Background/Purpose: Interleukin-17 (IL-17) and Tumor Necrosis Factor alpha (TNFa), when co-applied to RASF, induce synergistic expression of proinflammatory genes such as IL-6 and IL-8.  Work from our laboratory using co-cultures of cytokine-activated T cells and RASF has demonstrated that this synergy is largely contact-dependent (Tran et al., 2007).  Recently, Hot et al (2011) showed that PLD1 mRNA was similarly upregulated by IL-17A and the less potent IL-17F in RASF.  Another recent report (Sethu et al., 2010), used a mouse model of peritonitis to show that blocking PLD1 mitigates TNFa-driven inflammation in vivo.  In light of these observations, we sought to examine the potential role of PLD1 in pro-inflammatory gene expression by RASF stimulated with IL-17A and/or TNFa.

Methods: We used quantitative real-time PCR to characterize the cytokine-mediated effects on mRNA levels for a cluster of genes encoding cytokines, chemokines, and tissue remodeling enzymes important in the pathogenesis of RA.  Also, secretion of IL-6, IL-8, and CCL20 was measured by ELISA.  To investigate the relevance of PLD1 activity, we added various concentrations of the PLD1 inhibitor 1-butanol, knocked down PLD1 expression with siRNA, and used the two approaches together.

Results: PLD1 mRNA was weakly induced by IL-17 and/or TNFa (<2-fold increase).  1-butanol had complex effects on cytokine-induced target gene mRNA expression, in a manner that was dose-dependent and biphasic for all targets except ICAM1, IL-8, and MMP-14.  Compared with the 10 other targets studied, including PLD1 itself, ICAM1 mRNA expression showed the least sensitivity to treatment with 1-butanol.  When RASF were transfected with PLD1-specific siRNA, there was an effect on induction of mRNA and secreted protein, particularly for induction of IL-6, IL-8, and CCL20 when IL-17 was co-applied with TNFa.  Effects on mRNA and secreted protein were not always positively correlated.  For example, interference with PLD1 activity resulted in increased CCL20 mRNA, but inhibited CCL20 secretion. Effects of PLD1 knockdown were in part distinct from effects of 1-butanol.

Conclusion: PLD1 might be an important modulatory target for reducing cytokine-evoked expression of proinflammatory genes by RASF, because it exhibits gene-specific effects on mRNA levels and effects on efficacy of regulated secretion/exocytosis.  Stability of mRNAs and modulation of transcription efficiency are likely mechanisms by which PLD1 affects cytokine-induced expression of proinflammatory genes.  The effects of 1-butanol may reflect inhibition of not only PLD1 but also PLD2 and possibly other enzymes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-phospholipase-d1-pld1-in-the-expression-of-proinflammatory-genes-in-rheumatoid-arthritis-synovial-fibroblasts-rasf/