School of Biomedical Sciences - Research Publications
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ItemSorting nexin 5 selectively regulates dorsal-ruffle-mediated macropinocytosis in primary macrophagesLim, JP ; Gosavi, P ; Mintern, JD ; Ross, EM ; Gleeson, PA (COMPANY OF BIOLOGISTS LTD, 2015-12-01)The regulation of macropinocytosis, a specialised endocytosis pathway, is important for immune cell function. However, it is not known whether the biogenesis of macropinosomes involves one or more distinct pathways. We previously identified sorting nexin 5 (SNX5) as a regulator of macropinocytosis in macrophages. Here, we show that bone-marrow-derived macrophages from SNX5-knockout mice had a 60-70% reduction in macropinocytic uptake of dextran or ovalbumin, whereas phagocytosis and retrograde transport from the plasma membrane to the Golgi was unaffected. In contrast, deficiency of SNX5 had no effect on macropinocytosis or antigen presentation by dendritic cells. Activation of macrophages with CSF-1 resulted in a localisation of SNX5 to actin-rich ruffles in a manner dependent on receptor tyrosine kinases. SNX5-deficient macrophages showed a dramatic reduction in ruffling on the dorsal surface following CSF-1 receptor activation, whereas peripheral ruffling and cell migration were unaffected. We demonstrate that SNX5 is acting upstream of actin polymerisation following CSF-1 receptor activation. Overall, our findings reveal the important contribution of dorsal ruffing to receptor-activated macropinocytosis in primary macrophages and show that SNX5 selectively regulates macropinosomes derived from the dorsal ruffles.
ItemCriteria for Dendritic Cell Receptor Selection for Efficient Antibody-Targeted VaccinationReuter, A ; Panozza, SE ; Macri, C ; Dumont, C ; Li, J ; Liu, H ; Segura, E ; Vega-Ramos, J ; Gupta, N ; Caminschi, I ; Villadangos, JA ; Johnston, APR ; Mintern, JD (AMER ASSOC IMMUNOLOGISTS, 2015-03-15)Ab-targeted vaccination involves targeting a receptor of choice expressed by dendritic cells (DCs) with Ag-coupled Abs. Currently, there is little consensus as to which criteria determine receptor selection to ensure superior Ag presentation and immunity. In this study, we investigated parameters of DC receptor internalization and determined how they impact Ag presentation outcomes. First, using mixed bone marrow chimeras, we established that Ag-targeted, but not nontargeted, DCs are responsible for Ag presentation in settings of Ab-targeted vaccination in vivo. Next, we analyzed parameters of DEC205 (CD205), Clec9A, CD11c, CD11b, and CD40 endocytosis and obtained quantitative measurements of internalization speed, surface turnover, and delivered Ag load. Exploiting these parameters in MHC class I (MHC I) and MHC class II (MHC II) Ag presentation assays, we showed that receptor expression level, proportion of surface turnover, or speed of receptor internalization did not impact MHC I or MHC II Ag presentation efficiency. Furthermore, the Ag load delivered to DCs did not correlate with the efficiency of MHC I or MHC II Ag presentation. In contrast, targeting Ag to CD8(+) or CD8(-) DCs enhanced MHC I or MHC II Ag presentation, respectively. Therefore, receptor expression levels, speed of internalization, and/or the amount of Ag delivered can be excluded as major determinants that dictate Ag presentation efficiency in setting of Ab-targeted vaccination.
ItemModulation of antigen presentation by intracellular traffickingMintern, JD ; Macri, C ; Villadangos, JA (CURRENT BIOLOGY LTD, 2015-06-01)Processing and loading of antigen into major histocompatibility complex molecules (MHC) occurs in specific intracellular compartments. Accessing MHC loading compartments requires trafficking via specific pathways, some of which have yet to be fully characterized. For MHC I, cross-presentation involves antigen trafficking to a specialised compartment. We review the features of this compartment and how it is accessed by different mechanisms of antigen capture and internalization. We also summarize advances in understanding how antigen efficiently accesses the MHC II loading compartment, with particular focus on the role of autophagy. Understanding the mechanisms that control how antigen is trafficked to specific compartments for loading and presentation is crucial if these pathways are to be manipulated more effectively in settings of vaccination.
ItemDifferential use of autophagy by primary dendritic cells specialized in cross-presentationMintern, JD ; Macri, C ; Chin, WJ ; Panozza, SE ; Segura, E ; Patterson, NL ; Zeller, P ; Bourges, D ; Bedoui, S ; McMillan, PJ ; Idris, A ; Nowell, CJ ; Brown, A ; Radford, KJ ; Johnston, APR ; Villadangos, JA (TAYLOR & FRANCIS INC, 2015-06-01)Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed "cross-presentation." The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.