Tacrolimus Inhibits NF-κB Activation in Peripheral Human T Cells

Abstract

The calcineurin inhibitor, tacrolimus (TAC), inhibits the protein phosphatase activity of calcineurin, leading to suppression of the nuclear translocation of NFAT and inhibition of T cell activation. Apart from NFAT also the transcription factor NF-κB plays a key functional role in T cell activation. Therefore, blockade of the NF-κB activation cascade by immunosuppressive drugs prevents immune activation. Here we studied whether TAC blocks NF-κB activation in peripheral human T cells. After anti-CD3/CD28-activation of T cells from healthy volunteers, NF-κB (p65) phosphorylation was measured by flow cytometry in CD3+ T cells, CD4+ helper T cells and CD8+ cytotoxic T cells in the absence and presence of TAC 10 ng/mL, sotrastaurin 500 nM (positive control) and mycophenolic acid 10 µg/mL (negative control; n = 6). NF-κB transcriptional activity was measured by ELISA and intracellular TNFα protein, a downstream target, was measured by flow cytometry to assess the functional consequences of NF-κB blockade. Anti-CD3/28-activation induced NF-κB phosphorylation in CD3+ T cells, CD4+ T cells and CD8+ T cells by 34% (mean), 38% and 30% resp. (p<0.01). Sotrastaurin inhibited NF-κB activation in the respective T cell subsets by 93%, 95% and 86% (p<0.01 vs. no drug), while mycophenolic acid did not affect this activation pathway. Surprisingly, TAC also inhibited NF-κB phosphorylation, by 55% (p<0.01) in CD3+ T cells, by 56% (p<0.01) in CD4+ T cells and by 51% in CD8+ T cells (p<0.01). In addition, TAC suppressed NF-κB DNA binding capacity by 55% (p<0.05) in CD3+ T cells and TNFα protein expression was inhibited in CD3+ T cells, CD4+ T cells and CD8+ T cells by 76%, 71% and 93% resp. (p<0.01 vs. no drug), confirming impaired NF-κB signaling. This study shows the suppressive effect of TAC on NF-κB signaling in peripheral human T cell subsets, measured at three specific positions in the NF-κB activation cascade.

Pharmacokinetic considerations related to therapeutic drug monitoring of tacrolimus in kidney transplant patients

“…The effect of Tac on the NF-κB activation pathway was studied by quantitative analysis of NF-κB phosphorylation in primary T cell subsets. This study concluded that Tac has a suppressive effect on NF-κB signaling in peripheral T cell subsets [134].…”

Scoping systematic review of treatments for eczema

“…Both tacrolimus and pimecrolimus selectively inhibit the activation of T cells. 74,75 T cells play a key role in the characteristic inflammation of the skin in eczema. Pimecrolimus has also been trialled as an oral treatment for eczema, which is discussed later in this review (see Chapter 11).…”

Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry

“…By inhibiting the activity of calcineurin, TAC inhibits the T cell receptor-mediated activation of the nuclear factor of activated T cells (NFAT), one of the key signaling pathways controlling the immune response on a cellular level. In addition to TAC's effect on calcineurin and the NFAT signaling pathway, TAC has recently been described to also affect the NF-κB signaling pathway by inhibiting the phosphorylation of p65 at the serine 529 position (Vafadari et al, 2013). In order to demonstrate the applicability and relevance of IFC to simultaneously evaluate specific phosphorylation motifs and nuclear translocation, TAC exposure of healthy donor T cells was used as the experimental model system with the expectation that TAC induced inhibition of p65 at the serine 529 position should be detectable without affecting nuclear translocation of total p65.…”