Determining the role of CXCR5-expressing dendritic cells in immune function and TSE agent neuroinvasion from the intestine

Lead Research Organisation: University of Edinburgh
Department Name: Royal (Dick) School of Veterinary Scienc

Abstract

Transmissible spongiform encephalophathies (TSEs) are prolonged diseases which cause extensive degeneration in the brain. In the absence of a cure these diseases are invariably fatal. These diseases affect both animals and humans, and include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy ('mad cow disease') in cattle, chronic wasting disease in mule deer and elk, and scrapie in sheep and goats. Some animal species and humans have become infected with these diseases after eating TSE agent-contaminated food, or through transplantation of TSE agent-contaminated tissues or tissue products (eg: transfusion of blood from a variant CJD-infected donor). Many questions remain concerning the route the infectious TSE agent takes from the site of exposure (eg: intestine) to the brain where it causes damage to nerve cells. We have shown that soon after inoculation TSE agents first target and accumulate within lymphoid tissues (Peyer's patches in the intestine, lymph nodes and spleen) before they spread to the brain. Many TSE agents must accumulate in these lymphoid tissues before they can subsequently gain access to the brain (a process termed neuroinvasion) where they ultimately cause neurodegeneration and death. How TSE agents are delivered from the intestine to the lymphoid tissues where they accumulate before neuroinvasion is not known. The identification of the cells or mechanisms involved in TSE agent transport may identify an important process to which treatments can be directed to block neuroinvasion. Therefore, the aim of the research in this proposal is to study this important missing link and determine the mechanism/s involved in the initial delivery of TSE agents to lymphoid tissues. Migratory dendritic cells (DCs) continually circulate throughout the body. These sentinel cells sample foreign particles and microbes and deliver them to the lymphoid tissues to initiate an appropriate immune response. Our recent data suggest that migratory DCs also play a key role in the transportation of the TSE agents from intestine to the lymphoid tissues. However, whether DCs transfer TSE agents directly to the cells within the lymphoid tissues from which neuroinvasion subsequently occurs is not known. The status of DC is strongly influenced by the microbial contents and inflammatory conditions in the intestine, suggesting that these factors are likely to dramatically affect susceptibility to orally-acquired TSE agents. Therefore, further experiments are necessary to determine the precise role that DCs play in the early stages of TSE pathogenesis. Stimulation from chemokines through chemokine receptors play important roles in attracting cells such as DCs to lymphoid tissues and determining their location within them. For example, the chemokine CXCL13 is expressed within in B cell follicles and attracts cells that express the CXCR5 chemokine receptor into them. The follicular dendritic cells that reside within B cell follicles produce high levels of CXCL13 are critical sites of TSE agent accumulation within lymphoid tissues. Therefore, the major aim of the research in this proposal is to assess the role of CXCR5-expressing DCs in immune function and TSE agent neuroinvasion from the intestine. Specifically, the hypothesis will be tested that in the absence of CXCR5-expressing DCs, these DCs are unable to deliver the TSE agent to the FDCs within the B cell follicles of lymphoid tissues, and as a consequence, TSE agent neuroinvasion is blocked. A thorough understanding of the early events in TSE pathogenesis will aid the determination of risk and the development pre-clinical diagnostics and therapeutics.

Technical Summary

After oral exposure many transmissible spongiform encephalopathy (TSE) agents accumulate first upon follicular dendritic cells (FDCs) in the gut-associated lymphoid tissues (GALT). We have shown that TSE agent accumulation in the GALT, especially Peyer's patches, is obligatory for the transmission of disease to the brain. However, the mechanism through which TSE agents are initially conveyed from the gut lumen to the FDCs within the GALT is not known. Our recent data suggest that migratory dendritic cells (DCs, a distinct lineage from stromal-derived FDCs) play a key role in the translocation of the TSE agents from the gut lumen to the GALT. However, whether DCs transfer TSE agents directly to the FDCs from which neuroinvasion subsequently occurs is not known. As DC status is strongly influenced by microbial stimuli and inflammatory conditions in the intestine, these factors are likely to dramatically affect susceptibility to orally acquired TSE agents. Therefore, further experiments are necessary to determine the precise role that DCs play in the early stages of TSE pathogenesis. Chemokines and chemokine receptors play important roles in attracting lymphocytes and DCs to lymphoid tissues and controlling their positioning within them. The chemokine CXCL13 is expressed by FDCs and follicular stromal cells in B cell follicles and attracts CXCR5-expressing cells into them. The major aim, therefore, of the research in this proposal is to assess the role of CXCR5-expressing DCs in immune function and TSE agent neuroinvasion from the intestine. A unique transgenic mouse line will be generated that lacks CXCR5-expressing DCs. These mice will be used to test the specific hypothesis that in the absence of CXCR5-expressing DCs, TSE agent accumulation upon FDCs in the GALT is blocked and neuroinvasion impaired. A thorough understanding of the early events in TSE pathogenesis will aid the determination of risk and the development pre-clinical diagnostics and therapeutics.

Publications

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Bradford BM (2014) Human prion diseases and the risk of their transmission during anatomical dissection. in Clinical anatomy (New York, N.Y.)

