Exosome-like Nanoparticles from Ginger Rhizomes Inhibited NLRP3

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Exosome-like Nanoparticles from Ginger Rhizomes Inhibited NLRP3 Inflammasome Activation Xingyi Chen,† You Zhou,‡ and Jiujiu Yu*,† †

Department of Nutrition and Health Sciences, University of Nebraska Lincoln, 230 Filley Hall, Lincoln, Nebraska 68583-0922, United States ‡ Center for Biotechnology, University of Nebraska Lincoln, E117 Beadle Center, Lincoln, Nebraska 68588-0665, United States

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ABSTRACT: The nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome is a key regulator of innate immune responses, and its aberrant activation is implicated in the pathogenesis of many diseases such as Alzheimer’s disease and type 2 diabetes. Targeting the NLRP3 inflammasome could hold promise to combat these complex diseases, but therapies specifically inhibiting the NLRP3 inflammasome have not been developed for patient treatment. The current study aimed to identify food-borne exosomelike nanoparticles (ELNs) that inhibit NLRP3 inflammasome activity. Nine vegetables or fruits were selected to extract ELNs, which were examined for their inhibitory effects on activation of the NLRP3 inflammasome in primary macrophages. Although most of the tested ELNs posed minimal impacts, the ELNs from ginger rhizomes (G-ELNs) strongly inhibited NLRP3 inflammasome activation. The G-ELNs contained lipids, proteins, and RNAs and were easily taken up by macrophages. G-ELN treatment suppressed pathways downstream of inflammasome activation including caspase1 autocleavage, interleukin (IL)-1β and IL-18 secretion, and pyroptotic cell death. Apoptotic speck protein containing a caspase recruitment domain (ASC) oligomerization and speck formation assays indicated that G-ELNs blocked assembly of the NLRP3 inflammasome. The lipids in G-ELNs, rather than the RNAs or proteins, were responsible for the inhibitory activity observed. Together, the data suggested G-ELNs as new potent agents that block NLRP3 inflammasome assembly and activation. The unique features of G-ELNs including biomolecule protection and tissue bioavailability should facilitate the development of G-ELN-based therapy to target the NLRP3 inflammasome in the disease settings. KEYWORDS: ginger, exosomes, nanoparticles, NLRP3 inflammasome, inflammation cus,16 alum used as a vaccine adjuvant,17 or free fatty acids9 elevated in obesity, converges on a common pathway to generate direct ligands that activate the sensor NLRP3. Activated NLRP3 recruits ASC, which further recruits Casp1 to assemble the multiprotein inflammasome complex. The recruitment of Casp1 leads to its activation and autocleavage. This generates the proteolytic active Casp1 p10 and p20, which cleave pro-IL-1β and pro-IL-18 to generate the mature proinflammatory cytokines IL-1β and IL-18. Independent of cytokine production, active Casp1 in the NLRP3 inflammasome cleaves gasdermin D, which induces the pyroptotic cell death and thus causes the massive release of danger molecules that amplify inflammatory responses.18,19 The IL-1β-targeted protein therapies including recombinant IL-1 receptor antagonist or neutralizing IL-1β antibody have been used to treat NLRP3 inflammasome-related diseases such as type 2 diabetes in clinical trials.20,21 However, these therapies are costly, require frequent injections, and target

1. INTRODUCTION The nucleotide-binding domain and leucine-rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome is a cytoplasmic multiprotein complex consisting of the sensor NLRP3, adaptor apoptotic speck protein containing a caspase recruitment domain (ASC), and effector caspase 1 (Casp1).1,2 Activation of the NLRP3 inflammasome is a key function of the innate immune system, and recent advances have indicated that inappropriate inflammasome activity causes or contributes to the pathogenesis of a variety of diseases including autoinflammatory diseases [cryopyrinassociated periodic syndrome (CAPS),3 gout,4] neurodegenerative diseases (Alzheimer’s disease,5,6 multiple sclerosis7), and metabolic diseases (type 2 diabetes,8−10 atherosclerosis11,12). Targeting the NLRP3 inflammasome could delay or prevent disease progression and thus hold promise as a therapy to combat these complex diseases. Activation of the NLRP3 inflammasome requires two stepspriming and activating.13−15 The initial priming signal, such as lipopolysaccharide (LPS), upregulates transcription of NLRP3 and cytokine interleukin (IL)-1β. A second activating signal, such as extracellular ATP discharged from dying cells,16 nigericin derived from the bacterium Streptomyces hygroscopi© XXXX American Chemical Society

