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Title: ATM kinase and DNA-PKcs pathways regulate the expression of IL-23 in dendritic cells : interaction with ER-stress pathways and impact on Th17 responses
Author: Wang, Qunwei
ISNI:       0000 0004 6349 7595
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2014
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Dendritic cells (DCs) are key members of the immune system as they detect danger and reflect this during presentation of antigen to T cells. DC produce cytokines that influence both the innate and adaptive immune system. Notably they produce the IL- 12-family of cytokines known for their influence on T helper (Th) cell priming. Interleukin-23 is one such cytokine that is recognised for its roles in supporting Thl7 cell differentiation characterised by IL-17 and IL-22 production, in driving IL-17 secretion by y6 T cells, and in its contribution to anti-bacterial immunity and autoimmunity. A body of evidence exists that Dectin-l/TLR-2 Syk- CARD9-NFkB signalling pathways, p38 MAPK/Erk- NFk-B signalling pathways and PI3K/Akt pathways are involved in IL-23 production in their respective settings. However, little is known at the molecular level regarding the regulation of IL-23 secretion in human DC. Here we show for first time that the Ataxia Telangiectasia Mutated (ATM) pathway and DNA-Protein kinase catalytic subunit (DNA-PKcs), involved in DNA-damage-sensing and DNA double strand break (DSB) repair, act as repressors of IL-23 in DC. Inhibition of ATM with the highly-selective antagonist KU55933, or DNA-PKcs with NU7441, markedly increased IL-23 secretion human monocyte-derived DC (moDC) and freshly isolated CDlc+ myeloid DC (myDC). In contrast, inhibiting the closely related mammalian target of rapamycin (mTOR) had no effect on IL-23. Priming naive CD4+ T- cells in the presence of supernatant from ATM or DNA-PKcs-inhibited DC resulted in increased Thl7 responses. Whilst ATM or DNA-PKcs-blockade increased the abundance of pl9, p35 and p40 mRNA, IL- 12p70 secretion was much less affected. In order to further examine a role for ATM and DNA-PKcs in IL-23 regulation we exposed DC to low doses of ionizing radiation. Exposure of DC to X-rays resulted in a rapid increase (15min) in ATM phosphorylation and a corresponding depression of IL-23 production. Importantly, ATM-inhibition with KU55933 prevented radiation-induced ATM phosphorylation and abrogated the capacity of X-rays to suppress IL-23. Interestingly, the viability of DC exposed to X-rays after DNA-Pkcs inhibition was substantially affected but unaffected in DC exposed to IR after ATM inhibition, suggesting an important role for DNA-PKcs in the resistance of DC to DNA-damage. To explore how IL-23 was repressed by ATM or DNA-PKcs we examined the unfolded protein response (UPR) pathways by measuring generation of the spliced form of X-box protein-1 (XBPls), a key ER-stress transcription factor. When ATM or DNA-Pkcs were inhibited the abundance of XBPls mRNA increased and was followed 3hr later by increased p l9 transcription and subsequent IL-23 release. Unexpectedly, in DC deficient for the UPR-related CHOP transcription factor, inhibition of ATM or DNA-PKcs continued to secrete high levels of IL-23. However, inhibition of the PERK arm of the UPR reduced IL-23 production by matured DC, and importantly partially suppressed ATM- or DNA-PKcs-dependent IL-23. In summary, in addition to their canonical roles in DNA-repair, ATM and DNA-PKcs also regulate immune-activation by DC. Whilst their activation further represses IL-23-dependent responses, their inhibition permits markedly high levels of IL-23 to be generated. These pathways thus represent new therapeutic targets in autoimmunity and vaccine development.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available