The ataxia telangiectasia mutated kinase pathway regulates IL-23 expression by human dendritic cells.

Little is known of the regulation of IL-23 secretion in dendritic cells (DC) despite its importance for human Th17 responses. In this study, we show for first time, to our knowledge, that the ataxia telangiectasia mutated (ATM) pathway, involved in DNA damage sensing, acts as an IL-23 repressor. Inhibition of ATM with the highly selective antagonist KU55933 markedly increased IL-23 secretion in human monocyte-derived DC and freshly isolated myeloid DC. In contrast, inhibiting the closely related mammalian target of rapamycin had no effect on IL-23. Priming naive CD4(+) T cells with ATM-inhibited DC increased Th17 responses over and above those obtained with mature DC. Although ATM blockade increased the abundance of p19, p35, and p40 mRNA, IL-12p70 secretion was unaffected. To further examine a role for ATM in IL-23 regulation, we exposed DC to low doses of ionizing radiation. Exposure of DC to x-rays resulted in ATM phosphorylation and a corresponding depression of IL-23. Importantly, ATM inhibition with KU55933 prevented radiation-induced ATM phosphorylation and abrogated the capacity of x-rays to suppress IL-23. To explore how ATM repressed IL-23, we examined a role for endoplasmic reticulum stress responses by measuring generation of the spliced form of X-box protein-1, a key endoplasmic reticulum stress transcription factor. Inhibition of ATM increased the abundance of X-box protein-1 mRNA, and this was followed 3 h later by increased peak p19 transcription and IL-23 release. In summary, ATM activation or inhibition, respectively, inhibited or augmented IL-23 release. This novel role of the ATM pathway represents a new therapeutic target in autoimmunity and vaccine development.

Novel approach for interleukin-23 up-regulation in human dendritic cells and the impact on T helper type 17 generation.

Interleukin-23 (IL-23) is important for T helper type 17 (Th17) responses and strategies to regulate IL-23 in human dendritic cells (DC) are limited. This study describes a novel means to control IL-23 secretion by conditioning DC with a phosphatidyl inositol 3-kinase inhibitor Wortmannin (WM). Treatment of monocyte-derived DC with WM increased Toll-like receptor (TLR) -dependent IL-23 secretion 10-fold and IL-12p70 twofold, but IL-27 was unaffected. The effect of WM was restricted to TLR3/4 pathways, did not occur through TLR2, TLR7/8 or Dectin-1, and was characterized by increased p19, p35 and p40 transcription. These responses were not solely dependent on phosphatidyl inositol 3-kinase as the alternative inhibitor LY294002 did not modulate IL-23 production. The normal patterns of activation of mitogen-activated protein kinase pathways were unaffected by WM-conditioning but IL-23 secretion required p38, ERK and JNK pathways. Importantly, this effect was manifest in populations of blood DC. Conditioning freshly isolated myeloid DC with WM before TLR3 or TLR4 triggering resulted in high levels of IL-23 secretion and an absence of IL-12p70. These WM-conditioned myeloid DC were highly effective at priming Th17 responses from naive CD4(+) T cells. Our findings provide a novel means to generate IL-23-rich environments and Th17 responses and suggest as yet unidentified regulatory factors, identification of which will provide new approaches to control IL-23-dependent immunity in infectious disease, autoimmunity and malignancy.

Role of mitogen-activated protein kinase and PI3K pathways in the regulation of IL-12-family cytokines in dendritic cells and the generation of T H-responses.

Mitogen-activated protein kinases (MAPK) are targets for the immune-modulation of dendritic cells (DC). However, our knowledge of their role in the regulation of IL-12-family cytokines is limited. This study investigated the roles of p38, JNK, p44/42 and PI3K pathways in IL-12/23/27 production by human DC, and their impact on naïve T(H)-responses. We first identified TOP and UBC as robust DC housekeeping genes. Peak transcription of p35 and p40 occurred by 12h, p19 and p28 by 8h and EBI3 by 12-24h. Using selective antagonists, we showed that p38 was a positive regulator of IL-12, 23 and 27, JNK positively regulated IL-12 and IL-27, and inhibition of MEK1/2 had no marked effect. In contrast, the PI3K pathway markedly attenuated IL-23 responses and, to a lesser extent, IL-12, but not IL-27. To identify the role of these soluble factors, we co-stimulated naïve CD4+ T-cells in the presence of DC supernatant. The presence of mature DC supernatant induced not only strong IFNγ responses, but also IL-10 and IL-17A. Inhibition of p38 ablated T(H1), and IL-10 and IL-17A responses, whilst modestly enhancing IL-5 secretion. In contrast, inhibition of MEK1/2 abolished IL-17A production, whilst leaving other responses unaffected, whereas inhibition of JNK or PI3K had no discernable effect. In summary, we describe the expression of IL-12-family cytokines from DC and propose a modified model for their regulation. This study further clarifies the potential for therapeutic modulation through these mediators.