Activation Of NKT Cells Research Articles

Roles of Natural Killer T Cells and Natural Killer Cells in Kidney Injury.

Mouse natural killer T (NKT) cells and natural killer (NK) cells are innate immune cells that are highly abundant in the liver. In addition to their already-known antitumor and antimicrobial functions, their pathophysiological roles in the kidney have recently become evident. Under normal circumstances, the proportion of activated NKT cells in the kidney increases with age. Administration of a synthetic sphingoglycolipid ligand (alpha-galactosylceramide) further activates NKT cells, resulting in injury to renal vascular endothelial cells via the perforin-mediated pathway and tubular epithelial cells via the TNF-α/Fas ligand pathway, causing acute kidney injury (AKI) with hematuria. Activation of NKT cells by common bacterial DNA (CpG-ODN) also causes AKI. In addition, NKT cells together with B cells play significant roles in experimental lupus nephritis in NZB/NZW F1 mice through their Th2 immune responses. Mouse NK cells are also assumed to be involved in various renal diseases, and there may be complementary roles shared between NKT and NK cells. Human CD56+ T cells, a functional counterpart of mouse NKT cells, also damage renal cells through a mechanism similar to that of mice. A subpopulation of human CD56+ NK cells also exert strong cytotoxicity against renal cells and contribute to the progression of renal fibrosis.

Therapeutic targeting of the immunosuppressive axis in ovarian cancer using dietary supplements

Abstract Ovarian cancer is the deadliest gynecologic malignancy worldwide and mortality rates have remained steady despite advances in treatment. A major obstacle for current therapies is overcoming tumor immunosuppression, therefore targeting inhibitory factors produced in the tumor microenvironment could enhance antitumor responses. Natural Killer T (NKT) cells, a subset of T cells that recognize glycolipid antigens presented by CD1d, are important mediators of antitumor immune responses. We have previously shown that ovarian cancers shed the ganglioside GD3, which binds with high affinity to CD1d, thereby suppressing NKT cell activation. In addition to GD3, we have identified vascular endothelial growth factor (VEGF) as another immunosuppressive factor secreted by ovarian cancers. We found that VEGF inhibition results in decreased GD3 expression in human ovarian cancer cell lines and restoration of NKT cell responses. In addition, we found that treatment with VEGF can directly impair NKT cell functional responses. To therapeutically target the VEGF-GD3 signaling cascade, mouse and human ovarian cancer cell lines were treated with triptolide, a phytochemical which has demonstrated potent antitumor activity in vitro and in vivo. Treatment with triptolide reduces VEGF secretion by ovarian cancer cell lines and results in a concomitant decrease in the expression of GD3 synthase. In addition, pre-treatment of ovarian cancer cells with triptolide improves the ability of NKT cells to recognize and lyse the cancer cells. Thus, we have identified a novel therapeutic strategy that can be used to block the production of immunosuppressive factors, thereby restoring NKT cell-mediated antitumor immune responses in ovarian cancer.

Frontline Science: Concanavalin A-induced acute hepatitis is attenuated in vitamin D receptor knockout mice with decreased immune cell function.

The vitamin D receptor (VDR) is a nuclear receptor for the active form of vitamin D, 1α,25-dihydroxyvitamin D3 , and regulates various physiologic processes, such as bone and calcium metabolism, cellular proliferation and differentiation, and immunity. VDR is highly expressed in the intestine, kidney, bone, and macrophages, but is expressed at a low level in the liver. The liver is a major metabolic organ and also acts as an immune gateway for dietary nutrients and xenobiotics. In this study, we investigated the function of VDR in hepatic immune cells, such as Kupffer cells/macrophages, utilizing VDR knockout (KO) mice. We showed that VDR is functionally expressed in hepatic mononuclear cells, specifically resident Kupffer cells. We examined the role of VDR in acute hepatitis induced by concanavalin A (Con-A) and found that Con-A-induced hepatitis is attenuated in VDR-KO mice compared to wild-type (WT) mice. Con-A-induced hepatitis is known to be mediated by NKT cell activation, cytokine production, and reactive oxygen species (ROS) production in Kupffer cells/macrophages. However, the proportions of Kupffer cells/macrophages and the NKT cell activation were similar in the liver of WT and VDR-KO mice and inflammatory cytokine gene expression was increased in VDR-KO mice. On the other hand, plasma and hepatic ROS levels were decreased in the liver of VDR-KO mice compared to WT mice. The phagocytic activity of resident Kupffer cells and hepatic neutrophils were also decreased in VDR-KO mice. Therefore, VDR is necessary for Con-A-induced acute hepatitis and plays an important role in hepatic immune cell functions.

