982 - Toll-Like Receptor-9 Activation Regulates Macrophage Triggering Receptor Expressed on Myeloid Cells-1 Expression and shedding

Monday, November 7, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Yair Molad, Felsenstein Medical Reseach Center, Sackler Faculty of Medicine, Tel Aviv University, and Beilinson Hospital, Rabin Medical Center, Petah Tikva, Israel, Elisheva Shapira, Beilinson Hospital, Rabin Medical Center, Petah Tikva, Israel and Vered Carmon, Laboratory of Inflammation Research, Felsenstein Medical Research Center, Tel Aviv University, Petah Tikva, Israel
Presentation Number: 982

Background/Purpose: Toll-like receptor (TLR)-4-mediated immune response plays a pivotal role in the initiation and progression of the chronic and destructive stages of arthritis in both human disease and rodent models of rheumatoid arthritis (RA). In-addition, previous studies suggested TLR-9 plays a role in the pathogenesis of RA. For example, TLR-9 K.O. mice suffered of worse arthritis. Triggering receptor expressed on myeloid cells-1 (TREM-1) is a DAP12-associated receptor which its up-regulation is TLR-4-mediated and plays an essential role in innate immune response by augmenting the production of proinflammatory chemokines and cytokines.  Monocyte TREM-1 expression is increased in human and mouse synovium as well as its soluble form (sTREM-1) serum level. Moreover, sTREM-1 was shown to exert anti-inflammatory effects in animal models of RA. We hypothesize that anti-inflammatory effects of CpG-oligonucleotide (ODN), an inducer of TLR-9 activation, are mediated by regulating membrane TREM-1 expression and sTREM-1 shedding. Thus, the purpose of our study was to determine the in vitro effects of CpG-ODN-induced TLR-9 activation on macrophage TREM-1 expression and shedding.  

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.

Keywords: macrophages

Disclosure: Y. Molad, None; E. Shapira, None; V. Carmon, None.