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. Author manuscript; available in PMC: 2015 Apr 14.
Published in final edited form as: J Immunol. 2013 Mar 6;190(7):3579–3589. doi: 10.4049/jimmunol.1203170

Exosomes-like nanoparticles from intestinal mucosal cells carry prostaglandin E2 and suppress activation of liver natural killer T cells

Zhong-Bin Deng 2,*, Songwen Ju 2, Xiaoying Zhuang 2, Xiaoyu Xiang 2, Jingyao Mu 2, Yuelong Liu 4, Jiang Hong 2, Lifeng Zhang 2, James Mobley 6, Craig McClain 3, Wenke Feng 3, William Grizzle 4, Jun Yan 2, Donald Miller 2, Mitchell Kronenberg 7, Huang-Ge Zhang 1,2,*
PMCID: PMC4396634  NIHMSID: NIHMS441150  PMID: 23467936

Abstract

Regulation and induction of anergy in natural killer T (NKT) cells of the liver can inhibit autoimmune and anti-tumor responses by mechanisms that are poorly understood. We investigated the effects of prostaglandin E2 (PGE2), delivered by intestinal, mucus-derived, exosome-like nanoparticles (IDENs), on NKT cells in mice. Here, 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-GalCer stimulated liver NKT cells in a PGE2 EP2/EP4 receptor mediated manner. Pro-inflammatory 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.

Keywords: liver disease, mouse model, conA, immune regulation, IL-4

Introduction

The gut-associated immune system is complex and involves extensive communication with the liver(1, 2). Glycolipids derived from dietary metabolic products and lipids derived from some intestinal bacteria(3, 4) migrate constantly into the liver and these lipids can activate liver NKT cells through TCR engagement in vitro(5, 6). Liver NKT cells can also be activated in a TCR independent manner(7, 8). It is remarkable that liver NKT cells are normally quiescent even though they are constantly exposed to intestinal-derived products and are also thought to play an active role in systemic tumor surveillance and maintenance of tolerance(9).

Exosomes are microvesicles released naturally from a variety of cells(10). They carry a cargo of proteins, lipids, mRNAs and/or microRNAs and can transfer their cargo to recipient cells, thus serving as extracellular messengers to mediate cell-cell communication. Numerous recent reports indicate that exosomes have a significant effect on physiological and pathological processes in recipient cells(11). Intestinal epithelial cells release exosomes (30–100nm)(12) and have been shown to be capable of regulating the differentiation and activity of several different types of immune cells(12–14), which suggests the possibility that intestinal-derived exosome-like nanoparticles could play a key role in communication between the intestine and liver and in gut-related induction of immune tolerance or anergic-like state (12, 15).

In this study, we demonstrate that IDENs migrate into the liver and that inflammation promotes this migration. We also demonstrate that IDENs can induce an anergic-like state in liver NKT cells. Induction of a NKT cell anergic-like state by IDENs occurs through a previously undescribed mechanism where the transport of prostaglandin E2 (PGE2) by the IDENs(16) into the liver creates an environment that promotes the NKT cell anergic-like state.

Material and Methods

Isolation and purification of IDENs

Mice were euthanized and the small intestine removed. The luminal contents of the intestine were removed by gently flushing the intestine with 10 mL of ice-cold PBS. The intestine was then opened longitudinally, the mucus collected by mild physical separation using round forceps, and the mucus soaked in PBS before agitating on a rotator at 100 rpm for 15 min. The mucus-PBS mixture was processed by differential centrifugation and sucrose gradient centrifugation to isolate and purify the exosome-like nanoparticles or microparticles (pellets after 10,000g centrifugation) using a previously described protocol(17). The identical protocol was also used for isolation and purification of human intestinal IDENs from surgically removed tissue. Specimens were prepared for EM using a conventional procedure(18), stained with 1% uranyl acetate for 15 seconds, and observed in an FEI Tecnai Spirit T12 electron microscope operated at 200kV at a magnification of 38,000× and defocus of 2.5 μm. Photomicrographs were taken using a Gatan Ultrascan 4000 CCD camera. Protein concentration was determined using the Bio-Rad Protein Quantitation Assay kit with bovine serum albumin as a standard. The levels of PGE2 in IDENs were determined using an ELISA kit (Cayman Chemical).

Mice

C57BL/6 mice 6–12 weeks of age were obtained from Jackson Laboratories. All animal procedures were approved by the University of Louisville Institutional Animal Care and Use Committee.

Mice were administered by gavage or intravenous injection IDENs resuspended in PBS as described previously (19).

Autoimmune hepatitis was induced as described (20) using Con A from Vector Laboratories administered i.v. at 25 mg/kg or 37.5 mg/kg of body weight. For survival studies, mice were injected with 37.5 mg/kg of Con A. Colitis was induced using 3% solution of DSS with a molecular weight of 36,000–50,000 (MP Biomedicals) that was provided as drinking water to mice (18–25 g, 6 weeks old, male) for 5 days. In some experiments, mice were administered indomethacin (4mg/kg/day) (Sigma-Aldrich) in the drinking water.

Clinical Specimens

Human intestinal specimens were collected at the time of gastric bypass surgery. All samples were collected with the informed consent of the patients and the experiments were approved by the Institutional Review Board (IRB) Committee of the University of Alabama at Birmingham.

Cytokine detection

IFN-γ and IL-4 in culture supernatants and liver homogenates were quantified using ELISA kits (eBioscience).

Preparation of MNCs from the liver and flow cytometry analysis

MNCs were prepared from perfused livers (21) that were homogenized with a 70 μm pore filter. After washing extensively, the homogenates were resuspended in a 33% Percoll gradient at 22°C (25 mL per liver) and centrifuged at 2300 rpm for 20 min. The cell pellet was collected and red blood cells were removed using red cell lysis buffer.

