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Toll-like receptor group

Principal Investigator:

Identification and characterisation of physiologic and therapeutic regulators of Toll-like receptor mediated pro-inflammatory responses

Our laboratory’s research interest has centred on the molecular mechanisms underlying microbial recognition by immunocompetent cells and the regulation of such recognition. Our early research focused on the immunobiology of the co-receptor for Toll-like receptors (TLR), the CD14 molecule. More recently, the focus of our research has been extended to include the regulation of TLR triggering and that exerted by TLRs on the activity of other innate immune receptors.

The TLR family plays a crucial role in the innate immune response by mediating the recognition of, and response to a variety of microorganisms. Dysregulation of the TLR-mediated inflammatory response to severe microbial infections may, however, lead to serious pathological conditions including septic shock, and may result in death.

Our current research, therefore, involves the design and testing of novel strategies to blunt, but not abrogate, cellular responses to TLR ligands. It builds on our recent findings of naturally occurring soluble forms of TLR2 (sTLR2) capable of regulating cellular activation via transmembrane TLR2, and aims at generating novel therapeutics based on the capacity of sTLR2 to interact with CD14, and therefore to interfere with its co-receptor activity, as we demonstrated recently.

Such therapeutics aim at preventing and/or treating severe bacterial infections as well as other microbial-induced and sterile acute and chronic inflammatory conditions. More recently, we have extended our investigations to include the regulation that TLRs exerts on the activity of other innate immune receptors.

Our investigations on the regulatory mechanisms controlling TLR triggering extend to human breast milk, which is believed to promote a regulated and hence appropriate interaction between the neonatal gut and luminal bacteria. We have therefore sought TLR-specific mechanisms operating in milk that may modulate microbial recognition in the gut, thereby contributing to the beneficial effect of breast-feeding.

Hypothetical model of the interaction of sTLR2 with sCD14 and/or mCD14. This interaction, revealed by co-immunoprecipitation, FRET and chemical cross-linking experiments, may lead to a reduced CD14 co-receptor activity, which in turn may result in reduced signalling via TLR2/TLR1
sTLR2 interacts with sCD14. Molecular modeling of the interaction between the LRR domains of TLR2 (green) and those of CD14 (blue) as predicted by computational molecular docking experiments. A cylindrical supramolecular arrangement was deduced as the best assembly mode. Only the interior, hydrophobic part of the complex is shown. Within this predicted central hydrophobic pocket, bacterial lipopeptide may dock near to the N terminus (NT) end of the TLR2 LRR domains, stabilizing further a putative sTLR2-sCD14 interaction. The docking experiments indicate a C terminus-N terminus assembly of the two molecules (from E. Le Bouder et al. J. Immunol. 2003. 171: 6680.)

Our research in this area aims at the identification and characterisation of the human milk components that are involved in modulating TLR-mediated neonatal microbial recognition and absent from infant milk formulations. Through this investigation, we are not only defining fundamental molecular events that are of substantial relevance to innate immunity - as they relate to the mechanisms whereby TLRs are triggered and regulated -, but also helping to understand how milk components influence the quality of the interaction between microorganisms and gut immunocompetent cells. This, in turn, helps to better define the molecular basis of the beneficial effects long ascribed to breast milk.

Results of this study will inform the design of improved infant milk formulations. This may be beneficial to children for whom breast-feeding is not an option (e.g., HIV+ or tuberculosis+ mothers), to premature infants predisposed to inflammatory conditions of the gut, as well as to non-breastfed infants predisposed to other pathological conditions also believed to be related to the quality of exposure to microorganisms early in life, such as recurrent wheeze and atopy.

Main Published Findings

  1. The detection, biochemical and functional characterisation of two soluble forms of the TLR co-receptor, CD14 (sCD14).
  2. The demonstration that the soluble form of CD14 can enhance LPS-induced monocyte stimulation.
  3. The description of the unexpected expression of CD14 in human B cells, and demonstration of the functional activity of CD14 as mediator of B cell stimulation by LPS.
  4. The definition of the role that the TLR co-receptor CD14 plays in the phenomenon of cell tolerance to bacterial lipopolysaccharide by mediating the establishment of the tolerisation process, but not being responsible for the low responsiveness of the tolerised cells.
  5. The detection and first biochemical characterisation of a signalling receptor for sCD14.
  6. The description of the unexpected interaction of sCD14 with human T and B cells and its functional consequences, namely the negative regulation of T cell activation and differential regulation of immunoglobulin secretion by B cells (up-regulation of IgG and down-regulation of IgE).
  7. The detection, biochemical and functional characterisation of sCD14 in human breast milk and islet beta cells.
  8. The identification, biochemical and functional characterisation of naturally occurring soluble forms of TLR2 present in human plasma and breast milk, and capable of regulating inflammation in vitro and in vivo.
  9. Demonstration of the presence of protein milk components capable of enhancing TLR4 and TLR5 and inhibiting TLR2 and TLR3 signalling.

