Liver NKT Cells Research Articles

Bariatric-induced microbiome changes alter MASLD development in association with changes in the innate immune system.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects nearly 25% of the population and is the leading cause for liver-related mortality. Bariatric surgery is a well-known treatment for MASLD and obesity. Understanding the fundamental mechanisms by which bariatric surgery can alter MASLD can lead to new avenues of therapy and research. Previous studies have identified the microbiome's role in bariatric surgery and in inflammatory immune cell populations. The host innate immune system modulates hepatic inflammation and fibrosis, and thus the progression of MASLD. The precise role of immune cell types in the pathogenesis of MASLD remains an active area of investigation. The aim of this study was to understand the interplay between microbiota composition post-bariatric surgery and the immune system in MASLD. Eighteen morbidly obese females undergoing sleeve gastrectomy were followed pre-and post-surgery. Stool from four patients, showing resolved MASLD post-surgery with sustained weight loss, was transplanted into antibiotic treated mice. Mice received pre-or post-surgery stool and were fed a standard or high-fat diet. Bodyweight, food intake, and physiological parameters were tracked weekly. Metabolic parameters were measured post-study termination. The human study revealed that bariatric surgery led to significant weight loss (p > 0.05), decreased inflammatory markers, and improved glucose levels six months post-surgery. Patients with weight loss of 20% or more showed distinct changes in blood metabolites and gut microbiome composition, notably an increase in Bacteroides. The mouse model confirmed surgery-induced microbiome changes to be a major factor in the reduction of markers and attenuation of MASLD progression. Mice receiving post-surgery fecal transplants had significantly less weight gain and liver steatosis compared to pre-surgery recipients. There was also a significant decrease in inflammatory cytokines interferon gamma, interleukin 2, interleukin 15, and mig. This was accompanied by alterations in liver immunophenotype, including an increase in natural killer T cells and reduction of Kupfer cells in the post-surgery transplant group. Our findings suggest surgery induced microbial changes significantly reduce inflammatory markers and fatty liver progression. The results indicate a potential causal link between the microbiome and the host immune system, possibly mediated through modulation of liver NKT and Kupffer cells.

Multiparametric Flow Cytometry-Based Immunophenotyping of Mouse Liver Immune Cells.

The liver is a complex organ that governs many types of metabolisms, including energy metabolism and other cellular processes. The liver also plays a crucial role in important functions in immunity, and the activity of liver tissue-associated immunity affects the outcome of many liver pathologies. A thorough characterization of the liver immune microenvironment may contribute to a better understanding of immune signaling, the mechanisms of specific immune responses, and even to improved predictions about therapy outcomes. In this paper, we present an optimized, simple, and rapid protocol to characterize the liver-associated immune cell milieu. We believe that the most suitable technique for obtaining a complex immune cell suspension and for removing contaminating blood cells is to perform mouse liver perfusion, using only phosphate buffer saline. Combining an enzymatic digestion and a mechanical dissociation of liver tissue, followed by cell purification, improves downstream applications. This combination is an essential prerequisite for immune cell determination and characterization. We then demonstrate a flow cytometry-based multiparametric immunophenotyping along with a gating strategy to detect and quantify liver endothelial cells, T cells (helper and cytotoxic), B cells, NK cells, NKT cells, neutrophils, monocytes (subsets included), dendritic cells (subsets included), macrophages and Kupffer cells.

CXCR6 deficiency ameliorates ischemia-reperfusion injury by reducing the recruitment and cytokine production of hepatic NKT cells in a mouse model of non-alcoholic fatty liver disease