For flow cytometry analysis the cells were labeled using standard procedures(22). The following mAbs reactive with murine cells were purchased from Biolegend: CD69 (H1.2F3), CD40L (MR1), TCRαβ (H57-597), and IFN-γ (XMG1.2); these were used as direct conjugates to FITC, PE, PerCP, PE Cy7, or APC. Mouse CD1d tetramer loaded with the α-C-galactosylceramine analogue PBS-57 and conjugated with APC or PE and the unloaded control was provided by the NIH Tetramer Core Facility. Cells were analyzed by flow cytometry using a FACScan (BD Biosciences protocol; BD Pharmingen). Histogram analysis was performed using FlowJo software (Treestar, Ashland, OR).

In vivo imaging of IDENs

Mice were anesthetized with ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) via intraperitoneal injection, and inhaled isoflurane was used as necessary. To monitor the trafficking of IDENs administered by gavage, IDENs were first labeled with a near-infrared lipophilic carbocyanine dye-dioctadecyl-tetramethylindotricarbocyanine iodide (DiR, Invitrogen, Carlsbad, CA) using a previously described method(17). The mice were imaged over a 48-hour period using a Carestream Molecular Imaging system (Carestream Health, Woodbridge, CT). For controls, mice (five per group) received nonlabeled IDENs in PBS at the same concentration as for DiR dye-labeled exosomes. Images were collected using a high-sensitivity CCD camera with an exposure time for imaging of 2 minutes. Regions of interest were analyzed for signals and were reported in units of relative photon counts per second. The total photon count per minute was calculated (five animals) using Living Image software. The effects of DiR dye-labeled versus nonlabeled IDENs on mice was determined by dividing the number of photons collected for DiR dye-labeled IDEN treated mice by the number of photons collected for nonlabeled IDEN treated mice at different imaging time points. Results were represented as pseudocolor images indicating light intensity that were superimposed over grayscale reference photographs.

Assessment of liver damage

The quantity of ALT and AST in the sera of the mice was measured using the Infinity Enzymatic Assay Kit (Thermo Scientific). For histopathology, H&E staining was performed on paraffin-embedded liver sections. For immunofluorescence analysis, OCT (Sakura Finetek)-embedded tissue cryosections (9μm-thick) were fixed at −20°C in methanol/acetone (3:1). Slides were hydrated in PBS and blocked for 30 min at 25°C with Fc Block (10 μg/mL) and 5% (vol/vol) normal horse serum in PBS. After blockade, slides were incubated for 30 min at 25°C with ‘cocktails’ containing the following primary antibodies in PBS: anti-A33 (Santa Cruz Biotechnology), anti-CD31 (MEC 13.3, BD Biosciences), and anti-Lyve-1 (R&D Systems). Primary antibodies were detected with Alexa Fluor 647–donkey anti-goat, rabbit antibody to fluorescein isothiocyanate–Alexa Fluor 488 (A11090), (S21381; all from Invitrogen Life Sciences) or streptavidin-allophycocyanin (BD Biosciences). Slides were mounted with Slow Fade Gold Antifade plus DAPI (4,6-diamidino-2-phenylindole; S36938; Molecular Probes and Invitrogen Life Sciences). Tissues were assessed using a Zeiss LSM 510 confocal microscope equipped with a digital image analysis system (Pixera). Apoptosis was evaluated using the TUNEL assay performed on frozen liver sections using the in situ cell death detection kit (Roche).

Cell sorting

For measuring cytokine induction, NK1.1+CD5+ NKT cells were sorted from liver leukocytes using a method as described(23). The purity of the NKT cells collected, typically >90%, was assessed using CD1d tetramers loaded with α-galactosylceramide (provided by the NIAID Tetramer Facility, NIH). For proliferation assays, NKT cells were also enriched from liver by negative magnetic bead sorting (Miltenyi Biotec) using anti-CD11b, B220, CD8α, Gr-1, CD62L and CD11c antibodies using a standard protocol(24). Although CD4 is not depleted in this case, the specificity of NKT cell proliferation was determined by stimulation of α-GalCer which specifically activates NKT cells but not CD4 T cells. The purity of NKT and CD11c cells was determined by FACS analysis as described (24)

Cell proliferation and CFSE dilution analysis

NKT cells enriched by negative selection (4×105) were cultured with liver DCs (1×105) isolated by CD11c magnetic bead sorting (Miltenyi Biotec). In vitro cultures were incubated for 72 h under the indicated culture conditions and [3H]-thymidine was added for the last 16h. Incorporation of radioactivity was determined using a Betaplate liquid scintillation counter (Packard Bioscience). Results are expressed as mean cpm of triplicate wells ± SEM. For CFSE (Invitrogen) dilution analysis, splenocytes were labeled with 5 μM CFSE for 10 minutes at 37°C in PBS containing 0.1% BSA and washed twice with complete RPMI medium. Labeled NKT cells (4×105 cells/well) were cultured with liver DCs (5×104) under the indicated culture conditions for 96 h in complete RPMI1640 and stained with TCR-β and α-GalCer/CD1d. CFSE dilution was examined on gated α-GalCer/CD1d tetramer+ NKT cells.

Western Blot Analysis

Western blots were carried out as described previously (19). In brief, cells or exosomes were lysed in protein lysis buffer containing 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 5 mM sodium molybdate, and 20 mM phenylphosphate with protease and phosphatase inhibitors (1 mM PMSF, 10 μg/mL aprotinin, 20 μg/mL leupeptin, 20 μg/mL pepstatin A, 50 mM NaF, and 1 mM sodium orthovanadate). Proteins of lysed cells were separated on 10% polyacrylamide gels using SDS-PAGE and transferred to nitrocellulose membranes. The membranes were stained with Ponceau Red to validate that all samples contained similar amounts of protein.

