Volume 44, Issue 5, Pages 939-946 (May 2006) Toll-like receptor 9 (TLR9) is present in murine liver sinusoidal endothelial cells (LSECs) and mediates the effect of CpG-oligonucleotides  Montserrat Martin-Armas, Jaione Simon-Santamaria, Ingvild Pettersen, Ugo Moens, Bård Smedsrød, Baldur Sveinbjørnsson  Journal of Hepatology  Volume 44, Issue 5, Pages 939-946 (May 2006) DOI: 10.1016/j.jhep.2005.09.020 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Fate of intravenously injected 125I-CpGs: (A) Clearance of 125I-CpGs from blood in 3 mice. The half-life of 125I-CpGs in the circulation was aprox. 4min. (B) Organ distribution after 10min. The sum of the radioactivity in all the organs was taken as total recovery (100%). The organ distribution in the different animals varied with a standard deviation of 2%. Journal of Hepatology 2006 44, 939-946DOI: (10.1016/j.jhep.2005.09.020) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Cellular distribution of FITC-CpGs in vivo and in vitro. (A) Histology section of the liver 10min after intravenous injection of FITC-CpGs. The sinusoidal cells are clearly labeled. Phase contrast (B) and fluorescence micrographs (C) of cultured LSECs following incubation for 30min with FITC-CpGs. Journal of Hepatology 2006 44, 939-946DOI: (10.1016/j.jhep.2005.09.020) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Expression analysis of TLR9 receptor, MyD88 and β-actin in LSECs. The cell line RAW 247 and mouse spleen and kidney were included as positive controls. The figure shows that RT-PCR products of expected size were obtained. Molecular marker 100bp (M) ladder is indicated. N is the negative control sample. Journal of Hepatology 2006 44, 939-946DOI: (10.1016/j.jhep.2005.09.020) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 (A) Immunoperoxidase staining showing the presence of TLR9 in the liver. A clear staining can be observed along the sinusoids. (B) Cultured LSECs where TLR9 presence is visible by immunofluorescence. Journal of Hepatology 2006 44, 939-946DOI: (10.1016/j.jhep.2005.09.020) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 The picture shows cultured LSECs incubated for 30min with: (A) only media, (B) n-CpGs (5μg/ml) and (C and D) CpGs (5μg/ml). The pictures A, B and C show cells that are labeled with anti-NFκB p65 antibody. Of note, only cells in cultures incubated with CpGs show a clear staining of the nucleus. Labeling with phospho-specific NF-κB (D) confirm the translocation after activation with CpGs. Journal of Hepatology 2006 44, 939-946DOI: (10.1016/j.jhep.2005.09.020) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Production of IL-1β and IL-6 by LSECs: Cells were incubated for 24h with free serum media (control); with a concentration of 100μg/ml of AGE-BSA to confirm that the SR ligand does not have any effect in the IL-1β production; with LPS (100ng/ml) as a positive control; with n-CpG (5μg/ml) to demonstrate the importance of the CpG sequence; and with CpGs (5μg/ml) together with AGE-BSA (100μg/ml), collagen-α-chain (100μg/ml), chloroquine (100μM), monensin (10μM), and neutralizing TLR9 antibody (15μg/ml). The inhibition observed in the cultures pretreated with AGE-BSA, chloroquine and monensin, showed the necessity of the endocytosis of CpGs prior activation of the cells and that the receptor involved in the up-take is the LSECs SR. The controls taken as 100% have a mean value of 70pg/ml for ILb and 753pg/ml for IL-6. All the experiments are average of at least three experiments. Stars: P<0.05 compared with the control. Journal of Hepatology 2006 44, 939-946DOI: (10.1016/j.jhep.2005.09.020) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions