Method: Membrane TREM-1 expression was assayed by FACS and sTREM-1 level was assayed by ELISA. Zymography was used to determine metalloproteinase (MMP)-9 activity.
Result: We sought to determine whether CpG-ODN-mediated TLR-9 activation affects TREM-1 expression and sTREM-1 level using mouse peritoneal macrophages and mouse macrophage cell line RAW 264.7. We found that the expression of TREM-1 is not significantly altered by CpG-ODN alone whereas lipopolysaccharide (LPS) up-regulates TREM-1 as was previously shown. However, macrophage stimulation with both LPS and CpG-ODN significantly abrogated TREM-1 LPS-induced up-regulation (For peritoneal cells MFI= 86.03 ± 8.6 vs. 229 ± 19.4, p<0.005; and for RAW cells MFI= 55.7 ± 5.6. vs. 97.6 ± 7.5, p<0.05). Moreover, supernatant soluble TREM-1 level of CpG-ODN as well as LPS-stimulated cells was increased and sTREM-1 level as result of both LPS-and CpG-ODN macrophage stimulation was significantly higher compared with LPS or CpG-ODN alone (56.4 ± 5.36 pg/ml vs. 16.2 ± 2.74pg/ml, p = 0.05). The release of sTREM-1 was found to be positively correlated with MMP-9 activity (r=0.914, p<0.005) and was inhibited by chloroquine (a TLR-9 and MMP-9 inhibitor).
Conclusion: Our novel results suggest that CpG-ODN-induced TLR-9 activation abrogates the effect of LPS on macrophage TREM-1 up-regulation and induces either by itself or in conjunction with LPS a significant increase of sTREM-1 level through a mechanism that involves MMP-9-mediated TREM-1 shedding. Our results suggest that the anti-inflammatory effects of CpG-ODN might be mediated through decreased membrane TREM-1 expression and increased MMP-9-mediated TREM-1 shedding. Since both CpG-ODN and sTREM-1were found to exert anti-inflammatory effects in mouse models of RA, we suggest a novel pathway for CpG-ODN-mediated up-regulation of sTREM-1 that can be used to treat inflammatory arthritis.
Disclosure: Y. Molad, None; E. Shapira, None; V. Carmon, None.