RNA extraction and PCR

Total RNA was isolated from livers or NKT hybridomas with TRIzol according to the manufacturer’s specifications (Invitrogen) and the RNA repurified with an RNeasy mini kit (Qiagen). RNA (1μg) was reverse-transcribed with Superscript III and random primers (Invitrogen). For quantitation of genes of interest, cDNA samples were amplified in a CFX96 Realtime System (Bio-Rad Laboratories, Hercules, CA, USA) and SsoFast Evagreen supermixture (Bio-Rad Laboratories) according to the manufacturer’s instructions. Fold changes in mRNA expression between treatments and controls were determined by the δCT method as described(25). Differences between groups were determined using a two sided Student’s t-test and one-way ANOVA. Error bars on plots represent ± SE, unless otherwise noted. The data were normalized to a GAPDH reference. All primers were listed in the supplemental Table 1.

Agonists, antagonists, and inhibitors

IDEN lipids were extracted according to Bligh and Dyer(26) in chloroform/methanol/water (2.5:2.5:2.1, v/v/v). Lipids for treatment of cells were dried under nitrogen and sonicated in PBS at pH 7.4 and 0.05% (vol/vol) Tween-20 before use. Polyclonal anti-A33 antibody was purchased from Santa Cruz Biotechnology, cAMP analogs (dibutyryl cAMP, N6-Bnz-cAMP), PKA inhibitors (H-89, Rp-8-CPT-cAMPS and Rp-8-Br-cAMPS) were purchased from Sigma-Aldrich. PGE2, Butaprost, misoprostol, sulprostone and PGE2 ELISA kits were obtained from Cayman Chemical. EP4 agonists (ONO-AE1-329), EP1 antagonist (ONO-8713), EP3 antagonist (ONO-AE3-240), and EP4 antagonist (ONO-AE3-208) were kind gifts from Ono Pharmaceutical Company.

Cell cultures

Bone marrow-derived dendritic cells (BMDCs) were cultured as described previously(27). To study the activity of IDENs or IDEN lipid, total liver mononuclear (4 × 105 /well) or BMDCs cultured with FACS sorted NKT cells (1 × 105 BMDCs + 4× 105 NKTs/well) or in the presence of IDENs (30μg/mL) for 3h, and then stimulated by α-GalCer (100ng/mL) for 24–48 hr in round bottom 96-well plates containing RPMI supplemented with 10% FCS. In some experiments, sorted NKT cells pretreated with IDENs were stimulated by α-GalCer-tetramer. In some cases, 100 μM PGE2, 10 μM butaprost (EP2 agonist), 40 μM misoprostol (EP4 agonist), 10 μM sulprostone (EP1/EP3 agonist), or 100 μM of dibutyryl-cAMP, 100 μM N6-Bnz-cAMP, 3 mM H-89, 100 mM Rp-8-Br-cAMPS or 100 mM Rp-8-CPT-cAMPS were added to cocultures. Cells were assessed by flow cytometry or supernatants were used for cytokine measurement by ELISA.

Liposomes assembled from IDENs

Liposomes were assembled from IDENs used the protocol as described (28) with minor modification as described below: 1 mL of IDENs in PBS were mixed together with 3.75 mL of chloroform/methanol at a 1:2 ratio (v/v) in borosilicate glass test tubes, before adding 1.25 mL chloroform to the mixed sample and vortexing the mixture. Subsequently, 1.25 mL deinonized water was added and the mixture vortexed again. The sample was centrifuged at 1000 RPM in a table-top microcentrifuge for 5 min at 22°C. The bottom phased liquid that contained lipids was collected using a pasteur pipette. Solvent in the collected sample was completely removed via a constant flow of nitrogen gas at 60°C. Extracted IDEN lipids were hydrated with 800μL of Milli-Q water prior to bath sonication for 3 min, and then sonicated for an additional 2 min after addition of HEPES buffered saline (final contration, 10 mM Hepes, 50 mM NaCl). This resulted in a clear dispersion being produced that was stored at 4°C for 48 h prior to further experimentation or examination for nanoparticle formation using a conventional procedure(29).

Coupling of IDENs or microparticles to latex beads and immunofluorescence analysis

IDENs or microparticle-coated beads were prepared using a conventional procedure(29) and washed twice in PBS containing 10% FCS, stained with antibody directed against Annexin V (eBioscience), and analyzed using a BD Biosciences FACSCalibur. A “bead-only” control and an isotype-matched Ab control were prepared and fluorescence intensity was normalized based on these controls.

Statistical analysis

Survival data were analyzed using the logrank test. Student’s t-test was used for comparison of two samples with unequal variances. One-way ANOVA with Holm’s post hoc test was used for comparing means of three or more variables.

Results

IDENs migrate into the liver and inflammation promotes this migration

Using standard techniques(30), we isolated nanoparticles from the mucus of the small intestines of naïve C57BL/6 (B6) mice that were identifiable as exosome-like nanoparticles based on electron microscopic examination and their expression of exosomal markers (Fig. 1A). Western blot analysis for A33, a marker of intestinal epithelial cells (31), further demonstrated that the preparation contained a high proportion of intestinal epithelial cell-derived IDENs. These results were further validated by western blot analysis (Fig. 1A and B, right panel) of exosome makers including RAb5 and TSG101 or the microparticle marker, Annexin V(32, 33). Annexin V predominantly detected in microparticles was demonstrated by FACS analysis of Annexin V (Fig. 1B, left panel). Electron micrographs (Fig. 1C, left panel) show that IDENs were also present in the small intestine of human, and shared a number of exosomal protein markers identified on mouse IDENs (Fig. 1C, right panel).

