Session Type: Abstract Submissions (ACR)
Background/Purpose: Systemic Lupus Erythematosus (SLE) T cells display an aberrant response to CD3/ TCR stimulation. The CD3/TCR complex in SLE is characterized by the substitution of the CD3 associated kinase Zap70 by Syk. The objective of this study is to better understand the role of Syk in the dysregulated SLE T cell phenotype.
Methods: A two-step approach was followed: (i) to examine whether increased Syk expression creates an SLE-like phenotype Syk was overexpressed in healthy blood-donor T cells; and (ii) to examine whether aberrant gene expression changes in SLE T cells can be corrected by Syk downregulation, SYK was silenced using a SYK-specific siRNA. Cells were lysed following 72h of incubation and expression levels of 36 genes previously associated with SLE measured in real-time PCR. Protein was also collected and expression changes confirmed in flow cytometry and western blot. Expression was normalized towards CD3E and GPADH for PCR and β-actin for western blot.
Results: Forced expression of Syk in normal T cells reproduced many of the aberrant gene expression changes seen in SLE. Expression of cytokine IL-21, cell surface molecule CD44, and intracellular molecules PP2A and OAS2 were found to increase in cells overexpressing SYK (fold increase over control transfected ±SEM: IL-21, 5.7±1.5; CD44, 4.2±2.2; PP2A, 1.5±1; OAS2, 1.5±0.3). Silencing of Syk in SLE T cells normalized the expression of the above molecules (fold decrease over control transfected ±SEM: IL-21, -1.9±0.2; CD44, -2.8±0.4; PP2A, -2.7±1.9; OAS2, -1.1±0.8).
Findings were confirmed at the protein level using flow cytometry (mean positive cells ±SEM: (i) empty vector vs. SYK overexpressing, IL-21, 7.9±1 vs. 13±2.3, p=0.05; CD44v6, 10.7±1.2 vs. 16±2.1, p=0.05; (ii) control vs. SYK siRNA transfected, IL-21, 18.6±2.4 vs. 11.4±1.6, p=0.03; CD44v6, 11.7±0.7 vs.7.6±1.2, p=0.05) and western blot (mean relative expression ±SEM: (i) empty vector vs. SYK overexpressing, OAS2, 0.91±0.1 vs. 1.39±0.2, p=0.05; PP2A, 0.49±0.1 vs. 1.23±0.3, p=0.05; (ii) control vs. SYK siRNA transfected, OAS2, 1.39±0.3 vs. 1.06±0.2; PP2A, 1.14±0.3 vs. 0.69±0.1).
Conclusion: Our data show that overexpression of Syk in healthy T cells recapitulates at least part of the SLE T cell phenotype. Syk overexpressing T cells may provide more help to B cells through IL21 secretion, have enhanced migration to tissues through CD44 upregulation and produce pro-inflammatory rather than counter-inflammatory cytokines. Inhibiting Syk in SLE T cells leads to the opposite effect, further underscoring Syk’s potential as a therapeutic target in SLE.
A. P. Grammatikos,
V. C. Kyttaris,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/spleen-tyrosine-kinase-syk-regulates-systemic-lupus-erythematosus-t-cell-signaling/