Activation of CD11b+ Kupffer cells/macrophages as a common cause for exacerbation of TNF/Fas-ligand-dependent hepatitis in hypercholesterolemic mice

Abstract

We have reported that the mouse hepatic injury induced by either α-galactosylceramide (α-GalCer) or bacterial DNA motifs (CpG-ODN) is mediated by the TNF/NKT cell/Fas-ligand (FasL) pathway. In addition, F4/80(+) Kupffer cells can be subclassified into CD68(+) subset with a phagocytosing capacity and CD11b(+) subset with a TNF-producing capacity. CD11b(+) subset increase if mice are fed high-fat and cholesterol diet (HFCD). The present study examined how a HFCD affects the function of NKT cells and F4/80(+) CD11b(+) subset and these hepatitis models. After the C57BL/6 mice received a HFCD, high-cholesterol diet (HCD), high-fat diet (HFD) and control diet (CD) for four weeks, the HFCD mice increased surface CD1d and intracellular TLR-9 expression by the CD11b(+) population compared to CD mice. Hepatic injury induced either by α-GalCer or CpG-ODN was more severe in HCD and HFCD mice compared to CD mice, which was in proportion to the serum TNF levels. In addition, liver cholesterol levels but not serum cholesterol levels nor liver triglyceride levels were involved in the aggravation of hepatitis. The FasL expression of NKT cells induced by both reagents was upregulated in HFCD mice. Furthermore, the liver mononuclear cells and purified F4/80(+) CD11b(+) subset from HFCD mice stimulated with either reagent in vitro produced a larger amount of TNF than did those from CD mice. Intracellular TNF production in F4/80(+) CD11b(+) cells was confirmed. The increased number of F4/80(+) CD11b(+) Kupffer cells/macrophages by HFCD and their enhanced TNF production thus play a pivotal role in TNF/NKT cell/FasL dependent hepatic injury.

Figure 1. The serum ALT and TNF levels in mice after α-GalCer or CpG-ODN injection.

HFCD and CD mice were injected i.v. with either α-GalCer or CpG-ODN, and the serum levels of ALT (12 h after reagent injection) (a, b) and TNF (1 h after reagent injection) (c, d) were examined. The data shown are the means ± SE from eight mice in each group. *P<0.05 vs. CD and HFD, ** P<0.01 vs. CD and HFCD.

Figure 2. Serum IL-12, IL-10 and IL-6 levels after either α-GalCer or CpG-ODN injection.

The HFCD and CD mice were injected i.v. with either α-GalCer or CpG-ODN, and the serum levels of cytokines at the indicated times were examined. The data shown are the means ± SE from three mice in each group.*P<0.01 vs. CD and HFCD.

Figure 3. Histological findings of α-GalCer hepatic injuries.

The livers from a CD and a HFCD mouse 24 h after α-GalCer injection (100×). Livers were fixed in 10% formalin for hematoxylin-eosin staining. Data are the representative findings from five mice with similar results.

Figure 4. Histological findings of CpG hepatic injuries.

The livers from CD mice and HFCD mice 24 h after CpG injection (200×). The livers were fixed in 10% formalin for hematoxylin-eosin staining. Data are the representative findings from five mice with similar results.

Figure 5. F4/80 and either CD11b or CD68 expression of whole liver MNCs.

Liver MNCs were obtained from CD, HFD, HCD and HFCD mice and expression of F4/80 either with CD11b or CD68 were examined. The numbers were the means±SE from four mice in each group. F4/80 positive gate was determined by using the isotype control Ab.

Figure 6. The expression of CD68 and CD11b by liver F4/80⁺ Kupffer cells.

Liver MNCs were obtained from CD, HFD, HCD and HFCD mice, and Kupffer cells gated by F4/80 were analyzed for their expression levels of CD68 and CD11b. CD68 positive gate was determined by using the isotype control Ab. The numbers are the means±SE from six to eight mice in each group. *P<0.05 vs. CD and HFD.

Figure 7. CD1d expression by CD11b Kupffer cells/macrophages.

Liver MNCs were obtained from CD, HFD, HCD and HFCD mice, and Kupffer cells gated by F4/80 and CD11b were analyzed for their expression of CD1d. The data shown are representatives of five mice with similar results.

Figure 8. TLR-9 expression of CD11b Kupffer cells/macrophages.

Liver MNCs were obtained from CD, HFD, HCD and HFCD mice, and Kupffer cells gated by F4/80 and CD11b were analyzed for their expression of TLR-9. The data are the means±SE from five mice in each group. *P<0.05 vs. CD and HFD.

Figure 9. In vitro TNF production by liver MNCs or CD11b⁺ Kupffer cells/macrophages from CD and HFCD mice.

(a, b) Liver MNCs were obtained from the four groups of mice, and 5×10⁵ MNCs/200 µl were stimulated with either α-GalCer (100 ng/ml) or CpG (20 µg/ml) in a 96 well plate for 6 h. Supernatants were examined for TNF. The data shown are the means ± SE from five mice, *P<0.05 vs. CD and HFD, ** P<0.01 vs. CD and HFD. (c, d) Liver MNCs were obtained from eight to ten mice without collagenase digestion and F4/80⁺ cells (mostly CD11b⁺ Kupffer cells/macrophages) were purified by a MACS sorting system. A total of 5×10⁵ CD11b⁺ population/200 µl were stimulated with either α-GalCer or CpG in a 96 well plate for 6 h. The supernatants were examined for TNF. The data shown are the means ± SE from four independent experiments. *P<0.05 vs. CD and HFD, ** P<0.01 vs. CD and HFD.

Figure 10. Production of intracellular TNF by liver MNCs.

Liver MNCs from CD mice, HFCD mice and HFCD mice (3 months) were obtained 30 min after α-GalCer injection without collagenase treatment and liver F4/80 (CD11b)⁺ cells were examined for their intracellular TNF expression as described in materials and methods. Data are the representative findings from three to five mice with similar results.

Figure 11. The serum IFN-γ and IL-4 levels after α-GalCer or CpG injection in mice, and the FasL expression of NKT cells.

HFCD mice and CD mice were injected i.v. with α-GalCer and the serum levels of cytokines at 6 h after injection were measured (a, b). The data are the means±SE from five mice in each group. Mice were injected with CpG, and the FasL expression of NKT cells was examined at 1 h after injection (c). In other experiments, mice were injected i.p. with IL-12 (24 h before) and were injected i.v. with α-GalCer, and the FasL expression of NKT cells was examined 1 h after α-GalCer injection (d). The percentages are the means ± SE from four mice in each group. *P<0.05 vs.CD.

Figure 12. Improvement in the hepatic injury by HFCD/CD.

The mice received HFCD for four weeks and then the HFCD diet was changed to a CD diet (HFCD/CD). After four weeks, the mice were injected with either α-GalCer or CpG-ODN, and the serum ALT levels were examined. The data shown are the means ± SE from six to ten mice in each group. *P<0.05 vs. other groups.