Figure 1.

Figure 1

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Characterization of IDENs isolated from intestinal mucus. Intestine from B6 mice (A) or human (C) was used for isolation of nanoparticles or microparticles (B) by differential centrifugation as described previously (27). Sucrose-purified nanoparticles were examined by electron microscopy (A and C, left panel) (Bar = 100 nm). FACS analysis was performed to determine expression of Annexin V on the surface of IDENs or microparticles derived from mouse intestinal mucus (B, left panel). The presence of intestinal epithelial-specific marker (A33, villin-1) and/or exosome markers (CD63, CD9, CD81, RAB5, RAB11and HSP70) in the sucrose gradient-purified exosomes from the supernatants of 36-h cultured TS/A mouse breast tumor cells(51), mouse intestinal nanoparticles (A), mircoparticles (B) or human intestinal nanoparticles (C) were determined by western blot analysis (A, B and C, right panel). The presence of Annexin V was determined by FACS analysis (B, left panels). The imaging and western blot is representative of at least three experiments. (D) In vivo imaging of, and quantitation of signals in C57BL/6 mice gavage administered IDENs labeled with the infra-red dye DiR (100 μg/mouse in 100 μL PBS) or nonlabeled IDENs in PBS; and (E) images of the washed intestines (cut longitudinally) and other organs obtained 48 h after administration of IDENs. (F) Confocal image analysis of frozen sections of the livers of mice administered IDENs labeled with PKH26 red dye. Sections stained for A33, an intestinal epithelial cell-specific marker (green) and nuclei stained with DAPI. Distribution of IDENs in the intestines of mice 12 h after gavage administration of IDENs labeled with DiR dye (100 μg in 100 μL of PBS) assessed by (G) confocal image analysis of the frozen sections of intestine with immunofluorescent staining for A33 (green) and CD31 (red), original magnification ×40. (H) Confocal image analysis of the frozen sections of intestine with immunofluorescent staining for LYVE (green), original magnification ×60 (left panel) with enlargement of indicated area shown in the right panel. (I) Accumulation of DiR dye-labeled IDENs (100 μg/mouse in 100 μL PBS) administered by gavage 24 h after treatment with Con A, DSS, of PBS representative scan. Data are the mean ± SEM of three experiments (D) or representative of three experiments (E–I) (n = 10 mice per group).

Intestinal mucus is a physically cross-linked hydrogel with mesh sizes on the order of 10–100 nm(34, 35). Penetration of this barrier is largely dependent on the size of the particles (34, 35). The size of the IDENs is approximately 30–100 nm and we used gavage administration of IDENs labeled with an infrared fluorescent membrane dye (DiR) to track their migration patterns in vivo(17). Imaging revealed an accumulation of fluorescent signals in the gut over the first 24 h (Fig. 1D). At 48 h, signals were detectable in dissected liver and mesenteric lymph nodes but not the lung, spleen (Fig. 1E) or other organs (data not shown). The presence of IDENs in the liver was confirmed by immunohistology and costaining for A33 (Fig. 1F). The colocalization of the DiR and A33 signals with CD31, a marker of endothelial cells (Fig. 1G), and LYVE1, a marker of lymphatic capillaries (Fig. 1H), along the length of the intestinal endothelial and lymphatic vessels within 6 h of administration of IDENs suggests that the IDENs migrate into the liver from the gut primarily through the vascular and lymphatic vessels. The migration of A33-positive IDENs to the liver (Fig. 1I) was enhanced in mice treated with either concanavalin A (ConA), a lectin that induces inflammatory liver injury in mice and is a model of autoimmune liver disease(36), or dextran sodium sulfate (DSS), which induces colonic inflammation with characteristics of acute ulcerative colitis. Thus, IDENs can gain access and traffic within the vascular or lymphatic system of the liver. Inflammation in the liver or colon enhances this migration.

IDENs protect against inflammation-mediated liver damage

To explore the biological effects of the accumulation of IDENs in the liver, we tested the effects of pretreatment of mice with purified IDENs on ConA-induced liver injury(36). Mice that were pre-treated with vehicle only succumbed rapidly (Fig. 2A) and exhibited symptoms characteristic of Con A-induced liver injury, including elevated levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Fig. 2B). In addition there were histopathological changes in the liver (Fig. 2C) with extensive areas of confluent hepatocellular necrosis, marked sinusoidal hyperemia associated with hemorrhage, and mononuclear cell infiltrates scattered diffusely throughout the viable parenchyma. Pretreatment with IDENs markedly enhanced the survival of the mice (Fig. 2A), dampened the Con A-induced elevation in the levels of serum ALT and AST (Fig. 2B) and limited the liver histopathology to minor inflammatory infiltrates and small necrotic patches (Fig. 2C). Alleviation of liver injury appeared to be associated with a reduction in inflammatory responses because mice pretreated with IDENs had serum levels of the proinflammatory cytokines IFN-γ and TNF-α that were significantly lower than in the mice that were pretreated with vehicle only (Fig. 2D). Furthermore, the administration of IDENs reduced apoptosis of liver cells, as assessed by TUNEL staining (Fig. 2E).

Figure 2.