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Bradford BM (2012) Prion disease and the innate immune system. in Viruses

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Bradford, B (2010) Pathway Analysis of Integrin Alpha X/Beta 2 (CD11c/CD18) in the Murine Mononuclear Phagocyte Lineage in EMDS, European Macrophage and Dendritic Cell Society. 24th Annual Meeting

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Bradford, B (2013) Homeostatic vs inflammatory replenishment of tissue mononuclear phagocytes revealed by fate mapping CSF1R myeloid precursors in EMBO 2013 - Dr Jekyll and Mr Hyde: The Macrophage in Inflammation and Immunity

 
Description We have successfully created a unique transgenic mouse line enabling the conditional ablation of the CXCR5 gene in macrophages and dendritic cells.

After oral exposure to prions, the early replication of certain prion strains upon follicular dendritic cells (FDC) in the Peyer's patches in the small intestine is important for the efficient spread of disease to the brain (termed neuroinvasion). However, little is known of how prions are initially conveyed from the lumen of the gut to the FDC. Our data suggest prions are initially acquired from the gut lumen by M cells in the epithelia overlying the Peyer's patches. Following their transcytosis by M cells, particles and microorganisms typically exit into the intraepithelial pocket on the basolateral membrane where they are subsequently processed by the underlying leukocytes and lymphocytes. Migratory bone marrow-derived conventional dendritic cells (cDC) are centrally involved in the transport of antigens both within Peyer's patches and on into the mesenteric lymph nodes. A subset of cDC has also been identified that can migrate into the B cell follicles where FDC reside. We have previously show that in the transient absence of CD11c+ mononuclear phagocytes (such as cDC) at the time of oral exposure, the accumulation of prions in the Peyer's patches is impeded and disease susceptibility reduced. However, whether these mononuclear phagocytes convey prions directly to FDC within the B cell follicles of Peyer's patches is not known. Chemokines play important roles in attracting lymphocytes and leukocytes to lymphoid tissues and controlling their positioning within them. The chemokine CXCL13 is expressed by FDC and modulates the homing of CXCR5-expressing B cells towards them. Certain CD11c+ mononuclear phagocytes also express CXCR5 and can migrate into B cell follicles. We therefore determined whether CXCR5-expressing mononuclear phagocytes convey prions to FDC after oral exposure. Novel compound transgenic mice were created in which CXCR5-deficiency was specifically restricted to CD11c+ mononuclear phagocytes. When these mice were orally exposed to prions our data show that in the absence of CXCR5 expression in mononuclear phagocytes the early accumulation of prions upon FDC in Peyer's patches and the spleen was impaired, and disease susceptibility was significantly reduced. Together, these data suggest that after oral exposure CXCR5-expressing mononuclear phagocytes play an important role in the efficient transfer of prions towards FDC within Peyer's patches.

Infection with the gastrointestinal nematode Trichuris muris stimulates a TH2 dominated response in resistant mouse strains such as C57Bl/6, and infection is cleared within 21 days. We investigated the ability of CD11c-CXCR5-/- mice to mount an appropriate TH2 response to T. muris infection to facilitate clearance. Unlike wild-type CXCR5 expressing CXCR5fl/fl control mice, CD11c-CXCR5-/- mice were unable to clear T. muris after 30 days. Mouse strains susceptible to T. muris infection display a TH1 dominated response and remain persistently infected. Persistently T. muris infected CD11c-CXCR5-/- mice revealed increases in Interferon ?, IL1ß, IL2, IL6, IL10 and reduction in IL4, IL9 and IL25 cytokines when compared to T. muris cleared wild-type mice. Analysis of gene expression in the mesenteric lymph nodes of T. muris infected CD11c-CXCR5-/- mice revealed increases in both IL12 receptor subunits IL12RB1 and IL12RB2 and the co-stimulatory molecules CD80 and CD86. We have demonstrated that CXCR5 deficiency in CD11c+ cells alters the ability to form a coherent TH2 type response to T. muris infection, and further that the T-cell response is relatively unpolarised, displaying increases in both TH1 and TH2 associated markers. These data confirm that for the efficient formation of a TH2 response to infection with intestinal nematodes (either T.muris or H. polygyrus), antigen presenting (CD11c+) cells are required to localise to the B-cell follicle via expression of the chemokine receptor CXCR5.
Exploitation Route A thorough understanding of the cells and mechanisms involved in the propagation of prions within the gut will help identify novel factors that influence disease susceptibility or targets for intervention.

We have successfully created a unique transgenic mouse line enabling the conditional ablation of the CXCR5 gene in cells of interest. These mice have been shared with other scientists around the world for use in their own studies.

