Determining the role of CSF1R-dependent macrophages in of Paneth cells and the intestinal stem cell niche

The epithelium which lines the intestine is self-renewing and is continually replaced every 5-7 d. In order to do this, intestinal stem cells are situated at the base of each finger-like villous. These stem cells produce highly proliferative daughter cells which can differentiate into distinct intestinal epithelial cell populations, eg: goblet cells, tuft cells and Paneth cells. The differentiated cells travel along the villus epithelium and perform their physiological functions before being shed at the tip. A particular cell type known as the Paneth cell, remains at the base of these crypts nestled between the intestinal stem cells. A primary function of the Paneth cells is release antimicrobial products such as lysozymes and defensins which help to protect the crypt from bacterial infection. Paneth cells also produce molecules which are essential to maintain the intestinal stem cells. When Paneth cells are lost, this also leads to a loss of the intestinal stem cells, ultimately affecting the gut epithelium. Macrophages are abundantly positioned throughout the gut wall, These phagocytic cells help to protect the intestine against infection, but also help support the gut epithelium. We have shown that when these macrophages are lost, this leads to the subsequent loss of Paneth cells and intestinal stem cells. Our studies reveal a previously-unrecognised, essential role for macrophages in the constitutive maintenance of Paneth cells and intestinal stem cells. The disturbances to the Paneth cells and intestinal stem cells caused by macrophage-depletion also adversely affected the subsequent formation and function of other types of epithelial cells in the gut epithelium. The differentiation and function of specialised antigen sampling M cells was impaired in the Peyer's patches of the small intestine. In contrast, the differentiation and abundance of the mucous-secreting goblet cells was enhanced. This suggests that modification of the phenotype or abundance of macrophages in the gut wall, for example after CSF1R-blockade, pathogen infection or inflammation, could dramatically affect the development of the intestinal epithelium and the ability to sample gut antigens. Paneth cell-dysfunction is evident in Crohn's disease patients with small intestinal involvement, and may be a consequence of inadequate provision of support from monocytes. In this project we will study the role of macrophages in the maintenance of Paneth cells and intestinal stem cells. We will also determine whether the effects of macrophage-depletion on M cells affect the ability of the mucosal immune system to sample foreign particles in the intestine and mount a specific immune response to them. We also aim to determine whether a specific class of molecules known as Wnts are the important factors which the macrophages produce to support the Paneth cells and intestinal stem cells. Finally, we will determine whether the adverse effects of macrophage-depletion on Paneth cells and intestinal stem cells can be restored by treatment with a molecule known as R-spondin1. Pharmacological CSF1R-blockade has been proposed as a means to modulate certain cancers, and inflammatory, autoimmune and bone diseases. However, CSF1R-blockade also depletes macrophages. Therefore, CSF1R-blockade could indirectly compromise Paneth cells and intestinal stem cells, cell differentiation in the gut epithelium and the ability of the mucosal immune system to sample Ag and pathogens from the gut lumen. A thorough analysis of the role of macrophages in the maintenance of Paneth cells and intestinal stem cells is essential to help identify the macrophage factors which help support these cells. The information from this project will help develop new therapeutics to treat certain intestinal diseases associated with disturbances to Paneth cells and intestinal crypts.

Colony stimulating factor 1 (CSF1R)-CSF1 signalling controls macrophage growth and differentiation. We have revealed a novel role for macrophages in the maintenance of Paneth cells and the intestinal stem cell (ISC) niche. The disturbances to the ISC niche caused by macrophage-depletion after CSF1R-blockade affected the subsequent differentiation of gut epithelial cells, impeding M-cell, but enhanced goblet cell differentiation. Pharmacological CSF1R-blockade has been proposed to modulate certain cancers, and inflammatory, autoimmune and bone diseases. However, our data suggest that modification of the phenotype/abundance of macrophages in the gut after CSF1R-blockade could adversely affect the development of the intestinal epithelium, and the ability of the mucosal immune system to sample gut Ag/pathogens. Paneth cell-dysfunction is also evident in Crohn's disease patients with small intestinal involvement and may be a consequence of inadequate Wnt-ligand stimulation by monocytes. Our data imply that the Paneth cell-dysfunction in CD patients may similarly affect gut epithelial cell differentiation, lumenal Ag sampling and cause intestinal dysbiosis.