Fatty liver is used for transplantation due to organ shortage, but prone to cause complications like ischemia-reperfusion injury (IRI). NKT cells as a bridge between innate and adaptive immunity were reported to infiltrate the liver at the early phase of IRI induced in normal liver. However, the localization mechanism of NKT cells is not precise, and the role of NKT cells in fatty liver IRI is poorly understood. In present murine IRI model of non-alcoholic fatty liver disease, we demonstrated that although the number reduced in fatty liver, NKT cells still activated and accumulated to fatty liver following IRI, and contributed to IRI by producing inflammatory cytokine IFN-γ. We revealed that NKT cells in fatty liver expressed more CXCR6, a vital chemokine receptor; meanwhile, the ligand CXCL16 mRNA expression level in fatty liver was up-regulated. The up-regulation of the CXCR6/CXCL16 axis in fatty liver happened in IRI, which maybe endow NKT cells more chemotaxis. We further found CXCR6 deficiency reduced the recruitment of NKT cells in a tissue-dependent manner, and impaired the IFN-γ producing capacity of hepatic NKT cells. Serum ALT level and hepatic histology were both improved in CXCR6 deficient mice. The results provide evidence of the pathogenic role of NKT cells in fatty liver IRI, and important localization mechanism involving up-regulated CXCR6/CXCL16. Deficiency of CXCR6 protects the fatty liver from IRI by reducing the recruitment and cytokine production of hepatic NKT cells.

SAT-382-Crosstalk between liver NKT cells and liver sinusoidal endothelial cells

SAT-382-Crosstalk between liver NKT cells and liver sinusoidal endothelial cells

Abstract A204: A protective GD3-based vaccine increases NKT-cells in a C57BL/6 murine model

Abstract The disialyl gangliosides GD2/GD3 have been implicated in the enhancement of malignancy in a number of human and animal cancers and as a tumor antigen target for immunotherapy. Earlier we reported on a GD3-based vaccine, which protects canine melanoma patients when provided as an adjunct to standard of care (1). Despite a significant increase in the median survival time, the mechanism by which vaccine works remains unclear; therefore, we vaccinated C57BL/6 mice with the GD3-vaccine formulation and appropriate controls and evaluated changes in the immune cells. Methods: C57BL/6 mice were vaccinated weekly for 4 weeks followed by a week of rest. Mice were then euthanized to collect blood, liver, and spleen for evaluation of NKT-cells. Liver lymphocytes were collected by making mono-cellular suspension of liver obtained after enzymatic digestion. Collected lymphocytes were stained for CD3, CD4, TCRβ, NK1.1, CD49b and dead cells. In these experiments untreated and α-galactosylceramide (α-GalCer) treated mice were used as controls. Results: Mice treated with the subcutaneous injections of the GD3 vaccine showed an increase in NKT-cells (NK1.1+CD49b+CD3+CD4+TCRb+) in the liver but failed to show an increase in the spleen and blood when compared with the untreated mice. The mice treated with α-GalCer also showed an accumulation of NKT-cells in the liver; however, the increase in the livers obtained from vaccine-treated mice was 5-10-fold higher compared to the α-GalCer treated mice. Discussion: NKT-cells are the Swiss army knife of the immune system which are capable of producing different cytokines and chemokines to regulate the overall immune system. After their discovery two decades ago, activation of NKT-cells have shown to have a crucial protective role in various infectious, and non-infectious diseases. These cells form a bridge between the innate and adaptive immune cells. The activation and maintenance of these cells are dependent upon the presentation of lipid molecules on CD1 receptors by dendritic cells. Since the target antigen in the vaccine is a lipid (GD3) it is very likely it would be presented on CD1 receptors for the activation of NKT-cells. Due to the lack of validated and suitable canine CD1 receptor reagents, we were unable to define the mechanism behind the hypothetical protection found in the canine GD3 based vaccine (1). In the murine model NKT-cells are well characterized, and provided us with an opportunity to understand the possible selective activation of NKT-cells by the GD3 based vaccine. Our data found an increase in NKT-cells in the liver for mice vaccinated with GD3 and α-GalCer, but no discernible differences were found in blood and spleen between vaccinated mice and normal controls. Further investigation using the B16 melanoma cell line in C57BL/6 mice vaccinated with the GD3 vaccine and suitable controls may identify the NKT response in the tumor microenvironment. Conclusions: While the GD3-based vaccine and α-GalCer both increased NKT-cells in the murine liver, GD3 increased NKT counts 5-10 fold over α-GalCer. In addition, monitoring changes in NKT numbers in the peripheral blood may not be of benefit due to low cell counts. Acknowledgment: The study is funded by a grant from the American Kennel Club Health Foundation and The UF CVM Foundation. Reference: 1. Milner RJ. The immune response to disialoganglioside GD3 vaccination in dogs with spontaneous occurring melanoma: A large animal translational model. Poster # 123628_1 presented at: Proceeding American Association for Cancer Research, Tumor Immunology: Basic and Clinical Advances; 2010 Dec 30; Miami Beach, Florida. Citation Format: Rowan J. Milner, Bikash Sahay, Matthew Cascio, Marc Salute. A protective GD3-based vaccine increases NKT-cells in a C57BL/6 murine model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A204.

