Can microbiota modulate circadian oscillations to alter susceptibility to autoimmunity?

In our body exists an internal clock, known as the circadian rhythm (CR), which resets every 24 hours. The CR affects all cells in our body, including white blood cells, such as T cells, that make up our immune system. Thus, the immune system's response to "threats" can be altered by the CR. In addition, the beta cells that produce insulin in the pancreas are also controlled by CRs. In Type 1 diabetes (T1D), some T cells attack the insulin-producing beta cells by recognising small pieces of protein, called peptides, as foreign. It is currently unknown what triggers this attack but one area being explored relates to the germs (bacteria) that live in our body. Evidence in children suggests that those at greatest risk for developing T1D have different bacteria present in their intestine and that this can change how the immune system, such as T cells, respond to them. Recently, it has been shown that bacteria can change the CR in the body through the immune system. Thus, bacteria may be a useful target for altering the CR to promote a healthier immune system, preventing the development of T1D. This has not been studied before.

This research aims to understand how the bacteria in our bodies affect the CR of both T cells and the targeted insulin-producing beta cells. I hypothesise that bacteria can change the CR of both cell types, resulting in changes to the T cell's ability to destroy the beta cells. I will investigate this using the Non-obese diabetic (NOD) mouse model, which develops spontaneous T1D similar to humans. This work will be studied in 3 main aims:

1. Do bacteria change the CR in the insulin-producing beta cells making them more easily targeted by the T cells?
2. Do bacteria change the CR in the T cells making them better at destroying the beta cells?
3. Does disruption to the CR in the beta cells or CD8 T cells specifically alter the risk of developing T1D?

I will study both NOD mice with bacteria and NOD mice that do not have any bacteria, called Germ-free (GF) mice. These mice will be used to identify the role bacteria have on the CR of both beta cells and CD8 T cells. All mice will be studied at two different time points to identify changes caused by the CR.

Insulin-producing beta cells will be studied for their ability to produce insulin and will be investigated for the peptides they produce that may make them targets for the CD8 T cells. This will involve imaging NOD mice that have a fluorescent (coloured) signal after producing insulin, which allows them to be visualised. I will also introduce fluorescent bacteria into GF NOD mice allowing me to visualise whether the bacteria can enter the pancreas. I will also look at products bacteria produce that may alter the CR of the beta cells.

CD8 T cells will be studied as they are the most common T cell found in the pancreas of humans with T1D. I will study a specific CD8 T cell that can recognise both a peptide produced by the beta cells and a similar peptide produced by bacteria. Recognition of either of these peptides will result in the CD8 T cells destroying the beta cells. CD8 T cells will be studied for their interactions with beta cells, their ability to destroy peptide-coated target cells and changes in energy usage that may help the cells become more damaging. I will also study how responses to bacteria at different time points of the CR can also affect the CD8 T cells.

Finally, I will generate CR-deficient NOD mice and GF NOD mice in either beta cells or CD8 T cells specifically. These mice will be studied as mentioned above, as well as for their ability to develop spontaneous diabetes. This will help identify which cells (beta cells or CD8 T cells) may make better targets for future therapy. Understanding the CR and how it changes the cells may identify specific time points in which therapeutic success can be maximised. Given the increasing number of people diagnosed with T1D, and a lack of successful therapies, this is urgently needed.

