Liver Natural Killer T Cells Research Articles

Lipopolysaccharide preconditioning reduces liver metastasis of Colon26 cells by enhancing antitumor activity of natural killer cells and natural killer T cells in murine liver.

Lipopolysaccharide (LPS) preconditioning drastically augments bactericidal activity but reduces the host inflammatory response. Therefore, it may be beneficial to prevent postoperative infectious complications and mitigate host damage by surgical stress. Considering its clinical application, how LPS preconditioning influences the antitumor effect in the liver is an important issue. We then investigated the effect of LPS preconditioning on antitumor activity against Colon26 tumor cells in mice. Lipopolysaccharide preconditioning was induced in mice by the intraperitoneal injection of 5μg/kg LPS for three consecutive days. Intraportal inoculation of Colon26 cells, which express luminescent protein called Nano-lantern, was performed to evaluate the effect of LPS preconditioning on tumor liver metastasis. The antitumor activities of cytotoxic liver lymphocytes, especially natural killer (NK) cells and natural killer T (NKT) cells, against Colon26 cells were also examined in LPS preconditioned mice. Lipopolysaccharide preconditioning remarkably prevented liver metastasis of Colon26 cells, as observed by IVIS imaging system, and prolonged survival after tumor inoculation. LPS preconditioning increased the proportions and number of liver NK cells and NKT cells and augmented their intracellular perforin and granzyme B expression, while reducing their intracellular expression of IFN-γ. An in vitro antitumor cytotoxicity assay revealed that LPS preconditioning significantly augmented antitumor cytotoxicities of the liver NK cells and NKT cells, especially NKT cells, against Colon26 cells. Lipopolysaccharide preconditioning potently augmented antitumor cytotoxicity of liver NK cells and NKT cells, thereby improving mouse survival after intraportal inoculation of Colon26 tumor cells. It may be useful for perioperative care in oncological patients.

Hepatocyte CD1d protects against liver immunopathology in mice with schistosomiasis japonica.

Schistosomiasis is a neglected tropical disease with over 250 million people infected worldwide. The main clinically important species Schistosoma mansoni (S.mansoni) and Schistosoma japonicum (S.japonicum) cause inflammatory responses against tissue-trapped eggs, resulting in formation of granulomas mainly in host liver. Persistent granulomatous response results in severe fibrosis in the liver, leading to irreversible impairment of the liver and even death of the host. CD1d, a highly conserved MHC class I-like molecule, is expressed by both haematopoietic and non-haematopoietic cells. CD1d on antigen-presenting cells (APCs) of haematopoietic origin presents pathogen-derived lipid antigens to natural killer T (NKT) cells, which enables them to rapidly produce large amounts of various cytokines and facilitate CD4+ T helper (Th) cell differentiation upon invading pathogens. Noteworthy, hepatocytes of non-haematopoietic origin have recently been shown to be involved in maintaining liver NKT cell homeostasis through a CD1d-dependent manner. However, whether hepatocyte CD1d-dependent regulation of NKT cell homeostasis also modulates CD4+ Th cell responses and liver immunopathology in murine schistosomiasis remains to be addressed. Here, we show in mice that CD1d expression on hepatocytes was decreased dramatically upon S.japonicum infection, accompanied by increased NKT cells, as well as upregulated Th1 and Th2 responses. Overexpression of CD1d in hepatocytes significantly decreased local NKT numbers and cytokines (IFN-γ, IL-4, IL-13), concomitantly with downregulation of both Th1 and Th2 responses and alleviation in pathological damage in livers of S.japonicum-infected mice. These findings highlight the potential of hepatocyte CD1d-targeted therapies for liver immunopathology control in schistosomiasis.

Temporal Regulation of Natural Killer T Cell Interferon Gamma Responses by β-Catenin-Dependent and -Independent Wnt Signaling

Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator β-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. β-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and β-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of β-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of β-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/β-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of β-catenin activity. Our analyses in ICG001-treated mice confirmed a role for β-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and β-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of β-catenin to NKT cell functions.

