Activities of Salmonella flagellin: FliCing immunity on and off and from Th1 to Th2

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection

Abstract

Infections caused by Salmonella have a massive impact on human and live-stock health. In terms of disease they are amongst the leading causes of gastroenteritis in the UK, but their impact in other countries is much more serious where Salmonella kills over a million people every year. Clearing a primary Salmonella infection requires Salmonella-specific CD4 T cells to become activated. These T cells then polarize to become one of two types, a Th1 or Th2 cell. Th1 cells are best at activating cells that actively kill and digest bacteria (macrophages). When the same infection is re-encountered other cell-types including B cells that can develop to produce antibody are also important for optimal immunity. One of the most, if not the most, commonly recognized T cell antigen from Salmonella is flagellin and it is also a major target of B cell responses. Flagellin is the major constituent of long whip-like structures called flagella that are anchored into the cell wall and involved in bacterial motility. Although not required for virulence their extension far beyond the wall of the bacterial cell, and strong recognition by the innate and adaptive immune system make them important to study. Because so much of this protein is exposed to the host it makes a good target for the host to target, but their location outside the bacterium also offers a means by which the bacterium can interact with and affect the host. We found the immune response to flagellin can alter to be Th1 or Th2 depending upon how the protein is encountered by the immune system. Despite provoking a strong immune response immunization with FliC does not protect against Salmonella infection. Indeed when flagellin was given alongside an effective live bacterial vaccine we showed it reduced protection by impairing the production of a key Th1 molecule IFNg. Under some circumstances we noticed that flagellin can induce a hypersensitivity reaction in the host. This was seen when flagellin protein was given when small numbers of bacteria were present after a previous Salmonella infection. In contrast, when the infection had resolved this hypersensitivity reaction was not seen. This is of significance since flagellin has been proposed as a possible adjuvant in humans. Thus flagellin offers many paradoxes such as being the major target of the immune response to Salmonella yet these responses may sometimes not protect or may even do harm, and flagellin is even dispensable for virulence. This project wants to expand our preliminary and published findings to understand more about the complex relationship between the host, flagellin and Salmonella. We wish to identify: 1. Why does flagellin induce a Th2 response and whether the direction of the T cell response induced against isolated flagellin can be altered from a Th2 response to a Th1 response that then offers protection against infection? 2. How the intrinsic properties of flagellin may harm the host? 3. Whether the dominance of the T cell response to this one antigen enhances clearance of bacteria from the host is a mechanism by which the bacterium evades immunity? We will do this by using a mouse model of infection and vaccination. Microscopy will be used to examine tissues and allow the identification of where cells reside such as flagellin-specific T and B cells. More detail on the phenotype of cells will be attained using flow cytometry and gene expression will be assessed by the polymerase chain reaction. The work will offer insights into i) whether this potent protein may be used in a vaccine by altering how it is given to the host; ii) how effective vaccines to Salmonella should be designed and iii) whether potential problems such as hypersensitivity responses can be avoided during the administration of vaccines.

Technical Summary

Salmonella infections are major health and economic burdens in humans and live-stock. Resolution of primary infection requires CD4 Th1 cell interaction with macrophages whereas optimal immunity against reinfection also requires B cells. The TLR5 agonist flagellin is the immunodominant T cell antigen recognised during these infections, yet flagellin is dispensable for virulence. Our experiments suggest responses to flagellin can be double-edged. When given as a soluble protein a strong Th2 response is induced, yet the response to bacterial surface localised flagellin is Th1. Surprisingly, considering its immunodominance, vaccination with soluble flagellin is not protective even though high antibody titres are induced. Furthermore soluble flagellin can impair the protection afforded by a live vaccine in part by suppressing IFNg production from flagellin-specific CD4 T cells yet soluble FliC-primed Th2 transgenic T cells transferred into Rag1-/- mice can protect. Additionally flagellin can induce a hypersensitivity reaction in mice that have residual Salmonella infection. The aim of this project is to unravel the mechanisms by which these various positive and negative activities are mediated to improve vaccine design and reduce the disease burden caused by these infections in humans and live-stock. Specifically we wish to: 1. Identify if Th2 responses to soluble FliC can be redirected to Th1 and whether this improves protection to STm infection 2. To investigate the mechanism(s) by which flagellin's intrinsic properties can harm the host 3. To test whether the predominance of responses to flagellin in wild-type (WT) mice favours the host or the pathogen The purpose is to exploit the immunodominance of flagellin to improve the Th1 protection immune responses targeted against the protein offer. At the same time this needs to be achieved by understanding, and bypassing, the negative effects flagellin can have on the host.