Received: February 27, 2019 Revised: April 15, 2019 Accepted: April 18, 2019

A

DOI: 10.1021/acs.molpharmaceut.9b00246 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

of cilantro, aloe vera, dandelion, lavender, and cactus stem were purchased from the local stores. Each vegetable or fruit (2−10 g) was minced, soaked in ice-cold phosphate-buffered saline (PBS), and ground for 30−60 s in a kitchen blender. The juice was sequentially centrifuged at 500g for 10 min, 2000g for 20 min, and 10 000g for 30 min. The supernatant was ultracentrifuged at 100 000g for 2 h. The pellets were washed in ice-cold PBS, resuspended in the medium or PBS, and passed through a 200 nm filter (Acrodisc). Alternatively, the vesicles were further fractionated using sucrose gradient as described.42 The samples on the sucrose gradient were ultracentrifuged at 100 000g for 16 h. The ELN-containing fractions were diluted with PBS and ultracentrifuged at 100 000g for 2 h to pellet ELNs. The ELNs were analyzed and quantitated using the NanoSight NS300 instrument (Malvern) as described.42 2.4. Scanning Electron Microscopy Analysis of ELNs. The purified ELNs were resuspended with 2.5% glutaraldehyde (EMS #16320) and 1% tannic acid (EMS #21700) in 100 mM sodium cacodylate buffer (pH 7.2) and fixed for >30 min.43,44 The samples were further diluted in water (1:1) and loaded onto a Nuclepore track-etched Membrane (13 mm in diameter; 100 nm pore size; Whatman). The membrane was dried briefly on a Whatman filter paper, placed onto a scanning electron microscopy (SEM) stub, further air-dried for 2 hovernight, and sputter-coated with a layer (2−3 nm thick) of chromium using a Desk V sputter (Denton Vacuum Inc.). All ELN images were collected under a Hitachi S4700 SEM. 2.5. Lipid Extraction, Thin-Layer Chromatography Analysis, and Liposome Preparation. Total lipids of GELNs were extracted using the Folch method.38,45 A chloroform/methanol (Sigma, 2:1) solution was added to GELNs. The organic phase was collected, dried, and resuspended in chloroform. The lipid samples were separated on a Silica gel thin-layer chromatography (TLC) plate (EMD Millipore) using a mixture of chloroform/methanol/acetic acid (Sigma, 190:9:1) and visualized with CuSO4 phosphoric acid reagent (Sigma, 10% CuSO4 in 8% phosphoric acid). Liposomes were prepared as described.38 2.6. RNA Depletion in G-ELNs. The purified G-ELNs were resuspended in PBS with 10 μg/mL of RNase, subjected to bath sonication (Branson CPX5800H) at room temperature for 1.5 h, and then incubated at 37 °C for 1 h. The treated GELNs were washed with PBS and pelleted using ultracentrifugation (100 000g, 2 h). The RNA-depleted G-ELNs were incubated with macrophages or subjected to RNA extraction to evaluate the depletion efficiency. 2.7. Immunoblot Analysis and Antibodies. Cells were lysed in a SDS sampling buffer. The lysates were resolved on a 4−12% NuPAGE Bis−Tris protein gel (Novex) and transferred onto a 0.45 μm poly(vinylidene difluoride) membrane (GE Healthcare). Tubulin immunoblot was conducted in each experiment to confirm equivalent protein loading. Blots were probed with primary antibodies in Tris-buffered saline containing 0.1% Tween 20 (Sigma) and 5% nonfat milk (Nestle), followed by horseradish peroxidase-conjugated antirabbit antibody (Cell Signaling, 7074S, at 1:3000 dilution) or anti-mouse antibodies (Cell Signaling, 7076S, at 1:2500 dilution). Primary antibodies used included anti-NLRP3 mouse antibody (Adipogen, AG20B0014C100, 1:1000), antiASC rabbit antibody (Adipogen, AG25B0006C100, 1:1000), anti-Casp1 (p10) mouse antibody (Adipogen, AG20B0044C100, 1:1000), anti-NEK7 rabbit antibody

only one pathway downstream of the NLRP3 inflammasome activation. Some small-molecule chemicals, such as MCC95022 and Bay11-708223 or the metabolites β-hydroxybutyrate24 and 25-hydroxycholesterol,25 suppressed inflammasome activation in cell culture and improved NLRP3 inflammasome-mediated diseases including experimental autoimmune encephalomyelitis and CAPS in animal models.22−24 However, none of these has been translated into an inflammasome-targeting therapy in clinical practice. The aim of the current study was to use a nutraceutical approach to identify food-derived products that inhibit NLRP3 inflammasome activity. A variety of vegetables and fruits have recently been shown to contain exosome-like nanoparticles (ELNs).26−30 Dietary exosomes also have been identified in bovine milk.31−34 ELNs are 50−200 nm membrane-enclosed vesicles containing bioactive molecules such as proteins, lipids, and RNAs. Encapsulation of these biomolecules in the ELNs confers protection against degradation, and the vesicle structure of ELNs facilitates their intestinal absorption and tissue distribution.33,35,36 The biomolecules in dietary ELNs are bioavailable, regulate gene expression in mice and humans, and improve consumer health.31,32,37−39 With these unique features, dietary ELNs represent a promising new class of candidates for translational applications. Thus, we investigated whether any dietary ELNs regulate the NLRP3 inflammasome activity. We found that the ELNs from ginger rhizomes (GELNs) strongly inhibited NLRP3 inflammasome activity by blocking the assembly steps.