Figure 2

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IDENs protect mice against Con A–induced liver damage. (A–E) C57BL/6 mice were administered IDENs (100 μg/mouse in PBS, injected intravenously every two days for 14 days) prior to injection of Con A (37.5 mg/kg of body weight, injected intravenously 4 h after the last injection of exosomes) and assessed at specified times (n = 10 mice per group) for: (A) survival of mice post Con A injection and (B) levels of ALT and AST in the sera. **P < 0.01 as compared with mice injected with PBS (Student’s t-test); (C), H&E stained sections of the livers, original magnification ×20. (D) Levels of IFN-γ and TNF-α in the sera. **P < 0.05 and **P < 0.01 as compared with mice injected with PBS (Student’s t-test). (E) Apoptosis in the liver as assessed by TUNEL analysis.

IDENs induce hyporesponsiveness of liver NKT cells to α-GalCer

Con A-induced hepatitis is also dependent on NKT cell activity(37). Unlike CD4/CD8 T cells, NKT cells respond to glycolipid ligands. The glycolipid α-GalCer is a potent stimulator of NKT cell expansion when first administered but loses its effectiveness on repeated administration(9). To determine the effects of accumulation of IDENs in the liver on α-GalCer-driven NKT cell expansion, mice were injected intravenously with IDENs every two days for two weeks and 3 h after the last injection the mice were injected intravenously with α-GalCer (5μg/mouse). The rationale for choosing a two week treatment schedule was based on data published by others (24). In naïve mice, a single injection of α-GalCer caused TCR downregulation of NKT cells within 24h and a decrease in the percentages of TCR+ NKT cells recovered 3 days after the injection. This result is also consistent with what has been reported by others(24). In contrast, pretreatment with IDENs dampened the recovery of TCR+ NKT cells after α-GalCer injection (Fig. 3A) and reduced the α-GalCer-induced expression of the markers of early activation of NKT cells, i.e., CD69 and its costimulator CD40 ligand (Fig. 3B). Moreover, the α-GalCer-induced elevation of ALT and AST serum levels (Fig. 3C). production of IFN-γ and IL-4 (Fig. 3D) by liver NKT cells was reduced substantially in mice that had been pretreated with IDENs, indicating that IDENs can induce an anergic-like state in NKT cells exposed to inflammatory signals in vivo.

Figure 3.

Figure 3

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IDENs induce an anergic-like state in NKT cells. (A–D) C57BL/6 mice were injected intravenously with IDENs (100 μg/mouse in PBS) every 2 d for 14 d, and 4 h after the last injection of IDENs the mice were injected with α-GalCer (5μg/mL, intravenously). (A) Frequency of TCR-β+ CD-1d tetramer+ cells (NKT cells) in mononuclear cell preparations from the liver at indicated time points after α-GalCer treatment. (B) Expression of CD40L and CD69 on the TCR-β+ CD-1d tetramer+ cells after α-GalCer treatment for 24 h. (C and D) Levels of ALT and AST (C) and IFN-γ and IL-4 (D) in the serum. Data are representative of three experiments (A, B), or are the mean ± SEM of five experiments (C, D). *P < 0.05 and **P < 0.01 (Student’s t-test).

To directly test whether IDENs can induce an anergic-like state in NKT cells to glycolipids, we purified NKT cells from the livers of mice that had been administered IDENs or vehicle intravenously and cocultured NKT cells in vitro with dendritic cells from the livers of untreated mice in the presence of α-GalCer. The results confirmed that the NKT cells purified from the mice that had been administered IDENs were hyporesponsive to α-GalCer stimulation (Fig. 4A). Consistent with this, the addition of IDENs to liver NKT cells in culture suppressed the ex vivo proliferative response of the NKT cells to α-GalCer (Fig. 4B). We also analyzed the responses of FACS-sorted cells in vitro. Bone marrow-derived dendritic cells (BMDCs) were co-cultured with naïve liver NKT cells treated with IDENs or PBS for 3 h and then stimulated with α-GalCer for an additional 24 h (Fig. 4C); or FACS-sorted liver NKT cells were treated with IDENs or PBS for 3 h and subsequently stimulated with α-GalCer tetramer (Fig. 4D). Analysis of the supernatants indicated that the α-GalCer-induced production of IFN-γ and IL-4 by the NKT cells was significantly reduced when IDENs were added to either NKT cells co-cultured with DCs pulsed with α-GalCer (Fig. 4C) or purified NKT cells stimulated with tetramer (Fig. 4D).

Figure 4.

Figure 4

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Ex vivo analysis of NKT cell responses to IDENs and α-GalCer. (A) Sorted NKT cells from mice treated with PBS or IDENs and CD11c+ DCs sorted from livers of naïve mice were co-cultured for 3 d in the presence of α-GalCer. Cell division was assessed by labeling the NKT cells with CFSE (5 μM). (B) Proliferation of NKT cells from naïve mice were cultured with liver DCs in the presence of IDENs or PBS for 3h, and then stimulated by α-GalCer. The proliferation of the NKT cells was determined after a 16-hour pulse with 3H-thymidine and the cultured cells subsequently harvested. (C) Cytokine production by liver NKT cells from naïve mice cultured with BMDCs in the presence of IDENs or PBS for 3h, and then stimulated by α-GalCer (100ng/mL) for 24h. (D) Cytokine production by NKT cells from naïve mice pretreated by IDENs or PBS for 3h, and then stimulated by α-GalCer-tetramer for 24h. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of three experiments (A) or are the mean ± SEM of five experiments (B, C, D).