Our overall aim is to determine the role of macrophages in maintaining Paneth cells and the ISC niche and the consequences of CSF1R-blockade on crypt homeostasis. Our objectives are:

1: To determine the effects of CSF1R-blockade and inducible macrophage-depletion on Paneth cells and the ISC niche throughout the small and large intestines
2: To determine the effects of CSF1R-blockade on gut epithelial cell innate immunity
3: To determine whether macrophage-derived Wnts help maintain Paneth cells and ISC
4: To determine whether R-Spondin1 can protect Paneth cells against the effects of CSF1R-blockade

Our data will aid the development of novel therapeutics to counter the effects of CSF1R-blockade on the gut epithelium and to treat intestinal diseases associated with disturbances to crypts and Paneth cells.

Data supporting this application has revealed a previously unrecognised, essential role for CSF1R-depent macrophages in the constitutive maintenance of Paneth cells and the intestinal stem cell (ISC) niche. Therefore, scientists in many distinct disciplines (macrophage biology, mucosal immunology, stem cell biology) will significantly benefit from the novel data generated from this proposal which address an important gap in our understanding role of macrophages in the maintenance of the ISC niche.

Pharmaceutical companies may have significant interest in data from the objectives in this study data aimed at identifying the macrophage-derived factors which provide this homeostatic support to the ISC niche, and the influence that CSF1R blockade may have on Paneth cells and the crypts. These data may aid the development of novel therapeutics to treat certain intestinal diseases associated with disturbances to Paneth cells and intestinal crypts. Therefore, during the course of the project the applicant will consult with the Institute's Business Development and Commercialisation Department to seek potential Industry partners to exploit the project's data.

The effects of macrophage depletion on the ISC niche will be of particular interests to clinicians, especially gastroenterologists. Our data will aid the development of novel therapeutics to treat certain intestinal diseases associated with disturbances to intestinal crypts and Paneth cells. As outlined in our Pathways-to-Impact plan, the applicants will actively seek collaborations with clinical gastroenterologists to ensure translation of the findings from this project to clinical settings. Greater knowledge of the role of macrophages in maintaining Paneth cells and the ISC niche, will enhance our understanding of how therapeutic intervention of CSF1R and its downstream signalling pathways can be best applied in clinical disease settings including the treatment of certain cancers, as well as inflammatory and bone diseases, with therapeutic CSF1R intervention.

This project will impact on Patients treated with therapeutic CSF1R intervention. They will benefit from improved understanding and how to apply such treatments without affecting Paneth cells and ISC.

This study will enable the scientists working on the project to acquire many transferable skills. Important skills will be gained in sever major disciplines (macrophage biology, mucosal immunology), and for Dr Donaldson will provide an excellent opportunity to gain new important skills in (intestinal) stem cell biology. The scientist will also develop import skills in in vivo biology (a currently recognised research priority) and high resolution bioimaging, transcriptomics and bioinformatics.

As mentioned in our Pathways-to-Impact plan, a series of targeted workshops will be held to help disseminate the projects findings to key stakeholder groups. Project specific public engagement activities will also be created at delivered at Institute Open Days and other appropriate events. Data from this study will be also disseminated to the international scientific community in a timely manner using a combination of publication in quality peer-reviewed journals and presentation at scientific meetings eg: international and national scientific conferences, seminars at other research institutions and lectures to undergraduate students. Prof. Mabbott is regularly invited to present data at these events. Appropriate opportunities will be taken to communicate the project's findings to the public. The release of potential news-worthy publications would be discussed with the Institute and research council's press officers and press releases issued when appropriate. Many of Prof. Mabbott's recent studies were handled in such manner and have been regularly covered by the international media including international press (The Times etc.) and the BBC. The BBSRC has often featured data from Prof. Mabbott's lab. on its website.