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Malecka, Anna A. (2018) The impact of stroma and ionising radiation on the plasticity of dendritic cells and macrophages. PhD thesis, University of Nottingham.

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Abstract

The basic model of antigen-presenting cells (APC) activation describes a change of state from resting (immature) to activated (mature) upon encounter with a first signal. However, it is apparent that APC maturation is not binary but instead has a spectrum of outcomes. This likely reflects the complex and dynamic crosstalk resulting from their encounter with a wide variety of stromal cells. A deeper knowledge of these interactions, and how they are processed by APC to instruct immunity is required. This study explores the plasticity of the major APC, namely macrophage (Mφ) and dendritic cells (DC). We test the hypothesis that plasticity in APC function depends on the sequence of signals encountered during generation and activation. Human laboratory models of Mφ and DC were generated from monocytes as follows; monocyte-derived Mφ were cultured with M-CSF (M-Mφ) or GM-CSF (GM-Mφ) and DC with IL-4 and GM-CSF. These were characterised and the impact of cellular (e.g. fibroblasts (FB)) or external factors (e.g. ionising radiation (IR)) studied.

Changes in microenvironment considerably alter the functional-phenotype of Mφ however it remains to be shown if their initial polarisation restricts responses. Activation of GM-Mφ elicited IL-12 and -23 whilst M-Mφ expressed IL-10. However, the classic pro-inflammatory signal interferon γ (IFNγ) did not elicit M-Mφ that secreted cytokines to the level expected of GM- Mφ. The M-Mφ displayed mixed phenotype with both pro-inflammatory cytokines (albeit at lower levels, 75% less IL-23, 21% less IL-12) on a background of high IL-10. The IL-23 response of Mφ to re-stimulation was reduced or abrogated upon pre-treatment with LPS but enhanced in Mφ conditioned by IFNγ. These findings demonstrate limitations to Mφ plasticity reflecting initial conditioning and suggest the development of innate memory with a degree of specificity in Mφ responses to external factors.

We applied ionising radiation (IR) to our models at doses commonly used for radiotherapy (1-20Gy). Irradiation markedly suppressed IL-23 secretion by both Mφ and DC (p<0.01) in a dose-dependent manner without affecting viability. This was selective for IL-23 as there was no change in IL-1β, -6, -10, -27 or TNFα, and only a modest decrease in IL-12 secretion. To investigate the mechanism for IL-23 regulation we showed that IR activated Ataxia telangiectasia mutated kinase (ATM) which in turn inhibited the function of cAMP response element-binding protein (CREB) through phosphorylation of an inhibitory residue (S121). ATM activation suppressed IL-23 as inhibition of ATM with KU55933 increased its expression. Furthermore, the small-molecule CREB inhibitor C-Ci reduced IL-23 levels. As a consequence of modulation of IL-23 in APC, IR indirectly affected the generation of T-cell responses, namely Th-17. Cross-talk between APC and stromal cells was modelled by co-culture of DC with FB. The presence of FB prevented IL-23 downregulation by IR. This was dependent on TNFα and IL-1β secretion by DC which were unaffected by IR. In response, FB secreted PGE2 which resulted in a feed-back loop to enforce IL-23 secretion via activation of cAMP. Therefore, whilst IR could be seen to regulate IL-23 in a single cell culture system, this did not occur in co-cultures that simulated the interaction APC have with stroma.

In conclusion, we shown that the order in which APC receive conditioning signals ultimately affects how they respond to re-challenge. This is further influenced by a mixture of micro-environmental and external factors. Understanding the molecular pathways through which these cues manifest will result in in vitro models that better reflect the biology of APC and create opportunity for intervention.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Jackson, Andrew M. Patel, Poulam M.
Keywords:	Cell interaction; Crosstalk; Macrophages; Dendritic cells; Cell signals; Ionising radiation; Immune response; Stromal cells; Antigen-presenting cells
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QW Microbiology. Immunology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	50513
Depositing User:	Malecka, Anna
Date Deposited:	12 Jul 2018 04:40
Last Modified:	12 Jul 2020 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/50513

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