Mesenchymal stem cells protect from acute liver injury by attenuating hepatotoxicity of liver natural killer T cells in an inducible nitric oxide synthase- and indoleamine 2,3-dioxygenase-dependent manner.

The effects of mesenchymal stem cells (MSCs) on the phenotype and function of natural killer T (NKT) cells is not understood. We used concanavalin A (Con A) and α-galactosylceramide (α-GalCer)-induced liver injury to evaluate the effects of MSCs on NKT-dependent hepatotoxicity. Mouse MSCs (mMSCs) significantly reduced Con A- and α-GalCer-mediated hepatitis in C57Bl/6 mice, as demonstrated by histopathological and biochemical analysis, attenuated the influx of inflammatory [T-bet+ , tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ)-producing and GATA3+ , interleukin-4 (IL-4)-producing] liver NKT cells and downregulated TNF-α, IFN-γ and IL-4 levels in the sera. The liver NKT cells cultured in vitro with mMSCs produced lower amounts of inflammatory cytokines (TNF-α, IFN-γ, IL-4) and higher amounts of immunosuppressive IL-10 upon α-GalCer stimulation. mMSC treatment attenuated expression of apoptosis-inducing ligands on liver NKT cells and suppressed the expression of pro-apoptotic genes in the livers of α-GalCer-treated mice. mMSCs reduced the cytotoxicity of liver NKT cells against hepatocytes in vitro. The presence of 1-methyl-dl-tryptophan, a specific inhibitor of indoleamine 2,3-dioxygenase (IDO), or l-NG -monomethyl arginine citrate, a specific inhibitor of inducible nitric oxide synthase (iNOS), in mMSC-conditioned medium injected into α-GalCer-treated mice, counteracted the hepatoprotective effect of mMSCs in vivo and restored pro-inflammatory cytokine production and cytotoxicity of NKT cells in vitro. Human MSCs attenuated the production of inflammatory cytokines in α-GalCer-stimulated human peripheral blood mononuclear cells in an iNOS- and IDO-dependent manner and reduced their cytotoxicity against HepG2 cells. In conclusion, MSCs protect from acute liver injury by attenuating the cytotoxicity and capacity of liver NKT cells to produce inflammatory cytokines in an iNOS- and IDO-dependent manner.

Interleukin-15 receptor α on hepatic stellate cells regulates hepatic fibrogenesis in mice

Interleukin-15 (IL-15) and its high affinity receptor interleukin-15 receptor alpha (IL-15Rα) are widely expressed in immune cells and hepatic resident cells. IL-15 signaling has important functions in homeostasis of natural killer (NK), natural killer T (NKT) and cytotoxic T (CD8(+) T) cells, and in liver regeneration. We hypothesized that IL-15 has a protective role in liver fibrosis progression by maintaining NK cell homeostasis. Fibrosis was induced using two mechanistically distinct models. Congenic bone marrow transplantation was used to evaluate the contribution of IL-15 signaling from various compartments to NK, CD8(+) T and NKT cell homeostasis and fibrogenesis. The gene expression profile of hepatic stellate cell (HSC) from IL-15Rα knockout (IL-15RαKO) mice and wild-type mice were captured using microarray analysis and validated in isolated HSC. Quantitative real-time PCR was used to assess repressors of collagen transcription. IL-15RαKO mice exhibited more fibrosis in both models. IL-15 signaling from specific types of hepatic cells had divergent roles in maintaining liver NK, CD8(+) T and NKT cells, with a direct and protective role on radio-resistant non-parenchymal cells beyond the control of NK homeostasis. HSCs isolated from IL-15RαKO mice demonstrated upregulation of collagen production. Finally, IL-15RαKO HSC with or without transforming growth factor beta (TGF-β) stimulation exhibited increased expression of fibrosis markers and decreased collagen transcription repressors expression. IL-15Rα signaling has a direct anti-fibrotic effect independent of preserving NK homeostasis. These findings establish a rationale to further explore the anti-fibrotic potential of enhancing IL-15 signaling in HSCs. We investigated how a cellular protein, Interleukin-15 (IL-15), decreases the amount of scar tissue that is formed upon liver injury. We found that IL-15 and its receptor decrease the amount of scar tissue that is created by specialized liver cells (called stellate cells) and increase the number of a specific subgroup of immune cells (natural killer cells) that are known to eliminate stellate cells. GSE45612, GSE 68001 and GSE 25097.

