Cyclosporine A inhibits IL-15-induced IL-17 production in CD4⁺ T cells via down-regulation of PI3K/Akt and NF-κB

Abstract

Cyclosporine A (CSA) has various biological effects on T cells, including inhibition of interleukin (IL)-15-induced IL-17 production in CD4⁺ T cells from patients with rheumatoid arthritis (RA). However, the mechanism underlying this effect is not fully understood. Here, we tried to investigate the mechanism of CSA to inhibit IL-17 production induced by IL-15 in CD4⁺ T cells. Synovial fluid and serum levels of IL-15 and IL-17 were determined by ELISA. CD4⁺ T cells from RA patients were treated with IL-15 in the presence of CSA or several signal inhibitors. The concentration of IL-17 in culture supernatants was measured by ELISA and IL-17 mRNA expression was determined by RT-PCR. NF-κB binding activity for IL-17 transcription was assessed by electrophoretic mobility shift assay. IL-15 induced IL-17 production by CD4⁺ T cells in dose- and time-dependent manner. IL-15-stimulated IL-17 production and mRNA expression were inhibited by CSA in CD4⁺ T cells. Moreover PI3K/Akt inhibitor, NF-κB inhibitor, and FK506 significantly inhibited IL-15-induced IL-17 production in CD4⁺ T cells. Inhibition studies revealed the requirement of PI3K/Akt and NF-κB signal pathway for IL-15-induced IL-17 production. CSA down-regulated the phosphorylation of Akt and IκB. CSA inhibited binding of NF-κB to IL-17 promoter. The inhibitory effect of CSA on IL-15 induced IL-17 production partially depended on the increase in IL-10, since neutralizing anti-IL-10 antibodies were able to partially reverse this inhibition. CSA inhibits IL-17 production by CD4⁺ T cells and this effect is mediated by IL-15-activated NF-κB pathway in CD4⁺ T cells, which is possible mechanism of CSA in treating RA as NF-κB targeting strategy.

Introduction

Cyclosporine A (CSA) is an efficient inhibitor of the Ca²⁺/calmodulin-dependent phosphatase calcineurin. CSA forms complexes with low molecular weight cytosolic proteins, the immunophilins, and the resulting dimeric complexes bind to and inhibit the catalytic activity of the calcineurin A subunit [1]. Transfection studies have shown that calcineurin induces the activity of nuclear factor of activated T cells (NFAT) transcription factors, probably by dephosphorylating the inactive cytosolic forms of NFAT, which leads to the nuclear transport of NFAT factors in T cells [2].

Because NF-κB has many functions in the cell and in the inflammatory cascade, it has been implicated as a mediator of many immune suppressive agents including CSA, tacrolimus, and glucocorticoids [3]. Several studies have suggested that NF-κB is a target molecule of CSA and that inhibition of NF-κB by CSA in non-T cells might be more complete than was thought previously [4], [5], [6]. CSA interferes with the inducible degradation of NF-κB inhibitors and differentially inhibits the expression of key cell surface costimulatory molecules on dendritic cells by decreasing the nuclear translocation and DNA binding of NF-κB.

NF-κB is clearly one of the most important regulators of proinflammatory gene expression in rheumatoid arthritis (RA). Synthesis of cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 is mediated by NF-κB [7]. A study of T cells in a murine collagen-induced arthritis model showed that inhibition of NF-κB signaling in the T cells of transgenic mice substantially attenuates the incidence and severity of arthritis, demonstrating an important in vivo role of NF-κB in autoimmune disease [8].

IL-17 is of particular interest in RA because it contributes to joint inflammation and destruction [9]. IL-17 stimulates osteoclast and chondrocyte destruction of the collagen matrix [10], [11] and synoviocytes to secrete protease [12]—reactions that may destroy the joint mechanically, impair joint function, or aggravate inflammation in RA patients.

Considerably less is known about the regulation of IL-17 expression than the biological activities of IL-17. In human, IL-17 mRNA is found mainly in activated CD4⁺ memory T cells, although CD8+ T cells also secrete IL-17 at considerably lower levels [13], [14]. Pharmacological inhibitor studies have suggested that T cell receptor (TCR)-stimulated expression of IL-17 is both calcineurin and cAMP dependent [15]. We also reported TCR stimulation with anti-CD3 antibody activated the PI3K-Akt pathway and activation of the PI3K-Akt pathway resulted in a pronounced augmentation of NF-κB [16]. A recent study revealed that IL-15 triggered production of IL-17 in peripheral blood mononuclear cells (PBMC) from healthy blood donors via a CSA-sensitive mechanism [17]. The production of IL-17 is blocked completely by the presence of CSA, supporting the assumption that activation of calcineurin and further downstream dephosphorylation of NFAT and its translocation to the nucleus play a pivotal role in the induction of IL-17 transcription.

However, little is known about how IL-15 stimulates IL-17 production, the type of signaling molecules involved, and which target molecules are involved in the effects of CSA on IL-15-induced IL-17 production in T cells. A CSA-dependent relationship of IL-15 and IL-17 has been suggested, but has rarely been investigated, and the intracellular inhibitory action of CSA on IL-15-induced IL-17 production has much to be studied. The purpose of our study was to define the intracellular mechanism underlying CSA's inhibitory action. We investigated the effect of CSA on the IL-15-signaling mechanism for IL-17 production and identified the type of intracellular pathway by which CSA inhibits IL-15-dependent IL-17 production in T cells.

CSA down-regulated the phosphorylation of Akt and IκB and inhibited binding of NF-κB to the IL-17 promoter by IL-15 stimulation in CD4⁺ T cells of RA patients. Our data suggests that the CSA might have beneficial effects in treating RA by its actions of anti-inflammatory activity by down-regulating IL-17 production in T cells. NF-κB which was found to be related with the suppressive mechanism of CSA in CD4⁺ T cells could be therapeutic target in future. Moreover, CSA is nowadays a minor drug in treating RA but can be alternative drug in some clinical conditions such that CSA should be taken after organ transplantation or Behçet uveitis and so on.