Prometheus unbound: NKT cells inhibit hepatic regeneration
Authors: William M. Ridgway, Eric M. Gershwin
Journal: Hepatology (2014)

Although natural killer T (NKT) cells were discovered over 20 years ago, our understanding of their immunobiology continues to evolve and surprise. NKT cells are T lymphocytes: they arise in the bone marrow, are selected in the thymus, and express a T-cell receptor. Unlike classic T cells, however, they are not strictly "adaptive" immune cells: in particular, as a population they express a very narrow range of T-cell receptors. The vast majority of mouse NKT cells, for example, express the Vα14-J281 chain and only a finite number of Vβ chains.1 In addition, they express NK cell surface markers, such as NK 1.1. Moreover, unlike classical T cells, they are not restricted by major histocompatibility complex (MHC) Class I or Class II, but by an MHC-like molecule, CD1d.2 Furthermore, NKT cells do not recognize peptides in the context of CD1d, but rather specialized lipids.3 Functionally, NKT cells also reflect major differences from conventional T cells: they are able to produce both classic Th1 (interferon-gamma [IFN-γ]) and Th2 (interleukin [IL]-4) cytokines without prior peripheral stimulation, but when stimulated by their glycolipid antigens down-regulate T-cell receptor (TCR), expand, and divert to a Th1 phenotype.4 Like classical T cells, they are selected in the thymus by a self-molecule: however, it is not a protein, but a trihexosylceramide, iGb3, bound to CD1d.5 Mice deficient in iGb3 demonstrated a severe deficiency of NKT cells, illustrating its critical role in NKT cells selection and survival.5 These features of NKT cells place them into the expanding category of "innate-like" lymphocytes.6 "Innate" immunity has classically been defined by "stereotypical" responses mediated by invariant receptors to defined ligands: for example, the signaling and functional responses of TLR4 when bound to its ligand, lipopolysaccharide (LPS). Since the overall TCR repertoire of NKT cells is so limited, the population as a whole responds "innately" to just a few lipid antigens, rather than retaining a population-capability to respond to the full universe of T-cell antigens. Finally, and of great interest to the field of hepatic immunity, NKT cells do not circulate freely, but tend to home to and reside for life in specific tissues such as the liver, where they compose ∼30% of the intrahepatic lymphoid pool.7 The presence of large numbers of NKT cells in the liver, expressing only a finite repertoire of TCRs, raises perplexing questions: How did such large numbers arise? Any given classical TCR is present at vanishingly low numbers in the overall circulation, so how could this clonal TCR NKT cell expand so dramatically? Next, how did the NKT cells come to be restricted to the liver, and what was their function? The answer to the first question is that, unlike classical T cells, the NKT cell thymic precursor expands dramatically while still in the thymus, but only after entry into the NKT cell developmental pathway.8 This produces large numbers of NKT cells expressing only a few TCRs. In spite of many of the enigmas described above, there are numerous features of NKT liver residence that have been solved. NKT cells "patrolling" the hepatic sinusoids express CXCR6, and sinusoid epithelial cells produce and express the CXCR6 ligand, CXCL16. This CXCR6:CXCL16 interaction is important to hepatic NKT cell survival.9 The accumulation of NKT cells in the liver, however, was ultimately traced to the transcription factor-controlled lineage program that defined NKT cells, i.e., the expression of Promyelocytic Leukemia Zinc factor, PLZF.10 PLZF is induced at the time of thymic selection of NKT cells, and mice lacking PLZF do not develop the typical expanded, activated thymic NKT pool.11, 12 Finally, PLZF not only determine NKT cell differentiation, it is also responsible for homing and accumulation of NKT cells in liver. The latter has been shown by transgenically expressing PLZF in CD4+ T cells: these cells then homed to the liver and stayed there indefinitely, retained in the sinusoidal circulation by LFA:ICAM1 interactions.13 The early discovery of large numbers of resident hepatic NKT cells, as well as their production of large amounts of cytokines with a vast array of biologic effects, led to a massive research effort to dissect the influences of NKT cells on hepatic biology. The current article by Yin et al.14 continues this thread and importantly defines the effect of NKT cells on hepatic regeneration. We note that the first description of the influences of NKT cells on hepatic regeneration, using the partial hepatectomy (PH) model, appeared at least 15 years ago, when Matsushita et al.15 demonstrated that IL-12 drove NKT cell expansion and liver damage after PH. Subsequently, Ito et al.16 demonstrated that the IL-12 effect in PH was due to NKT cells; they further showed decreased liver injury in the PH model Ja281 knockout mice that lack NKT cells. Finally, IFN-γ mediated many of the deleterious NKT cell inflammatory effects on hepatic regeneration. There have been other studies that have evaluated the effects of α-galcer in the PHx model that address not only liver injury, but