Molecular characterisation of Toll-like receptor 4 biased signalling through the TIR-domain-containing adapter-inducing interferon-beta

Development of successful vaccination strategies in humans of all ages is important not only for healthy ageing across the lifecourse, but also to reduce antimicrobial resistance. Vaccination of animals both to prevent animal diseases and to reduce the burden of zoonotic pathogens is critical to improve animal health and welfare as well as increasing food security. Adjuvants are molecules used to to improve the efficiency of vaccines in man and animals. Many adjuvants target a class of receptors that recognise pathogenic micro-organisms called Pattern Recognition Receptors (PRRs). One PRR, Toll-like Receptor 4 (TLR4), is important for recognising Gram negative bacteria and protecting the host against infections with these bacterial species. Over activity of this receptor, however, leads to severe inflammation and it is thought that increased activation of TLR4 as we age underpins many of the conditions commonly seen in an ageing population. TLR4 activates separate, but linked arms of the immune response. One arm, through a protein called Toll-Interleukin 1 Receptor (TIR)-domain-containing adapter-inducing interferon-beta (Trif), is very efficient at facilitating the development of protective vaccine responses. The other, through a protein called Myeloid Differentiation Primary Response 88 (MyD88), protects animals against Gram negative bacterial infections. An adjuvant molecule monophosphoryl lipid A (MPLA) selectively activates Trif in humans, but the mechanisms by which this occurs are not understood. In this grant we will determine how MPLA activates TLR4-Trif by changing the structure of TLR4 and determining whether this alters activation of Trif and/or MyD88. We will also determine the protein complexes formed by Trif after MPLA activation of immune cells. Finally we will try and identify molecules that will selectively target TLR4 and Trif driven immune responses to generate new compounds for vaccine adjuvants and other therapeutic applications.

Development of successful vaccination strategies in humans of all ages is important not only for healthy ageing across the lifecourse, but also to reduce antimicrobial resistance. Vaccination of animals both to prevent animal diseases and to reduce the burden of zoonotic pathogens is critical to improve animal health and welfare as well as increasing food security. The adjuvant monophosphoryl lipid A (MPLA) is an agonist at the Pattern Recognition Receptor Toll-like receptor 4 (TLR4). In humans MPLA selectively activates TLR4 signaing through the Trif-related adaptor molecule (Tram) / Toll-Interleukin 1 Receptor (TIR)-domain-containing adapter-inducing interferon-beta (Trif) signaling pathway, but the molecular basis by which this occurs is not understood. In our recent work we have determined: 1) the molecular basis for bacterial lipid recognition at TLR4 2) the stoichiometry of TLR4-induced signaling complexes and how this relates to TLR4-dependent signaling induced by lipids in living cells 3) the structural basis for regulation of lipid-dependent TLR signaling. Here we will build upon this work to explore the hypothesis that there are discrete amino acid differences in the extra cellular and TIR domains of TLR4 that will preferentially divert signals through Tram and Trif, rather than Mal and MyD88, that can be exploited to develop molecules with selective activity at this signaling pathway. We will use structure-function analysis to characterise the mechanistic basis for the Trif-biased activation of TLR4 by MPLA and to determine the molecular nature of the MyD88- and Trif-dependent signaling complexes formed. In the final part of the proposal we will identify small molecules and/or cell permeable peptides that selectively target TLR4/Tram/Trif specific signaling which could be used for a range of therapeutic applications in man and animals.

Our research program aims to determine how the adjuvant molecule monophosphoryl lipid A (MPLA) interacts with the pattern recognition receptor Toll-like receptor 4 (TLR4). Activation of TLR4 by infectious agents, such as bacteria, induces an innate immune response and inflammation through two signalling pathways via the adaptor proteins Trif or MyD88. Effective adjuvant activity is driven through the Trif pathway whereas signaling through MyD88 controls Gram negative bacterial infections. MPLA selectively activates Trif signaling in humans, but the mechanisms by which this occurs are not well understood. Determining the structural mechanisms by which this occurs in man and animals offers the opportunity to use this information to develop new vaccine adjuvants which is important in a world where antimicrobial resistance is increasing and vaccination of man and animals to reduce the pathogen burden is one mechanism to tackle this problem. We also hope to find molecules that selectively inhibit TLR4-Trif as potentially novel anti-inflammatory therapeutic interventions for the many chronic inflammatory conditions that afflict the elderly whilst leaving the protective TLR4-MyD88 signaling against bacterial infection untouched so our work may well have very wide therapeutic impact.

Short term impact: The molecular mechanisms by which MPLA selectively activates TLR4 will be of immediate interest to the academic community, particularly in immunology and infectious diseases. Innate immunity is a field of fast moving and intensive research so our mechanistic work will be of particular interest to scientists working in this area. Innate immunity and infectious diseases are also areas of intense interest to the pharmaceutical industry. The Immunology Catalyst at GSK (of which Clare Bryant is a member) is focused on innate immunity as it is thought that the next generation of immunotherapies will come from this area of research. Our work will therefore be of immediate interest to the pharmaceutical industry

Medium and long term impact: We have many long standing collaborations with the pharmaceutical industry (for example Astra Zeneca, Genentech, Apollo Therapeutics, Zoetis Animal Health and GSK). In the course of the project we will attempt to identify small molecule or peptide modulators of TLR4 activity. We already have a number of candidate molecules which if selective for TLR4-Trif signalling are likely to be of interest to our pharmaceutical colleagues allowing for rapid translation of this work for potential development of future medicines. We are already developing small molecule TLR4 antagonists with Apollo Therapeutics and we expect that identification of further molecules that target this pathway will be of interest to a number of pharmaceutical companies.

Other forms of impact: with the emergence of antimicrobial resistance and the increased burden of chronic inflammatory disease in an ageing population our work is likely to be of interest to the media. We are very experienced in public engagement and all our major advances will be discussed with the Cambridge University Communications office. Our postdoctoral workers will be working in a multidisciplinary team so will have opportunities to learn new techniques, such as cryoelectron and light sheet microscopy, to broaden their skill base. They will also undertake professional development training within the university to develop the skills necessary for the effective conduct and management of this research, research leadership, the integrity and ethical conduct of the research and the management and communication of open data.