Glanville, James Robert William; (2025) Characterisation and utilisation of complementary murine and human models of inflammation to investigate pro-resolution/anti-inflammatory pathways in rheumatoid arthritis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Rheumatoid arthritis (RA) is the most common primary autoimmune inflammatory arthropathy worldwide. Inflammation is a combination of inflammatory ‘go’ signals counteracted and regulated by internal ‘off’ signals, leading to the clearance of the inflammatory stimulus and the initiation of resolution. Despite an increasing appreciation of the role of inflammation and its resolution in the development of autoimmune disease, this remains a relatively understudied field of research. This thesis describes novel murine models of both non-resolving and resolving inflammation with a complementary human model in both health and in RA patients using the same inflammatory stimulus; UV-killed Escherichia coli (UV-KEc). Collectively, these can be used to investigate potential pro-resolution/anti-inflammatory signals in RA. The intradermal injection of UV-KEc in both mouse and humans resulted in a significant inflammatory response, characterised by the influx of neutrophils. In mouse, at a dose of 1.5 x 108, this process was non-resolving at the end of the time-course (72h), defined by the absence of neutrophil clearance. At a dose of 1.5 x 107, inflammation had resolved with neutrophil numbers returning to baseline levels. At both doses, significant changes were identified in bone marrow neutrophil subsets, with an emergency release of developmentally immature neutrophils. A significant expansion of Ly6Cint monocytes was also seen using both doses of UV-KEc. Furthermore, key endocannabinoids were detectable in both inflamed murine skin and plasma, with marked differences seen compared to un-injected controls; notably 2-arachidonoylglycerol (2-AG), N-palmitoylethanolamine (PEA) and N-arachidonoylethanolamine (AEA). I have also shown that the use of a traditional anti-inflammatory medication used in RA (naproxen) and a novel investigational hydrogen sulfide-releasing (H2S) non-steroidal anti-inflammatory drug (NSAID), ATB-346, can be safely used in this system to investigate inflammatory pathways following the injection of UV-KEc into the skin of healthy volunteers. ATB-346 and naproxen were shown to be potently anti-inflammatory, as evidenced by a significant reduction in neutrophils at the inflamed site at 4h compared to healthy controls. ATB-346 also significantly increased the expression of cluster of differentiation 14 (CD14) on both classical and intermediate monocyte subsets at 48h, suggesting a switch towards an anti-inflammatory phenotype. ATB-346 and naproxen also suppressed cyclo oxygenase (COX) equally, as measured by a significant reduction in prostaglandin E2 (PGE2) at 4h. Finally, the effect of UV-KEc-triggered dermal inflammation in RA patients who were in disease remission was investigated. RA patients demonstrated significantly increased levels of plasmacytoid dendritic cells at the site of inflammation at 48h. Using Uniform Manifold Approximation and Projection (UMAP) analysis, a unique population of interleukin-7 (IL-7) receptor (CD127)+ macrophages was found in RA patients in disease remission at the site of inflammation, which was absent in all healthy controls. Two RA patients who did not have this population flared soon after the completion of the study, suggesting that these cells may be important for the maintenance of remission in RA. Counterintuitively, RA patients also reported significantly reduced pain and tenderness throughout the inflammatory time-course. The concept of harnessing mediators involved in the resolution of inflammation to treat patients with autoimmune disease is an attractive strategy, potentially negating the use of immunosuppressive therapies, which increase the risk of infection and malignancy. The work presented here aimed to identify anti-inflammatory/pro-resolution targets and the modulation of CD127/IL-7 signalling may represent one such novel pathway.

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