Intestinal mucus-derived nanoparticle–mediated activation of Wnt/β-catenin signaling plays a role in induction of liver natural killer T cell anergy in mice

The Wnt/β-catenin pathway has been known to play a role in induction of immune tolerance, but its role in the induction and maintenance of natural killer T (NKT) cell anergy is unknown. We found that activation of the Wnt pathways in the liver microenvironment is important for induction of NKT cell anergy. We identified a number of stimuli triggering Wnt/β-catenin pathway activation, including exogenous NKT cell activator, glycolipid α-GalCer, and endogenous prostaglandin E2 (PGE2). Glycolipid α-GalCer treatment of mice induced the expression of wnt3a and wnt5a in the liver and subsequently resulted in a liver microenvironment that induced NKT cell anergy to α-GalCer restimulation. We also found that circulating PGE2 carried by nanoparticles is stable, and that these nanoparticles are A33(+) . A33(+) is a marker of intestinal epithelial cells, which suggests that the nanoparticles are derived from the intestine. Mice treated with PGE2 associated with intestinal mucus-derived exosome-like nanoparticles (IDENs) induced NKT cell anergy. PGE2 treatment leads to activation of the Wnt/β-catenin pathway by inactivation of glycogen synthase kinase 3β of NKT cells. IDEN-associated PGE2 also induces NKT cell anergy through modification of the ability of dendritic cells to induce interleukin-12 and interferon-β in the context of both glycolipid presentation and Toll-like receptor-mediated pathways. These findings demonstrate that IDEN-associated PGE2 serves as an endogenous immune modulator between the liver and intestines and maintains liver NKT cell homeostasis. This finding has implications for development of NKT cell-based immunotherapies. (HEPATOLOGY 2013).

The role of NKT cells in immune evasion of liver metastases arising from intraocular tumors (74.12)

Abstract Uveal melanoma is the most common intraocular tumor in adults within the United States. Half of the patients develop liver metastases, which is almost universally fatal. We have previously demonstrated that liver natural killer (NK) cell-antitumor immunity was suppressed in mice bearing ocular tumors, and natural killer T (NKT) cells were responsible, in part, for liver NK cell suppression. This study examines the role NKT cells in promoting liver metastases arising from intraocular tumors. We showed that when wild type and NKT-deficient C57BL/6 mice were injected with B16LS9 melanoma cells, NKT-deficient mice developed less liver metastasis than wild type mice. The cytotoxicity of liver NK cells was also assessed. Results indicated that NK cells from NKT-deficient mice were more cytotoxic compared to NK cells from wild type mice. However, in-vitro assays suggested that NKT cells were unable to directly suppress NK cell-mediated cytotoxicity. To investigate the potential role of a third-party cell, myeloid derived suppressor cells (MDSCs) were depleted by anti-GR1 antibody. Surprisingly, tumor bearing MDSC-depleted mice developed more liver metastases than isotype-treated mice. Our results imply that liver NKT cells suppress NK cell cytotoxicity through a GR1+ third-party cell population other than MDSCs. We are currently investigating whether liver NKT cells impart their immunosuppressive function through GR1+ neutrophils.