Circadian Rhythms (CRs) reflect daily oscillations of gene activation/repression, influencing metabolic and immune responses. Recently, microbiota have been shown to modulate CRs but this has not been explored in autoimmunity. Most studies are conducted in the daytime, whereas animals are nocturnal and humans are diurnal, and thus studied in different CR phases. Thus, CR differences may be responsible for a lack of successful translation of therapies. This proposal will study the effect of CR on both the target pancreatic insulin-producing beta cells and the pathogenic islet-reactive CD8 T cells, in a mouse model of Type 1 diabetes (T1D). I hypothesise that microbiota modulate CR-induced beta cell functions, including metabolic perturbations and immunological changes in T cells, altering disease susceptibility to T1D. This will be studied in specific pathogen-free and germ-free (GF) mice by investigating:
1. Beta cell functions e.g. insulin secretion and mitochondrial respiration and the immunopeptidome at different CR times to identify changes in beta cell immunogenicity. Microbial translocation and bacteria-derived metabolites will be studied as a mechanism of microbially-induced beta cell changes.
2. Antigen-specific CD8 T cell metabolism and functions e.g. cytotoxicity, proliferation and cytokine responses at different CR times, to identify the best intervention times. Microbial mimic peptides of islet antigens will be studied in vitro and in vivo following colonisation of GF mice with molecular mimic-encoding bacteria to assess how different T cell receptor avidities alter CR responses.
3. Cell-specific CR-deficient beta cells and CD8 T cells to identify intrinsic CR-mediated effects as in 1 and 2.
In summary I will identify the importance of CR-mediated effects and how microbiota can modulate them. This work is pivotal for developing future studies using modulators of CRs to improve translational success of current and new preventative treatments for T1D.

This proposal aims to investigate the circadian rhythm (CR) influences on pancreatic beta cells and CD8 T cells, in addition to how they may be modulated by the microbiota, focusing on bacteria of the gut. The study of animal models enables researchers to investigate questions in greater depth than they may be able to do in humans. However, this research, particularly in the development of preventative immunotherapies does not always translate well into human studies. One reason for this is mice are nocturnal and humans are diurnal and thus have CRs which are oscillating differently. This novel research may provide pivotal information that could dramatically influence current immunotherapy or broader therapy work in the treatment or prevention of autoimmune diseases and other diseases including infections and cancer. This proposal will identify key times in the CR in both islet beta cells and antigen-specific CD8 T cells for potential pharmacological interventions to maximise the desired outcomes. This is incredibly important and valuable to many pharmaceutical companies, which experience huge financial costs in developing new biologics, which may not perform sufficiently well in early clinical studies and thus may never be continued. This proposal will raise awareness of factoring in the time of administration of therapies and therefore may dramatically alter the design and implementation of clinical studies. Clinical studies often have a greater variability within the cohort compared to mouse studies and thus, standardising the time at which the therapy is administered in an individual's CR will help minimise this variation to maximise therapeutic primary and secondary proposed outcomes. Given the interest in microbiota modulation of CR in this proposal, it is also feasible that government agencies and the NHS may reconsider the guidance for administration of antibiotics or antivirals in order to maximise the therapeutic impact without encouraging prolonged antibiotic/antiviral use and the development of antibiotic resistance. As this work is set in the context of type 1 diabetes, it will be directly useful for diabetes charities to raise awareness of CR within the field and to the broader scientific community. However, studies in other disease settings will need to be conducted to confirm the best CR time for intervention, as the CR will not impact every disease to the same extent. I hope to study this in the future. It is important however to consider CR in many aspects of research, translation and therapy development and administration. I believe that addressing CR is an important factor when conducting research studies and may indirectly benefit the economy globally, as understanding CR-induced effects or CR modulation may enable the development of more successful therapies. This would result in greater investment for pharmaceutical companies. In the short-term conducting experiments at multiple time points would be costlier and thus there is a greater expenditure in materials required for the experiments, benefitting multiple suppliers and requiring greater investment in workforces. While this basic science research is in mice, the next stage would be to begin further modulating the CR and testing immunotherapies such as anti-CD3, shown to be previously successful in mice at different times. In addition, I would also begin translating this proposal into humans with CR-modulation investigation. It is hoped within the next 10 years to have identified CR impacts on the human immune system and beta cell function, and to have identified ways to use the CR to improve therapeutic success. This research proposal will also enable me and my staff to develop new skills important for future studies including in vivo and ex vivo live cell imaging, conducting and analysing the immunopeptidome and pathway analysis of the RNA-seq data to explore new ways to modulate pathways/molecules influenced by the CR to improve clinical efficacy.