Background/Purpose: CD4 T cells from patients with rheumatoid arthritis (RA) are metabolically reprogrammed, diverting glucose away from glycolysis towards the production of biosynthetic precursors. Several of the pro-inflammatory effector functions of such CD4 T cells are metabolically controlled; including T cell longevity, differentiation into Th1 and Th17 cells and the formation of tissue-invasive membrane ruffles.

Methods: T cells from seropositive RA patients with active disease and age-matched healthy controls were examined. Intra- and extracellular metabolites were quantified, and energy sensing was assessed by pAMPK and pS6K1 analysis. Lysosomal localization of mTORC1 and AMPK was examined by confocal microscopy and immunoblotting. T cell differentiation into pro-inflammatory effector cells was determined through lineage-determining transcription factors and intracellular cytokines. Tissue inflammation in human synovium was evaluated in NSG chimeric mice engrafted with human synovium and reconstituted with human T cells.

Results: In response to T cell receptor triggering, RA T cells hyperproliferated while intracellular ATP concentrations were significantly lower than in control T cells. Despite a high AMP/ATP ratio, RA T cells failed to activate the energy senor AMPK, but mTORC1 activation was sustained. AMPK activation occurs on the lysosomal surface where the kinase colocalizes with mTORC1 to enable cross-regulation of both energy sensors. Immunoblotting and imaging analysis revealed a lack of lysosomal AMPK in RA T cells, while mTORC1 was maintained on the outer lysosomal membrane. Lysosomal anchoring requires AMPK lipidation through posttranslational myristoylation. RA T cells were deficient in N-myristoyltransferase 1 (NMT1), the enzyme which catalyzes the transfer of myristate from CoA to AMPK. Restoring NMT1 expression rescued the lysosomal recruitment of AMPK, secured the activation of the kinase, inhibited the unopposed activation of mTORC1 and prevented the differentiation of T cells into cytokine producing, pro-inflammatory effector cells in vitro and in vivo. We devised multiple therapeutic interventions to overcome the lack of lysosomal AMPK and tested their tissue-protective effects in the human synovium-NSG chimeras; including forced overexpression of NMT1 in RA T cells; treatment with A769662, an AMPK activator that acts independently of the lysosome, and injection of the mTORC1 inhibitor Rapamycin. All interventions were equally potent in suppressing synovitis.

Conclusion: Pro-inflammatory effector functions of T cells are highly dependent on the subcellular localization and cross-regulation of the energy sensors AMPK and mTORC1. Deficiency of protein myristoylation due to loss-of-function of NMT1 deviates AMPK away from the lysosomal surface, prevents its proper activation and enables unopposed mTORC1 activity. Protein trafficking and subcellular localization of the energy sensors AMPK and mTOC1 may be druggable by novel therapeutic strategies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/lysosomal-placement-of-the-energy-sensors-ampk-and-mtorc1-controls-tissue-inflammation-in-rheumatoid-arthritis/