2. MATERIALS AND METHODS 2.1. Mice. C57BL/6J mice (Jackson Laboratory) were housed under specific pathogen-free conditions. Animal care was conducted following the Institute for Laboratory Animal Research guidelines. Animal experiments were carried out in accordance with the protocol (project ID 1421) approved by the Institutional Animal Care and Use Committee of University of Nebraska Lincoln. 2.2. Macrophage Cell Culture. Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were obtained as described.40,41 Peritoneal macrophages were collected from the peritoneal cavities of C57BL/6J mice 3 days after the mice were intraperitoneally injected with 3% (v/v) of brewer thioglycollate medium (Sigma). Immortalized BMDMs (iBMDMs) from C57BL/6J mice were obtained from Dr. Kate Fitzgerald (University of Massachusetts, Worcester, MA). The macrophages were cultured in the RPMI medium (Corning) containing 10% fetal bovine serum (FBS) (Atlanta Biologicals), 20% L929 cell-conditioned medium, 50 μg/mL PenStrep (Corning), 2 mM glutamine (Corning), 1 mM sodium pyruvate (Corning), and 10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid buffer (Corning). To examine the effects of dietary ELNs on inflammasome activation, macrophages were pretreated with dietary ELNs for 16 h and then primed with ultrapure LPS (InvivoGen, tlrl-peklps) for 3 h followed by stimulation of inflammasome activators. ATP (Sigma, 5 mM, 30 min), nigericin (Enzo Life Sci, 5 μM, 30 min), and alum (Thermo Scientific, 0.5% v/v, 5 h) were used to activate the NLRP3 inflammasome. Transfected calf DNA (Sigma, D3664, 2 μg/well, 2 h) was used to activate the absent in melanoma 2 (AIM2) inflammasome. 2.3. Isolation of ELNs. ELNs were isolated from vegetables or fruits as previously described.27,38,42 Briefly, grapefruit, rhizomes of ginger and turmeric, garlic cloves, leaves B


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Molecular Pharmaceutics (Abcam, ab133514, 1:1000), anti-tubulin rabbit polyantibody (Santa Cruz, SC-5286, 1:200), and anti-IL-1β goat antibody (R&D systems, AF401NA, 1:2000). 2.8. Labeling and Uptake of G-ELNs. The membrane lipids, proteins, and RNAs of G-ELNs were labeled with PKH26 (Sigma), ExoGlow Protein EV Labeling Kit (System Biosciences), and ExoGlow RNA EV Labeling Kit (System Biosciences), respectively, as per manufacturer’s protocol. The fluorescence-labeled G-ELNs were incubated with BMDMs for 16 h. The cells were washed 3−4 times with PBS and fixed with 4% paraformaldehyde (Sigma). Images were acquired with an A1R-Ti2 confocal system (Nikon). 2.9. Enzyme-Linked Immunosorbent Assay and Lactate Dehydrogenase Release. The cell medium was centrifuged at 300g for 5 min to remove cells or cell debris. The supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) analysis to measure the levels of secreted cytokines IL-1β (eBioscience, 88701388), IL-18 (MBL, D042-3), IL-6 (BioLegend, 431301), and tumor necrosis factor α (TNFα) (BioLegend, 430901). The CytoTox 96 Nonradioactive Cytotoxicity Assay kit (Promega) was used for lactate dehydrogenase (LDH) release assay. The 10× lysis solution from the kit was used to measure maximum LDH release. The percent cytotoxicity was calculated using the following formula: 100× experimental LDH release/maximum LDH release. 2.10. RNA Extraction and Quantitative RT-PCR. The RNAs from the G-ELNs were extracted using miRNeasy Mini kit (Qiagen) and RNAs from cells were extracted using RNAbee (Tel-Test) as per manufacturer’s protocol. The highcapacity cDNA Reverse Transcription Kit (Applied Biosystems) was used for cDNA synthesis. A CFX Connect real-time system (Bio-Rad) was used for quantitative polymerase chain reaction. The relative mRNA level was obtained by normalizing the gene of interest to the hypoxanthine guanine phosphoribosyl transferase (Hprt) gene. 2.11. ASC Speck Staining and ASC Oligomer CrossLinking. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with PBS containing 2% bovine serum albumin (VWR), and stained with anti-ASC antibody (Adipogen, AG25B0006C100, 1:200) and Alexa Fluor-594-conjugated secondary antibody (Invitrogen, A-11037, 1:200). 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain nuclei. Cell images were taken using an A1R-Ti2 confocal system (Nikon). ASC oligomer cross-linking analysis was conducted as described.46,47 Disuccinimidyl suberate (2 mM) was used as the cross-link reagent. 2.12. Statistics. Statistics were calculated using Excel. Comparisons of two groups were analyzed using two-tailed ttest as indicated. Data were presented as the mean ± standard deviation. P values < 0.05 were considered significant, as indicated by *. p < 0.01 was indicated by **. All cell culture data shown in the manuscript are representative of experiments conducted at least thrice.

