Session Type: Abstract Submissions (ACR)
Background/Purpose: CD4+CD28- T cells are enriched in chronic inflammatory diseases like rheumatoid arthritis (RA) and lupus. They are cytotoxic and resistant to apoptosis. Compared to CD28+ cells, CD28- CD4 T cells over-express killer immunoglobulin-like receptors (KIRs) and other pro-inflammatory molecules. These genes are regulated by DNA methylation, so they are over-expressed by CD4 T cells that are demethylated in vitro. This is a result of decreased signaling through the ERK and JNK pathways and, consequently, decreased activity of the DNA methyltransferase enzymes (DNMTs) responsible for DNA methylation. Protein phosphatase 5 (PP5) is a stress induced regulator of gene expression in multiple signaling pathways, including those involved in aging. It is expressed in CD4+CD28-, but not CD4+CD28+ T cells, and it inhibits both ERK and JNK signaling. We hypothesized that PP5 is over-expressed in CD4+ T cells in patients with RA and lupus, and that over-expressing PP5 in CD4 T cells from healthy donors will induce expression of methylation sensitive genes unique to CD4+CD28- T cells.
Methods: CD4+ T cells were isolated from healthy controls and patients, and PP5 mRNA was measured by RT-PCR. To study the effects of PP5 on gene expression, PBMCs from healthy donors were stimulated with phytohemagglutinin and cultured for 3 days with IL-2. CD4+ T cells were then isolated by negative selection, transfected (Amaxa Nucleofector) with constructs encoding GFP and PP5 or GFP alone, and cultured 24-72 hours. Expression of DNMT1 and methylation sensitive genes was assessed by RT-PCR in sorted CD4+GFP+ T cells. DNMT1 expression was measured 24 hours after transfection, and the other genes were analyzed 72 hours after transfection. Cell surface protein expression was measured by flow cytometry 72 hours after transfection.
Results: Compared to CD4+ T cells from healthy donors, PP5 mRNA is over-expressed in patients with lupus (1.97 fold change ±0.18 SEM, p=0.03) and RA (1.6±0.2, p=0.06). When transfected into CD4+ T cells from healthy donors, PP5 increased mRNA levels of KIR (2DL4 gene, 2.4±0.7, n=3, p=0.04), perforin (1.38±0.07 fold, p=0.03, n=3), CD11a (1.2±0.1 fold, p=0.047, n=5), and CD70 (10.5±4.1 fold, p=0.03, n=7). PP5 also increased the percentage of cells expressing surface KIRs (33±7% with control vs. 62±7% with PP5, n=7, p<0.01), CD70 (18.3±7% with control vs. 34±10.8% with PP5, n=3, p=0.06), and CD40L (15±6.1% with control vs. 27.3±9% with PP5, n=3, p<0.05). Finally, PP5 caused a corresponding 20±8% decrease (n=5, p=0.02) in DNMT1 mRNA expression.
Conclusion: CD4+CD28- T cells, which are enriched in lupus and RA, over-express pro-inflammatory methylation sensitive genes. These data demonstrate, for the first time, that PP5 contributes to the regulation of these genes (KIR, perforin, CD70, CD40L, and CD11a) in CD4+ T cells. PP5 is hypothesized to accomplish this by demethylating regulatory elements in the promoters for these genes, and this is currently being tested. PP5 expression is induced by oxidative and replicative stress, so it could provide a mechanistic link between those physiologic stressors and autoimmune disease flares.
D. R. Patel,
B. C. Richardson,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/protein-phosphatase-5-pp5-regulates-methylation-sensitive-gene-expression-in-cd4-t-cells-3/