A holistic study of the effect of the murine microbiome on metabolism and systemic inflammation using integrated molecular imaging technologies

Adams, Lauren (2024) A holistic study of the effect of the murine microbiome on metabolism and systemic inflammation using integrated molecular imaging technologies. PhD thesis, University of Glasgow.

Abstract

Inflammatory bowel disease (IBD) is a multifactorial disease involving genetic susceptibility, impaired barrier function, alterations in the microbiome, and a dysfunctional immune response. Current therapeutic treatments are aimed at controlling symptoms; however, symptoms recur, and patients can become susceptible to infection, increasing the need for new treatments. There has been a growing interest in the role host-microbe interactions play in the development of IBD and many studies have uncovered distinct shifts in small molecule classes in patients compared to healthy controls. Microbial and host small molecules are able to interact with host immune cells and polarize them towards either a pro- or anti-inflammatory phenotype. This immunometabolism/ immunomodulation is an emerging concept that plays an important role in human health and disease. Furthermore, IBD patients frequently experience extraintestinal manifestations, commonly affecting organs such as the liver, lung, eyes, kidney, and spleen, and often results in a loss of proper function. The cause of systemic inflammation is still poorly understood; however, studies have suggested that small molecules originating from the intestinal bacterial community may play a role. Firstly, we aimed to discover small molecular changes in the intestine (ileum and colon) and systemic sites (liver and eye) in a mouse model after infection with an IBD associated pathobiont, adherent-invasive Escherichia coli (AIEC). We applied a powerful analytical technique known as mass spectrometry imaging (MSI) to map the spatial distribution and relative abundance of molecules across a sample surface in a label-free manner. This allows the distribution of known and unknown molecules including proteins, metabolites, and lipids to be determined. Our infectious mouse model revealed specific molecular changes across the different organs that were either microbial or host derived, and many play a role in the onset or perpetuation of inflammation. Despite, producing informative host-microbe interaction data, results of this proof-of-concept study were not fully reflective of human IBD. Therefore, we investigated metabolomic adaptation in intestinal and systemic sites in a dextran sodium sulphate (DSS) mouse model. This model has been shown to reflect human disease, including disrupted barrier function, microbial dysbiosis and immune cell dysregulation. MSI revealed 30 molecules in the colon, 88 molecules in the ileum, 239 molecules in the liver, 65 molecules in the spleen and 16 molecules in the kidney differed in abundance between DSS colitis and uninflamed control mice. This study confirmed the identity of some intestinal molecules including creatine, docosahexaenoic acid (DHA), and 1- methylnicotinamide (1-MNA). However, identifying the location and abundance of specific molecules does not provide an overall assessment of the tissue environment that may lead to inflammation. Therefore, we applied imaging mass cytometry (IMC) to tissue regions of interest (ROI), where molecules of interest had been located. IMC found markers such as CD4 and major histocompatibility complex class II (MHCII) were decreased in the inflamed ileum, whereas CD4 and others (e.g. NKp46, granzyme B) were increased in the inflamed colon compared to the control. Hence, our study combined two imaging technologies to gain a better understanding of how region-specific molecules may be influencing the immunological profile in the ileum, colon, and liver. Rheumatoid arthritis (RA) is another inflammatory condition with a poorly understood aetiology and has been linked to IBD. We applied MSI and IMC to investigate metabolomic and immunological changes in a mouse model of RA, collagen induced arthritis (CIA). We found 9 molecules in the lung, 3 molecules in the kidney, 2 molecules in the liver, and 16 molecules in the spleen could differentiate between diseased and non-diseased mice. In the lung, only one molecule was increased in the diseased group compared to the control groups and was identified as lysophosphatidylcholine 18:0 (LysoPC 18:0). IMC revealed cells expressing the markers B220 and CD19 (B cells) were increased in the lung of CIA mice compared to controls. Therefore, LysoPC (18:0) may promote the recruitment of B cells in the lung. As microbes play an important role in immune priming and homeostasis, this study wanted to identify metabolomic and immune profile differences in mice with (specific pathogen free) and without (germ-free) a microbiome. MSI revealed 9 molecules in the colon, 1 molecule in the ileum, 255 molecules in the liver, 5 molecules in the spleen and kidney, and 6 molecules in the lung that discriminate between germ-free (GF) and specific pathogen free (SPF) mice. We were able to confirm the identity of two polyamine molecules, spermidine and spermine, that were decreased in the SPF liver compared to GF liver. In vitro experimentation showed that spermidine reduced the percentage of macrophages expressing MHCII in a dose dependent manner. Impaired macrophage MHCII expression has been linked to dysfunctional adaptive immune activation; thus, reducing spermidine might be a microbial adaptation to support the development of the adaptive immune response. This study highlights the usefulness of MSI and IMC as metabolomic and immunological discovery tools that can be used together to identify how molecules and cells within an environment may be interacting under different conditions. This allows us to hypothesise and test various host-microbe and immunometabolism mechanisms that may be implicated in inflammatory processes during IBD and RA.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: I have to also thank the BBSRC and AstraZeneca for their award of my industrial CASE PhD studentship.
• Subjects: Q Science > QR Microbiology > QR180 Immunology; R Medicine > R Medicine (General); R Medicine > RB Pathology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Funder's Name: Biotechnology and Biological Sciences Research Council
• Supervisor's Name: Wall, Dr. Donal, Burchmore, Dr. Richard and Goodwin, Dr. Richard
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84322
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 21 May 2024 11:08
• Last Modified: 21 May 2024 11:14
• Thesis DOI: 10.5525/gla.thesis.84322
• URI: https://theses.gla.ac.uk/id/eprint/84322