vers of HFD-fed rats and in the free fatty acid-treated rat hepatocyte BRL-3A cell line, indicating possible regulatory effects of honey on the NLRP3 inflammasome. However, the direct effects of honey on the NLRP3 inflammasome had not been demonstrated. In our studies, we found that VLNs from honey specifically inhibited activity of the NLRP3 inflammasome (Figure 2) by preventing formation of the inflammasome complex ( Figure 3). Therefore, our research has clearly defined the role of H-VLNs in suppressing activity of the NLRP3 inflammasome and has elucidated that honey inhibits inflammation through a new target -the NLRP3 inflammasome. It is plausible that the anti-inflammatory bioactivities of honey identified in previous studies are mediated at least partially through inhibition of NLRP3 inflammasome activity. miRNAs in dietary VLNs have been found to play various roles. miRNA-7267-3p in ginger-derived VLNs has been found to suppress expression of the ycnE gene in gut microbiota Lactobacillaceae, leading to a change of tryptophan metabolism in these bacteria (Teng et al., 2018). miR-167a, another miRNA in ginger-derived VLNs, inhibited transcription of a Pili gene, SpaC in Lactobacillaceae and therefore reduced bacterial mobilization from the gut to other organs (Teng et al., 2018). miRNAs from ginger-derived VLNs, including miR-159a, miR-166u, and miR-166p, have been shown to inhibit proliferation of a periodontal pathogen, Porphyromonas gingivalis (Sundaram et al., 2019). Ultrasonication of bovine skim milk leads to a >98% depletion of miRNAs in the milk EVs and a 20% depletion of milk EVs (Leiferman et al., 2018;Wu et al., 2019). When such ultrasonicated skim milk was supplemented in a mouse diet, the expressions of approximately 10 genes in skeletal muscle were altered, compared to mice fed with a diet supplemented with untreated skim milk (Leiferman et al., 2018). The same dietary depletion of milk EVs and their miRNA cargos elevated intestinal inflammation and altered transcription of 16 immune genes in Mdra -/mice. However, it is not clear whether depletion of miRNAs in the milk EVs influences directly transcription in consumers or through other mechanisms (such as reshaping the microbiota in the gut). Our studies demonstrated that RNAs in H-VLNs are necessary and sufficient for suppressing NLRP3 inflammasome activity ( Figure 5). Further screening of miRNAs revealed that miR-4057 in H-VLNs suppressed NLRP3 inflammasome activation in macrophages ( Figure 6). Therefore, our research has demonstrated that miRNAs from another dietary source, H-VLNs, possess intrinsic anti-inflammatory activities.
The role of dietary miRNAs in consumers is highly controversial. An early report suggested that rice-originated miR-168a was enriched in mouse serum and liver after rice consumption, accompanied by decreased levels of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) in the liver samples (Zhang et al., 2011). However, because of conflicting follow-up data in this area, the concept of dietary transfer of miRNAs has been the subject of intense debate (Witwer & Hirschi, 2014;Witwer & Zhang, 2017). Although miRNAs in dietary VLNs seem to regulate the functions and/or population of microbiota in the gut of consumers (Teng et al., 2018;Zhou et al., 2019), corroborating and proverbial evidence is needed to support their direct regulatory role in consumers. In our studies, intraperitoneal injection of H-VLNs alleviated inflammation in experimentally induced acute liver injury in mice (Figure 4), providing proof-of-principle demonstration of the anti-inflammatory functions of H-VLNs in vivo. However, further studies are warranted to investigate whether oral administration of H-VLNs and their bioactive component miRNAs could be absorbed through the gastrointestinal tract and exert any anti-inflammatory function in consumers.
Despite the well-documented nutraceutical values of honey, some adverse effects of honey have been reported due to contamination from pesticides, antibiotics, and heavy metals introduced through disease control, environmental exposure, and processing (Ajibola et al., 2012;Bogdanov et al., 2008). The high sugar content of honey poses the theoretical risk of increasing blood glucose levels in diabetic patients. Indeed, in clinical trials, consumption of honey for 8 weeks increased the level of blood haemoglobin A1c (HbA1c) in patients with type 2 diabetes (Bahrami et al., 2009;Sadeghi et al., 2019), suggesting that extra caution is needed when honey is consumed by diabetic patients. The H-VLN extraction process removes sugars and likely most of the contaminated agrochemicals in honey, although H-VLNs retain their potent anti-inflammatory bioactivities and possibly other functions. Therefore, H-VLNs could be potentially used to achieve at least some of the medicinal functions of honey in vulnerable populations such as diabetic patients.
In conclusion, VLNs with strong anti-inflammatory functions represent a new type of bioactive agent identified in honey. This finding opens a new avenue for studying honey, a medicinal food since ancient time. Many questions await further investigation. Besides the studies raised earlier, further studies are necessary to determine the molecular mechanisms by which H-VLNs prevent inflammasome formation and activation. It would be also important and interesting to investigate other potential functions of H-VLNs and their possible interactions with microbiota in the gut.

