Outer Membrane Vesicles Released from Garlic Exosome-like Nanoparticles (GaELNs) Train Gut Bacteria that Reverses Type 2 Diabetes via the Gut-Brain Axis.

Gut microbiota function has numerous effects on humans and the diet humans consume has emerged as a pivotal determinant of gut microbiota function. Here, a new concept that gut microbiota can be trained by diet-derived exosome-like nanoparticles (ELNs) to release healthy outer membrane vesicles (OMVs) is introduced. Specifically, OMVs released from garlic ELN (GaELNs) trained human gut Akkermansia muciniphila (A. muciniphila) can reverse high-fat diet-induced type 2 diabetes (T2DM) in mice. Oral administration of OMVs released from GaELNs trained A. muciniphila can traffick to the brain where they are taken up by microglial cells, resulting in inhibition of high-fat diet-induced brain inflammation. GaELNs treatment increases the levels of OMV Amuc-1100, P9, and phosphatidylcholines. Increasing the levels of Amuc-1100 and P9 leads to increasing the GLP-1 plasma level. Increasing the levels of phosphatidylcholines is required for inhibition of cGas and STING-mediated inflammation and GLP-1R crosstalk with the insulin pathway that leads to increasing expression of Insulin Receptor Substrate (IRS1 and IRS2) on OMV targeted cells. These findings reveal a molecular mechanism whereby OMVs from plant nanoparticle-trained gut bacteria regulate genes expressed in the brain, and have implications for the treatment of brain dysfunction caused by a metabolic syndrome.

Rab1A-Mediated Exosomal Sorting of miR-200c Enhances Breast Cancer Lung Metastasis.

Background: Recent therapeutic approaches have improved survival rate for women with breast cancer, but the survival rate for metastatic breast cancer is still low. Exosomes released by various cells are involved in all steps of breast cancer development. Methods: We established the multimodal imaging report expression in breast cancer cells with lentivirus vectors pGluc and pBirA to investigate the secreted exosomes. Comparative microRNA (miRNA) analysis was performed with miRNA qPCR array in mice with breast cancer lung metastasis. The co-immunoprecipitation and chromatin immunoprecipitation assays were used to identify the mechanism of miRNA sorting to exosomes. The potential therapeutic strategy using an anti-sorting antibody was used to investigate breast cancer lung metastasis. Results: We identified 26 high- and 32 low-expression level miRNAs in exosomes from metastasis compared to those from primary tumors and normal tissues. The tumor suppressors, including miR-200c and let-7a, were reduced in tumor tissues and metastasis but increased in the respective exosomes compared to normal tissues. Furthermore, the Ras-related protein (Rab1A) facilitated miR-200c sorting to exosomes circumventing the influence of tumor suppressor miR-200c on tumor cells, while the metastatic exosome cargo miR-200c inhibited F4/80+ macrophage immune response. Administration of anti-Rab1A antibody significantly repressed the trafficking of miR-200c to exosomes and breast cancer lung metastasis. Conclusion: Our study has identified a novel molecular mechanism for breast cancer lung metastasis mediated by exosome cargo miRNAs and provided a new therapeutic strategy for cancer immunotherapy.

Lactobacillus rhamnosus GG alleviates colitis caused by chemotherapy via biofilm formation.

BACKGROUND AND AIM: Severe colitis is a common side effect of chemotherapy in cancer patients. In this study, we attempted to enhance the viability of probiotics in a gastric acid environment and improve the colitis induced by dextran sulfate sodium (DSS) and docetaxel. METHODS: We purified Lactobacillus from yogurt and estimated their growth at pH 6.8 and pH 2.0. In the further investigation, the bacterial biofilm formation was used to define the mechanism by which administration of Lactobacillus rhamnosus (LGG) via oral gavage alleviates the colitis and intestine permeability of the mice induced by DSS and docetaxel. The potential benefit of probiotics on the treatment of breast cancer metastasis has been assessed as well. RESULTS: Lactobacillus from yogurt growth was unexpectedly faster in the pH 2.0 than in the neutral pH medium during the first hour. LGG administered in the fasting state via oral gavage significantly improved the preventive effect in the colitis caused by DSS and docetaxel. LGG reduced the permeability of the intestine and decreased the expression of proinflammatory cytokines, TNF-α, IL-1ß, and IL-6, in colitis by biofilm formation. Increasing the docetaxel dose may reduce breast tumor growth and metastasis in the lung but did not benefit survival due to severe colitis. However, the LGG supplement significantly improved the survival of tumor-bearing mice following a high dose of docetaxel treatment. CONCLUSIONS: Our findings provide new insights into the potential mechanism of probiotic protection of the intestine and provide a novel therapeutic strategy to augment the chemotherapeutic treatment of tumors.

