Toll-like receptor 4 | EPFL Graph Search

Toll-like receptor 4 is a protein that in humans is encoded by the TLR4 gene. TLR4 is a transmembrane protein, member of the toll-like receptor family, which belongs to the pattern recognition receptor (PRR) family. Its activation leads to an intracellular signaling pathway NF-κB and inflammatory cytokine production which is responsible for activating the innate immune system. TLR4 expressing cells are myeloid (erythrocytes, granulocytes, macrophages) rather than lymphoid (T-cells, B-cells, NK cells). Most myeloid cells also express high levels of CD14, which facilitates activation of TLR4 by LPS. It is most well known for recognizing lipopolysaccharide (LPS), a component present in many Gram-negative bacteria (e.g. Neisseria spp.) and selected Gram-positive bacteria. Its ligands also include several viral proteins, polysaccharide, and a variety of endogenous proteins such as low-density lipoprotein, beta-defensins, and heat shock protein. Palmitic acid and lauric acid are also TLR4 agonists, and chronic inflammatory responses via cytokine release can result from high dietary intake of these nutrients. However, unsaturated omega-3 and omega-6 fatty acids serve as TLR4 antagonists and can negate the inflammation caused by a high-fat diet. TLR4 has also been designated as CD284 (cluster of differentiation 284). The molecular weight of TLR4 is approximately 95 kDa. TLR4 is a member of the toll-like receptor (TLR) family, which plays a fundamental role in pathogen recognition and activation of innate immunity. They recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, and mediate the production of cytokines necessary for the development of effective immunity. TLRs are highly conserved from plants to Drosophila to humans and share structural and functional similarities. The various TLRs exhibit different patterns of expression. This receptor is most abundantly expressed in placenta, and in myelomonocytic subpopulation of the leukocytes.

Engineering the Extra Domain A of fibronectin as a vaccine adjuvant and study of its role in tissue regeneration

Ziad Julier

The extracellular matrix (ECM) protein fibronectin (FN) is a remarkably multifaceted molecule, including numerous fibronectin type III (FNIII) repeats carrying out different functions, which despite being extensively studied for over thirty years still presents certain functions that remain incompletely elucidated. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4) and appears to be involved in the progression of different skin diseases while studies also suggest that FNIII EDA is involved in tissue repair. The aim of this thesis is thus to better understand some of the immunological and regenerative functions of FNIII EDA and to translate these findings to develop new vaccination strategies based on endogenous proteins. One important task in vaccinology is to develop delivery strategies by which to present antigen, along TLR agonists, so as to induce a desirable adaptive immune response. The use of TLR agonists is associated with sometimes severe constraints and concerns about safety, causing inflammatory responses. Thus, the development of safe and effective vaccines with fewer side effects is a strong need and requires the development of new adjuvants with a correct balance between a strong activation of the immune system and a low toxicity for the patient. Here, by producing various FN fragments containing FNIII EDA, we show that the immunological activity of FNIII EDA depends on its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with its neighboring FNIII repeats enhanced its activity in agonizing TLR4, while C-terminal extension abrogated it. In addition, we reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Moreover, as FNIII EDA is naturally present within the ECM, we incorporated FNIII EDA bound in a fibrin matrix to mimic the in vivo situation and create an adjuvant-rich microenvironment. Such matrices were shown to induce functional cytotoxic CD8+ T cell responses in two murine cancer models. Furthermore, combinations of specific TLR-agonists have the ability to synergize to induce immune responses that are greater than the sum of their individual effect. Hence, we demonstrate that combination of the TLR9 agonist CpG with FNIII EDA synergizes to induce an efficient immune response while keeping the dose of the toxic CpG at a minimum. Here, the combination of both adjuvants induced a potent immune response sufficient to slow down tumor growth in a murine tumor model as well as to break immune tolerance to eradicate circulating hepatitis B virus (HBV) in a transgenic HBV mouse model. Finally in the last part of this thesis we revealed that FNIII EDA is a growth factor binding domain of FN, with a strong affinity for the platelet-derived growth factor-AA (PDGF-AA). We also demonstrated that FNIII EDA and PDGF-AA have together the ability to enhance fibroblasts proliferation and differentiation into myofibroblasts. In addition, we explored the effects of fibrin matrices functionalized with variants of FNIII EDA on wound closure.

EPFL2016

Engineering the Extra Domain A of fibronectin as a vaccine adjuvant and study of its role in tissue regeneration

Ziad Julier

EPFL2016