A new experimental murine model for lipopolysaccharide-mediated lethal shock with lung injury

We have recently established a new experimental murine model for lipopolysaccharide (LPS)-mediated lethal shock with lung-specific injury. Severe lung injury is induced by administration of LPS into α-galactosylceramide (α-GalCer)-sensitized mice; the mice died with acute lung injury and respiratory distress within 24 h. α-GalCer activates natural killer T (NKT) cells in the lungs and liver, and induces the production of interferon (IFN)-γ. However, IFN-γ signaling is only triggered in the lungs and makes them susceptible to LPS. On the other hand, IFN-γ signaling is inhibited in liver and results in few hepatic lesions. Unlike liver NKT cells, lung NKT cells fail to produce interleukin (IL)-4, which down-regulates the IFN-γ signaling, in response to α-GalCer. The differential cytokine profile between lung and liver NKT cells may lead to organ-specific lung lesions. The experimental system using α-GalCer sensitization could be a useful experimental model for clinical endotoxic or septic shock as it presents respiratory failure, a typical manifestation in severe septic patients. In this review, key evidence and the introducuction of the detailed mechanism of LPS-mediated lung-specific injury in α-GalCer-sensitized mice is provided. In particular, the molecular background of organ-specific development of lung injury in the model is focused on.

NKT Cell Exacerbation of Liver Metastases Arising from Melanomas Transplanted into Either the Eyes or Spleens of Mice

To explore the role of natural killer T (NKT) cells in the development of liver metastases in mice harboring intraocular melanomas. Cells derived from the cutaneous B16 melanoma cell line (B16LS9) were transplanted either into the vitreous body or under the spleen capsules of wild-type C57BL/6 mice and NKT-cell-deficient Jα18(-/-) and CD1d(-/-) mice. The development of liver metastases was evaluated by histopathology. The effect of NK cells on liver metastases was determined by selective depletion with anti-asialo-GM1 antiserum in vivo and NK-cell-mediated cytolysis of B16LS9 melanoma cells in vitro. The role of IL-10 and transforming growth factor (TGF)-β in the inhibition of liver NK resistance to liver metastases was determined by in vivo and in vitro neutralization with monoclonal antibodies. Liver NKT cells, especially type I NKT cells, enhanced liver metastases arising from intraocular melanomas. NKT-cell-deficient mice developed significantly fewer liver metastases that were NK-cell dependent. Tumor-induced liver NKT cells, especially type I NKT cells, inhibited liver NK-cell cytotoxicity by an IL-10-dependent process. NKT cells exert protective effects in many murine tumor models. However, the present results reveal that NKT cells exacerbate liver metastases arising from intraocular melanomas. To the authors' knowledge, this is the first report that liver NKT cells, especially type I NKT cells, inhibit liver NK-cell antimetastatic activity by the production of IL-10. These results suggest that hepatic NKT cell activity can have an important effect in the immune surveillance of liver metastases.

Accelerated liver fibrosis in hepatitis B virus transgenic mice: Involvement of natural killer T cells

The immunopathogenic process from hepatitis B virus (HBV) infection to liver fibrosis is incompletely understood because it lacks an animal model. In this study we observed the development of liver fibrosis in HBV transgenic (HBV-tg) mice and found the roles of natural killer T (NKT) cells in HBV-related liver fibrosis. We found liver fibrosis spontaneously developed in HBV-tg mice with the elevated transcription of col1a1, matrix metalloproteinase (MMP)2, and tissue inhibitor of metalloproteinase (TIMP)1. Mice were then injected with repetitive hepatotoxin carbon tetrachloride (CCl(4) ) to induce prominent liver fibrosis. After chronic CCl(4) treatment, the serum alanine aminotransferase (ALT) was higher, the liver regenerative nodules became more and bigger, and the fibrosis area was remarkably increased in HBV-tg mice than in C57BL/6 mice. Moreover, the increase in col1a1 and MMP2 transcription was greater, with a sustaining high level of TIMP1 and a greater activation of hepatic stellate cells (HSCs) in the livers of CCl(4) -treated HBV-tg mice. Our data also showed that there were more liver mononuclear cells (MNCs) in HBV-tg mice after CCl(4) injection, and Rag1(-/-) mice adoptive transferred lymphocytes from HBV-tg mice displayed increased collagen deposition. Further study demonstrated the number of liver NKT cells increased after CCl(4) treatment and NKT cells were overactivated in HBV-tg mice in the long term. It was further confirmed that NKT cells were critical for HSCs activation by depletion of NKT cells of HBV-tg mice and adoptive transfer of purified NKT cells from HBV-tg mice into recipient Rag1(-/-) mice. The inflammatory cytokines IL-4 and IL-13 produced by NKT cells played a pivotal role in HSCs activation in an in vitro coculture experiment. These data suggest that NKT cells from HBV-tg mice induce the HSC activation in liver fibrogenesis.