An extracellular vesicular mutant KRAS-associated protein complex promotes lung inflammation and tumor growth.

Extracellular vesicles (EVs) contain more than 100 proteins. Whether there are EVs proteins that act as an 'organiser' of protein networks to generate a new or different biological effect from that identified in EV-producing cells has never been demonstrated. Here, as a proof-of-concept, we demonstrate that EV-G12D-mutant KRAS serves as a leader that forms a protein complex and promotes lung inflammation and tumour growth via the Fn1/IL-17A/FGF21 axis. Mechanistically, in contrast to cytosol derived G12D-mutant KRAS complex from EVs-producing cells, EV-G12D-mutant KRAS interacts with a group of extracellular vesicular factors via fibronectin-1 (Fn1), which drives the activation of the IL-17A/FGF21 inflammation pathway in EV recipient cells. We show that: (i), depletion of EV-Fn1 leads to a reduction of a number of inflammatory cytokines including IL-17A; (ii) induction of IL-17A promotes lung inflammation, which in turn leads to IL-17A mediated induction of FGF21 in the lung; and (iii) EV-G12D-mutant KRAS complex mediated lung inflammation is abrogated in IL-17 receptor KO mice. These findings establish a new concept in EV function with potential implications for novel therapeutic interventions in EV-mediated disease processes.

Gut bacterial isoamylamine promotes age-related cognitive dysfunction by promoting microglial cell death.

The intestinal microbiome releases a plethora of small molecules. Here, we show that the Ruminococcaceae metabolite isoamylamine (IAA) is enriched in aged mice and elderly people, whereas Ruminococcaceae phages, belonging to the Myoviridae family, are reduced. Young mice orally administered IAA show cognitive decline, whereas Myoviridae phage administration reduces IAA levels. Mechanistically, IAA promotes apoptosis of microglial cells by recruiting the transcriptional regulator p53 to the S100A8 promoter region. Specifically, IAA recognizes and binds the S100A8 promoter region to facilitate the unwinding of its self-complementary hairpin structure, thereby subsequently enabling p53 to access the S100A8 promoter and enhance S100A8 expression. Thus, our findings provide evidence that small molecules released from the gut microbiome can directly bind genomic DNA and act as transcriptional coregulators by recruiting transcription factors. These findings further unveil a molecular mechanism that connects gut metabolism to gene expression in the brain with implications for disease development.

Garlic exosome-like nanoparticles reverse high-fat diet induced obesity via the gut/brain axis.

Background: Obesity is becoming a global epidemic and reversing the pathological processes underlying obesity and metabolic co-morbidities is challenging. Obesity induced chronic inflammation including brain inflammation is a hallmark of obesity via the gut-brain axis. The objective of this study was to develop garlic exosome-like nanoparticles (GaELNs) that inhibit systemic as well as brain inflammatory activity and reverse a HFD induced obesity in mice. Methods: GELNs were isolated and administrated orally into HFD fed mice. GaELNs were fluorescent labeled for monitoring their in vivo trafficking route after oral administration and quantified the number particles in several tissues. The brain inflammation was determined by measuring inflammatory cytokines by ELISA and real-time PCR. Mitochondrial membrane permeability of microglial cells was determined using JC-10 fluorescence dye. The in vivo apoptotic cell death was quantified by TUNEL assay. The brain metabolites were identified and quantified by LC-MS analysis. Memory function of the mice was determined by several memory functional analysis. The effect of GaELNs on glucose and insulin response of the mice was determined by glucose and insulin tolerance tests. c-Myc localization and interaction with BASP1 and calmodulin was determined by confocal microscopy. Results: Our results show that GaELNs is preferentially taken up microglial cells and inhibits the brain inflammation in HFD mice. GaELN phosphatidic acid (PA) (36:4) is required for the uptake of GaELNs via interaction with microglial BASP1. Formation of the GaELNs/BASP1 complex is required for inhibition of c-Myc mediated expression of STING. GaELN PA binds to BASP1, leading to inhibition of c-Myc expression and activity through competitively binding to CaM with c-Myc transcription factor. Inhibition of STING activity leads to reducing the expression of an array of inflammatory cytokines including IFN-γ and TNF-α. IFN-γ induces the expression of IDO1, which in turn the metabolites generated as IDO1 dependent manner activate the AHR pathway that contributes to developing obesity. The metabolites derived from the GaELNs treated microglial cells promote neuronal differentiation and inhibit mitochondrial mediated neuronal cell death. GaELNs treated HFD mice showed improved memory function and increased glucose tolerance and insulin sensitivity in these mice. Conclusion: Collectively, these results demonstrate how nanoparticles from a healthy diet can inhibit unhealthy high-fat diet induced brain inflammation and reveal a link between brain microglia/diet to brain inflammatory disease outcomes via diet-derived exosome-like nanoparticles.