Our unique CXCR5fl/fl were provided to two key collaborators and these have been used in important studies that were published in the Journal of Experimental medicine. One study (Melo-Gonzalez et al. J Exp Med 2019 in press) describes the role of ILC3 cells in intestinal IgA responses. A second one (Denton et al. J Exp. Med 2019; PMID: 30723095) describes the role of CXCR5 in the formation of ectopic germinal centres in the lung.
Sectors Agriculture, Food and Drink,Healthcare

 
Description Role of distinct mononuclear phagocyte subsets in oral prion disease pathogenesis
Amount £550,023 (GBP)
Funding ID BB/S005471/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2019 
End 04/2022
 
Description Study of the role of macrophages in prion disease pathogenesis
Amount £3,000 (GBP)
Funding ID GR002413 
Organisation University of Edinburgh 
Sector Academic/University
Country United Kingdom
Start 03/2016 
End 12/2017
 
Title CXCR5-flox mice 
Description We have created a novel transgenic mouse line in which the expression of the gene Cxcr5 can be conditionally ablated in target cells. To enable conditional deletion of Cxcr5 in specific cell populations without affecting the CXCL13-CXCR5-dependent events required for normal lymphoid tissue development, mice with a conditional Cxcr5 allele were created by introducing loxP sites flanking exon 2. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2014 
Provided To Others? Yes  
Impact Previously, it was necessary to use bone marrow irradiation chimeras to study the in vivo role of Cxcr5-expression conventional dendritic cells. These mice provide a refined method to study the role of Cxcr5-expressing conventional dendritic cells without the requirement to first create bone marrow irradiation chimeras. 
URL http://jvi.asm.org/content/early/2017/03/02/JVI.00124-17.abstract
 
Description Study of role of sialoadhesin in prion disease pathogenesis 
Organisation University of Dundee
Department Division of Population Health Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided the mouse prion disease pathogenesis models
Collaborator Contribution Prof. Paul R. Crocker provided expertise in glycoimmunology as well as CD169-deficient mice
Impact Immunology, 143, 120-129 (2014); doi:10.1111/imm.12294
Start Year 2012
 
Description Study of the role of Cxcr5 in ectopic germinal centre formation in the lung 
Organisation Babraham Institute
Department Lymphocyte Signalling
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Our major contribution was the provision of our unique Cxcr5-floxed mice to this project. These mice were originally created on BBSRC grant BB/F019726/1.
Collaborator Contribution Our lead partner, Dr. Michelle Linterman at the Babraham Institute used these mice to study the role of Cxcr5 in ectopic germinal centre formation in the lung.
Impact Paper published in the Journal of Experimental Medicine in 2019 "Type I interferon induces CXCL13 to support ectopic germinal center formation" JEM 03-04-2019 issue, vol. 216 no. 3. http://doi.org/10.1084/jem.20181216
Start Year 2016
 
Description Study role of CXCR5 expression in ILC3 cells 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided the unique CXCR5-floxed mice created in this project to Dr. Matthew Hepworth at the University of Manchester.
Collaborator Contribution The CXCR5-floxed mice were crossed to ID2 ER2 Cre - ROSA-RFP mice to enable the study of effects of CXCR5-deficiency in ILC3 cells.
Impact Manuscript published in the Journal of Experimental Medicine in February 2019. PMID:30814299 "Antigen presenting ILC3 regulate T cell-dependent IgA responses to colonic mucosal bacteria" by Felipe Melo-Gonzalez, Hana Kammoun, Elza Evren, Emma Dutton, Markella Papadopoulou, Barry Bradford, Ceylan Tanes, Fahmina Fardus-Reid, Jonathan Swann, Kyle Bittinger, Neil Mabbott, Bruce Vallance, Tim Willinger, David Withers, and Matthew Hepworth
Start Year 2017
 
Description Keeping bugs at Bay: a Public Engagement Activity at the Roslin Institute 2014 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This activity aims to teach people how the immune system fights bugs.
Year(s) Of Engagement Activity 2014
 
Description Keeping bugs at Bay: a Public Engagement Activity at the Royal Highland Show 2015 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact This activity aims to teach people how the immune system fights bugs.
Year(s) Of Engagement Activity 2015
 
Description Media comments on identification of case of BSE in a cow on an Aberdeenshire farm 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact I was contacted by the media to provide my professional expertise and opinion on the identification of a case of bovine spongiform encephalopathy (BSE) on a Scottish farm in 2018. I provided a press statement with some information, as well as a detailed Q&A document. These were used by the media in both print and on-line articles. For example, The Herald, Daily Telegraph, Daily Mail, etc.
Year(s) Of Engagement Activity 2018
URL https://www.ed.ac.uk/roslin/news-events/latest-news/comment-on-bse-case-aberdeenshire
 
Description Press release on Denton et al 2019-J Exp Med: Lung tissue forms immune cell hubs when needed 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A press statement was issued describing our study published in the Journal of Experimental Medicine: Denton et al. 2019 J Exp Med; PMID: 30723095
Year(s) Of Engagement Activity 2019
URL https://www.ed.ac.uk/roslin/news-events/latest-news/lung-tissue-immune-cell-hubs?platform=hootsuite&...
 
Description Visit to Iowa State University to give talk and discuss my research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact On October 8th 2018 I gave a talk to approx. 100 post-graduate students at Iowa State University in the Immunology and Neurobiology research programmes. I also attended small workshop group sessions with the students to discuss my science and also give career advice etc.
Year(s) Of Engagement Activity 2018