Selected Publications

  1. Raby A.C., LeBouder E., Colmont C., Davies J.,Richards P., Coles B., Jones S., Brennan P., Topley N., LabétaM.O. Soluble TLR2 reduces inflammation without compromising bacterial clearance by disrupting TLR2 triggering. J. Immunol. 2009. 183: 506.
  2. LeBouder E, Rey-Nores JE, Raby AC, Affolter M, Vidal K, ThorntonCA, Labéta MO. Modulation of neonatal microbial recognition: TLR-mediated innate immune responses are specifically and differentially modulated by human milk. J. Immunol. 2006. 176: 3742.
  3. LeBouder E, Rey-Nores JE, Rushmere NK, Grigorov M, Lawn SD, Affolter M, Griffin GE, Ferrara P, Schiffrin EJ, Morgan BP, Labéta MO. Soluble forms of Toll-like receptor (TLR) 2 capable of modulating TLR2 signaling are present in human plasma and breast milk. J. Immunol. 2003. 171: 6680, and commentary 171: 6321.
  4. Labéta MO, Vidal K, Nores JE, Arias M, Vita N, Morgan BP, Guillemot JC, Loyaux D, Ferrara P, Schmid D, Affolter M, Borysiewicz LK, Donnet-Hughes A, Schiffrin EJ. Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14. J. Exp. Med . 2000. 191: 1807.
  5. Arias MA, Rey Nores JE, Vita N, Stelter F, Borysiewicz LK, Ferrara P, Labéta MO. Human B cell function is regulated by interaction with soluble CD14: opposite effects on IgG1 and IgE production. J. Immunol. Cutting edge , 2000. 164: 348.
  6. Rey Nores JE, Bensussan A, Vita N, Stelter F, Arias MA, Jones M, Lefort S, Borysiewicz LK, Ferrara P, Labéta MO. Soluble CD14 acts as a negative regulator of human T cell activation and function. Eur. J. Immunol. 1999. 29: 265.
  7. Vita N, Lefort S, Sozzani P, Reeb R, Richards S, Borysiewicz LK, Ferrara P, Labéta MO. Detection and biochemical characteristics of the receptor for complexes of soluble CD14 and bacterial lipopolysaccharide. J. Immunol. 1997. 158: 3457.

Collaborations

  1. Sanofi-Aventis, Labége, France: P. Ferrara, J.-C. Guillemot
  2. Nestlé S.A.Lausanne, Switzerland: E. Schiffrin, M. Grigorov, M. Affolter
  3. School of Applied Sciences, University of Wales Institute, Cardiff, U.K.: J. E. Rey-Nores
  4. School of Medicine, University of Wales, Swansea, U.K.: C. A. Thornton
  5. Dept. of Medicine, School of Medicine, Cardiff University, U.K.: C. George.

 Funding

The work summarised above has been funded by the Wellcome Trust, The British Council, the Medical Research Council (MRC)-Cardiff University, The Welsh Office of Research and Development for Health and Social Care (WORD), the Cardiff Partnership Fund (CPF), and has also benefited from continuous and generous funding by industrial collaborators, Nestlé S.A. (Switzerland) and Sanofi-Aventis (Labége, France). Productive collaborative work with these two industrial partners included the detection, biochemical and functional characterisation of sCD14 in human milk (ref. 4), the identification and characterisation of soluble forms of TLR2 (sTLR2) in plasma and milk (ref. 3), and the description of TLR-enhancing and TLR-inhibitory proteins present in milk (ref. 2; in collaboration with Nestlé S.A.). These recent findings resulted in two patents filed in collaboration with Nestlé S.A. In vivo and in vitro studies funded by the Wellcome Trust, the MRC-Cardiff University and CPF aim to examine the molecular details of the interaction of TLR2 with the co-receptor CD14 (Wellcome Trust), and to describe the molecular mechanism underlying the regulatory capacity of sTLR2 (MRC-Cardiff University, CPF) that our group has recently reported. Results from this work have led to a third patent recently filed.