Methods: Subcutaneous chambers were implanted in wild type and knockout mice, in which the gene encoding for the killer receptor Ncr1 was replaced with a green fluorescent reporter gene (GFP). Following challenge with 106 bacteria / chamber, the cellular contents of the exudates were analyzed by FACS and the supernatants by ELISA. In vitro: Binding of Ncr1 to oral bacteria was analyzed using the Ncr1-Ig fusion protein. The ability of oral bacteria to activate cells through the Ncr1 receptor was verified in various functional assays.
Results: By tracking the GFP, we observed rapid accumulation of NK cells following the bacterial challenge. The TNF-á levels rapidly increased in chambers of wild type mice 2 hours post F. nucleatum challenge, but were markedly lower in the homozygotes knockout mice. In contrast, similar levels of TNF-á were observed when P. gingivalis was injected in both mice species. In agreement with the in vivo results indicating that Ncr1 specifically recognizes F. nucleatum, binding of Ncr1-Ig fusion protein was observed only to F. nucleatum but not to P. gingivalis. The unknown Ncr1 ligand expressed on F. nucleatum was sensitive to heat, proteinase K and pronase treatment, but not to neuraminidase or galactose inhibition. Finally, we show that F. nucleatum induced functional activation of the Ncr1 in various in vitro assays.
Conclusions: Our results demonstrate, for the first time, direct recognition of an oral bacterium by a NK cells receptor. These findings provide an insight into the relationship between the innate immune response to periodontopathic bacteria.
Acknowledgements: Supported by the Israel Science Foundation, grant no. 774/07