also regeneration.17 The data by Yin et al. expands these observations in much greater depth by studying the kinetics and also by demonstrating that α-galcer inhibits regeneration only if administered prior to or up to 24 hours post-PHx. The current article further attempts to settle the role and timing of the effect of NKT cells on hepatic regeneration, demonstrating that two different strains lacking NKT cells had normal regeneration after PH. Although this appears to contradict the original Ito et al. study, here they studied not just liver injury (i.e., elevated aspartate aminotransferase [AST] as Ito et al. showed) but actual regeneration in the tissue. To further resolve contradictions in the literature, they simulated hepatic inflammation at various times before or after PH by treatment with the NKT cell specific agonist, α-galcer. This is a relevant clinical scenario, since many diseases that require vigorous liver regeneration (e.g., chronic liver viral infections such as hepatitis B or C, or liver transplant) occur in the context of inflammation. α-galcer treatment illustrated the potential of NKT-mediated inflammation on regeneration. In this setting, activated NKT cells caused decreased regeneration. What is the mechanism of these results? Yin et al. repeated their experiments in the setting of mice lacking the ability to produce either IFN-γ or IL4, the canonical NKT cytokines (Fig. 1). They treated both IFN-γ and STAT1 KO mice with α-galcer and showed restoration of liver regeneration compared to wild-type mice. Surprisingly, they also showed restoration of liver regeneration in IL4 and STAT6 knockout (KO) mice. This effect of IL4 was then shown to be an indirect effect of IL-4 on expansion of NKT cells, thus increasing total IFN-γ production (Fig. 1). These data provide a model wherein the clinical inflammatory hepatic microenvironment is critical to the effect of NKT cells on hepatic regeneration. These and the other observations presented by Yin et al. should be presented in the context of similar data elsewhere. First, other groups using CD1d−/− and bb2M−/− mice demonstrated comparable liver regeneration to wild-type mice.18, 19 In addition, although PHx leads to an increase in iNKT cell number, the relative subsequent production of both IFN-γ and IL-4 are minimal.20 Additionally, two other articles indicate that liver regeneration is reduced in IL-4 KO mice or in mice deleted of IL-4 by monoclonal therapy.20, 21 In contrast, the study herein reflects that IL-4 contributes to the inhibitory effect of α-galcer on liver regeneration. We suggest that these discrepancies reflect the dichotomy of the effects of IL-4 at baseline compared to the values during immunopathology. Of course, there are further complexities that could not be addressed in the current context. It is now appreciated that mice with cytokine knockout from conception are somewhat blunt instruments, since the lack of cytokines throughout immune system development affects multiple cell lineages and changes the entire immune system in complex ways. For example, IL-4 plays a major role in intrathymic NKT cell development8; furthermore, loss of IL-4 might affect any number of innate lymphoid cell lineages22, 23; thus, the IL4 knockout mice themselves might have altered NKT cell functions. This issue could be addressed by conditional deletion of IL-4 in adult mice and testing its effect on hepatic regeneration in the setting of a normal immune system. Similarly, the mice used herein have the gene deletions in all organs and cells, so it is hard to localize the effect to a specific cell subset—which might be addressed in the future by cell-specific knockouts of these cytokines. Nonetheless, the current findings clarify some of the issues in the field of the effect of NKT cells on liver physiology and present a framework for future studies. Participation of activated iNKT cells in the inhibition of liver regeneration following partial hepatectomy (PH). Top panel: After PH, in untreated wild-type mice or in mice lacking iNKT, hepatocytes recover with the help of cytokines, TNF-α, and IL-6, along with hormones and growth factors that induce expression of STAT3,C/EBP and other factors.24, 25 As a result, hepatocytes are resistant to apoptosis and necrosis. Cyclins are up-regulated and cell division regenerates the liver. Lower panel: α-galcer treatment activates NKT cells and inhibits liver regeneration. α-Galcer-activated iNKT drives IFN-γ production that up-regulates hepatocyte STAT1, producing vulnerability to apoptosis and necrosis. Nucleic acid synthesis is inhibited and overall cell proliferation is decreased, resulting in inhibition of liver regeneration. α-Galcer also drives IL-4 production that can indirectly decrease hepatic regeneration by way of expansion of NKT cell numbers and thus further IFN-γ production. iNKT activation can also induce NK cells to produce even more IFN-γ in a negative feedback cycle of decreased hepatic regeneration. William M. Ridgway, M.D.1M. Eric Gershwin, M.D.2 1Division of Immunology Allergy and Rheumatology, University of Cincinnati College of Medicine, Cincinnati, OH 2Division of Rheumatology Allergy and Clinical Immunology University of California at Davis School of Medicine Davis, CA