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II-14 disturbed clearance of apoptotic debris in pristane-treated tlr9ko mice leads to accumulation of a unique macrophage population
  1. Kerstin Nundel,
  2. Anette Christ,
  3. Shruti Sharma and
  4. Ann Marshak-Rothstein
  1. University of Massachusetts Medical School, Department of Medicine, Worcester, USA

Abstract

Background Nucleic acid binding TLRs have been found to play a critical role in the production of autoantibodies and disease development in animal models of SLE. Intriguingly, TLR9 appears to play both a protective and disease promoting role. While TLR9 is required for the production of anti-dsDNA autoantibodies, TLR9KO autoimmune-prone mice develop more severe disease than their TLR9-sufficient counterparts. Studies from our group and others have pointed to B cell expression of TLR9 as a key determining factor. However, our recent studies point to an additional role for TLR9in myeloid lineage cells.

Materials and methods Pristane injected BALB/c wildtype (WT) and TLR9KO mice were analysed for disease severity at 5 months. Kidney sections were stained for IgG deposition and Ly6G positive cells. Single cell suspensions of different tissues were analysed using flow cytometry. Mixed BM chimaeras (50% WT: 50% TLR9KO) were injected with pristane and myeloid populations were analysed. For apoptotic cell clearance, WT and TLR9KO bone marrow derived macrophages (BMDM) were stimulated with CFSE labelled apoptotic cells and analysed 24 hours later by confocal microscopy.

Results We have found that TLR9-deficiency dramatically exacerbates the onset of renal disease resulting in decreased survival. Increased levels of IgG accumulate in pristane treated TLR9KO glomeruli compared to WT glomeruli, and the increased IgG deposits are associated with an increased myeloid infiltrate. Moreover, this myeloid infiltrate contained an increased frequency of granulocytes a well as an unusual CD11b+ Ly6Cint Ly6Gint (Ly6CGint) subset. To better understand the origin of these populations, the myeloid subsets of pristane-treated mixed (TLR9WT + TLR9KO) BM chimaeras were analysed. Remarkably, the Ly6CGint population was entirely derived from the TLR9KO stem cells. Morphologic analysis revealed that the Ly6CGint population are macrophages containing large lipid droplets, suggesting a role for TLR9 in degradation of pristane. Further in vitro analysis of BMDMs stimulated with apoptotic cells showed that most WT BMDMs cleared apoptotic cells by 24 h. However, a large fraction of TLR9-deficient BMDMs still had un-degraded apoptotic cells in the lysosomal compartment, suggesting a role for TLR9 in clearance.

Conclusions These data demonstrate a direct effect of TLR9-deficiency on the expansion of a unique CD11b+ population, and further suggest that these cells play a major and direct role in the accelerated disease characteristic in TLR9KO mice. Furthermore, a specific role for TLR9 in the clearance of apoptotic cells may be the underlying cause for the accumulation of this CD11b+ subset.

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