Molecular Investigations into Novel Nuclear Receptor Modulators as Therapeutics in Inflammatory Disease

Abstract

Orphan nuclear receptor subfamily 4A (NR4As) have emerged as pivotal regulators of inflammatory responses, primarily through their interactions with NF-κB. Over the last decade a number of agents have emerged as NR4A modulators and show potential as therapeutics. However, more research is required into their cytotoxicity profile and how they affect varied cell types and inflammatory outcome downstream of multiple inflammatory ligands, and this is the focus of this thesis. Using NR4A agonists Csn-B and C-DIM12, we have first examined their cytotoxicity profile, and second their ability to alter inflammatory outcome of six ligands, lipopolysaccharide (LPS), tumour necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), flagellin (FL), lipoteichoic acid (LTA) and zymosan (ZY), in both epithelial and myeloid cells lines, Caco-2 and THP-1. Cytotoxicity analysis harnessed a variety of experimental approaches using both direct / indirect measurements of cytotoxicity. Inflammatory outcome analysis encompassed the use of a cytokine/chemokine array, ELISAs, qRT-PCR, NF-κB transcriptional activity and metabolic parameters. Cytotoxicity analysis in Caco-2 cells revealed Csn-B at concentrations up to 100μM, and C-DIM12 up to 10μM, does not adversely affect cellular toxicity, and in turn viability. While 50μM C-DIM12 increases caspase-3/7 activity. In THP-1 cells, Csn-B at 1.0 and 10μM, does not result in cytotoxicity in any parameter measured, while 100μM affects membrane integrity. Regarding C-DIM12 in THP-1 cells, all experimental approaches show at 0.1 and 1.0μM no cytotoxic affects are observed, while at 50μM, all approaches show cytotoxic affects. While some changes were observed following C-DIM12 exposure at 10μM, with a decline in the reducing capacity of the cells and caspase-3/7 activity increasing modestly at 24h. However, following in depth analysis of the experimental approaches, we concluded that changes observed with C-DIM12 10μM, may very well reflect altered metabolism and not solely cytotoxicity. In Caco-2 cells key inflammatory mediators, TNFα, MCP-1, IL-8 and IL-1β, were examined following exposure with the six inflammatory ligands, with/without Csn-B (1.0μM) or C-DIM12 (10μM). Importantly, these are known NR4A targets and have been shown to be modulated by Csn-B and/or C-DIM12 previously. Data obtained shows that neither Csn-B or C-DIM12 are able 7 to significantly modulate these targets downstream of TNFα, LPS, ZY, FL or LTA in Caco-2 cells. While a significant potentiation of TNFα and MCP-1 gene expression was observed following IL-1β exposure with C-DIM12 at 4h, this was not reflected by ELISA analysis at 24h. In THP-1 cells, we reveal that Csn-B (1.0μM) and C-DIM12 (10μM), while can attenuate some targets induced by these ligands (MCP-1, CCL5 and CXCL1), these targets are principally unique to each NR4A agonist. Furthermore, it is revealed that, 10μM C-DIM12, and not 1μM C-DIM12 or Csn-B, can significantly attenuate the increased NF-κB transcriptional activity observed following the exposure to multiple inflammatory ligands (LPS, FL, TNFα, LTA and ZY). And hypothesis metabolic changes observed within this thesis (present in 10μM and not 1μM C-DIM12) may be important in its ability to attenuate these pro-inflammatory responses. Thus, data obtained within this thesis contributes significantly to the understanding of the cytotoxicity associated with NR4A agonists, and their ability to attenuate inflammatory responses, in both epithelial and immune cells.