Figure 1. G-ELNs inhibited the activation of NLRP3 inflammasome. (A) Schematic showing dietary ELN incubation followed by inflammasome activation in BMDMs. (B) Effects of G-ELNs on NLRP3 inflammasome activation. BMDMs were incubated with GELNs for 16 h, followed by NLRP3 inflammasome activation using LPS + ATP. Tubulin is a loading control. BMDMs treated with LPS alone were used as a negative control for inflammasome activation. **p < 0.01 relative to BMDMs treated with LPS + ATP (black bar).

release, and the cells were lysed to assess Casp1 autocleavage by measuring the Casp1 p10 level, which were used to evaluate NLRP3 inflammasome activation.16,48 Most of ELNs tested had mild inhibitory or stimulatory effects on IL-1β release and Casp1 p10 level (Table 1). However, G-ELNs strongly suppressed both IL-1β release and Casp1 p10 level upon NLRP3 inflammasome activation (Table 1, Figure 1B). Thus, G-ELNs were selected for further studies. 3.2. Detailed Characterization of G-ELNs. G-ELNs were characterized in detail. Consistent with the size of most dietary ELNs,32,49,50 G-ELNs was approximately 130 nm in diameter (Figure 2A). Approximately 0.5 to 2 × 1011 vesicles/g were purified from fresh ginger rhizomes. The purified G-ELNs were subjected to SEM analysis, which revealed that the G-ELNs had sphere-shaped morphology within the diameter range of 120−150 nm (Figure 2B). Dietary ELNs, including G-ELNs, were shown to contain lipids, RNAs, and proteins.26,27,38 The yields of RNAs, proteins, and lipids in the G-ELNs purified in our laboratory were 30.0 ± 10.8 ng/1010 vesicles, 2.8 ± 0.3 μg/ 1010 vesicles, and 32 ± 2.9 μg/1010 vesicles, respectively. The RNAs in G-ELNs were primarily small-sized RNAs (Figure 2C). RNase treatment caused complete degradation of the nucleic acids extracted from G-ELNs, confirming that they were mainly RNAs. The proteins from G-ELNs were in the range of 10−70 kDa (Figure 2D). TLC analysis showed that a variety of lipids were present in G-ELNs (Figure 2E). Thus, the G-ELNs purified in our laboratory were verified as nanoparticles containing biomolecules including RNAs, proteins, and lipids. During our studies, we noticed that the G-ELNs from different sources had differential potency for inhibiting NLRP3 inflammasome activity. Thus, a screening of fresh ginger rhizomes was conducted when new ginger rhizomes were purchased. Figure S1A shows the results from a representative ginger screening. The G-ELNs from four ginger rhizome samples inhibited the IL-1β secretion and Casp1 autocleavage, although G-ELN potency was slightly different. However, the G-ELNs from one ginger rhizome sample promoted IL-1β secretion but still inhibited Casp1 autocleavage. Thus, the GELNs extracted from fresh ginger rhizomes were assessed for

3. RESULTS 3.1. G-ELNs Inhibited NLRP3 Inflammasome Activation. To determine whether any dietary ELNs regulate NLRP3 inflammasome activity, ELNs were extracted from cilantro, aloe vera, grapefruit, garlic, turmeric, dandelion, lavender, cactus, and ginger, then incubated with BMDMs, followed by NLRP3 inflammasome activation (Figure 1A). The cell-free culture medium was collected to measure IL-1β C


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Molecular Pharmaceutics Table 1. Summary of the Effects of Nine Dietary ELNs on NLRP3 Inflammasome Activationa dietary ELNs

cilantro

aloe vera

grapefruit

garlic

turmeric

dandelion

lavender

cactus

ginger

IL-1β release Casp1 p10

no/↑ ↓

↓ no/↑

↓ no/↑

↓ ↓

↑ no/↓

↑ ↓

↓ no/↓

↑↑ ↓

↓↓↓ ↓↓↓

a ↑: indicates dietary ELNs increased IL-1β release or Casp1 p10 level. ↓: indicates dietary ELNs decreased IL-1β release or Casp1 p10 level. More arrows mean stronger effects.

Figure 2. Characterization of G-ELNs. (A) Size and yield of G-ELNs were measured using NanoSight NS300. (B) Morphology of G-ELNs under SEM. (C) RNA gel of G-ELN RNAs. The RNAs extracted from G-ELNs were treated with or without RNase and run on a 2.5% agarose gel. The RNA ladder of 80−1000 nucleotides (nt) was used. (D) Protein gel of G-ELN proteins. The proteins extracted from G-ELNs were separated on a Bis−Tris protein gel and stained with Coomassie blue staining. (E) TLC analysis of G-ELN lipids. The lipids from G-ELNs were separated on a TLC silica gel plate and visualized with CuSO4 phosphoric acid solution.

Figure 3. G-ELNs were taken up by BMDMs. The membrane lipids (A), proteins (B), and RNAs (C) of G-ELNs were labeled with PKH26, ExoGlow-Protein EV Labeling Kit, and ExoGlow-RNA EV Labeling Kit, respectively. BMDMs were incubated with 3 × 1010/mL of PKH26-labeled G-ELNs, 0.5 × 1010/mL of protein-labeled G-ELNs, or 3 × 1010/mL of RNA-labeled G-ELNs for 16 h. Prot: protein. DAPI was included to stain nuclei.