Immunization-Expanded NKT Follicular Helper Cells Drive IgG1 Isotype Switch against an Exogenous T-Independent Polysaccharide but Do Not Promote Recall Responses.

The CD1d-binding glycolipid α-galactosylceramide (α-GC) is a potent adjuvant that activates NKT cells and in turn enhances T-dependent humoral immunity. Very little is known about how NKT cells and the NKT follicular helper (NKTfh) subset influence the immune response to T-independent polysaccharides. In this study, we used a Cre-Lox approach to generate mice devoid of the Bcl6 master transcription factor in CD4 lineage cells and thus devoid of NKTfh cells but not total NKT cells. It was observed that α-GC-driven IgG1 class switch against a polysaccharide Ag was dependent on the NKTfh subset. However, α-GC was unable to stimulate a polysaccharide-specific Ab recall response. It was observed that NKT-derived IL-21 was able to exert limited influence on the IgG1 response and was therefore likely to work in concert with other factors. This work shows that α-GC-driven NKTfh cells can direct polysaccharide-specific B cell responses by promoting IgG1 class switch but do not provide signals needed for generation of polysaccharide-specific B cell memory.

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.

Analyzing Antigen Recognition by Natural Killer T Cells.

Natural killer T (NKT) cells are a subset of T lymphocytes that recognize a wide variety of lipid antigens presented by the atypical MHC class I molecule CD1d. NKT cells exhibit rapid activation after recognition of cognate antigens, secrete abundant amounts of T helper (Th) 1, Th2, and Th17 cytokines within hours of activation and shape the immune response through subsequent activation of dendritic, NK, T, and B cells. NKT cells therefore play central roles in antimicrobial and anticancer immunity and in the modulation of various autoimmune disorders. Consequently, recent research has focused on the discovery of microbial and self-antigens involved in NKT cell activation. In this chapter, we will discuss different strategies for studying antigen recognition by NKT cells including CD1d tetramer-based approaches and in vitro assays characterizing NKT cell activation in response to lipid antigen presentation. While Toll-like receptor (TLR) agonists and cytokines such as IL-12 are critical for NKT cell activation in vivo, particularly in the context of microbial infection, methods for detection of TLR- and cytokine-dependent NKT cell activation will not be discussed in this section.

CD3+CD56+ natural killer T cell infiltration is increased in cervical cancer and negatively correlated with tumour progression

This study investigated the phenotype and function of natural killer T (NKT) cells infiltrating cervical cancer tissues, in an attempt to understand the regulation of NKT cells by cervical cancer cells. Forty-two patients with cervical cancer were included. Flow cytometry was used to analyze the percentage of NKT cells in tumour tissues and its correlation with clinical staging and lymphatic metastasis. The expression of surface receptor and effector molecules on infiltrating NKT cells was determined. The changes of the phenotype, subtype and cytotoxicity of NKT cells were investigated. The regulation of NKT cells by cervical cancer cells was investigated by co-culture with HeLa or SiHa cells. The effect of TGF-β1 on NKT activity regulated by cervical cancer cells was studied. The results showed that the infiltration of NKT cells in cervical cancer tissues was significantly higher than that in tumour-adjacent tissues, and the degree of infiltration was negatively correlated with the progression of the disease. The activity and killing effect of infiltrating NKT were inhibited in cervical cancer tissues, leading to increase of CD4+NKT cells and decrease of CD8−CD4−NKT cells. The activity of NKT cells was down-regulated by co-culture with cervical cancer cells, but subtypes of NKT cells were not altered. Cervical cancer cells inhibited the function of NKT cells by secreting TGF-β1. The study demonstrates that the infiltration of NKT cells in cervical cancer tissues is increased significantly and negatively correlated with tumour progression.