GPBAR1 Functions as Gatekeeper for Liver NKT Cells and provides Counterregulatory Signals in Mouse Models of Immune-Mediated Hepatitis.

Background & AimsGPBAR1, also known as TGR5, is a G protein–coupled receptor activated by bile acids. Hepatic innate immune cells are involved in the immunopathogenesis of human liver diseases and in several murine hepatitis models. Here, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation in rodent models of hepatitis.Material and methodsHepatitis was induced by concanavalin A (Con A) or α-galactosyl-ceramide (α-GalCer). 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective agonist of GPBAR1, was administrated by o.s.ResultsIn the mouse models of hepatitis, the genetic ablation of Gpabar1 worsened the severity of liver injury and resulted in a type I NKT cells phenotype that was biased toward a NKT1, a proinflammatory, IFN-γ producing, NKT cells subtype. Further on, NKT cells from GPBAR1–/– mice were sufficient to cause a severe hepatitis when transferred to naïve mice. In contrast, GPBAR1 agonism rescued wild-type mice from acute liver damage and redirects the NKT cells polarization toward a NKT10, a regulatory, IL-10 secreting, type I NKT cell subset. In addition, GPBAR1 agonism significantly expanded the subset of IL-10 secreting type II NKT cells. RNAseq analysis of both NKT cells type confirmed that IL-10 is a major target for GPABR1. Accordingly, IL-10 gene ablation abrogated protection afforded by GPBAR1 agonism in the Con A model.ConclusionPresent results illustrate a role for GPBAR1 in regulating liver NKT ecology. Because NKT cells are an essential component of liver immune system, our data provide a compelling evidence for a GPBAR1-IL-10 axis in regulating of liver immunity.

Chlordecone potentiates auto-immune hepatitis and promotes brain entry of MHV3 during viral hepatitis in mouse models

Chlordecone is an organochlorine used in the 1970’s as a pesticide in banana plantations. It has a long half-life in the soil and can potentially contaminate humans and animals through food. Chlordecone targets, and mainly accumulates in, the liver, leading to hepatomegaly and neurological signs in mammals. Chlordecone does not cause liver injuries or any inflammation by itself at low doses, but it can potentiate the hepatotoxic effects of other chemicals and drugs. We studied the impact of chlordecone on the progression of acute hepatitis in mouse models of co-exposure to chlordecone with Concanavalin A or murine hepatitis virus type 3. We examined the progression of these two types of hepatitis by measuring hepatic transaminase levels in the serum and inflammatory cells in the liver, liver histological studies. Amplified tremors presented in the MHV3- chlordecone mouse model had led us to study the expression of specific genes in the brain. We show that chlordecone amplifies the auto-immune hepatitis induced by Concanavalin A by increasing the number of liver NKT cells, which are involved in liver damage. Chlordecone also accelerated the death of mice infected by murine hepatitis virus and enhanced the entry of the virus into the cervical spinal cord in infected mice, leading to considerable neurological damage. In conclusion, chlordecone potentiates both the Concanavalin A-induced hepatitis and brain damage caused by an hepatotropic/neurotropic virus.