their effects on IL-1β secretion and Casp1 autocleavage upon NLRP3 inflammasome activation before they were used for any other experiments. We also tested the storage conditions for ginger rhizomes and G-ELNs. Remarkably, the G-ELNs from fresh ginger slices and frozen ginger slices stored at −80 °C showed comparable inhibition of both IL-1β secretion and Casp1 autocleavage (Figure S1B). Freshly isolated G-ELNs and frozen G-ELNs stored at −80 °C had similar inhibitory effects on both IL-1β secretion and Casp1 autocleavage (Figure S1C). Therefore, the

ginger rhizomes with inhibitory effects were sliced and stored at −80 °C, or the purified G-ELNs with inhibitory effects were stored at −80 °C for other experiments to ensure the quality and consistency of the vesicles. 3.3. G-ELNs Were Taken up by BMDMs. To monitor the uptake of G-ELNs by BMDMs, the membrane lipids, proteins, and RNAs of G-ELNs were labeled with lipophilic membrane dye PKH26, the ExoGlow-Protein EV Labeling Kit, and the ExoGlow-RNA EV Labeling Kit, respectively, and the labeled G-ELNs were incubated with BMDMs. The membrane lipidD


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Figure 4. G-ELNs inhibited NLRP3 inflammasome-mediated IL-1β and IL-18 secretion and pyroptosis. (A) G-ELNs inhibited both IL-1β and IL18 secretion. (B) G-ELNs had no significant impact on IL-6 and TNFα secretion. (C) G-ELNs inhibited pyroptosis. BMDMs were incubated with G-ELNs for 16 h, followed by NLRP3 inflammasome activation using LPS + ATP. The cell-free culture medium was collected to measure the level of IL-1β, IL-18, IL-6, TNFα, or LDH. For LDH release assay, the culture medium with 5% FBS was used. BMDMs treated with LPS alone were shown as a negative control (gray bar) for inflammasome activation. *p < 0.05, **p < 0.01 relative to BMDMs treated with LPS + ATP (black bar).

G-ELNs suppressed both IL-1β release and Casp1 autocleavage stimulated by either nigericin or alum (Figure 5A).

labeled (Figure 3A), protein-labeled (Figure 3B) or RNAlabeled (Figure 3C) G-ELNs were detected in BMDMs, indicating that G-ELNs, including the bioactive molecules inside the vesicles, were easily taken up by BMDMs. 3.4. G-ELNs Inhibited NLRP3 Inflammasome-Mediated IL-1β and IL-18 Secretion and Pyroptosis. NLRP3 inflammasome activation leads to secretion of not only IL-1β but also IL-18.2,13 Therefore, we tested the effects of G-ELNs on IL-18 release upon NLRP3 inflammasome activation. The G-ELNs strongly inhibited the release of both IL-1β and IL-18 in a dose-dependent manner (Figure 4A). In contrast, G-ELNs had no significant effects on the levels of LPS-induced cytokines IL-6 and TNFα (Figure 4B), indicating that GELNs had a marginal impact on the general inflammatory responses of macrophages, but specifically suppressed NLRP3 inflammasome activity. NLRP3 inflammasome activation leads to pyroptotic cell death, which is often evaluated by measuring LDH release.18,41 Using this method, we found that G-ELNs strongly inhibited pyroptosis (Figure 4C). To exclude the possibility that G-ELNs inhibited NLRP3 inflammasome activation only in BMDMs, we tested the functions of G-ELNs in two additional macrophage cell lines. G-ELNs substantially suppressed NLRP3 inflammasome activation in both peritoneal macrophages (Figure S2A) and iBMDMs (Figure S2B). Together, these data indicated that GELNs inhibited NLRP3 inflammasome activity in a variety of macrophage cells. 3.5. G-ELNs Inhibited NLRP3 Inflammasome Activated by a Variety of Stimuli but Had No Impact on AIM2 Inflammasome-Mediated Casp1 Autocleavage. The sensor NLRP3 triggers assembly and activation of the NLRP3 inflammasome in response to a diverse set of danger molecules.2,13,15 To determine whether G-ELNs inhibited NLRP3 inflammasome activation under different conditions, we used two other NLRP3 activators, nigericin and alum, to activate the NLRP3 inflammasome in BMDMs. Remarkably,

Figure 5. G-ELNs inhibited NLRP3 inflammasome activated by different stimuli but had no effects on Casp1 autocleavage upon AIM2 inflammasome activation. (A) G-ELNs inhibited NLRP3 inflammasome activated by nigericin and alum. (B) G-ELNs had no effects on AIM2 inflammasome-mediated Casp1 autocleavage. BMDMs were incubated with 3 × 1010/mL of G-ELNs for 16 h, followed by activation of NLRP3 or AIM2 inflammasome. *p < 0.05, **p < 0.01 relative to the macrophages treated with LPS + different stimuli (black bar).

