Circadian clocks in arthritis: the inflamed joint and gut microbiome around the clock

Abstract

Circadian clocks impose rhythms on the host, which coordinate physiological processes with environmental cues and are important regulators of the immune system. Rheumatoid arthritis (RA) is an autoimmune disease, with daily oscillations in symptoms and disease markers. This thesis investigates 24h rhythmicity in mouse models of arthritis, both in the inflamed joint and in the gut, a key site of microbial and immunological activity which is known to be impacted in human chronic inflammatory disease. Firstly, we aimed to investigate the effects of chronic joint inflammation induced by collagen induced arthritis (CIA) on the rhythmicity of the host gut and microbiome. RNA sequencing of colon samples revealed temporal reorganisation of the gut transcriptome with notable changes in immunoglobulin A (IgA) production and tight junction pathways. Assessment of faecal albumin indicated disrupted barrier permeability in CIA, though further methods to demonstrate barrier disruption were unsuccessful. 16s rRNA sequencing revealed that in CIA there was reorganisation of rhythmicity in microbiota at the phyla, genus and species level, including species of the Lactobacilli genera which are associated with autoimmunity. Analysis of circulating microbial metabolites showed an increase in bile acids associated with immunity and barrier dysfunction in CIA. We then aimed to assess disease rhythmicity in a second model of inflammatory arthritis, antigen induced arthritis (AIA). Assessment of immune cells within the joint (flow cytometry) and cytokine concentration in periphery (Bioplex assays) across the day determined no diurnal variation in immune cell populations, but increased peripheral cytokine expression at zeitgeber time (ZT)6 (rest phase). Further work utilised the AIA model to investigate the importance of cell intrinsic clocks within fibroblast-like synoviocytes (FLS, resident joint cells and key players in local joint inflammation). Reduction in Bmal1 expression in FLS (Col6a1-Bmal1- /-) did not affect the AIA symptoms or FLS activation marker expression, suggesting that in AIA the FLS clock does not influence disease development, nor responses by FLS. The monoarthritic AIA model was used to further investigate changes to the rhythmicity of the host gut and the microbiome in arthritis. In contrast to the polyarthritic CIA model, we showed that in AIA there were no changes in rhythmic IgA production, or changes to barrier permeability. However, despite this 16s rRNA sequencing revealed reorganisation of rhythmicity of the microbiota at the phyla, genera and species level as early as 3 days post intra-articular injection. This suggests that reorganisation of the microbiome in the setting of arthritis can occur in the absence of changes to rhythmic IgA production and barrier permeability. Interestingly, we showed that some species lost rhythmicity at the peak of disease and then re-gained rhythmicity at the chronic phase, suggesting that microbial changes in AIA are transient. Overall, data in this project provides new insights into rhythmicity in mouse models of arthritis. We have revealed perturbations within the rhythmic transcriptome, and alterations in the daily oscillations of the microbial community within the gut in the setting of arthritis. This work might help us better understand microbial dysbiosis in human RA and highlights the need for a better understanding of how microbial changes might affect host gut processes.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Ray (Supervisor), Julie Gibbs (Supervisor) & David Bechtold (Supervisor)