Proteomic definition of dendritic cell phenotypes matured in response to helminth antigens

Dendritic cells (DCs) efficiently recognise molecules from microbial pathogens stimulating them to undergo a process of maturation which governs their ability to prime the adaptive immune response. The character of the adaptive response depends on how DCs have been matured by the microbial pathogens leading to the development of Th1 or Th2 lymphocytes. However, little is known about how DCs drive Th2 responses. Our project therefore employed a combination of the latest gel-based and gel-free technologies to specifically define the proteome (i.e. the composition of all proteins) of DCs that drive Th2 versus Th1 lymphocyte responses. In order to do this, we examined the soluble (i.e. largely cytosolic) proteome of DCs stimulated with helminth or bacterial products, known to induce Th1, or Th2-type immune responses respectively.

Patterns of spots (proteins) separated by 2D electrophoresis (2DE) of DCs matured with 0-3hRP (molecules released by schistosome cercariae = pro-Th2) and LPS (bacterial lipopolysaccharide = pro-Th1) were largely similar with ~93 % of spots matching. The major changes in protein expression affected cytoskeletal proteins, reflecting morphological differences between the types of differentially matured DCs observed by microscopy. Indeed, immature Medium-DCs displayed an elongated bipolar morphology representative of highly migratory cells. In contrast, LPS-DCs showed the characteristics of mature DCs, with a more rounded shape, while 0-3hRP-DCs showed an intermediate morphology.

Our proteomic analyses of pro-Th2 DCs stimulated with 0-3hRP demonstrate that they are not immature, since they have a proteome distinct to medium DCs, and does not support the ‘default’ hypothesis for the induction of Th2 responses. We were also unable to identify a ‘signature’ pro-Th2 DC proteome that might be expected if they maturated along a specialised pathway. However, our study clearly establishes that, in terms of their proteome, 0-3hRP-DCs exhibit a proteome intermediate between immature Medium-DCs and pro-Th1 LPS-DCs. The proteome of our pro-Th2 DCs is closer to that of immature Medium-DCs than to pro-Th1 LPS-DCs. Therefore, our proteomic study supports the view that Th2 differentiation results from the ‘limited’ maturation of DC.

In our final studies, we wished to examine those proteins not in the soluble (i.e. cytosolic) component of lysed DCs, and so we performed iTRAQ experiments on plasma membrane enriched fractions of differentially matured DCs. Such proteins are separated by liquid chromatography, rather than 2DE, and then subject to analysis by mass spectroscopy. Our studies have revealed the identification of a number of well-known immune-related membrane associated molecules such as integrins, C-type lectins, MHCII molecules, as well as some intracellular markers indicative of lysosome activity that normally follows the process of phagocytosis. These molecules all need to be verified using conventional assays of marker identification such as flow cytometric analysis of antibody-labelled cells. However, at this early stage it appears that pro-Th2 DCs are not as well activated, or mature as pro-Th1 DCs, and that the machinery associated with phagocytosis is suppressed in pro-Th2 DCs.