Molecular mechanisms of autophagic memory in pathogenic T cells in human arthritis.

T-cell resilience is critical to the immune pathogenesis of human autoimmune arthritis. Autophagy is essential for memory T cell generation and associated with pathogenesis in rheumatoid arthritis (RA). Our aim here was to delineate the role and molecular mechanism of autophagy in resilience and persistence of pathogenic T cells from autoimmune arthritis. We demonstrated "Autophagic memory" as elevated autophagy levels in CD4+ memory T cells compared to CD4+ naive T cells and in Jurkat Human T cell line trained with starvation stress. We then showed increased levels of autophagy in pathogenic CD4+ T cells subsets from autoimmune arthritis patients. Using RNA-sequencing, transcription factor gene regulatory network and methylation analyses we identified MYC as a key regulator of autophagic memory. We validated MYC levels using qPCR and further demonstrated that inhibiting MYC increased autophagy. The present study proposes the novel concept of autophagic memory and suggests that autophagic memory confers metabolic advantage to pathogenic T cells from arthritis and supports its resilience and long term survival. Particularly, suppression of MYC imparted the heightened autophagy levels in pathogenic T cells. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target.

Increased autophagy in CD4+ T cells of rheumatoid arthritis patients results in T-cell hyperactivation and apoptosis resistance.

Rheumatoid arthritis (RA) is an autoimmune disease hallmarked by aberrant cellular homeostasis, resulting in hyperactive CD4+ T cells that are more resistant to apoptosis. Both hyperactivation and resistance to apoptosis may contribute to the pathogenicity of CD4+ T cells in the autoimmune process. A better knowledge of the mechanisms determining such impaired homeostasis could contribute significantly to both the understanding and the treatment of the disease. Here we investigated whether autophagy, is dysregulated in CD4+ T cells of RA patients, resulting in disturbed T-cell homeostasis. We demonstrate that the rate of autophagy is significantly increased in CD4+ T cells from RA patients, and that increased autophagy is also a feature of in vitro activated CD4+ T cells. The increased apoptosis resistance observed in CD4+ T cells from RA patients was significantly reversed upon autophagy inhibition. These mechanisms may contribute to RA pathogenesis, as autophagy inhibition reduced both arthritis incidence and disease severity in a mouse collagen induced arthritis mouse model. Conversely, in Atg5flox/flox -CD4-Cre+ mice, in which all T cells are autophagy deficient, T cells showed impaired activation and proliferation. These data provide novel insight into the pathogenesis of RA and underscore the relevance of autophagy as a promising therapeutic target.

HPLC determination of erythrocyte methotrexate polyglutamates after low-dose methotrexate therapy in patients with rheumatoid arthritis.

BACKGROUND: Methotrexate (MTX) may produce antiarthritic effects through polyglutamation to methotrexate polyglutamates (MTXPGs), a process that covalently attaches sequential gamma-linked glutamic residues to MTX. We sought to develop an innovative HPLC method for the quantification of these metabolites in erythrocytes. METHODS: Two alternative approaches were developed. In the first approach, MTXPGs from 50 micro L of packed erythrocytes were converted to MTX in the presence of plasma gamma-glutamyl hydrolase and mercaptoethanol at 37 degrees C. In the second approach, MTXPG species (up to the hepta order of glutamation) from 100 micro L packed erythrocytes were directly quantified in a single run. In both methods, the MTXPGs were extracted from the biological matrix by a simple perchloric acid deproteinization step with direct injection of the extract into the HPLC. The chromatography used a C(18) reversed-phase column, an ammonium acetate/acetonitrile buffer, and postcolumn photo-oxidation of MTXPGs to fluorescent analytes. RESULTS: Intra- and interday imprecision (CVs) were <10% at low and high concentrations of analytes for both methods. The limit of quantification was 5 nmol/L. In 70 patients with rheumatoid arthritis receiving weekly low-dose MTX, the mean (SD) total MTXPG concentration measured after conversion of MTXPGs to MTX was similar to the total MTXPG concentration calculated from the sum of individual MTXPG species [117 (56) vs 120 (59) nmol/L; r = 0.97; slope = 1.0]. The triglutamate predominated over all other MTXPG species (36% of total), the pentaglutamate was the highest order of glutamation detected, and a stability study revealed no change in the polyglutamation pattern in erythrocytes 48 h after phlebotomy when the specimen was stored at 2-8 degrees C. CONCLUSION: The proposed method for quantification of erythrocyte MTXPGs is rapid, sensitive, and accurate and can be applied to the routine monitoring of MTX therapy.