Lipid-loaded Hepatocytes fail to Suppress IL-4 Production by Human CD4+ iNKT Cells

Background:

Invariant natural killer T (iNKT) cells recognising glycolipids presented by CD1 d are implicated in the inflammatory cascade associated with hepatic steatosis and its progression to steato hepatitis (NASH). Therefore, we established an in vitro steatosis model of HepaRG cells to elucidate the contribution of iNKT cells to initiation of inflammation after steatosis.

Methods:

Deposition of 0.5 mM free fatty acid (FFA) on HepaRG was achieved by treating the cells with 2:1 oleic acid and palmitic acid for 24 hours. Next, the effect of steatosis on iNKT cells was investigated by differentiating freshly isolated iNKT cells with FFA-loaded HepaRG for 7 days. Thereafter, cytokine profile and activation status of differentiated iNKT cells was analyzed by flow cytometry.

Results:

The direct coculture of iNKT and HepaRG prevented the spontaneous production of IL-4 in differentiated iNKT cells (HepaRG vs. medium alone: 16% vs. 31 %, p = 0.02). However, the presence of FFA in HepaRG significantly affected this suppressive effect (HepaRG vs. FFA-HepaRG 16% vs. 20%, p = 0.03). Furthermore, expansion of iNKT cells in transwell separated from HepaRG indicated that this suppressive effect requires a direct contact between both cells (HepaRG vs. HepaRG+TW: 16% vs. 33%, p = 0.004and FFA-HepaRG vs. FFA-HepaRG+TW: 20% vs. 34%; p = 0.04). Luminex analysis of the coculture supernatants revealed expression of IL-15 by hepatocytes in contact with iNKT, which was hijacked by the iNKT cells to promote the expansion of IL-4+population. Nevertheless, the unloaded control employed an unknown mechanism disrupted by FFA accumulation to maintain the suppression of IL-4+ iNKT even in the presence of IL-15. Finally, analysis of intrahepatic and peripheral iNKTs from patients with and without steatosis indicated a higher activation state of iNKT (Fatty vs. non-fatty PBL HLA-DR+iNKT: 30% vs. 15%, p = 0.0001 and IHL HLA-DR+ iNKT: 23% vs. 10%, p = 0.013) and frequency of IL-4+PBL iNKT (Fatty vs. non-fatty PBL IL-4+iNKT: 13% vs. 28%, p = 0.038) in patients with steatosis.

Conclusion:

In summary, the results show that IL-4+iNKT might contribute to inflammation in hepatic steatosis because a suppressive mechanism of liver cells is compromised by fat accumulation.