Amelioration of hepatic fibrosis via beta-glucosylceramide-mediated immune modulation is associated with altered CD8 and NKT lymphocyte distribution.
BACKGROUND
While CD8 lymphocytes possess pro-fibrogenic properties and NK (non-T) cells are anti-fibrogenic, the role of NKT lymphocytes in liver fibrosis is still unclear. Beta-glucosylceramide (GC), a naturally occurring glycolipid, exerts modulatory effects on these cells.


OBJECTIVE
To explore the role of NKT cells in hepatic fibrosis via GC.


METHODS
Hepatic fibrosis was induced by biweekly intra-peritoneal (IP) carbon tetrachloride (CCl(4)) administrations for 7 weeks in 5 groups (A-E) of male C57Bl/6 mice. Mice were treated with daily IP GC injections in groups A and C, or daily oral doses in groups B and D. GC was administered either for the duration of the study period (in groups A and B), or for the last 3 weeks of CCl(4) induction (groups C and D). GC-treated mice were compared with non-treated fibrotic controls (group E) and naive rodents (group F). Liver fibrosis, injury parameters and FACS analysis of lymphocytes were assessed.


RESULTS
Marked amelioration (P < 0.0001) of hepatic fibrosis observed in all GC-treated mice without altering reactive oxygen species production. As determined by Sirius red-stained liver tissue sections and measured by Bioquant morphometry; all CCl(4)-administered groups significantly (P < 0.0001) increased the relative fibrosis area compared with naive animals. The increases were 14.4 +/- 1.03-fold in group A, 7.9 +/- 0.37-fold in group B, 5.2 +/- 0.2-fold in group C, 10.3 +/- 0.4-fold in group D and 23.8 +/- 1.9-fold in group E. Western blot analysis for alpha smooth muscle actin from liver extracts followed a similar pattern, increasing in groups A-E. A significant decrease in liver damage was observed in all GC-treated groups, as noted by a decrease in transaminase serum levels (P < 0.005). The beneficial effect of GC was associated with a significant decrease in the intra-hepatic NKT and CD8 lymphocytes as well as their attenuation of both T(h)1 and T(h)2 cytokines.


CONCLUSIONS
Administration of GC had a significant anti-fibrotic effect following CCl(4) administration. This effect was associated with an altered NKT and CD8 lymphocyte distribution and a cytokine shift. Immune modulation using GC may have a role in the treatment of fibrosis and other immune-mediated disorders.
Safadi
Rifaat
R
Liver Unit, Department of Medicine, Haassah Medical Center, Jerusalem, Israel. safadi@hadassah.org.il
Zigmond
Ehud
E
Pappo
Orit
O
Shalev
Zvi
Z
Ilan
Yaron
Y
D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002251:Carbon Tetrachloride; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D005963:Glucosylceramides / Q000276:immunology / Q000494:pharmacology*; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism; D008099:Liver / Q000276:immunology* / Q000473:pathology; D008106:Liver Cirrhosis, Experimental / Q000139:chemically induced / Q000276:immunology* / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016176:T-Lymphocyte Subsets / Q000276:immunology*
D016207:Cytokines; D005963:Glucosylceramides; D002251:Carbon Tetrachloride