TRAPC : A novel Triggering Receptor expressed on Antigen Presenting Cells

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology

Sammanfattning: The triggering receptor expressed on myeloid cells (TREM) family of receptors plays an important role in innate immunity. They have been described to regulate the course of sepsis, DC maturation, bone modulation and microglial function, and as markers for bacterial infections. We describe a novel activating murine TREM family receptor that is expressed in cells of both myeloid and lymphoid origin, including DCs and monocytes/macrophages and B-cells, but not in T- or NK cells. The molecule was designated triggering receptor expressed in antigen presenting cells (TRAPC). TRAPC belongs to the immunoglobulin superfamily, and associates with the adaptor-signaling molecule DAP12. TRAPC displays the highest identity to human TLT-4 and NKp44. Treating the macrophage cell lines J774.1 and RAW 264.7 with LPS led to increased expression of TRAPC. Cross-linking of TRAPC on macrophages and DCs induced nitric oxide production in both cell types, and up-regulated expression of CD40 on the DCs. Taken together, these results show that TRAPC is an activating receptor that may be involved in regulating immune responses, e.g. during bacterial infections. In order to investigate if TRAPC indeed plays a regulatory role during bacterial infections we have analyzed the role of TRAPC for its ability to regulate macrophage responses to infections with Gram-negative bacteria and lipopolysaccharide (LPS). Cross-linking of TRAPC on murine macrophage-like J774-A.1 cells resulted in an increase in the expression of iNOS and production of nitric oxide (NO), and in degradation of IkappaB-alpha. A more pronounced NO production was achieved when exposing J774-A.1 cells to LPS, or by infecting the cells with Escherichia coli or S. enterica serovar Typhimurium. Surprisingly, cross-linking of TRAPC on J774-A.1 cells prior to infection with E. coli or S. enterica serovar Typhimurium or exposure to LPS reduced the amounts of iNOS and production of NO. For S. enterica serovar Typhimurium, the reduced NO levels were accompanied by a substantial increase in intracellular bacterial replication. These results imply a regulatory role for TRAPC during innate immune responses to bacterial infections, acting on the NF-kappaB signaling pathway. To gain further insight in the function of TRAPC in immune responses we set out to identify the ligand for TRAPC. Using a soluble TRAPC Fc-fusion protein and flow cytometry, a potential ligand was detected on B cells, macrophages and DCs, i.e. on cells that also express TRAPC. In a first step to identify the ligand, cells positive for the potential ligand were lysed and immunoprecipitated with the TRAPC-Fc fusion protein. After separation in a denaturing polyacrylamide gel, a band potentially containing the ligand was detected. The identification of the ligand for TRAPC is crucial in understanding the function of TRAPC in innate immune responses. Given the pronounced effect of TRAPC on the response of macrophages to bacterial infections, it is tempting to speculate that bacteria have evolved to use TRAPC and other DAP12 associated receptors to their advantage. In this scenario, by inducing DAP12 mediated inhibition of antibacterial macrophage responses, bacteria would promote their own survival. It remains to be investigated what mechanism(s) underlies the TRAPC-mediated regulation of innate immune effector functions.

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