Histone demethylases UTX and JMJD3 are required for NKT cell development in mice

BackgroundNatural killer (NK)T cells and conventional T cells share phenotypic characteristic however they differ in transcription factor requirements and functional properties. The role of histone modifying enzymes in conventional T cell development has been extensively studied, little is known about the function of enzymes regulating histone methylation in NKT cells.ResultsWe show that conditional deletion of histone demethylases UTX and JMJD3 by CD4-Cre leads to near complete loss of liver NKT cells, while conventional T cells are less affected. Loss of NKT cells is cell intrinsic and not due to an insufficient selection environment. The absence of NKT cells in UTX/JMJD3-deficient mice protects mice from concanavalin A‐induced liver injury, a model of NKT‐mediated hepatitis. GO‐analysis of RNA-seq data indicates that cell cycle genes are downregulated in UTX/JMJD3-deleted NKT progenitors, and suggest that failed expansion may account for some of the cellular deficiency. The phenotype appears to be demethylase‐dependent, because UTY, a homolog of UTX that lacks catalytic function, is not sufficient to restore their development and removal of H3K27me3 by deletion of EZH2 partially rescues the defect.ConclusionsNKT cell development and gene expression is sensitive to proper regulation of H3K27 methylation. The H3K27me3 demethylase enzymes, in particular UTX, promote NKT cell development, and are required for effective NKT function.

MicroRNA-21 in Natural Killer T cell α-galactosylceramide activation

Abstract Natural Killer T (NKT) cells are innate-like lymphocytes that rapidly produce cytokines upon activation. However, following primary stimulation with the potent ligand α-galactosylceramide (αGC), NKT cells become hyporesponsive, defined as a severe reduction in effector cytokines upon re-stimulation. The purpose of this project is to investigate the role of microRNAs (miRNAs) during NKT cell transition from effector response, expansion and hyporesponsiveness. MiRNAs are ~22bp non-coding RNAs that associate with the RNA-induced silencing complex (RISC) to inhibit the translation of target transcripts. We performed a Nanostring microRNA analysis to determine global expression of microRNAs in NKT cells after α-GC stimulation. MicroRNA-21 (miR-21) was found to be highly upregulated in splenic NKT cells at 3-hours, 3-day, and 30-days post-activation compared to unstimulated controls. Our preliminary data show that MiR-21KO/KO splenic and liver NKT cells have a lower frequency at day-3 compared to WT-littermate controls. We hypothesize that miR-21 regulates the NKT cell transition from activation to hyporesponsiveness and that miR-21KO/KO NKT cells exhibit an interrupted hyporesponsive state.

Correction: Exosome-like Nanoparticles from Intestinal Mucosal Cells Carry Prostaglandin E2 and Suppress Activation of Liver NKT Cells.

Correction: Exosome-like Nanoparticles from Intestinal Mucosal Cells Carry Prostaglandin E2 and Suppress Activation of Liver NKT Cells.

NKT cells in the liver are important for periferal tolerance induction

Abstract NKT cells are mainly located in the liver in mice. The role of liver inherent NKT cells are still unclear. Method In this study, we employed mouse orthotopic liver transplantation and heterotopic heart transplantation models to critical examine the role of liver NKT cells in liver and peripheral tolerance induction. Results NKT cells were increased in the tolerated liver grafts which expressed higher levels of CD95L and PD-L1, while the CD4+CD25+Foxp3+ regulatory T cells (Treg) were markedly increased in the both liver and spleen at day 7 post transplantation. The heart allograft survival were prolonged significantly in the recipient which accepted donor spleen cells by portal vein (p.v.) injection in contract to the recipient which received the donor spleen cells by tail vein (i.v.) injection. In vitro immunological assay revealed that the number of NKT cells in the liver was increased and Treg were increased in both liver and spleen from the p.v. treated mice significantly. The IL-2 expression was decreased, IL-4 and IL-10 were increased. Further, significantly increased IL-4, IL-10, and IFN-γ production was detected from liver NPCs under aGalCer stimulation. Conclusion NKT cells are important to liver transplant tolerance induction, appear to play a key role in down regulation of peripheral immune responses and facilitate CD4+CD25+Foxp3+ Treg induction.

Candida albicans up-regulates the Fas-L expression in liver Natural Killer and Natural Killer T cells.