However, when DNA was transfected in BMDMs to activate the AIM2 inflammasome, which detects cytosolic DNA during the bacterial and viral infection,51−53 G-ELNs had no impact on Casp1 autocleavage (Figure 5B). However, IL-1β release was still reduced by the G-ELN treatment, likely because GELNs reduced the level of pro-IL-1β proteins (Figure 5B). Thus, G-ELNs specifically inhibited Casp1 autocleavage mediated by the NLRP3 inflammasome but not by the AIM2 inflammasome. E


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Molecular Pharmaceutics 3.6. G-ELNs Inhibited ASC Oligomerization and Speck Formation. Upon NLRP3 activation, ASC oligomerizes and forms a high-molecular-mass multiprotein complex with NLRP3 in the cytoplasm. 54−56 The protein levels of inflammasome subunits NLRP3, ASC, and Casp1 were comparable in BMDMs without or with G-ELN treatment (Figure 6A). In addition, the protein level of a key

and Figure 6D). Thus, G-ELN strongly blocked assembly of the NLRP3 inflammasome. 3.7. Long-Term Incubation of G-ELNs was Required to Inhibit NLRP3 Inflammasome Activation and Il1b Gene Expression. To gain more insights into how G-ELNs exert the anti-inflammasome function in macrophages, GELNs were incubated with BMDMs for different periods of time, followed by NLRP3 inflammasome activation. Short incubation (16 h) strongly suppressed both IL-1β release and Casp1 autocleavage (Figure 7B). Notably, the proIL-1β protein level gradually decreased when the G-ELN incubation time was extended (Figure 7B). However, the protein levels of the NLRP3 inflammasome subunits NLRP3, Casp1, and ASC were not affected by short or long incubation of G-ELNs (Figure 7A,B). The significant decrease in pro-IL-1β protein level caused by extended G-ELN treatment led us to examine whether GELNs regulate expression of the Il1b gene, which is highly induced by LPS priming.58,59 Short G-ELN incubation (12 h) strongly inhibited the expression of the Il1b gene (Figure 7D). Therefore, long-term G-ELN incubation inhibited both NLRP3 inflammasome activity and Il1b gene expression. 3.8. Lipids in G-ELNs were Responsible for Inhibiting NLRP3 Inflammasome Activity. To determine which of the biomolecules in G-ELNs inhibits NLRP3 inflammasome activity, G-ELNs were subjected to different treatments. Heat treatment was used to denature the proteins in G-ELNs. Remarkably, heat-treated G-ELNs still strongly suppressed IL1β secretion and Casp1 autocleavage upon NLRP3 inflammasome activation (Figure 8A), indicating that the heat-sensitive proteins in G-ELNs were not the biomolecules that inhibited NLRP3 inflammasome activity. Bath sonication and RNase treatment were used to deplete RNAs in the G-ELNs. Bath sonication compromises integrity of the vesicle membrane and thus facilitates RNase in reaching RNAs in the vesicles to degrade them.37,60 Approximately 77% of the RNAs in GELNs were removed by this treatment (Figure S3). However, loss of the majority of RNAs did not affect the ability of these G-ELNs to inhibit NLRP3 inflammasome activity (Figure 8B), suggesting that most of the RNAs in G-ELNs were not essential in inhibiting the NLRP3 inflammasome. The dispensability of most proteins or RNAs in G-ELNs for the anti-inflammasome function pointed to the possible active role of lipids in G-ELNs in suppression of inflammasome activity. To directly evaluate the function of lipids in G-ELNs, total lipids were extracted from G-ELNs, dried, reassembled into liposomes, and then incubated with BMDMs. As shown in Figure 8C, these liposomes substantially suppressed the NLRP3 inflammasome activity. These data suggest that the lipids in G-ELNs were the active biomolecules that inhibited the NLRP3 inflammasome activity.

Figure 6. G-ELNs had no effects on the protein levels of inflammasome subunits or mediator but inhibited ASC oligomerization and speck formation. BMDMs were incubated with G-ELNs at different doses (A) or at the concentration of 3 × 1010/mL (B−D) for 16 h, followed by NLRP3 inflammasome activation using LPS + ATP. The cells were collected for immunoblot analysis (A,B) or fixed for ASC immunofluorescence staining (C,D). (A) G-ELNs did not affect the protein levels of inflammasome mediator Nek7 or subunits NLRP3, ASC, Casp1. (B) G-ELNs inhibited ASC oligomerization. TX: Triton X-100. (C) Representative images of ASC immunofluorescence staining in BMDMs. BMDMs were treated with Casp1 inhibitor VX765 (10 μM) for 30 min before ATP incubation to stabilize the inflammasome complex. The inflammasome was stained as a single speck (pointed by arrows) in the cells upon inflammasome activation. 1st Ab: anti-ASC rabbit antibody; 2nd Ab: Alexa Fluor594-conjugated anti-rabbit antibody. (D) Quantification of the speck positive cells. **p < 0.01 relative to BMDMs treated with LPS + ATP (black bar).

inflammasome mediator, never in mitosis gene a (NIMA)related kinase 7 (Nek7),46,47,57 was not affected by G-ELN treatment (Figure 6A). However, G-ELN treatment led to a failure of ASC oligomerization (Figure 6B). The large inflammasome complex could be visualized under confocal microscopy as a single speck in BMDMs, when an anti-ASC antibody was used for immunofluorescence staining.47,48 Consistent with the literature,47,48 we found that many macrophages contained a single ASC speck upon NLRP3 inflammasome activation (Figure 6C, middle panel and Figure 6D). Remarkably, the ASC speck formation was largely abolished by G-ELN treatment (Figure 6C, bottom panel,

4. DISCUSSION In summary, we have shown that G-ELNs strongly inhibited the release of cytokine IL-1β and IL-18, Casp1 autocleavage, and pyroptosis during NLRP3 inflammasome activation. The G-ELNs contain lipids, proteins, and RNAs. They were easily F