Ginger nanoparticles mediated induction of Foxa2 prevents high-fat diet-induced insulin resistance.

Rationale: The obesity epidemic has expanded globally, due in large part to the increased consumption of high-fat diets (HFD), and has increased the risk of major chronic diseases, including type 2 diabetes. Diet manipulation is the foundation of prevention and treatment of obesity and diabetes. The molecular mechanisms that mediate the diet-based prevention of insulin resistance, however, remain to be identified. Here, we report that treatment with orally administered ginger-derived nanoparticles (GDNP) prevents insulin resistance by restoring homeostasis in gut epithelial Foxa2 mediated signaling in mice fed a high-fat diet (HFD). Methods: Ginger-derived nanoparticles (GDNP) were added into drinking water to treat high-fat diet fed mice for at least one year or throughout their life span. A micro array profile of intestinal, liver and fat tissue of GDNP treated mice was used to analyze their gene expression profile. Genes associated with metabolism or insulin signaling were further quantified using the real time polymerase chain reaction (RT-PCR). Surface plasmon resonance (SPR) was used for determining the interaction between Foxa2 protein and phosphatic acid lipid nanoparticles. Results: HFD-feeding inhibited the expression of Foxa2; the GDNPs increased the expression of Foxa2 and protected Foxa2 against Akt-1 mediated phosphorylation and subsequent inactivation of Foxa2. Increasing expression of Foxa2 leads to altering the composition of intestinal epithelial cell (IEC) exosomes of mice fed a HFD and prevents IEC exosome mediated insulin resistance. Collectively, oral administration of GDNP prevents insulin resistance in HFD mice. Interestingly, oral administration of GDNP also extended the life span of the mice and inhibited skin inflammation. Conclusion: Our findings showed that GDNP treatment can prevent HFD-induced obesity and insulin resistance via protecting the Foxa2 from Akt-1 mediated phosphorylation. GDNP treatment provides an alternative approach based on diet manipulation for the development of therapeutic interventions for obesity.

Exosome-like nanoparticles from Mulberry bark prevent DSS-induced colitis via the AhR/COPS8 pathway.

Bark protects the tree against environmental insults. Here, we analyzed whether this defensive strategy could be utilized to broadly enhance protection against colitis. As a proof of concept, we show that exosome-like nanoparticles (MBELNs) derived from edible mulberry bark confer protection against colitis in a mouse model by promoting heat shock protein family A (Hsp70) member 8 (HSPA8)-mediated activation of the AhR signaling pathway. Activation of this pathway in intestinal epithelial cells leads to the induction of COP9 Constitutive Photomorphogenic Homolog Subunit 8 (COPS8). Utilizing a gut epithelium-specific knockout of COPS8, we demonstrate that COPS8 acts downstream of the AhR pathway and is required for the protective effect of MBELNs by inducing an array of anti-microbial peptides. Our results indicate that MBELNs represent an undescribed mode of inter-kingdom communication in the mammalian intestine through an AhR-COPS8-mediated anti-inflammatory pathway. These data suggest that inflammatory pathways in a microbiota-enriched intestinal environment are regulated by COPS8 and that edible plant-derived ELNs may hold the potential as new agents for the prevention and treatment of gut-related inflammatory disease.

Restoring Oat Nanoparticles Mediated Brain Memory Function of Mice Fed Alcohol by Sorting Inflammatory Dectin-1 Complex Into Microglial Exosomes.

Microglia modulate pro-inflammatory and neurotoxic activities. Edible plant-derived factors improve brain function. Current knowledge of the molecular interactions between edible plant-derived factors and the microglial cell is limited. Here an alcohol-induced chronic brain inflammation model is used to identify that the microglial cell is the novel target of oat nanoparticles (oatN). Oral administration of oatN inhibits brain inflammation and improves brain memory function of mice that are fed alcohol. Mechanistically, ethanol activates dectin-1 mediated inflammatory pathway. OatN is taken up by microglial cells via ß-glucan mediated binding to microglial hippocalcin (HPCA) whereas oatN digalactosyldiacylglycerol (DGDG) prevents assess of oatN ß-glucan to dectin-1. Subsequently endocytosed ß-glucan/HPCA is recruited in an endosomal recycling compartment (ERC) via interaction with Rab11a. This complex then sequesters the dectin-1 in the ERC in an oatN ß-glucan dependent manner and alters the location of dectin-1 from Golgi to early endosomes and lysosomes and increases exportation of dectin-1 into exosomes in an Rab11a dependent manner. Collectively, these cascading actions lead to preventing the activation of the alcoholic induced brain inflammation signing pathway(s). This coordinated assembling of the HPCA/Rab11a/dectin-1 complex by oral administration of oatN may contribute to the prevention of brain inflammation.