Publications

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Flores-Langarica A (2011) T-zone localized monocyte-derived dendritic cells promote Th1 priming to Salmonella. in European journal of immunology

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Gil-Cruz C (2009) The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response. in Proceedings of the National Academy of Sciences of the United States of America

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Hitchcock JR (2015) Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets. in The Journal of clinical investigation

 
Description In this research project we examined how the immune response develops to a bacterial protein called flagellin. This particular flagellin comes from Salmonella and triggers an immune response through a molecule called TLR5.

What we were able to show is that the immune response to flagellin shows some plasticity. This means it starts in one form but can then change. This can help improve the rate of clearance of infection if the protein is used as a vaccine. Using this protein we were able to show that it can be used as a "Trojan Horse" to drive immune responses in the gut when the protein is given as an injection. This is a significant property of flagellin because it is notoriously difficult to do this. This means we can use this protein to "carry" other antigens to the gut and enhance responses to them. This will make this a very powerful way to deliver new vaccines. research that has developed from this project is examining how we can best do this.
Exploitation Route This is explained above. We have now used these findings to improve vaccine delivery to the gut and enhance the size of a response to a vaccine.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description project grant from BBSRC
Amount £600,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2014 
End 01/2017
 
Description wellcome trust CRF
Amount £250,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Research Training Fellowship
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description Babraham 
Organisation Babraham Institute
Country United Kingdom 
Sector Private 
PI Contribution We contribute to the collaboration through our immunohistology skills
Collaborator Contribution They provide unique mouse models and reagents
Impact The RNA-binding protein HuR is essential for the B cell antibody response. Diaz-Muñoz MD, Bell SE, Fairfax K, Monzon-Casanova E, Cunningham AF, Gonzalez-Porta M, Andrews SR, Bunik VI, Zarnack K, Curk T, Heggermont WA, Heymans S, Gibson GE, Kontoyiannis DL, Ule J, Turner M. Nat Immunol. 2015 Apr;16(4):415-25. doi: 10.1038/ni.3115. Epub 2015 Feb 23. PMID: 25706746 Free PMC Article
Start Year 2011
 
Description MRC Centre for Immune Regulation 
Organisation University of Birmingham
Department MRC Centre for Immune Regulation
Country United Kingdom 
Sector Public 
PI Contribution Contribution to a theme in the Centre: Induction and regulation of immunity in secondary lymphoid tissues. My work examining how immune responses develop to pathogens and their components offers broad cross-theme collaborations.
Collaborator Contribution Access to material and equipment as well as MRC students
Impact From this collaboration nearly all of my publications and patent are due, in part, to this collaboration.
 
Description Minnesota 
Organisation University of Minnesota
Country United States 
Sector Academic/University 
PI Contribution This was a joint project where we investigated the role of TLR5 in the maintenance of host homeostasis and T cell activation. Experiments were reproduced in both laboratories.
Collaborator Contribution This was a joint project where we investigated the role of TLR5 in the maintenance of host homeostasis and T cell activation. Experiments were reproduced in both laboratories.
Impact A paper in JI (21451112).
Start Year 2010
 
Description University of Lund 
Organisation Lund University
Country Sweden 
Sector Academic/University 
PI Contribution The post-doctoral fellow Dr Adriana Flores-Langarica made two visits to Lund to work collaboratively on the project. The resources used (animal models, consumables etc) were covered by Lund. This is a significant cost and research benefit to us as it made available unique resources.
Collaborator Contribution They provided unique models for our studies
Impact The first manuscript from this work is in preparation
Start Year 2013
 
Description Vaccine development with GSK Vaccines for Global Health 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution We are developing a novel generation of vaccines. We have the expertise to assess how they function
Collaborator Contribution Our partners generate the vaccine candidates
Impact This has just started
Start Year 2017
 
Description Secondary school student work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact 1 student for work experience.

This has developed into a longer-term relationship. For instance, I will host another student next year and have arranged to talk at the school next year.
Year(s) Of Engagement Activity 2012
 
Description school visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talks at local schools about animal experimentation and immunology, vaccinology and infectious disease. During this it became clear it is important alos to offer career advice. This is something also pursued by members of my group

Increased awareness and involvement with other schools
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014