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Cross-talk between NK, APC and regulatory T cells

Final Report Summary - NK-APC-TREG X-TALK (Cross-talk between NK, APC and regulatory T cells)

Project context and objectives

Very little is known about the correlation of activation states of immune cells and their metabolism. Recently, emerging data focused on the change of metabolic pathways involved in T-cell proliferation and activation, while very few studies exist on dendritic cells (DCs). Do dangerous signals affect changes in cellular metabolic pathways in DCs to ensure sufficient bioenergetic and metabolic resources to support new gene expression, maturation programming and migration capacity? In other words, does the DC metabolic activity, like mitochondrial respiration, change during DC activation?

Project results
1. Oxygen consumption (OC) increased during early activation in DCs induced by Toll-Like Receptor (TLR) ligands in vitro and in vivo. No mitochondrial mass augmentation occurs, rather morphological changes with higher density and increasing thickness of mitochondrial christae.
2. Pharmacological inhibition of mitochondrial respiration by rotenone affect DC immunogenicity, in the following functions.
(a) DC-phenotype and T-cell proliferation

Rotenone-LPS co-treated CD8+ DCs showed stronger reduction of stimulatory molecules than CD11b+ DCs, while the major histocompatibility complex (MHC) class I surface expression level is not affected. The overall effects on splenic DCs affect T proliferation in vitro and in vivo.
(b) DC migration in micro-channels and in chemotaxis assays. Our experiments showed for the first time that sorted DCs differ to the well-characterised bone-marrow-derived DCs (BMDC) in speed and motion. Splenic DCs migrate faster (20 um/min) than BMDCs (5 um/min), with a continuous migratory-unique direction. In addition, sorted CD8+ and CD11b+ splenic DCs have very similar migratory proprieties in micro-channels, while applying to different cell sizes.
(c) Ag internalisation
Phagocytosis efficiency is affected in a dose-dependent manner in BMDCs, while endocytosis is strongly affected in CD8?+ DCs and slightly in CD11b+ DCs.
(d) Cell adhesion
The morphology of splenic DCs changes during TLR activation in vitro in 3D analysis. Indeed, activated DCs spread quickly and extensively showing long dendrites (area = 1944 um2 ± 117). In the presence of rotenone, DCs are strongly constrained into a smaller area (529 um2 ± 163, where p < 0.0001).
3. Mitochondria polarisation in Dcs

Imaging experiments showed polarisation of mitochondria in DCs toward CD8+-antigen-specific T cells in vitro as well as towards CD4+-specific T cells in vivo in draining lymph nodes. During migration in micro-channels, mitochondria are mainly located close to the nucleus on the rear front of cells. Mitochondria distribute along microtubule networks and follow Lamp+ and ER compartments while Golgi compartments are excluded. As described in neuron, DCs reach the most distal portion in the dendrites of DCs. It would be interesting to see if the role of distal mitochondria in the dendrite of activated DCs is linked to the secretion of cytokines as reported in neuron during exocytosis of neurotransmitters.

Conclusions
1. Mitochondrial respiration as oxygen consumption rate (OCR) increases during early activation of DCs by TLR agonists. This phenomena does not rely on change in mitochondrial mass augmentation, rather in mitochondria morphology. We think that normal DC immunogenicity may require activation by mammalian target of rapamycin (mTOR), which increases the OCR, and the pharmacological inhibition of mitochondrial respiration may foster tolerogenic DC development.
2. Mitochondrial functions are implied in DC migration, phagocytosis, endocytosis and T cell proliferation. CD8a+ DCs are more sensitive to rotenone then CD11b+ DCs.

Our findings suggest that: (a) agents that directly or indirectly enhance mitochondrial function in DC should promote DC immunogenicity, and hence might be useful as adjuvants; (b) agents that prevent or reverse mitochondrial activation upon DC stimulation should be useful to generate tolerogenic DCs; and (c) monitoring OCR in DCs can provide an assay to screen for immunogenic and/or tolerogenic agents and pathways that could be used clinically for immune modulation in patients.