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Figure 7. Long-term incubation of G-ELNs inhibited both NLRP3 inflammasome activation and Il1b gene expression. (A,B) BMDMs were incubated with 3 × 1010/mL of G-ELNs for different periods, followed by NLRP3 inflammasome activation using LPS + ATP. Tubulin is a loading control. **p < 0.01 relative to the macrophages treated with LPS + ATP (black bar). (C,D) BMDMs were incubated with 3 × 1010/mL of G-ELNs for different periods, followed by LPS treatment. The untreated (un) BMDMs were used as a negative control for LPS treatment. The Hprt gene was used as a housekeeping gene. *p < 0.05, **p < 0.01 relative to the macrophages treated with LPS (black bar).

Figure 8. Lipids in G-ELNs were responsible for inhibiting NLRP3 inflammasome activation. (A) G-ELNs were either untreated (un) or subjected to heat treatment at 95 °C for 10 min (heated) to denature proteins. 3 × 1010/mL of G-ELNs were used. (B) G-ELNs were either untreated (un) or subjected to bath sonication and RNase treatment (S/R-treated) to remove the majority of RNAs. 3 × 1010/mL of G-ELNs were used. (C) Lipids in G-ELNs were extracted, dried, and reassembled into liposomes (G-lip). The G-ELNs or liposomes were incubated with BMDMs for 16 h, followed by NLRP3 inflammasome activation using LPS + ATP. Tubulin is a loading control. *p < 0.05, **p < 0.01 relative to macrophages treated with LPS + ATP (black bar).

found that G-ELNs blocked NLRP3 inflammasome assembly and activation, and by doing so, inhibited not only IL-1β release but also other pathways downstream of NLRP3 inflammasome activation including IL-18 secretion, Casp1 autocleavage, and pyroptosis. Therefore, G-ELNs represent a new promising class of NLRP3 inflammasome inhibitors. Endogenous exosomes are potent vehicles of intercellular communication because they transport the biomolecules from donor cells to recipient cells in a broad range of organisms.35,36 Accumulating evidence suggests that the biomolecules in dietary ELNs or exosomes are bioavailable and exert beneficial effects in animal models and humans. For example, the microRNAs in milk exosomes were detected in intestinal mucosa, spleen, liver, heart, and brain after the milk exosomes were orally administrated.39 Grape-derived ELNs were taken up by intestinal stem cells,62 and grapefruit ELNs were detected in liver Kupffer cells.38 G-ELNs were shown to activate nuclear factor erythroid derived 2 like 2 (Nfe2l2) and protect mice against alcohol-induced liver damage.38 The ELNs from grapes, broccoli, and ginger improved the disease condition of colitis in mouse models.26,30,62,63 The unique

taken up by macrophages. The inhibitory effect of G-ELNs on the NLRP3 inflammasome was specific because G-ELNs did not suppress AIM2 inflammasome activation. G-ELNs had no effects on the protein levels of inflammasome subunits or mediator but remarkably inhibited ASC oligomerization and inflammasome formation. The lipids in G-ELNs actively inhibited activation of the NLRP3 inflammasome. Collectively, these studies have identified G-ELNs as new potent agents that block NLRP3 inflammasome assembly and activation. Currently, the NLRP3 inflammasome-related therapy used in clinical trials is the protein therapy that targets cytokine IL1β, one downstream product of NLRP3 inflammasome activation.20,21 Using Il18r-null mice, Il1r-null mice, or double knockout mice, the Hoffman group discovered that other events downstream of NLRP3 inflammasome activation, such as IL-18 secretion and pyroptosis, also contributed to the inflammation caused by NLRP3 inflammasome activation.61 Thus, inhibitors that target activation of the NLRP3 inflammasome should inhibit all of the downstream pathways of the NLRP3 inflammasome and may have more significant clinical outcomes compared with IL-1β-targeted therapy. We G


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TNFα.26,30 In another study, 24 h incubation of G-ELNs with the macrophage cell line RAW264.7 increased transcription of the Tnf and Il6 genes.27 In our laboratory, G-ELNs usually had no significant effects on LPS-induced cytokine secretion of IL6 and TNFα after they were incubated with BMDMs for 16 h (Figure 4B). The exact causes for these discrepancies were not clear, but plausible reasons could be the quality of the ginger rhizomes, as well as the different experimental conditions and systems used in these studies. Overall, our research and other studies underscore the important role of G-ELNs in the regulation of inflammation. In conclusion, our studies have established G-ELNs as new potent agents that inhibit NLRP3 inflammasome assembly and activation. With unique features of biomolecule protection and tissue bioavailability, G-ELNs represent a new class of NLRP3 inflammasome inhibitor with high potential for translational application. Further investigation is necessary to identify the active lipid biomolecules in the G-ELNs that mediate the inhibitory effects on NLRP3 inflammasome activation. GELNs did not affect the protein levels of NLRP3 inflammasome subunits or mediator but prevented inflammasome assembly. Thus, further studies are warranted to investigate how G-ELNs block inflammasome assembly and activation at the molecular level.