Immune modulation of ovalbumin-induced lung injury in mice using β-glucosylceramide and a potential role of the liver

CD1d-restricted natural killer T (NKT) cells are implicated in the pathogenesis of asthma. β-Glucosylceramide (GC), a naturally occurring lipid, was previously shown to alter NKT cell distribution in the liver. We hypothesized that GC can affect lung and liver NKT cell distribution and ameliorate asthma. Mice were sensitized by intra-peritoneal injection of ovalbumin (OVA) for 2 weeks followed by repeated intranasal OVA challenges to induce lung injury mimicking asthma. OVA induced asthma groups were either treated by intranasal instillation of normal saline, intranasal instillation of GC or inhaled budesonide. To investigate the role of the liver, hepatic fibrosis was induced using carbon tetrachloride prior to asthma induction. Allergen induced bronchoconstriction was measured prior to sacrifice. Isolated lymphocytes from lungs, livers and spleens were analyzed for OVA induced proliferation and flow cytometry. Liver and lung histology, serum aminotransferase and anti-OVA antibodies level were assessed. Treatment with GC significantly reduced OVA induced airway responsiveness ( p < 0.001) similar to inhaled budesonide. GC significantly reduced the peri-bronchial and peri-vascular inflammatory infiltration mainly through an effect on T cells, as suggested by decreased T cell proliferation ( p = 0.009). Liver CD4 and NKT cells significantly increased after GC treatment suggesting liver involvement. Inducing hepatic fibrosis blunted the propagation of asthma in spite of sufficient increase of serum anti-OVA titers. GC has an immunomodulatory effect on a murine model of experimental asthma. We also suggest that the liver acts as an immunomodulatory organ and might have a regulatory effect on pulmonary diseases.

Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Vα14-Jα18 TCRα gene

AbstractEstablishment of a system with efficient generation of natural killer T (NKT) cells from embryonic stem (ES) cells would enable us to identify the cells with NKT-cell potential and obtain NKT cells with desired function. Here, using cloned ES (NKT-ES) cells generated by the transfer of nuclei from mature NKT cells, we have established a culture system that preferentially developed functional NKT cells and also identified early NKT progenitors, which first appeared on day 11 as a c-kit+ population in the cocultures on OP9 cells with expression of Notch ligand, delta-like1 (OP9/Dll-1) and became c-kitlo/− on day 14. Interestingly, in the presence of Notch signals, NKT-ES cells differentiated only to thymic CD44lo CD24hi NKT cells producing mainly interleukin-4 (IL-4), whereas NKT cells resembling CD44hi CD24lo liver NKT cells producing mainly interferon γ (IFN-γ) and exhibiting strong adjuvant activity in vivo were developed in the switch culture starting at day 14 in the absence of Notch. The cloned ES culture system offers a new opportunity for the elucidation of the molecular events on NKT-cell development and for the establishment of NKT-cell therapy.