After Candida albicans arrival to the liver, the local production of proinflammatory cytokines and the expanded intrahepatic lymphocytes (IHL) can be either beneficial or detrimental to the host. Herein we explored the balance between protective inflammatory reaction and liver damage, focusing our study on the contribution of TNF-α and Fas–Fas-L pathways in the hepatocellular apoptosis associated to C. albicans infection. A robust tissue reaction and a progressive increase of IL-1β, IL-6 and TNF-α were observed in infected animals. Blocking the biological activity of TNF-α did not modify the number of apoptotic cells observed in C. albicans infected animals. Fas-L molecule was up regulated on purified hepatic mononuclear cells and its expression progressed with the infection. In the IHL compartment, the absolute number of Fas-L+ NK and NKT cells increased on days 1 and 3 of the infection. C. albicans was also able to up regulate Fas-L expression in normal liver NK and NKT cells after in vitro contact. The innate receptor TLR2 was involved in this phenomenon. In the interplay between host factors and evasion strategies exploited by pathogens, the mechanism supported here could represent an additional way that allows this fungus to circumvent protective immune responses in the liver.

NKT cells act through third party bone marrow-derived cells to suppress NK cell activity in the liver and exacerbate hepatic melanoma metastases.

Uveal melanoma (UM) is the most common intraocular tumor in adults and liver metastasis is the leading cause of death in UM patients. We have previously shown that NKT cell-deficient mice develop significantly fewer liver metastases from intraocular melanomas than do wild-type (WT) mice. Here, we examine the interplay between liver NKT cells and NK cells in resistance to liver metastases from intraocular melanomas. NKT cell-deficient CD1d(-/-) mice and WT C57BL/6 mice treated with anti-CD1d antibody developed significantly fewer liver metastases than WT mice following either intraocular or intrasplenic injection of B16LS9 melanoma cells. The increased number of metastases in WT mice was associated with reduced liver NK cytotoxicity and decreased production of IFN-γ. However, liver NK cell-mediated cytotoxic activity was identical in non-tumor bearing NKT cell-deficient mice and WT mice, indicating that liver metastases were crucial for the suppression of liver NK cells. Depressed liver NK cytotoxicity in WT mice was associated with production of IL-10 by bone marrow-derived liver cells that were neither Kupffer cells nor myeloid-derived suppressor cells and by increased IL-10 receptor expression on liver NK cells. IL-10(-/-) mice had significantly fewer liver metastases than WT mice, but were not significantly different from NKT cell-deficient mice. Thus, development of melanoma liver metastases is associated with upregulation of IL-10 in the liver and an elevated expression of IL-10 receptor on liver NK cells. This impairment of liver NK activity is NKT cell-dependent and only occurs in hosts with melanoma liver metastases.

Lymphocytes in atherosclerosis.

Atherosclerosis is the major cause of cardiovascular disease, and cardiovascular disease remains the primary cause of death in the United States. The primary complication for patients with type 2 diabetes mellitus (T2D) is cardiovascular disease.1–8 Because the incidences of T2D and obesity are drastically increasing in the United States and now worldwide, understanding how atherosclerosis is accelerated in these diseases becomes even more important to discern. Although many scientific advances in research on atherosclerosis have been made by multiple groups during the past 30 years, particularly about the function of endothelial and smooth muscle biology and macrophage foam cell formation in atherosclerotic plaque development, the roles of immune cells in regulating atherosclerosis onset and progression are still being elucidated. It was widely thought that immune cells played little part in atherogenesis until 1986, when Hansson et al9–12 reported the presence of lymphocytes within atherosclerotic lesions; yet it was many years later until it was widely accepted that immune cells play an important role in atherogenesis. Since 1986, the viewpoint has changed significantly, but details on lymphocyte subsets and mechanisms are still emerging. Moreover, controversy still exists on the roles that some lymphocyte subsets play in atherosclerosis. These discrepancies in findings are at least partly because of differences in mouse models used, diet composition, and length of time on diet for atherosclerosis measurements. However, despite a few ongoing controversies, there are many studies on immune cells in atherogenesis in mice that show clear findings.10,12–23 For example, CD4+ T lymphocytes have been shown to accelerate atherosclerosis.18,24–29 Activated CD4 T cells can be distinguished based on their phenotype, and the predominant CD4+ effector T cells studied to date in the context of atherosclerosis are Th1, …

Isolation of Non-parenchymal Cells from the Mouse Liver.