Natural killer T cells from peripheral blood of patients with pregnancy-induced hypertension inhibit the proliferation and migration of vascular endothelial cells by secreting interleukin-17

The aim of this study was to investigate the phenotype and function of NKT cells and the effect of NKT cells on the functions of vascular endothelial cells. The study included 30 pregnant women with hypertension and 20 pregnant women with normal delivery. Peripheral blood and placental tissues were collected from all patients and control subjects. Lymphocytes were isolated from peripheral blood and tissues. NKT cell ratio, expression of surface receptors and expression of effector molecules were detected by flow cytometry. Culture supernatant of NKT cells was collected and co-cultured with human umbilical vein endothelial cells (HUVECs). Proliferation was determined by CCK-8 assay, and migration, by transwell assay. The results showed elevated ratios of NKT and NK cells in peripheral blood and placental tissues from women with pregnancy-induced hypertension (PIH) compared with women with normal pregnancies. The expression of NKp30, NKG2D and NKG2A receptors on the surface of NKT cells from women with PIH was altered: the ratio of CD4+ NKT cells was increased but the ratio of CD8+ NKT cells was decreased. The ratios of GranB+ NKT cells in peripheral blood, IFN-γ-positive T cells in placental tissues and IL-17-positive NKT cells in both peripheral blood and placental tissues were elevated in PIH patients. The present study demonstrated that the ratio and activity of NKT cells from peripheral blood and placental tissues of women with PIH were elevated. In addition, NKT cells from peripheral blood of PIH patients inhibited the proliferation and migration of vascular endothelial cells by secreting IL-17.

Autophagy and the Immune Response.

Innate immunity and adaptive immunity play critical roles in maintaining normal physiological functions and the development of diseases. In innate immune responses, heterogeneous autophagy can directly remove intracellular pathogens while activating PRRs, including TLRs and NLRs, to trigger their signal transduction pathways and promote NKT cell activation, cytokine secretion, and phagocytosis. In adaptive immune responses, the autophagy reaction has an important effect on the homeostasis, function, and differentiation of T lymphocytes, the survival, and development of B lymphocytes and the survival of plasma cells. This review highlights the key role that autophagy plays in the innate immune system and the acquired immune system. Further clarifying the mechanism by which autophagy regulates the immune system is essential for elucidating the precise mechanisms of various diseases and for developing new treatment methods.

Vascular Endothelial Cells Activate Peripheral Natural Killer T Cells and Participate in Regulation of Downstream Immune Cascades in Patients with Sepsis.

BackgroundThis study investigated the effect of supernatant of endothelial cells stimulated by peripheral blood serum from sepsis patients on phenotype and function of peripheral NKT cells.Material/MethodsTwenty-one patients with sepsis and 21 healthy subjects were included. Peripheral blood (5 ml) was collected from all patients and healthy subjects. To isolate peripheral blood mononuclear cells (PBMCs), Ficoll lymphocyte separation solution was used. Flow cytometry was carried out to determine NKT cell ratio, activity, and cytokine secretion. Human umbilical vein endothelial cells were cultured with serum from sepsis patients for 48 h before changing to fresh medium, and supernatant was collected. The supernatant was used to co-culture PBMCs before analyzing NKT activity and cytokines.ResultsThe ratios of CD3-CD56+NK cells and CD3+CD56+NKT cells were increased in peripheral blood from sepsis patients. Surface receptors p30, G2D, and p44 of CD3+CD56+NKT cells were elevated, while inhibitory receptors NKG2A and 158b were decreased. CD4+ NKT cells in peripheral blood from sepsis patients were enhanced. GranB, IFN-γ, IL-4, and IL-17 in NKT cells from sepsis patients were up-regulated. After co-culture with vascular endothelial cells treated with sepsis serum, expression of p30 and G2D in NKT cells was upregulated, and number of TCRVα24-positive cells was increased. In addition, ratio of CD4+NKT cells was increased, and intracellular expression of IL-4 and IFN-γ was elevated.ConclusionsThe study demonstrates that the level of NKT cells in peripheral blood from sepsis patients is increased, and their activity is enhanced. In addition, vascular endothelial cells from sepsis patients can regulate the activity of NKT cells.

Activation of NKT Cells in an Anti-PD-1-Resistant Tumor Model Enhances Antitumor Immunity by Reinvigorating Exhausted CD8 T Cells.