Lemon exosome-like nanoparticles enhance stress survival of gut bacteria by RNase P-mediated specific tRNA decay.

Diet and bile play critical roles in shaping gut microbiota, but the molecular mechanism underlying interplay with intestinal microbiota is unclear. Here, we showed that lemon-derived exosome-like nanoparticles (LELNs) enhance lactobacilli toleration to bile. To decipher the mechanism, we used Lactobacillus rhamnosus GG (LGG) as proof of concept to show that LELNs enhance LGG bile resistance via limiting production of Msp1 and Msp3, resulting in decrease of bile accessibility to cell membrane. Furthermore, we found that decline of Msps protein levels was regulated through specific tRNAser UCC and tRNAser UCG decay. We identified RNase P, an essential housekeeping endonuclease, being responsible for LELNs-induced tRNAser UCC and tRNAser UCG decay. We further identified galacturonic acid-enriched pectin-type polysaccharide as the active factor in LELNs to increase bile resistance and downregulate tRNAser UCC and tRNAser UCG level in the LGG. Our study demonstrates a tRNA-based gene expression regulation mechanism among lactobacilli to increase bile resistance.

miR-375 prevents high-fat diet-induced insulin resistance and obesity by targeting the aryl hydrocarbon receptor and bacterial tryptophanase (tnaA) gene.

Background: Diet manipulation is the basis for prevention of obesity and diabetes. The molecular mechanisms that mediate the diet-based prevention of insulin resistance are not well understood. Here, as proof-of-concept, ginger-derived nanoparticles (GDNP) were used for studying molecular mechanisms underlying GDNP mediated prevention of high-fat diet induced insulin resistance. Methods: Ginger-derived nanoparticles (GDNP) were isolated from ginger roots and administered orally to C57BL/6 high-fat diet mice. Fecal exosomes released from intestinal epithelial cells (IECs) of PBS or GDNP treated high-fat diet (HFD) fed mice were isolated by differential centrifugation. A micro-RNA (miRNA) polymerase chain reaction (PCR) array was used to profile the exosomal miRs and miRs of interest were further analyzed by quantitative real time (RT) PCR. miR-375 or antisense-miR375 was packed into nanoparticles made from the lipids extracted from GDNP. Nanoparticles was fluorescent labeled for monitoring their in vivo trafficking route after oral administration. The effect of these nanoparticles on glucose and insulin response of mice was determined by glucose and insulin tolerance tests. Results: We report that HFD feeding increased the expression of AhR and inhibited the expression of miR-375 and VAMP7. Treatment with orally administered ginger-derived nanoparticles (GDNP) resulted in reversing HFD mediated inhibition of the expression of miR-375 and VAMP7. miR-375 knockout mice exhibited impaired glucose homeostasis and insulin resistance. Induction of intracellular miR-375 led to inhibition of the expression of AhR and VAMP7 mediated exporting of miR-375 into intestinal epithelial exosomes where they were taken up by gut bacteria and inhibited the production of the AhR ligand indole. Intestinal exosomes can also traffic to the liver and be taken up by hepatocytes, leading to miR-375 mediated inhibition of hepatic AhR over-expression and inducing the expression of genes associated with the hepatic insulin response. Altogether, GDNP prevents high-fat diet-induced insulin resistance by miR-375 mediated inhibition of the aryl hydrocarbon receptor mediated pathways over activated by HFD feeding. Conclusion: Collectively our findings reveal that oral administration of GDNP to HFD mice improves host glucose tolerance and insulin response via regulating AhR expression by GDNP induced miR-375 and VAMP7.

High-fat diet-induced upregulation of exosomal phosphatidylcholine contributes to insulin resistance.