Hepatocytes comprise the majority of liver mass and cell number. However, in order to understand liver biology, the non-parenchymal cells (NPCs) must be considered. Herein, a relatively rapid and efficient method for isolating liver NPCs from a mouse is described. Using this method, liver sinusoidal endothelial cells, Kupffer cells, natural killer (NK) and NK-T cells, dendritic cells, CD4+ and CD8+ T cells, and quiescent hepatic stellate cells can be purified. This protocol permits the collection of peripheral blood, intact liver tissue, and hepatocytes, in addition to NPCs. In situ perfusion via the portal vein leads to efficient liver digestion. NPCs are enriched from the resulting single-cell suspension by differential and gradient centrifugation. The NPCs can by analyzed or sorted into highly enriched populations using flow cytometry. The isolated cells are suitable for flow cytometry, protein, and mRNA analyses as well as primary culture.

Gal-3 regulates the capacity of dendritic cells to promote NKT-cell-induced liver injury.

Galectin-3 (Gal-3), an endogenous lectin, exhibits pro- and anti-inflammatory effects in various disease conditions. In order to explore the role of Gal-3 in NKT-cell-dependent pathology, we induced hepatitis in C57BL/6 WT and Gal-3-deficient mice by using specific ligand for NKT cells: α-galactosylceramide, glycolipid Ag presented by CD1d. The injection of α-galactosylceramide significantly enhanced expression of Gal-3 in liver NKT and dendritic cells (DCs). Genetic deletion or selective inhibition of Gal-3 (induced by Gal-3-inhibitor TD139) abrogated the susceptibility to NKT-cell-dependent hepatitis. Blood levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-12) and their production by liver DCs and NKT cells were also downregulated. Genetic deletion or selective inhibition of Gal-3 alleviated influx of inflammatory CD11c(+) CD11b(+) DCs in the liver and favored tolerogenic phenotype and IL-10 production of liver NKT and DCs. Deletion of Gal-3 attenuated the capacity of DCs to support liver damage in the passive transfer experiments and to produce pro-inflammatory cytokines in vitro. Gal-3-deficient DCs failed to optimally stimulate production of pro-inflammatory cytokines in NKT cells, in vitro and in vivo. In conclusion, Gal-3 regulates the capacity of DCs to support NKT-cell-mediated liver injury, playing an important pro-inflammatory role in acute liver injury.

Diverse Roles of Natural Killer T Lymphocytes in Liver and Spleen in Mice with Experimental Autoimmune Uveitis

Natural killer T lymphocytes (NKT cells) have diverse roles in different organs. However, it is still unclear whether NKT cells play different roles in the liver and spleen in mice with experimental autoimmune uveitis. In the present study, we explored their diverse roles of NKT cells in the liver and spleen and found that liver-derived NKT cells could efficiently inhibit Th1 and Th17 differentiation, whereas the function of spleen-derived NKT cells was less potent. Meanwhile, the occurrence of the peak ratios of NKT cells/T cells in the spleen and liver was markedly asynchronous after immunization. Moreover, different methods of immunization could result in different immune responses in mice. Furthermore, the memory response was also found in the generation of NKT cells in mice when they received the same antigen. These results indicate that the functional roles of NKT cells possess diversity in the spleen and liver in mice with experimental autoimmune uveitis.