PD-1-based cancer immunotherapy is a successful example of immune checkpoint blockade that provides long-term durable therapeutic effects in patients with cancer across a wide spectrum of cancer types. Accumulating evidence suggests that anti-PD-1 therapy enhances antitumor immunity by reversing the function of exhausted T cells in the tumor environment. However, the responsiveness rate of patients with cancer to anti-PD-1 therapy remains low, providing an urgent need for optimization and improvement. In this study, we designed an anti-PD-1-resistant mouse tumor model and showed that unresponsiveness to anti-PD-1 is associated with a gradual increase in CD8 T-cell exhaustion. We also found that invariant natural killer T cell stimulation by the synthetic ligand α-galactosylceramide (αGC) can enhance the antitumor effect in anti-PD-1-resistant tumors by restoring the effector function of tumor antigen-specific exhausted CD8 T cells. IL2 and IL12 were among the cytokines produced by αGC stimulation critical for reinvigorating exhausted CD8 T cells in tumor-bearing mice and patients with cancer. Furthermore, we observed a synergistic increase in the antitumor effect between αGC-loaded antigen-presenting cells and PD-1 blockade in a therapeutic murine tumor model. Our study suggests NKT cell stimulation as a promising therapeutic strategy for the treatment of patients with anti-PD-1-resistant cancer.Significance: These findings provide mechanistic insights into the application of NKT cell stimulation as a potent adjuvant for immunotherapy against advanced cancer. Cancer Res; 78(18); 5315-26. ©2018 AACR.

Natural killer T cell ligand alpha-galactosylceramide protects against gut ischemia reperfusion-induced organ injury in mice

Background & AimsGut ischemia reperfusion (I/R) injury is a life-threatening condition. The immune response plays an important role in I/R-induced organ injury. Alpha-galactosylceramide (α-GalCer) is a potent natural killer T (NKT) cell stimulator. Activation of NKT cells by α-GalCer has been shown to reduce I/R-induced injury in the liver and heart. However, whether α-GalCer has any protective effects on gut I/R-induced organ injury remained unknown. The aim of this study was to test the hypothesis that α-GalCer attenuates gut I/R-induced local and remote organ injury through modulating immune responses. MethodsGut ischemia was induced by placing a microvascular clip across the superior mesenteric artery for 30 min in male adult mice. After removing the clip, α-GalCer (2 µg/mouse) or normal saline containing 0.5% Tween 20 (Vehicle) was administered intraperitoneally. Blood, gut, lung and mesenteric lymph node (MLN) samples were collected 4 h after reperfusion to detect bacterial translocation, tight junction protein, tissue damage, edema, apoptosis, IL-4, IL-10, IFN-γ and TNF-α levels. Resultsα-GalCer significantly reduced bacterial translocation to the MLN, restored tight junction protein and attenuated gut and lung injury after gut I/R. α-GalCer markedly stimulated the production of IL-4, IL-10 and IFN-γ, but had no obvious effects on TNF-α production in gut I/R mice. Pretreatment with anti-CD1d, IL-4 or IL-10, but not IFN-γ blocking antibodies abolished the protective effects of α-GalCer in gut I/R. Conclusionsα-GalCer treatment improved gut barrier function and attenuated gut and lung injury after gut I/R. The beneficial effects of α-GalCer in gut I/R were NKT cell dependent and mediated through upregulation of IL-4 and IL-10. Thus, activation of NKT cells by α-GalCer may serve as a novel option in the treatment of gut I/R injury.

Natural killer T cells mediate inflammation in the bile ducts.

Cholangiocytes function as antigen-presenting cells with CD1d-dependent activation of natural killer T (NKT) cells in vitro. NKT cells may act both pro- and anti-inflammatory in liver immunopathology. We explored this immune pathway and the antigen-presenting potential of NKT cells in the bile ducts by challenging wild-type and Cd1d-/- mice with intrabiliary injection of the NKT cell activating agent oxazolone. Pharmacological blocking of CD1d-mediated activation was performed with a monoclonal antibody. Intrabiliary oxazolone injection in wild-type mice caused acute cholangitis with significant weight loss, elevated serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase and bilirubin, increased histologic grade of cholangitis and number of T cells, macrophages, neutrophils and myofibroblasts per portal tract after 7 days. NKT cells were activated after intrabiliary injection of oxazolone with upregulation of activation markers. Cd1d-/- and wild-type mice pretreated with antibody blocking of CD1d were protected from disease. These findings implicate that cells in the bile ducts function as antigen-presenting cells in vivo and activate NKT cells in a CD1d-restricted manner. The elucidation of this biliary immune pathway opens up for potentially new therapeutic approaches for cholangiopathies.

