuate podosome assembly, even when reduced to levels that resulted in a defect in static adhesion. As previously reported, WASp and WIP expression was demonstrated to be necessary for podosome formation. Furthermore, a role for the cortactin homolog HS1 in CD18 activation in myeloid cells is suggested, as HS1 knock-down resulted in defective CD18-dependent adhesion and reduced podosome formation when cells were plated on ICAM but not on fibronectin. The results presented here define a robust method for manipulating immature DC for cytoskeletal studies and advance our current understanding of the regulation of podosome assembly in human DC.

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