Kupffer Cell and Interleukin-12–Dependent Loss of Natural Killer T Cells in Hepatosteatosis

Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-12, major T helper (Th) 1 cytokines, and reduced hepatic natural killer T (NKT) cell numbers. The relationship between lipid accumulation, cytokine expression, and hepatic NKT cells is not known. This study was conducted to assess the role of IL-12 in the development of hepatic steatosis and its potential impact on liver NKT cells. Male C57Bl/6 wildtype (WT) and IL-12-deficient (IL-12(-/-)) mice were fed a choline-deficient diet (CDD) for 0, 10, or 20 weeks. CDD led to marked hepatosteatosis, reduced hepatic but not splenic NKT cell numbers and function, and increased hepatic expression of the T(h)1-type cytokines IL-12, interferon gamma (IFN-gamma), and TNF-alpha in WT mice. The absence of IL-12 resulted in similar CDD-induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFN-gamma and TNF-alpha expression. Treatment of CDD-fed mice with lipopolysaccharide led to a significant increase in hepatic IL-12 expression, and Kupffer cell (KC) depletion reduced liver IL-12 expression and restored NKT cells in CDD-induced fatty liver. Interestingly, KCs from CDD-fed mice failed to produce increased quantities of IL-12 upon activation in vitro when compared to similarly treated KCs from control fed mice, suggesting that secondary factors in vivo promote heightened IL-12 production. Finally, human livers with severe steatosis showed a substantial decrease in NKT cells. Hepatosteatosis reduces the numbers of hepatic NKT cells in a KC-and IL-12-dependent manner. Our results suggest a pivotal and multifunctional role of KC-derived IL-12 in the altered immune response in steatotic liver, a process that is likely active within human nonalcoholic fatty liver disease.

Widespread natural variation in murine natural killer T‐cell number and function

Natural killer T (NKT) cells comprise a novel T-lymphocyte subset that can influence a wide variety of immune responses through their ability to secrete large amounts of a variety of cytokines. Although variation in NKT-cell number and function has been extensively studied in autoimmune disease-prone mice, in which it has been linked to disease susceptibility, relatively little is known of the natural variation of NKT-cell number and function among normal inbred mouse strains. Here, we demonstrate strain-dependent variation in the susceptibility of C57BL/6J and BALB/cJ mice to NKT-mediated airway hyperreactivity, which correlated with significant increases in serum interleukin-4 (IL-4) and IL-13 elicited by the synthetic glycosphingolipid alpha-galactosylceramide. Examination of NKT-cell function revealed a significantly greater frequency of cytokine-producing NKT cells in C57BL/6J versus BALB/cJ mice as well as significant differences in the kinetics of NKT-cell cytokine production. Extension of this analysis to a panel of inbred mouse strains indicated that variability in NKT-cell cytokine production was widespread. Similarly, an examination of NKT-cell frequency revealed a significantly greater number of liver NKT cells in the C57BL/6J mice versus BALB/cJ mouse livers. Again, examination of a panel of inbred mouse strains revealed that liver NKT-cell numbers were quite variable, spanning over a 100-fold range. Taken together, these results demonstrate the presence of widespread natural variation in NKT-cell number and function among common inbred mouse strains, which may have implications for the examination of the influence of NKT cells in immune responses and disease pathogenesis among different genetic backgrounds.

Functional alterations of liver innate immunity of mice with aging in response to CpG-oligodeoxynucleotide

