The present study established a model of oral steroid administration for eight weeks to simulate the insulin resistance and the associated liver injury which is similar to that noted in patients with metabolic syndrome. Steroid-treated mice exhibited insulin resistance, hypertriglyceridemia, and hepatocellular injury. These results support previously published data on the effect of steroids on the liver7, 11. CD1d−/− mice were used for determining the potential role of CD1d in mediating steroid damage. The steroid-associated hyperglycemia and hypertriglyceridemia were independent of CD1d, however, CD1d−/− mice were relatively resistant to steroid-induced steatosis. The relative resistance to steatosis was independent of the steroid-induced liver damage in these mice manifested by the increased liver enzymes and hepatocytes necrosis on histology. The present study supports a role for CD1d in the development of steatosis per se, while not preventing the steroid-mediated hepatocyte damage or metabolic derangements. These results suggest a partial role for the activation of CD1d-positive cells in NAFLD development.
Hepatic steatosis is associated with activation of the innate immune system30. Leptin-deficient ob/ob mice exhibit metabolic and inflammatory features that mimic human NAFLD31–34. These mice exhibit a decrease in liver NKT cells35, 36, suggesting that these lymphocytes may regulate the development of NAFLD30. In this model, leptin deficiency modulates inflammation independently of hepatic steatosis. An obese/steatosis HFD model, associated with both weight gain and hepatic steatosis, is also associated with a reduction in liver NKT cells30, 37 38. These findings can be interpreted as a lack of a full protective effect of NKT deficiency, wherein a decrease of NKT cells is associated with greater damage, independent of steatosis. These observations may also result from post activation-mediated cell death, wherein NKT activation leads to greater steatohepatitis and a subsequent decrease in NKT cells27. Endogenous IL-12 released by Kupffer cells (KC) in obesity30 39, may promote the release of IFN-γ by activated NKT cells, with a subsequent decrease of intrahepatic NKTs30, 40. Dietary fatty acids were also linked to hepatic NKT cell deficiency41, showing that dietary saturated fatty acids (SFA) or monounsaturated fatty acids (MUFA), but not polyunsaturated fatty acids (PUFA), deplete liver NKT lymphocytes30, 41.
Obesity is characterized by low-grade systemic inflammation and the adipocyte-macrophage interaction contributes to its development42, 43. An interplay between insulin resistance and inflammation is thought to contribute to the development of hepatic steatosis and steatohepatitis. Anti-inflammatory agents are beneficial in animal models and in patients with NASH25, 44. The results of the present study support the lack of a direct relationship between steroid-induced metabolic changes and hepatocyte injury and steatosis, implying a pathogenesis dichotomy between steatosis and liver damage in NAFLD. NAFLD comprises a spectrum from pure steatosis to NASH. The present data suggests that the mechanism that induces liver damage in NASH may be independent of steatosis, and can further explain the liver damage in lean NASH patients45, and the fact than the majority of patients with simple steatosis do not develop NASH46.
GC has been shown to act as liver-protecting agent, preventing drug-induced liver injury (DILI) of several hepatotoxic drugs18. In the present study, administering GC both prevented and treated the steroid-mediated liver damage as observed by a reduction of ALT and AST serum levels and a decrease in the levels of ballooning and apoptosis upon histological examination. GC alleviated steroid-induced hyperglycemia, hyperinsulinemia, and hypertriglyceridemia. The protective effect of GC was independent of CD1d. However, while CD1d−/− mice showed less steroid-mediated steatosis, GC treatment exerted a more profound effect on reducing steatosis in these mice as compared with WT mice. These data support a dichotomy in GC function for reducing steroid-mediated steatosis, which is prevented by CD1d, which was not seen for the effect of GC on the hepatocellular damage.
Manipulations of NKT cells are beneficial in the settings of diabetes, fatty liver disease, and NASH in animal models16–20, 39–41, 47–50 and in humans23. GC treatment exerts a fat-reducing effect in animal models and humans23, 33. The present data support CD1d+ lymphocyte plasticity which underlines the differences noted between the different models. NKT cells were shown to be either useful or associated with liver damage under different conditions in an environment- and/or disease-dependent manner47, 48. GC treatment alleviates immunologically incongruous disorders, suggesting it is associated with a "fine tuning" of immune responses by altering NKT-plasticity48, 49, and this effect may be CD1d-mediated50, 51. The CD1d-independence of the GC-fat reduction effect under steroid therapy in the present study may also suggest an alternative CD1d-independent liver protective effect of GC. A CD1d evading mechanism, similar to the one described for infectious agents may also play a role under these conditions52. Interleukin-1 is an important target of steroid therapy53, and promotes the inflammation in NASH51; a possible link via IL1 and GC may explain these findings.
GC therapy has been associated with increased IL10 and IL17 serum levels, suggesting its effect on the systemic immune system. Serum IL-10 levels are significantly lower in patients with NASH54, while IL-17 exacerbates hepatic steatosis and inflammation in NAFLD55. As only two cytokines were measured in the present study, an overall alteration of the cytokine milieu in an anti-inflammatory direction may underlie the noted beneficial clinical effect. Future studies will need to dissect the subsets of cells mediating these effects.
In conclusion, our study established a model of NAFLD and metabolic derangements by steroid administration for eight weeks in which the steroid-mediated steatosis was CD1d-dependent while steroid-induced liver necrosis and inflammation, insulin resistance, and hyperlipidemia were CD1d-independent. The results further support a pathogenesis dichotomy between steatosis and steatohepatitis in NAFLD. CD1d−/− mice were relatively resistant to steroid-induced steatosis, suggesting that NKT cells play a role in fat accumulation. The data further support the lack of a direct association between steatosis per se and liver damage. The beneficial effect of GC to alleviate the steroid induced-metabolic changes and liver damage were independent of CD1d, while CD1d prevented the ability of GC to reduce steroid-mediated steatosis. Overall, the data supports the potential use of GC as both a liver protector to be used with steroid therapy and as drug to treat NAFLD. Our results also support the need for a combination therapy in patients with NASH wherein both inflammatory and metabolic pathways need to be targeted56.
Table 1
Group | Mice | Treatment | Duration (weeks) |
A | C57BL/6 | Vehicle | 8 |
B | C57BL/6 | Dexamethasone | 8 |
C | C57BL/6 | Dexamethasone + β-glucosylceramide | 8 |
D | C57BL/6 | Vehicle | 12 |
E | C57BL/6 | Dexamethasone | 12 |
F | C57BL/6 | β-glucosylceramide | 12 |
G | C57BL/6 | Dexamethasone for 6 weeks followed by Dexamethasone + β-glucosylceramide additional 2 weeks | 6 weeks for induction and 2 weeks for treatment |
H | CD1d-/- | Vehicle | 8 |
I | CD1d-/- | Dexamethasone | 8 |
J | CD1d-/- | Dexamethasone + β-glucosylceramide | 8 |