exosome-like structure of G-ELNs, which encloses the biomolecules in nanoparticles, protects the biomolecules from protease- or nuclease-mediated degradation and confers resistance to the stomach-like and intestine-like solutions.26,27 It has been shown that G-ELNs were detected in the intestinal macrophages27 and liver and mesenteric lymph nodes38 after oral administration in mice. Thus, G-ELNs seem to be absorbed through the gastrointestinal system and transported to different tissues. The unique features of G-ELNs including biomolecule protection and tissue bioavailability may facilitate translation of G-ELNs into a dietary or therapeutic intervention. We found that the lipids in G-ELNs, rather than proteins or RNAs, were the active biomolecules that inhibited NLRP3 inflammasome activity. This finding is highly novel because, in most previous studies, the proteins or RNAs within endogenous exosomes, dietary exosomes, or dietary ELNs were identified as the active biomolecules. For example, the integrin proteins in tumor exosomes were identified in promoting tumor metastasis.64 The miR155 in exosomes secreted from the adipose tissue macrophages of obese mice suppressed expression of the transcription factor peroxisome proliferator-activated receptor gamma and exacerbated insulin resistance.65 Depletion of the microRNAs in bovine milk exosomes led to moderate changes in gene expression in mouse skeletal muscle.37 The ath-miR167a and mdo-miR7267 in G-ELNs targeted the spaC and ycnE gene, respectively, in the gut bacterium Lactobacillus rhamnosus.30 In this study, we have demonstrated that the lipids in dietary ELNs could serve as active biomolecules to regulate the function of recipient cells. The G-ELNs purified in our laboratory were characterized by their size and yield using a NanoSight machine and by morphology and integrity using SEM (Figure 2A,B). The size of G-ELNs were around 120−150 nm in diameter, similar to the size of most dietary ELNs31,32,49 but smaller than G-ELNs characterized in two previous studies (average 300 nm in diameter).26,38 It is likely that most vesicles larger than 200 nm were removed because we applied a 200 nm filter in the last step of ELN isolation. The G-ELNs we purified contained RNAs, proteins, and lipids (Figure 2C−E), which were consistent with the contents of G-ELNs from other research groups.26,27,30,38 The exosomes from mammalian cells, mammalian tissues, or bovine milk are often further characterized by immunoblot analysis of surface markers.50,65−67 However, the specific surface markers of ELNs from ginger or other plants have not been established and will require further studies. G-ELNs were shown to decrease the IL-1β level and disease severity in the inflammatory bowel disease (IBD) and colitisassociated cancer in mouse models.26,30 The microRNAs in GELNs regulate the composition, function, and localization of gut microbiota and thus protect the animals from IBD.30 Zhuang et al. showed that oral administration of G-ELNs in mice protected the liver from alcohol-induced injury.38 Our study identified a new function of G-ELNsthey strongly inhibited NLRP3 inflammasome activation and expression of the Il1b gene in macrophages. Considering the important involvement of IL-1β and the NLRP3 inflammasome in IBD,68,69 the beneficial effects of G-ELNs on IBD could at least partially arise from their inhibition of the NLRP3 inflammasome and suppression of the Il1b gene expression. In the mouse IBD model, G-ELNs decreased the level of cytokine

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.9b00246.

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Quality and storage test of G-ELNs; inhibitory effects of G-ELNs on NLRP3 inflammasome activity in two other macrophage cell lines; and RNA depletion efficiency in G-ELNs (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1 402 472 7013. Fax: +1 402 472 1587. ORCID

Jiujiu Yu: 0000-0001-9927-8720 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Institutes of Health (NIH) 1P20GM104320 Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules (NPOD) Seed Grant and Project Leader Grant, and the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) Hatch Project 1015948. The authors acknowledge the use of the Biomedical and Obesity Research Core (BORC) of the NPOD and the Systems Biology (Microscopy) Core of the Nebraska Center for Integrated Biomolecular Communication (NCIBC) at the University of Nebraska Lincoln. We thank the assistance of Dr. Steven Kachman (NPOD) for statistical analysis and Terri Fangman (NCIBC) for confocal operation. We also thank Dr. Janos Zempleni and his laboratory members for the technical assistance and invaluable discussions and Dr. Soonkyu Chung for critical review of the manuscript. H


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ABBREVIATIONS ASC, apoptotic speck protein containing a caspase recruitment domain; AIM2, absent in Melanoma 2; BMDMs, bone marrow-derived macrophages; CAPS, cryopyrin-associated periodic syndrome; Casp1, caspase 1; ELISA, enzyme-linked immunosorbent assay; ELNs, exosome-like nanoparticles; FBS, fetal bovine serum; G-ELNs, ELNs from ginger rhizomes; Hprt, hypoxanthine guanine phosphoribosyl transferase; IBD, inflammatory bowel disease; iBMDMs, immortalized BMDMs; IL, interleukin; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; NIMA, never in mitosis gene a; Nek7, NIMA-related kinase 7; NLRP3, nucleotide-binding domain and leucine-rich repeat containing family, pyrin domain containing 3; Nfe2l2, nuclear factor erythroid derived 2 like 2; TLC, thin-layer chromatography

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Exosome-like Nanoparticles from Ginger Rhizomes Inhibited NLRP3

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