Regulation of arthritis by butyrate-mediated activation of regulatory B cells

Regulatory B cells (Bregs) are a type of B cell (a type of white blood cell) that in healthy individuals help the immune system to protect the body against excessive inflammation. They function by releasing chemicals, in particular one called Interleukin-10 (IL-10), that block the function of potentially harmful white blood cells and also by converting those harmful cells into protective cells. In autoimmune diseases, such as arthritis or lupus, Bregs don't work properly and their malfunctioning may contribute to disease.

Recently, we have identified that a protein called the Aryl-hydrocarbon-Receptor (AhR) appears to be present at high levels in IL-10 producing mouse and human Bregs, and may in fact control IL-10 production by these cells. Ahr is of particular interest because it is a molecule that detects toxins, nutrients, and bacterial components in parts of the body that are exposed to the external signals, for instance the gut. We think that one of the ways that Bregs are activated is by sensing bacterial and food components in the gut by using Ahr, which then primes the Bregs to become protective cells.

In support of this idea, we have so far found that if you genetically modify a mouse so that none of its B cells produce Ahr, then that mouse will lack functional Bregs. As a consequence, these mice get worse disease when we give them an experimental form of arthritis. In addition, we have found that butyrate, a molecule produced by bacteria when they digest fibre-rich foods, enhances the production of Ahr by Bregs, and also suppresses arthritis when fed to mice. This suggests that butyrate produced by bacteria in the gut might activate Bregs through Ahr to produce IL-10 and as consequence protect against arthritis. What we want to do now is unravel exactly how butyrate and Ahr interact with each other, and prove what effect this has on Bregs.
We aim to understand:

1- Whether oral administration of butyrate suppresses autoimmune disease by enhancing or increasing the number of Ahr producing Bregs.
2- What genes are turned on in Ahr producing Bregs when they sense butyrate, and how activation of these genes will control Breg function.

Plan:
To address the first aim, we will feed butyrate to mice that have Bregs that produce Ahr and to mice that have Bregs that don't produce Ahr. We will compare the severity of arthritis, the production of IL-10 by Bregs, and the effect on inflammatory white blood cells in these mice. We will look in multiple organs in the two groups of mice to determine where butyrate exert its anti-inflammatory effect and how this effect depends upon the presence of AhR in Bregs. We will also test the effect of butyrate in suppressing the development of other autoimmune diseases, such as lupus and an experimental multiple sclerosis-like disease. Finally, we will test whether the amelioration in clinical symptoms is accompanied by an increase of Ahr producing Bregs.

To address the second aim, we will use a number of powerful computational techniques that focus on what happens to the DNA in a Breg that do or don't produce Ahr after mice have received butyrate treatment. These techniques will allow us to identify a) which genes are activated by AhR in Bregs; b) which genes are turned on; c) which genes are primed to be turned on if necessary, and d) how butyrate enhances this activation.

Value:
Possible interactions between AhR and butyrate may represent an important link between environmental molecules in the gut and the immune system. Clarifying the mechanisms by which specific molecules are sensed by AhR and can regulate immune responses in different cell types will provide a foundation for how AhR can be targeted to treat a range of autoimmune and inflammatory diseases.

Regulatory B (Breg) cells restrain the severity of inflammation associated with immunological-disorders and help to limit collateral damage following unresolved infections. They act by directly suppressing the proliferation of effector T cells and promoting the differentiation of regulatory T cells, as well as inhibiting pro-inflammatory cytokine secretion by innate immune cells. Although it is known that the cytokine interleukin-10 (IL10) is crucial for these functions, the lack of a Breg cell specific marker has hindered the detailed characterization, and potential clinical utility, of these cells. We have recently identified the aryl hydrocarbon receptor (AhR) as a critical transcription factor for the differentiation of IL-10+Bregs. We have shown that arthritic mice lacking AhR expression in B cells display a profound decrease in Bregs in both spleen and in Gut-Associated Lymphoid Tissues (GALT). We have discovered that amongst a wide panel of microbiota-derived metabolites tested, the short-chain fatty acid, and AhR ligand, butyrate suppresses arthritis in a B cell dependent manner. Considering these findings alongside previous data showing that AhR ligands are abundant in the gut we hypothesize that: butyrate produced by gut bacteria, by enhancing the activity of the transcription factor AhR in B cells, promotes the differentiation of Breg. Our aims are: to identify whether butyrate promotes the differentiation of AhR+Bregs which can suppress autoimmunity, and whether butyrate acts directly on B cells or via an intermediate cell or bacteria; to understand the molecular mechanisms by which AhR promotes regulatory B cell function and how this is modulated by butyrate treatment.

These experiments will result in a deeper understanding of how anatomical environmental signals are crucial for the generation of Bregs, and the detailed study of the Breg transcriptome will lead to new ways to identify the Bregs and reveal the multiple ways they may inhibit diseases.

Academic beneficiaries: The knowledge gained by this project would most directly benefit academics working in the fields of Bregs, immunology, inflammation, rheumatology as well as molecular and cellular biology. However, since B cells are pivotal players in other immune processes and human diseases, this work is likely to be of interest to academics working on other immune-related diseases, as well as people in the fields of infection, cancer and vaccine development. Our results will also be of interest to those interested in how bacterial metabolites, and in particular butyrate, interact with the immune system. In order to inform these beneficiaries of our work we will present our data at local departmental and divisional meetings as well at national and international meetings, in particular at meetings such as The British Society for Immunology, The British Society for Rheumatology, The European Congress of Immunology and The American College of Rheumatology and the Keystone Symposia. We will have the opportunity to present our findings at least 2-3 times a year at internal meetings, and we would hope to present our data at least once a year at international meetings from the secondyear of the proposed work.

Non-academic Beneficiaries:
If this project is successful, we believe that subsequent research projects could greatly increase the effectiveness of health services in several ways. 1) using short chain fatty acids to manipulate the immune system covers administration of prebiotics, probiotics, and even faecal microbial transplants, all of which change the balance of bacterial metabolites. Our work if successful, will show whether targeting the intestinal microbiota by for example enhancing the production of butyrate, represents a promising therapy for rheumatoid arthritis. 2) Much as mTOR inhibitors such as rapamycin are being used to stabilise Treg function for cellular therapy, out results may pave the way for butyrate, and potentially other AhR ligands, to be used to stabilise Breg function for cellular therapy. 3) Our sequencing experiments in the second part of the plan of work will potentially provide novel targets for distinguishing Bregs from non-Bregs. This could allow the development of targeted therapies enhancing Breg function in autoimmunity, or removing it in cancer or infection settings.

Industry Beneficiaries: If our research were to lead to improved health services, we will make contact with industry in order to facilitate the translation of our work in accordance with Good Manufacturing Practice (GMP) principles. Further, if our research results in commercially exploitable findings we will involve UCL Advances (The Centre for Entrepreneurship and Business Interaction at UCL) to achieve the highest possible impact.