Immune functions of liver natural killer T (NKT) cells induced by the synthetic ligand alpha-galactosylceramide enhanced age-dependently; hepatic injury and multiorgan dysfunction syndrome (MODS) induced by ligand-activated NKT cells were also enhanced. This study investigated how aging affects liver innate immunity after common bacteria DNA stimulation. Young (6 weeks) and old (50-60 weeks) C57BL/6 mice were injected with CpG oligodeoxynucleotides (CpG-ODN), and the functions of liver leukocytes were assessed. A CpG-ODN injection into the old mice remarkably increased tumor necrosis factor (TNF) production in Kupffer cells, and MODS and lethal shock were induced, both of which are rarely seen in young mice. Old Kupffer cells showed increased Toll-like receptor-9 expression, and CpG-ODN challenge augmented TNF receptor and Fas-L expression in liver NKT cells. Experiments using mice depleted of natural killer (NK) cells by anti-asialoGM1 antibody (Ab), perforin knockout mice, and mice pretreated with neutralizing interferon (IFN)-gamma Ab demonstrated the important role of liver NK cells in antitumor immunity. The production capacities of old mice for IFN-gamma, IFN-alpha, and perforin were much lower than those of young mice, and the CpG-induced antitumor cytotoxicity of liver NK cells lessened. Lethal shock and MODS greatly decreased in old mice depleted/deficient in TNF, FasL, or NKT cells. However, depletion of NK cells also decreased serum TNF levels and FasL expression of NKT cells, which resulted in improved hepatic injury and survival, suggesting that NK cells are indirectly involved in MODS/lethal shock induced by NKT cells. Neutralization of TNF did not reduce the CpG-induced antitumor effect in the liver. Hepatic injury and MODS mediated by NKT cells via the TNF and FasL-mediated pathway after CpG injection increased, but the antitumor activity of liver NK cells decreased with aging.

Ito Cells Are Liver-Resident Antigen-Presenting Cells for Activating T Cell Responses

Here we identified Ito cells (hepatic stellate cells, HSC), known for storage of vitamin A and participation in hepatic fibrosis, as professional liver-resident antigen-presenting cells (APC). Ito cells efficiently presented antigens to CD1-, major histocompatibility complex (MHC)-I-, and MHC-II-restricted T cells. Ito cells presented lipid antigens to CD1-restricted T lymphocytes such as natural killer T (NKT) cells and promoted homeostatic proliferation of liver NKT cells through interleukin-15. Moreover, Ito cells presented antigenic peptides to CD8(+) and CD4(+) T cells and mediated crosspriming of CD8(+) T cells. Peptide-specific T cells were activated by transgenic Ito cells presenting endogenous neoantigen. Upon bacterial infection, Ito cells elicited antigen-specific T cells and mediated protection. In contrast to other liver cell types that have been implicated in induction of immunological tolerance, our data identify Ito cells as professional intrahepatic APCs activating T cells and eliciting a multitude of T cell responses specific for protein and lipid antigens.

NKT cells get specialized

A report on page 1279 suggests that, for antitumor immunity, all natural killer T (NKT) cells are not created equal. Crowe and colleagues show that a subset of liver-derived NKT cells is uniquely equipped to fight off tumors. NKT cells are innate immune cells known for their rapid production of both T helper (Th) 1 and Th2 cytokines upon stimulation. NKT cells can either promote or suppress immune responses, likely depending upon which cytokines the cells predominantly produce. Subsets of NKT cells have been identified (based on the expression of CD4) that have distinct cytokine production profiles in vitro. But whether these subsets are functionally distinct in vivo had not been tested. In their previous work, this group had shown that NKT cells from the liver were able to combat tumors in mice. They now show that it is only CD4− NKT cells from the liver than can promote tumor rejection. CD4+ NKT cells from the liver and both CD4+ and CD4− NKT cells derived from the spleen or thymus were ill equipped for the job. The authors are now trying to determine what sets the CD4− liver NKT cells apart from the rest, as cytokine production among the NKT cell subsets was comparable. In the meantime, the identification of a specialized antitumor subset of NKT cells may provide a way to improve upon current NKT cell–based tumor therapies.

Dietary Factors Alter Hepatic Innate Immune System in Mice With Nonalcoholic Ratty Liver Disease * #