IDEN-associated prostaglandin E2 inhibits α-GalCer-induction of NKT cell activation

We found that liver NKT cells stimulated in vitro with αGalCer in the presence of lipids extracted from IDENs produced much less IFN-γ and IL-4 (Fig. 5A), suggesting that the lipid content of the IDENs plays a role in the IDEN-induced effects on NKT cells. Liposomes assembled from IDENs also have a similar effect as IDENs and IDEN lipids (Supplemental Fig. 1), suggesting that the structure of liposomes assembled from IDEN lipids may not be essential for IDEN mediated inhibition of activation of NKT cells stimulated by αGalCer. Our data(38) and that of others(39) indicate that PGE2 from various sources is enriched on exosomes and we found that the IDENs carried significant amounts (greater than 80 ng/μg) of PGE2 (Fig. 5B). PGE2 is known to regulate T cell activation through the modulation of expression of PGE2 receptors on T cells(40). Whether the expression of PGE2 receptors on NKT cells are regulated by α-GalCer-stimulation is not known. RT-PCR analysis of the expression of the four known PGE2 receptor subtypes indicated selective upregulation of the EP2 and EP4 receptors on α-GalCer-stimulated liver NKT cells (Supplementary Fig. 2). This suggested the possibility that the IDEN-associated PGE2 could play a role in the induction of the NKT cell anergic-like state. Next, we tested the effects the amount indomethacin (a cyclooxygenase 2 inhibitor that blocks the generation of PGE2) would have on the association of PGE2 with IDENs. ELISA results indicated that indomethacin treatment reduced the amount of PGE2 associated with the IDENs (Fig. 5B). When comparing the IDENs isolated from the intestine of naïve mice pretreated with vehicle only to the IDENs isolated from the intestine of indomethacin treated mice, the IDENs from indomethacin treated mice were less potent at inducing the anergic-like state of NKT cells exposed to α-GalCer stimulation (Fig. 5C). Indomethacin treatment resulted in promoting Con-A-induced mouse mortality (Fig. 5D), more severe hepatocellular necrosis (Fig. 5E), and higher sera levels of IFN-γ and ALT (Fig. 5F). Our data (Fig. 6A) and the data published by others suggest that there was a selective decrease in liver NKT cells in DSS induced colitis(41). Whether IDENs isolated from DSS induced colitis have less capacity to inhibit live NKT cell activation is unknown. We found that in comparison with IDENs isolated from the intestine of naïve mice, the IDENs isolated from the intestine of mice pretreated with 3% DSS in drinking water are less potent in inducing the IDEN-mediated anergic-like state of NKT cells to α-GalCer stimulation (Fig. 6B). This reduction appears to be associated with a decreased amount of IDEN PGE2 (Fig. 6C). PGE2 is also known to modulate CD4 T cell activation, and our data indicated that IDENs or PGE2 treatment leads to a significant reduction of both IFN-γ and IL-2 produced by ConA induced CD4 T cells activation (Fig. 6D), suggesting that IDEN PGE2 may play a role in the inhibition of CD4 T cell activation, as well as in the ConA induced mouse liver inflammation model.

Figure 5. IDEN PGE2 induces an anergic-like state in NKT cells.

Figure 5

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(A) Levels of IFN-γ and IL-4 in supernatants from sorted liver NKT cells cultured with BMDCs in the presence of IDENs or total lipids isolated from IDENs for 3 h and then stimulated by α-GalCer (100ng/mL) for 24 h. (B) ELISA analysis of PGE2 levels in IDENs from naïve or indomethacin-treated C57BL/6 mice. (C) Levels of IFN-γ and IL-4 in supernatants of sorted liver NKT cells cultured with BMDCs in the presence of IDENs isolated from naïve C57BL/6 mice or indomethacin-treated mice for 3 h, and then stimulated by α-GalCer (100ng/mL) for 24 h. (D–F) Exacerbation of liver injury by indomethacin in mice treated with Con A. C57BL/6 mice (n=5) were administered indomethacin (4mg/kg/day) orally in the drinking water for one week. Con A was administered intravenously at 37.5 mg/kg of body weight. (D) Effects of indomethacin pretreatment on survival in mice treated with Con A. Survival curves are depicted as time after Con A injection. (E) H&E-stained sections of livers from Con A-injected mice pretreated with PBS or indomethacin, original magnification ×20. (F) Levels of IFN-γ and ALT were assessed in serum at different time points in Con A-injected mice pretreated by PBS (PBS-Con A) or indomethacin (indomethacin-Con A). *P < 0.05 and **P < 0.01 (one-tailed unpaired Student’s t-test). Data (A–C, D, F) are the mean ±SEM of 3 experiments (n=7). (E) is representative of at least three independent experiments (n=7) (E).

Figure 6.

Figure 6

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IDENs derived from DSS induced colitis mouse model have significantly less suppression of liver NKT cell activation. (A) Frequency of TCR-β+ NK1.1+ cells (NKT cells) in mononuclear cell preparations from the liver at day 8 after mice were treated with 3% DSS in drinking water. (B) Levels of IFN-γ and IL-4 in supernatants of sorted liver NKT cells cultured with BMDCs in the presence of IDENs isolated from naïve C57BL/6 mice or DSS-treated mice for 3 h, and then stimulated with α-GalCer (100ng/mL) for 24 h. (C) ELISA analysis of PGE2 levels in IDENs from naïve or DSS-treated C57BL/6 mice. Data are representative of three experiments (A), or are the mean ± SEM of five experiments (B, C). **P < 0.01 (Student’s t-test). (D) Levels of IFN-γ and IL-2 in supernatants of sorted naïve CD4 T cells in the presence of IDENs or PGE2 for 3 h, and then stimulated by Con A (20μg/mL) for 24 h. Data are the mean ± SEM of five experiments. **P < 0.01 (Student’s t-test).