Anti-cancer activity of Angelica gigas by increasing immune response and stimulating natural killer and natural killer T cells

BackgroundThe polysaccharide component of Angelica gigas induces immuno-stimulatory effects on innate immune cells. However, it is unclear whether A. gigas’ adjuvant activity on the immune system can elicit anti-cancer responses.MethodsA water-soluble immuno-stimulatory component of A. gigas was prepared. How this ISAg modulated the activation of innate immune cells such as dendritic cells (DCs) was examined. ISAg-induced cytotoxic activity via natural killer (NK) and NKT cells was also tested using a tumor-bearing mouse model.ResultsISAg treatment induced nitric oxide (NO) production and cytokine gene expression involved in innate immune responses. ISAg activated macrophages and DCs to secrete cytokine IL-12, through the TLR4 signaling pathway. IL-12 plays a crucial role in ISAg-mediated NK and NKT cell activation. Thus, the anti-cancer activity of NK and NKT cells induced ISAg-mediated cytotoxicity of B16 melanoma cells in mice.ConclusionsThese results indicated that the natural ingredient, ISAg, has adjuvant activity to induce strong anti-cancer activity of NK and NKT cells in vivo.

Activation of natural killer T cells contributes to triptolide-induced liver injury in mice.

Triptolide (TP) is the main active ingredient of Tripterygium wilfordii Hook.f, which has attracted great interest due to its promising efficacy for autoimmune diseases and tumors. However, severe adverse reactions, especially hepatotoxicity, have restricted its approval in the market. In the present study we explored the role of hepatic natural killer T (NKT) cells in the pathogenesis of TP-induced liver injury in mice. TP (600 μg/kg/day, i.g.) was administered to female mice for 1, 3, or 5 days. We found that administration of TP dose-dependently induced hepatotoxicity, evidenced by the body weight reduction, elevated serum ALT and AST levels, as well as significant histopathological changes in the livers. However, the mice were resistant to the development of TP-induced liver injury when their NKT cells were depleted by injection of anti-NK1.1 mAb (200 μg, i.p.) on days -2 and -1 before TP administration. We further revealed that TP administration activated NKT cells, dominantly releasing Th1 cytokine IFN-γ, recruiting neutrophils and macrophages, and leading to liver damage. After anti-NK1.1 injection, however, the mice mainly secreted Th2 cytokine IL-4 in the livers and exhibited a significantly lower percentage of hepatic infiltrating neutrophils and macrophages upon TP challenge. The activation of NKT cells was associated with the upregulation of Toll-like receptor (TLR) signaling pathway. Collectively, these results demonstrate a novel role of NKT cells contributing to the mechanisms of TP-induced liver injury. More importantly, the regulation of NKT cells may promote effective measures that control drug-induced liver injury.

Naturally produced type I IFNs enhance human myeloid dendritic cell maturation and IL-12p70 production and mediate elevated effector functions in innate and adaptive immune cells

There has recently been a paradigm shift in the field of dendritic cell (DC)-based immunotherapy, where several clinical studies have confirmed the feasibility and advantageousness of using directly isolated human blood-derived DCs over in vitro differentiated subsets. There are two major DC subsets found in blood; plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), and both have been tested clinically. CD1c+ mDCs are highly efficient antigen-presenting cells that have the ability to secrete IL-12p70, while pDCs are professional IFN-α-secreting cells that are shown to induce innate immune responses in melanoma patients. Hence, combining mDCs and pDCs poses as an attractive, multi-functional vaccine approach. However, type I IFNs have been reported to inhibit IL-12p70 production and mDC-induced T-cell activation. In this study, we investigate the effect of IFN-α on mDC maturation and function. We demonstrate that both recombinant IFN-α and activated pDCs strongly enhance mDC maturation and increase IL-12p70 production. Co-cultured mDCs and pDCs additionally have beneficial effect on NK and NKT-cell activation and also enhances IFN-γ production by allogeneic T cells. In contrast, the presence of type I IFNs reduces the proliferative T-cell response. The mere presence of a small fraction of activated pDCs is sufficient for these effects and the required ratio between the subsets is non-stringent. Taken together, these results support the usage of mDCs and pDCs combined into one immunotherapeutic vaccine with broad immunostimulatory features.

