The novel roles of DNA damage repair proteins ATM and DNA-PK in the regulation of the ER stress response and IL-23 expression in dendritic cells

Shah, Sabaria (2017) The novel roles of DNA damage repair proteins ATM and DNA-PK in the regulation of the ER stress response and IL-23 expression in dendritic cells. PhD thesis, University of Nottingham.

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Dendritic cells (DC) are the principal, cross-priming antigen-presenting cell responsible for naïve T cell activation and differentiation. Specific programmes of cytokine secretion by DC are particularly important for the polarisation of CD4+ T-cells to discrete effector populations i.e. Th1, Th17, Tr1 etc. Our knowledge of control for some of these cytokine signals is currently limited and modulating the polarisation of defined Th-subsets remains a challenge. Improved control of Th-differentiation has considerable potential in the fields of immune disorders and infectious disease. We describe a novel immune-regulatory function of two DNA-repair proteins signal to modulate IL-23 secretion, a key signal for Th17 differentiation.

ATM and DNA-PK, are two protein kinases classically regarded for their paramount importance in repair of DNA double strand breaks. We characterised an alternate immune-regulatory function for ATM and DNA-PK for IL-23 production by DC. Therefore, constitutive expression of activated ATM and DNA-PK in DC, but not in CD14+ monocytes, related not only to their ability to repair damaged DNA, but to secrete IL-23. Increased IL-23 secretion was observed in activated human monocyte derived-DC (moDC), mouse bone marrow derived-DC and macrophages following inhibition of ATM and DNA-PK using the highly selective antagonists KU55399 and NU7441, respectively. In contrast, inhibiting ATR, a closely related single-strand DNA repair kinase, had no effect on IL-23. Interestingly, whilst inhibition of ATM only activated IL-23 secretion, inhibition of DNA-PK also led to increased IL-6, IL-1β and IL-10. IL-23 regulation through ATM/DNA-PK only occurred following toll-like receptor 4 (TLR4) activation and not with TLR1/2, 3, 5, 7/8, Dectin-1 and Dectin-1/TLR2 agonists. In support of a proposed immune-regulatory role for DNA-repair proteins, ionising radiation activated ATM and DNA-PK and consequently repressed IL-23. Radiation-dependent ATM/DNA-PK phosphorylation was prevented by KU55399 and NU7441, however only the ATM inhibitor restored IL-23 levels.

To determine the mechanism of action we tested the hypotheses that increased IL-23 resulted from

a) Altered activation of the Unfolded Protein Response (UPR) pathways,

b) Increased IL-23 was IL-1β dependent, or

c) Differential activation of NF-κB.

These were not supported by experimental evidence as

a) whilst inhibition of ATM/DNA-PK activated the UPR pathways they could not be shown to regulate IL-23

b) blocking IL-1β receptor with IL-1RA did not attenuate ATM/DNA-PK regulated IL-23.

c) Evidence for prolonged activation of NF-κB following DNA-PK inhibition was obtained, however its role remained unclear.

A role for the CREB transcription factor was established for ATM-dependent IL-23 regulation. Activated ATM phosphorylated CREB at Ser121, a residue associated with negative CREB activity and consequently ATM inhibition enhanced CREB activity in a luciferase reporter assay. The addition of an inhibitor of the interaction of CREB with its transcriptional co-activator, CBP, abolished ATM-enhanced IL-23 response.

Control of IL-23 dependent immune responses is increasingly important in autoimmunity and cancer. This study identifies a novel immune-regulatory role for DNA-repair systems in DC resulting in the regulation of IL-23 and other Th17 associated cytokines (IL-1, IL-6). Further studies could explore the potential of either small molecule inhibitors of ATM/DNA-PK to enhance, or stress signals (e.g. radiation) to repress IL-23 responses.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Jackson, A.
Patel, P.
Keywords: Dendritic cell, IL-23
Subjects: QS-QZ Preclinical sciences (NLM Classification) > QW Microbiology. Immunology > QW501 Immunology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 43550
Depositing User: Shah, Sabaria
Date Deposited: 04 Oct 2017 12:49
Last Modified: 06 May 2020 12:03

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