. Honey bees, honey, and other hive products
Twenty colonies of European honey bees (Apis mellifera L.) were maintained with standard Langstroth equipment at the University of Nebraska-Lincoln East Campus Pollinator Garden (Lincoln NE, USA). Beehive samples, including fresh unprocessed honey, royal jelly, and freshly collected forage (nectar and pollen), were harvested from 3 to 4 randomly selected queenright and healthy colonies and immediately subjected to VLN extraction. Five commercially available honeys, including manuka honey (unprocessed, product of New Zealand, distributed by Wedderspoon, Malvern, PA, USA); two local Nebraska honeys (unprocessed or lightly heated and filtered, product of Nebraska USA, distributed by It's all about bees!, Ralston, NE, USA); two organic honeys (unfiltered or filtered, product of Brazil and Mexico, distributed by Wholesome Sweeteners, Sugar Land, TX, USA) were purchased from the local Whole Foods Market (Lincoln, NE, USA).

. Isolation of VLNs
Honeys, other hive products, and grapefruit were subjected to VLN extraction as described with minor modifications (Chen et al., 2019;Mu et al., 2014;Wang et al., 2018). A total of 2 g of pollen or grapefruit pulps were ground for 15 sec in cold PBS in a kitchen blender, while 2 g of honey, nectar, or royal jelly were diluted with cold PBS. The diluent underwent sequential centrifugation at 500 × g for 10 min, 2000 × g for 20 min and 10,000 × g for 30 min. The supernatant was subjected to ultracentrifugation at 100,000 × g for 2 h. The nanoparticle pellets were washed with cold PBS, resuspended in PBS or medium, and filtered through a 200 nm Acrodisc filter (Pall Laboratory, Port Washington, NY, USA). Alternatively, VLNs in PBS were loaded on the top of a sucrose gradient (8%: 30%: 60% from the top to bottom) and underwent ultracentrifugation at 150,000 × g for 16 h. Fractions were carefully collected at 1 ml each (Fractions 1-11 from top to the bottom). The particle number in each fraction was measured. The pellet at the bottom of the tube was washed twice with PBS and resuspended in PBS for further analysis. The fractionated particles were diluted with PBS and ultracentrifuged at 100,000 × g for 2 h to collect the nanoparticles.

. Characterization of VLNs
A NanoSight NS300 instrument (Malvern, Westborough, MA, USA) was used to measure size and yield of VLNs as described (Chen et al., 2019). RNAs were extracted from VLNs using miRNeasy Mini kit (Qiagen, Germantown, MD, USA) and run on a 2.5% agarose gel. Proteins were extracted from VLNs using lysis buffer containing 150 mM NaCl, 0.5% NP-40, 50 mM Tris-HCl (pH7.5), resolved on a Bis-Tris protein gel (Invitrogen, Carlsbad, CA, USA), and visualized with Coomassie blue staining. Lipids were purified from VLNs using the Folch method (Folch et al., 1957;Zhuang et al., 2015), run on a silica gel TLC plate (EMD Millipore, Burlington, MA, USA) using a solvent mixture of chloroform /methanol /acetic acid (190:9:1, Sigma, St. Louis, MO, USA), and stained with 10% CuSO 4 in 8% phosphoric acid (Sigma). Total lipids of H-VLNs were used to prepare liposomes as described (Chen et al., 2019). For the Triton X-100 lysis experiment, H-VLNs in PBS were aliquoted into 5 tubes, to which were added 0, 0.01%, 0.025%, 0.05% or 0.1% (v/v in PBS) of Triton X-100, followed by vigorous agitation using a vortex mixer at highest speed for 30 sec. The mixture was incubated at room temperature for 30 min. The samples were vortexed again