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Functional genomics of innate immune pathways in mammalian cells

Final Activity Report Summary - INNATE IMMUNITY (Functional genomics of innate immune pathways in mammalian cells)

The overall goal of the project was to identify novel key players in innate immune pathways by functional genomics. Our focus was to obtain a better understanding of an inflammatory pathway in monocytes / macrophages that is mediated by the immunoreceptor tyrosine based activation motif (ITAM) containing adapter DAP12 that couples to the cell surface receptor triggering receptor expressed by myeloid cells-1 (TREM-1). Triggering via TREM-1 results in the production of pro-inflammatory cytokines, chemokines, reactive oxygen species (ROS), and leads to rapid degranulation of neutrophilic granules, and phagocytosis. Furthermore, application of a TREM-1 / Ig fusion protein in an animal model of experimentally induced sepsis increases survival. In order to target components of the TREM-1 / DAP12 pathway as therapeutic strategy for the treatment of inflammatory diseases, it is of great importance to obtain a better understanding of signalling components downstream of TREM-1 (Tessarz et al., 2008, Immunology Letters). In order to fulfil our goal an international team was recruited.

In our project we applied several approaches to uncover novel signalling components in the TREM-1/DAP12 pathway. We established a pro-myelocytic cell line U937 overexpressing TREM-1 and DAP12 and refined methods to visualize proteins that become tyrosine phosphorylated after TREM-1 triggering. We discovered that one of the phosphorylated proteins with a MW of 30kD was the non-T cell activation linker NTAL. We showed that one of the NTAL associated phosphoproteins was the adapter molecules Grb-2. In subsequent studies the requirements of the 3 distal tyrosines in NTAL for this interaction was identified. NTAL knock-down U937-TD cells were successfully established. In U937-DT cells, in which NTAL expression was decreased by RNAi, increased inflammatory cytokine production was observed revealing that NTAL is a negative regulator of TREM-1 / DAP12 signalling (Tessarz et al., 2007, Journal of Immunology). In order to prove the specificity of the knock-down of NTAL, a shRNA insensitive mutant of NTAL was introduced into NTAL knock-down cell lines. In these 'rescue cell lines' the enhancement of cytokine production was abrogated. In addition, we introduced mutated forms of NTAL which did not bind to Grb-2. These experiments revealed that the binding to Grb-2 was required for NTAL's negative effect on cytokine production (manuscript in preparation).

Additional candidate tyrosine phosphorylated proteins after TREM-1 triggering were identified. One protein was identified to be the Tec kinase family member Btk. Btk knock-down U937-TD cells were successfully established. In U937-DT cells in which Btk expression is decreased by RNAi, significantly decreased inflammatory cytokine production was observed revealing that Btk is a positive regulator of TREM-1 / DAP12 signalling (manuscript in preparation).

In order to gain a better understanding of TREM-1 and its so far unidentified ligand(s) we performed a comprehensive study of expression patterns of TREM-1 and TREM-1-ligand in the steady state and during inflammation in mice using a monoclonal antibody that was produced in the lab. These experiments revealed that in mice TREM-1 is upregulated on inflammatory monocytes and myeloid derived suppressor cells. TREM-1 ligand that we detected with a TREM-1-Ig-fusionprotein was only detectable on granulocytes after stimulation with TLR ligands (Zanzinger et al, 2009, Immunology in press).

In summary, our study so far identified and characterised one negative (NTAL) and one positive (Btk) regulator in the pathway and revealed novel aspects of TREM-1 expression on myeloid cells in the mouse. In addition, the project yielded exciting preliminary results that were essential for the preparation of a new proposal that is now submitted to the local funding agency DFG. Several collaborations with industry were initiated. The knowledge gained by the project might contribute to the development of novel improved strategies for the treatment of inflammatory diseases and septic shock.