CD1d-dependent natural killer T cells attenuate angiotensin II-induced cardiac remodelling via IL-10 signalling in mice.

CD1d is a member of the cluster of differentiation 1 (CD1) family of glycoproteins expressed on the surface of various antigen-presenting cells, which is recognized by natural killer T (NKT) cells. CD1d-dependent NKT cells play an important role in immune-mediated diseases; but the role of these cells in regulating cardiac remodelling remains unknown. Cardiac remodelling was induced by angiotensin (Ang) II infusion for 2 weeks. Ang II-induced increase in hypertension, cardiac performance, hypertrophy and fibrosis, inflammatory response, and activation of the NF-kB and TGF-β1/Smad2/3 pathways was significantly aggravated in CD1d knockout (CD1dko) mice compared with wild-type (WT) mice, but these effects were markedly abrogated in WT mice treated with α-galactosylceramide (αGC), a specific activator of NKT cells. Adoptive transfer of CD1dko bone marrow cells to WT mice further confirmed the deleterious effect of CD1dko. Moreover, IL-10 expression was significantly decreased in CD1dko hearts but increased in αGC-treated mice. Co-culture experiments revealed that CD1dko dendritic cells significantly reduced IL-10 mRNA expression from NKT cells. Administration of recombinant murine IL-10 to CD1dko mice improved hypertension, cardiac performance, and adverse cardiac remodelling induced by Ang II, and its cardioprotective effect was possibly associated with activation of STAT3, and inhibition of the TGF-β1 and NF-kB pathways. These findings revealed a previously undefined role for CD1d-dependent NKT cells in Ang II-induced cardiac remodelling, hence activation of NKT cells may be a novel therapeutic target for hypertensive cardiac disease.

Lactobacillus Pentosus Strain S-PT84 Attenuates Insulin Resistance and Steatohepatitis by Maintaining Gut Permeability and Polarizing M2 Macrophages

Obesity or high-fat diet enhances gut permeability and metabolic endotoxemia, which can trigger insulin resistance and NASH. However, there are few promising treatments targeting lipotoxicity-mediated endotoxemia in NASH. The Lactobacillus pentosus strain S-PT84 has immunomodulatory functions including Th1/Th2 balance modulatory effects and natural killer (NK)/NKT cell activation. In this study, we examined the effect of S-PT84 on diet-induced NASH. C57BL/6 mice were fed a high-cholesterol/high-fat diet (CL) alone or with 1×1010 S-PT84 (CL + S-PT) for 22 weeks. We quantified intrahepatic immune cells using a fluorescence-activated cell sorter (FACS). S-PT84 administration improved hepatic steatosis by decreasing TG and FFA levels by 34% and 37%, respectively. S-PT84 also inhibited the development of hepatic inflammation and fibrosis, lowering F4/80+ macrophage/Kupffer cell infiltration and the hydroxyproline content of the liver. S-PT84 administration in mice fed a CL diet led to improved hyperinsulinemia as well as enhanced hepatic insulin signal assessed by phospho-Akt. FACS analysis revealed that mice fed the CL+S-PT had 71% more CD11c-CD206+ M2 macrophages than mice fed the CL diet, resulting in a predominance of the M2 over the M1 macrophage population. S-PT84, however, showed little effect on NK/NKT cell and T regulatory cell populations in the liver. Furthermore, CL diet increased intestinal permeability as measured with the carboxyfluoresce assay and increased plasma lipopolysaccharide binding protein (LBP) levels. S-PT84 diminished increased intestinal permeability and plasma LBP levels. Additionally, S-PT84 reduced IL-17 producing T cells and increased ZO-1, occludin, and claudin proteins constituting tight junction in colon. In conclusion, S-PT84 treatment attenuated lipotoxicity-induced insulin resistance and NASH by maintaining gut permeability and polarizing M2 liver macrophages. Disclosure Y. Sakai: None. M. Nagashimada: None. T. Ota: None.

SAT-016 - Liver sinusoidal endothelial cells are involved in the aggravation of liver injury induced by the activated NKT cells in aged mice

SAT-016 - Liver sinusoidal endothelial cells are involved in the aggravation of liver injury induced by the activated NKT cells in aged mice

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.