Although the immunomodulatory effects of PGE2 are well known(42, 43), PGE2 has not been implicated previously in modulation of NKT cell activity. We found that addition of exogenous PGE2 to monocytes isolated from the livers of naïve mice (Fig. 7A) or to cocultures of mature BMDCs with NKT cells purified from the livers of naïve mice (Fig. 7B) led to inhibition of α-GalCer-induced production of both IFN-γ and IL-4 in a PGE2 dose dependent manner. Similar results were obtained using the NKT1.2 NKT hybridoma (Supplementary Fig. 3). Addition of either EP2- or EP4- selective agonists to cocultures of FACS-purified liver NKT cells with BMDCs in the presence of α-GalCer suppressed the secretion of IFN-γ and IL-4 (Fig. 7C); whereas, agonists selective for EP1 and EP3 had no effect (data not shown). Furthermore, the addition of either the EP4 antagonist, ONO-AE3-208 (Fig. 7D), or the EP2 antagonist, AH6809 (Fig. 7E), partially reversed the PGE2 or IDEN-mediated inhibition of IFN-γ and IL-4 production, whereas EP1 and EP3 antagonists had no effect (data not shown). These findings lead to the novel conclusion that PGE2 can inhibit the production of IFN-γ and IL-4 in α-GalCer-stimulated NKT cells and that these effects are mediated by the EP2 and EP4 receptors.

Figure 7. IDEN PGE2 induces an anergic-like state in NKT cells via the EP2/EP4 signaling pathway.

Figure 7

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Levels of IFN-γ and IL-4 in supernatants of liver leucocytes (A) or sorted hepatic NKT cells (B–E) co-cultured with BMDCs that were pretreated for 3 h with the following agents, and then stimulated with α-GalCer for 24 h. The agents were diluted PGE2 (A, B), EP2 (10 μM butaprost) or EP4 (100 nM ONO-AE1-329) agonist (C) or in combination with PGE2 or IDEM with/without EP4 (1μM ONO-AE3-208) or EP2 (AH6809) antagonist (D–E). *P < 0.05 and **P < 0.01 (one-tailed unpaired Student’s t-test). Data (A–E) are the mean ±SEM of 3 experiments (n=5).

The cAMP-PKA pathway plays an essential role in the IDEN-associated PGE2-dependent induction of NKT cell anergy

Both EP2 and EP4 are Gαs protein–coupled receptors, which upon binding PGE2 act to increase the levels of intracellular cAMP(44). We found that addition of a dibutyryl cAMP analogue (dB-cAMP), a PKA-specific agonist (N6-Bnz-cAMP), or forskolin, a downstream activator of the cAMP-mediated pathway, suppressed the in vitro production of IFN-γ and IL-4 by NKT cells (Fig. 8A). Furthermore, both the IDEN- (Fig. 8B) and PGE2- (Fig. 8C) mediated suppression of IFN-γ and IL-4 production was inhibited by H-89, a PKA inhibitor, or Rp-8-Br-Camps and Rp-8-CPT-cAMPS, type I and type II PKA inhibitors, respectively, confirming that the IDEN-mediated inhibition of IFN-γ and IL-4 production by NKT cells requires PGE2-EP2/EP4 signaling and is dependent on the cAMP/PKA pathway.

Figure 8. IDEN PGE2 induces an anergic-like state in NKT cells via EP2/EP4 mediating the cAMP-PKA pathway.

Figure 8

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Levels of IFN-γ and IL-4 in supernatants of sorted hepatic NKT cells co-cultured with BMDCs that were pretreated for 3 h with the following agents, and then stimulated with α-GalCer for 24 h. The agents were db-cAMP (100 μM), N6-Bnz-cAMP (100 μM), Forskolin (10 μM) (A), or in combination with IDENs (B) or PEG2 (C) with/without H-89 (3 μM), Rp-8-Br-cAMPS (100 μM) or Rp-8-CPT-cAMPS (100 μM). ELISA of IFN-γ and IL-4 in the cultured supernatants. *P < 0.05 and **P < 0.01 (one-tailed unpaired Student’s t-test). Data (A–C) are the mean ±SEM of 4 experiments (n=5).

Discussion

In this study, we demonstrate that IDENs migrate to the liver where they induce a NKT cell anergy-like state. We also establish for the first time that a PGE2-mediated pathway plays a role in the induction of the anergic-like state in NKT cells, which can occur through a cAMP/PKA activity-dependent mechanism. The rapid enhancement of the activity occurs through a traditional positive feedback mechanism in which the PGE2-induced enhancement of expression of the EP2 and EP4 receptors can amplify PGE2 signaling in NKT cells. Notably, the activation of the PGE2 pathway also appears to create in the liver a microenvironment in which naïve NKT cells are resistant to activation by α-GalCer stimulation, which is one of the major obstacles in the use of α-GalCer for the treatment of certain types of cancer.

It is a reasonable assumption that control of the balance between the anergic-like state and activation of NKT cells is a complex process involving the integration of numerous signals. The current studies pin-point a regulatory mechanism by which IDENs can influence this balance through mechanisms that involve considerable cross-talk between intestinal IDENs, PGE2 and NKT cells. For example, in the current studies we found that inflammation induced by either Con A injection or drinking DSS water can alter the amount of PGE2 carried by the IDENs. Specifically, our data suggest that although inflammation induced by Con A or DSS induced colitis resulted in increasing amounts of IDENs accumulating in the liver and significant reduction of PGE2 carried by IDENs which made them less potent for inhibiting α-GalCer stimulated liver NKT cell activation. This suggests that different microenvironmental conditions may tailor the ability of the IDENs to either promote or suppress NKT cell activation. Although two acute inflammatory mouse models were used in this study, IDENs derived from chronic inflammatory driven disease models such as colon cancer may have different phenotypes, which we have not addressed in this study. In addition, although the regulator that controls the amounts of PGE2 carried by IDENs has not been identified, we are interested in determining whether IDENs prefer carrying PGE2 rather than other prostaglandins regulated by Cox1 and Cox2. Furthermore, COX-1 is constitutively expressed in most tissues including intestine. In contrast, COX-2 is induced in inflammation by various stimuli including cytokines and growth factors. Whether IDENs have a higher capacity in preferentially binding prostaglandins regulated by Cox1 than Cox2 needs to be addressed by comparing lipidomic profiles of IDENs isolated from Cox1 and Cox2 knockout mice.

