Although NKT cells have been shown to be involved in immune mediated diseases [
27], asthma [
28], antitumor immune responses [
29] and inflammatory liver disease [
14,
30,
31], their role in liver regeneration is not fully elucidated. In the current study we aimed to explore one aspect of NKT in liver regeneration.
To investigate the contribution of NKT cells to liver regeneration we first choose to activate NKT cells by ConA. ConA is a plant lectin that induces hepatitis in a well-characterized model of NKT cell-mediated liver disease [
32]. Administration of moderate doses of ConA is a well-accepted tool to activate NKT cells. In 8–15 h significant rise in aminotransferases is observed but at later time points, usually after 48 h, liver function is reconstituted [
33]. Previous studies have demonstrated the critical contribution of NKT cells to ConA-induced hepatitis by showing that CD1d deficient mice were highly resistant to ConA injection compared to wt mice [
13,
14]. In the current study we showed in CD1d-/- mice and in anti CD1d-treated mice by well-accepted mitotic markers (Ki-67 and PCNA) that hepatocyte proliferation 48 h after PH is markedly reduced. In contrast, CD1-/- mice on balb.c background have been shown to demonstrate almost normal liver regeneration after PH [
9,
34]. It is important to stress out that NKT cell numbers are highly variable between mouse strains [
35].
Early enhanced regeneration due to ConA pretreatment was attributed to the disappearance of NKT by activation-induced cell death (AICD). Within the current model, ConA induces the rapid activation and depletion of NKT cells, which serve to initiate the injury response. Forty eight hours or more after PH NKT cells can repopulate the liver and are therefore capable of contributing to the regenerative response [
36]. Huang et al showed that ConA pretreatment did not accelerate liver regeneration 48 h after PH, although at earlier time points ConA significantly induced liver regeneration rate. In this study NKT cells disappeared 24 h after ConA stimulation which was the time point of PH [
15]. We observed that pre-treatment with ConA 4 days before PH revealed lower hepatic proliferation 48 h after PH, compared to non-ConA experiments. Similar observations were obtained by Hines and colleagues that showed under the same experiment setting 40% reduced cell proliferative response after ConA challenge. Four days after ConA administration NKT cells repopulate the hepatic parenchyma [
36]. Collectively the negatively regulated role of activated NKT cells diminished along the restoration of NKT cells 48 h after PH. Tekada et al first demonstrated the critical contribution of NKT cells to ConA-induced hepatitis by showing that CD1d deficient mice were highly resistant to ConA injection compared to wt mice [
14]. Nevertheless, the role of NKT cells in liver regeneration is less clear. Impaired liver regeneration by using ConA may provide a tool of exploring their role. We showed significant decreased liver injury in CD1d-/- mice and in anti CD1d-treated mice that were pre-treated with ConA 4 days before PH. High ALT levels 3 h and 48 h after PH are completely attributed to ConA treatment, as without ConA ALT levels in the 2 models were similar to wt or isotype-control mice. When ConA was not applied, targeting CD1d receptor by mAb was reflected by significantly reduced hepatic proliferation, which may be associated with possible blunting of CD1d receptors on hepatocytes. One cannot rule out that impaired liver regeneration is associated with reduced liver injury caused by the absence of NKT cells. The expression of several cyclins is correlated with hepatocyte cell cycle progression after PH [
17,
24,
25]. Hines and colleagues, who attributed the mechanisms of ConA pre-treatment to the modulation of oval cells, have also studied the effect of impaired liver regeneration on cell cycle. They showed that in ConA pretreated mice the expression of Cyclin D1 was reduced 6 and 24 h after PH and of Cyclin E after 48 h. They further showed that phosphorylated Stat3 and IL-6 were reduced in ConA-induced hepatitis, whereas p21 and Smad2 increased. These findings are in agreement with a recent report showing that depletion of NK and NKT cells by anti NK.1.1 antibody resulted in reduced BrdU uptake along with decreased expression of PCNA and Cyclin D, 48 h after PH [
9]. However, under the context of NKT deficiency cell cycle modifiers were not thoroughly investigated. In our study we show decreased expression of hepatic Cyclin B in CD1d-/- mice and in anti- CD1d-treated mice 48 h after PH. These results were observed in impaired liver regeneration with ConA or without ConA (data not shown). Surprisingly, we found that the levels of p21 in CD1d1-/- mice and in anti- CD1d treated mice were lower compared to wt and isotype control mice. Although this level expression was measured 48 h after PH and not earlier, we cannot rule other mechanistic options. Indeed, p21 was demonstrated as a key inhibitor of G
1 to S phase progression in hepatocytes [
37] and its expression inhibits several genes involved in cell cycle [
38], nevertheless, recent discoveries suggest that p21 has additional activities that are unrelated to its function as CDK inhibitors, especially in light of the identification of new targets as well as evidence of Cip/Kip cytoplasmic re-localization [
39‐
41]. However, more investigation is needed to explore the connection between NKT deficiency and p21.
The effect of IL-6 on various forms of liver injury, including ConA and liver regeneration has been well documented. In our study we found that the reduced hepatocyte proliferation in the absence of NKT cells was accompanied with significant decreased levels of serum IL-6 levels 3 and 48 h after PH. This observation was regardless of ConA challenge. Accordingly, in mice treated with NK1.1 antibody, IL-6 mRNA levels following PH were blunted significantly [
9]. Also in NKT cell-deficient mice, the diminished ConA-induced liver injury was restored by the adoptive transfer of liver mononuclear cells or NKT cells from wild-type mice, but not from IL-6-treated mice [
42]. In the study of Sun et al [
43] IL-6 prevented ConA-induced hepatitis via the suppression of NKT cells. This effect was partly due to the suppression of Fas ligand expression on NKT cells. Furthermore, adoptive transfer of hepatic mononuclear cells restored ConA-induced liver in jury in anti NK1.1 treated mice. However, adoptive transfer of hepatic mononuclear cells treated with IL-6 failed to restore such injury [
43]. Taken together from our investigation, it is apparent that the presence and activity of NKT cells are needed for proper liver regeneration. Impairment of this process by ConA does not change this trend. Further studies are warranted to explore specifically the role of the multiple types of NKT cells. We also show that the impaired Cyclin B1 and p21 expression within the absence of NKT cells or when NKT cells are hampered is in conjugation with reduced liver regeneration and IL-6 levels.