Glucocorticoid receptor-dependent immunomodulatory effect of ursodeoxycholic acid on liver lymphocytes in mice

Although ursodeoxycholic acid (UDCA) has long been used for patients with chronic cholestatic liver diseases, particularly primary biliary cirrhosis, it may modulate the host immune response. This study investigated the effect of UDCA feeding on experimental hepatitis, endotoxin shock, and bacterial infection in mice. C57BL/6 mice were fed a diet supplemented with or without 0.3% (wt/vol) UDCA for 4 wk. UDCA improved hepatocyte injury and survival in concanavalin-A (Con-A)-induced hepatitis by suppressing IFN-γ production by liver mononuclear cells (MNC), especially NK and NKT cells. UDCA also increased survival after lipopolysaccharide (LPS)-challenge; however, it increased mortality of mice following Escherichia coli infection due to the worsening of infection. UDCA-fed mice showed suppressed serum IL-18 levels and production of IL-18 from liver Kupffer cells, which together with IL-12 potently induce IFN-γ production. However, unlike normal mice, exogenous IL-18 pretreatment did not increase the serum IFN-γ levels after E. coli, LPS, or Con-A challenge in the UDCA-fed mice. Interestingly, however, glucocorticoid receptor (GR) expression was significantly upregulated in the liver MNC of the UDCA-fed mice but not in their whole liver tissue homogenates. Silencing GR in the liver MNC abrogated the suppressive effect of UDCA on LPS- or Con-A-induced IFN-γ production. Furthermore, RU486, a GR antagonist, restored the serum IFN-γ level in UDCA-fed mice after E. coli, LPS, or Con-A challenge. Taken together, these results suggest that IFN-γ-reducing immunomodulatory property of UDCA is mediated by elevated GR in the liver lymphocytes in an IL-12/18-independent manner.

Liver natural killer T cells enhance, rather than inhibit, liver metastases arising from intraocular melanomas in mice. (P2087)

Abstract Uveal melanoma (UM) is the most common intraocular tumor in adults and liver metastasis is the leading cause of death in UM patients. We have shown that NKT cell-deficient mice develop significantly less liver metastasis from intraocular melanomas than wild-type (WT) mice. Here we examine the interplay between liver NKT cells and NK cells in resistance to liver metastases from intraocular melanomas. NKT cell-deficient CD1d-/- mice and WT C57BL/6 mice treated with anti-CD1d antibody developed significantly fewer liver metastases than WT mice following either intraocular or intrasplenic injection of B16LS9 melanoma cells. Increased metastases in WT mice was associated with reduced liver NK cytotoxicity. However, liver NK cytotoxic activity was identical in non-tumor-bearing NKT cell-deficient mice and WT mice, indicating that liver metastases were crucial for suppression of liver NK cells. Depressed liver NK cytotoxicity in WT mice was associated with increased IL-10 production by bone marrow-derived liver cells and increased IL-10 receptor expression on liver NK cells. IL-10-/- mice had significantly fewer liver metastases than WT mice, but were not significantly different from NKT cell-deficient mice. Thus, development of melanoma liver metastases is associated with upregulation of IL-10 in the liver and an elevated expression of IL-10 receptor on liver NK cells. Impaired liver NK activity is NKT cell-dependent and only occurs in hosts with melanoma liver metastases.

Exosome-like Nanoparticles from Intestinal Mucosal Cells Carry Prostaglandin E2 and Suppress Activation of Liver NKT Cells

Regulation and induction of anergy in NKT cells of the liver can inhibit autoimmune and antitumor responses by mechanisms that are poorly understood. We investigated the effects of PGE2, delivered by intestinal, mucus-derived, exosome-like nanoparticles (IDENs), on NKT cells in mice. In this study, we demonstrate that IDENs migrate to the liver where they induce NKT cell anergy. These effects were mediated by an IDENs' PGE2. Blocking PGE2 synthesis attenuated IDENs inhibition of induction of IFN-γ and IL-4 by α-galactosylceramide (α-GalCer)-stimulated liver NKT cells in a PGE2 E-type prostanoid 2/E-type prostanoid 4 receptor-mediated manner. Proinflammatory conditions enhanced the migration of IDENs to the liver where α-GalCer and PGE2 induced NKT anergy in response to subsequent α-GalCer stimulation. These findings demonstrate that IDENs carrying PGE2 can be transferred from the intestine to the liver, where they act as immune modulators, inducing an anergic-like state of NKT cells. These reagents might be developed as therapeutics for autoimmune liver diseases.