We also speculate that exosome or exosome-like nanoparticle recipient cells may also interact in a coordinated manner with the exosome producing cells. For an example, when the host microenvironment is under “fire” such as in infection or when there is an accumulation of damaged cells, inflammation is required for removing these cells. In this case, immune-suppressors such as PGE2 carried by exosomes or exosome-like nanoparticles must be reduced for the receipt cells to be activated. Coordinated interactions of exosome or exosome-like nanoparticle producing cells with recipient cells may cause a subsequent release of particles predominantly packed with pro-inflammatory molecules that further amplify inflammatory responses via paracrine and autocrine pathways until the infected or damaged cells are removed. As a result, the newly achieved balance between pro-inflammatory and anti-inflammatory factors may be more related to the ratio of pro-inflammatory and anti-inflammatory factors to each other than absolute quantities of each. Therefore, it is conceivable that the data presented in Figure 2D, where the sera levels of TNF-α and IFN-γ are higher than those from naïve mice, that the overall balance or ratio of pro-inflammatory and anti- inflammatory factors is achieved such that liver damage due to Con A injection was prevented. Dysregulation of this balance may contribute to the development of disease. This assumption is supported by recent results reported by another groups. Kornek showed that activated T-cells(45) released microparticles and that the levels of released microparticles correlated with the histological severity of patients with active hepatitis C. Another study (46) indicated that microparticles derived from CD14+ monocytes and NKT cells are critical to adipose liver inflammation of patients with NAFL/NASH. Further research is required to determine whether IDEN-mediated inhibition of liver NKT activation also has an inhibiting effect on the releasing of pro-inflammatory microparticles from NKT.

A large number of glycolipids that can activate NKT cells have been studied; whereas, only a few of the NKT inhibitory lipids have been studied. Earlier studies demonstrated that neutral glycolipid, Gg3Cer shed from a tumor cell line can inhibit CD1d1-mediated antigen presentation to NKT cells(47). Di-palmitoyl-phosphatidyl-ethanolamine (DPPE) (48) covalently attached to polyethyleneglycol (PEG) acts as an antagonist to inhibit the α-GalCer-induced phosphorylation of ERK tyrosine kinase in NKT cells, thus blocking NKT TCR signaling and activation of NKT cells. However, under physiological conditions these lipid complex are not detected in vivo, and mechanism(s) underlying how the anergic-like state in liver NKT cells is maintained is not known. Data presented in this study suggest that intestinal mucus derived PGE2 carried by exosome-like nanoparticle-mediated can induce the NKT cell anergy-like state in mouse models. This finding opens up a new avenue to further study whether other intestinal-related antigens carried by intestinal mucus derived exosome-like nanoparticles play a role in induction of immune tolerance.

Although the role of IDEN associated PGE2 in the induction of the NKT cell anergic-like state are addressed in this study, we speculate that this finding may also be applied to other types of cells that express PGE2 receptors. A large body of evidence suggests that PGE2 receptors are expressed on not only NKT, but also CD4 T cells(49) and other types of cells (50) as well. However, if those cells do not have the in vivo capacity of taking up IDENs, PGE2 carried by IDENs may not have an effect on these cells even if these cells are PGE2 receptor positive.

In summary, to our knowledge this is the first study to demonstrate a PGE2 role in inducing NKT cell anergy. Our data support a model in which the PGE2 carried by IDENs is taken up by antigen presenting cells or NKT cells of the liver and then initiates a unique suppressive mechanism. This mechanism does not require recognition of lipid antigens. In addition, the IDENs work as a functional bridge between the intestinal and liver immune systems. We propose that this mechanism of suppression is a major pathway that counterbalances intestinal lipid-mediated activation of NKT cells in the liver.

Supplementary Material

1

Acknowledgments

This work was supported by grants from the National Institutes of Health (NIH) (RO1CA137037, R01CA107181, RO1AT004294, and RO1CA116092); the Louisville Veterans Administration Medical Center (VAMC) Merit Review Grants (H.-G.Z.); and a grant from the Susan G. Komen Breast Cancer Foundation.

We thank the NIH Tetramer Facility for providing PBS-57 ligand complexed to CD1d monomers or tetramers, Ono Pharmaceutical Company for providing EP4 (ONO-AE1-329), EP1 antagonist ONO-8713, EP3 antagonist ONO-AE3-240, EP4 antagonist (ONO-AE3-208), and Dr. Mitchell Kronenberg who provided the NKT1.2 hybridoma. We thank Drs. Fiona Hunter and Jerald Ainsworth for editorial assistance.

Footnotes

Contribution: Z.D. and H.-G.Z. designed the research, analyzed and interpreted data, and drafted the manuscript; D.M., M.K., C.M., W.G interpreted findings and drafted the manuscript; and X.Z., S.J., X.X., Y.L., J.M., J.H., L.Z., J.M., W.F., and J.Y., performed experiments and interpreted data.

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1
NIHMS441150-supplement-1.pdf (208.8KB, pdf)

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