Dietary factors promote obesity and obesity-related disorders, such as fatty liver disease. Natural killer T (NKT) cells are components of the innate immune system that regulate proinflammatory (Th-1) and anti-inflammatory (Th-2) immune responses. Previously, we noted that NKT cells are selectively reduced in the fatty livers of obese, leptin-deficient ob/ob mice and demonstrated that this promotes proinflammatory polarization of hepatic cytokine production, exacerbating lipopolysaccharide (LPS) liver injury in these animals. In the current study, we show that hepatic NKT cells are also depleted by diets that induce obesity and fatty livers in wild-type mice, promoting Th-1 polarization of hepatic cytokine production and sensitization to LPS liver injury despite persistent leptin. Adult male C57BL6 mice fed diets containing high amounts of either fat or sucrose, or combined high-fat, high-sucrose, develop increased hepatic NKT cell apoptosis and reduced liver NKT cells. The hepatic lymphocytes are more Th-1 polarized with increased intracellular interferon gamma and tumor necrosis factor alpha. Mice fed high-fat diets also exhibit more liver injury, reflected by 2-fold greater serum alanine aminotransferase (ALT) than control animals after receiving LPS. In conclusion, when otherwise normal mice are fed with high-fat or sucrose diet, they become obese, develop fatty livers, and acquire hepatic innate immune system abnormalities, including increased NKT cell apoptosis. The latter reduces liver NKT cell populations and promotes excessive hepatic production of Th-1 cytokines that promote hepatic inflammation. These diet-induced alterations in the hepatic innate immune system may contribute to obesity-related liver disease.

Contribution of Va24+Vb11+ natural killer T cells in Wilsonian hepatitis

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. There is no evidence that the WD patient's immune system attacks copper accumulated hepatocytes. Here we describe that the frequency and absolute number of Valpha24+Vbeta11+ natural killer T (NKT) cells were significantly increased in 3 cases of WD, whereas those of CD3+CD161+ NKT cells were within the normal range. Patients no. 1 and 2 had a presymptomatic form of WD. Their tissue specimens showed pathological changes of mild degeneration of hepatocytes with a few infiltrating mononuclear cells and a low degree of fatty change. Patient no. 3 displayed fulminant hepatitis with Coombs-negative haemolytic anaemia. The tissue specimens of patient no. 3 showed macronodular cirrhosis with thick fibrosis, inflammatory infiltrates and spotty necrosis. Human Valpha24+Vbeta11+ NKT cells are almost equal to CD1d-restricted NKT cells. Therefore we investigated CD1d-restricted NKT cells in the LEC rat as an animal model of WD. In LEC rats before hepatitis onset, the number and phenotype of liver NKT cells were normal. At about 4 months of age all LEC rats developed acute hepatitis accompanied by acute jaundice, and CD161high NKT cells developed in their livers. CD161highalphabetaTCRbright NKT cells developed in some of them. Their hepatitis was severe. CD161highalphabetaTCRbright NKT cells expressed an invariant rat Valpha14-Jalpha281 chain, which is CD1d-restricted. Furthermore, liver lymphocytes in the acute jaundiced LEC rats with CD161highalphabetaTCRbright NKT cells had significant and CD1d-specific cytotoxic activity.

Concanavalin a injection activates intrahepatic innate immune cells to provoke an antitumor effect in murine liver.

Concanavalin A (ConA), directly injected into mice, induces T cell-mediated liver injury. However, it remains unclear whether ConA injection can activate innate immune cells, including natural killer (NK) cells and natural killer T (NKT) cells, both of which exist abundantly in the liver. Here we report that ConA injection stimulated interferon (IFN)-gamma production from liver NKT cells as early as 2 hours after injection and augmented YAC-1 cytotoxicity of liver NK cells. ConA-induced NK cell activation required other types of immune cells and critically depended on IFN-gamma. Because a nonhepatotoxic low dose of ConA was capable of fully activating both NKT cells and NK cells, we next addressed the possibility of ConA injection displaying an antitumor effect in the liver without liver injury. A nonhepatotoxic low-dose ConA injection augmented the cytotoxicity of liver NK cells against Colon-26 colon cancer cells and suppressed hepatic metastasis of Colon-26 cells in a NK cell- and IFN-gamma-dependent manner. In conclusion, a nonhepatotoxic low dose of ConA might serve as an immunomodulator that can preferentially activate the innate immune cells to induce an antitumor effect against metastatic liver tumor.