The immune / inflammatory receptors TLR4 and TLR2

CRISTIAN JORGE ALEJANDRO ASENSIO

Trieste

Simposio; CONFERENCIA EN SIMPOSIO ICGEB 4th ICGEB Alumni Meeting'' 2012; 2012

ICGEB , TRIESTE, ITALIA.

The presentation will show different sets of results related to the modulation of innate immune responses by toll-like receptors (TLRs), in particular TLR4 and TLR2. The first part of the talk will show results concerning the use of Bordetella pertussis outer membrane vesicles (OMVs) as vaccine candidates against whooping cough. The parental bacteria from which the nano-vesicles were obtained were genetically manipulated, by the incorporation of a plasmid, to express a different LPS (TLR4 ligand) structure which results less toxic (pro-inflammatory) than the WT structure. Nevertheless, these nano-vesicles maintain their immunogenic capacity since their protein antigens are not altered. LPS is necessary as an adjuvant but it can be toxic if its concentration and/or activity are not properly regulated or if it is given to some genetically susceptible people. The genetic manipulation of parental bacteria is better than the traditional reduction of the LPS concentration from the WT OMVs by using detergents. Thus, ?genetically engineered OMVs?, having a native structure without detergents, are useful technical platforms to study innate and adaptive immune responses to bacteria by genetically altering their surface components in quality and/or quantity. By using OMVs it can be studied the contribution to the immune responses of individual bacterial surface molecules. The application of OMVs in vaccinology has being considered with increasing interest in the last couple of years. The second part is related to the proteomic screening of protein biomarkers involved specifically in the late phase and sustained innate immune responses to bacteria. It will be presented a low-abundant cytoplasmic protein biomarker specifically involved in such kind of responses to any kind of TLR2 ligand. We found that human macrophages constantly check, during many days, the presence and concentration of TLR2 ligands. At least at the level of the said biomarker, they can keep the ?count? of the concentration of ligands regardless of their molecular structures, sources, identities or routes of incorporation in the cells. The biomarker allows the screening and validation of TLR2 ligands giving also information about their half-life in the cells. It also informs about the interaction between the intracellular TLR2 molecules and the cytoskeleton. On the other hand, the biomarker will allow novel diagnostic systems to study responses to TLR2 ligands in different infectious and non-infectious pathologies. Interestingly, so far, the biomarker was not altered or modulated by the concomitant activation of other signaling pathways different from TLR2 including the ER stress (or UPR), TLR4, phagocytosis, apoptosis, autophagy, etc. Mycobacteria, Borrelia and E. coli were not able to prevent or alter this default and automatic response to TLR2 ligands. The biomarker might be located downstream of the earlier endocytosis and phagocytosis pathways/compartments. The data suggest that this protein could be a TLR2-specific cytosolic accessory protein that is part of a cytosolic machinery specifically involved in the control of the late-phase and sustained trafficking, signaling and availability of pools of intracellular TLR2 molecules. This checkpoint would be proportional to the ligands concentration. So far, no other protein having these spatio-temporal properties was described for any TLR and it is also the first cytosolic biomarker specific for any TLR type. Our proteome screening method, so far, is the only able to sensitively, reproducibly and quantitatively monitor the said biomarker and per se is also a general method allowing the quantitative monitoring of other low-abundant protein biomarkers. The third part is about Tristetraprolin, an RNA binding protein involved in the control of the stability and translation of AU-rich mRNAs. In contrast to the previous, this is a protein behaving as a point of convergence of many pathways, including TLRs. Some results about the control of its activity and intracellular location by post-translational modifications will be mentioned.