High-fat diet (HFD) decreases insulin sensitivity. How high-fat diet causes insulin resistance is largely unknown. Here, we show that lean mice become insulin resistant after being administered exosomes isolated from the feces of obese mice fed a HFD or from patients with type II diabetes. HFD altered the lipid composition of exosomes from predominantly phosphatidylethanolamine (PE) in exosomes from lean animals (L-Exo) to phosphatidylcholine (PC) in exosomes from obese animals (H-Exo). Mechanistically, we show that intestinal H-Exo is taken up by macrophages and hepatocytes, leading to inhibition of the insulin signaling pathway. Moreover, exosome-derived PC binds to and activates AhR, leading to inhibition of the expression of genes essential for activation of the insulin signaling pathway, including IRS-2, and its downstream genes PI3K and Akt. Together, our results reveal HFD-induced exosomes as potential contributors to the development of insulin resistance. Intestinal exosomes thus have potential as broad therapeutic targets.

Lemon Exosome-like Nanoparticles-Manipulated Probiotics Protect Mice from C. d iff Infection.

Clostridioides difficile (C. diff) is the leading cause of antibiotic-associated colitis. Here, we report that lemon exosome-like nanoparticles (LELNs) manipulated probiotics to inhibit C. diff infection (CDI). LELN-manipulated Lactobacillus rhamnosus GG (LGG) and Streptococcus thermophilus ST-21 (STH) (LELN-LS) decrease CDI mortality via an LELN-mediated increase in bile resistance and gut survivability. LELN-LS treatment increases the AhR ligands indole-3-lactic acid (I3LA) and indole-3-carboxaldehyde (I3Ald), leading to induction of IL-22, and increases lactic acid leading to a decrease of C. diff fecal shedding by inhibiting C. diff growth and indole biosynthesis. A synergistic effect between STH and LGG was identified. The STH metabolites inhibit gluconeogenesis of LGG and allow fructose-1,6-bisphosphate (FBP) to accumulate in LGG; accumulated FBP then activates lactate dehydrogenase of LGG (LGG-LDH) and enhances production of lactic acid and the AhR ligand. Our findings provide a new strategy for CDI prevention and treatment with a new type of prebiotics.

Plant-Derived Exosomal Nanoparticles Inhibit Pathogenicity of Porphyromonas gingivalis.

Plant exosomes protect plants against infection; however, whether edible plant exosomes can protect mammalian hosts against infection is not known. In this study, we show that ginger exosome-like nanoparticles (GELNs) are selectively taken up by the periodontal pathogen Porphyromonas gingivalis in a GELN phosphatidic acid (PA) dependent manner via interactions with hemin-binding protein 35 (HBP35) on the surface of P. gingivalis. Compared with PA (34:2), PA (34:1) did not interact with HBP35, indicating that the degree of unsaturation of PA plays a critical role in GELN-mediated interaction with HBP35. On binding to HBP35, pathogenic mechanisms of P. gingivalis were significantly reduced following interaction with GELN cargo molecules, including PA and miRs. These cargo molecules interacted with multiple pathogenic factors in the recipient bacteria simultaneously. Using edible plant exosome-like nanoparticles as a potential therapeutic agent to prevent/treat chronic periodontitis was further demonstrated in a mouse model.

Plant-Derived Exosomal MicroRNAs Shape the Gut Microbiota.

The gut microbiota can be altered by dietary interventions to prevent and treat various diseases. However, the mechanisms by which food products modulate commensals remain largely unknown. We demonstrate that plant-derived exosome-like nanoparticles (ELNs) are taken up by the gut microbiota and contain RNAs that alter microbiome composition and host physiology. Ginger ELNs (GELNs) are preferentially taken up by Lactobacillaceae in a GELN lipid-dependent manner and contain microRNAs that target various genes in Lactobacillus rhamnosus (LGG). Among these, GELN mdo-miR7267-3p-mediated targeting of the LGG monooxygenase ycnE yields increased indole-3-carboxaldehyde (I3A). GELN-RNAs or I3A, a ligand for aryl hydrocarbon receptor, are sufficient to induce production of IL-22, which is linked to barrier function improvement. These functions of GELN-RNAs can ameliorate mouse colitis via IL-22-dependent mechanisms. These findings reveal how plant products and their effects on the microbiome may be used to target specific host processes to alleviate disease.

Blood exosomes regulate the tissue distribution of grapefruit-derived nanovector via CD36 and IGFR1 pathways.

Tumor-specific delivery of therapeutics is challenging. One of the major hurdles for successfully delivering targeted agents by nanovectors is the filtering role of the liver in rapidly sequestering nanovectors from the circulation. Exosomes, a type of endogenous nanoparticle, circulate continuously in the peripheral blood and play a role in intercellular communication. The aim of this study was to determine whether the level of endogenous exosomes has an effect on nanovector delivery efficiency of targeted agents. Methods: Exosomes were isolated from peripheral blood and intravenously (I.V.) injected into tumor-bearing mice. Subsequently, 1,1-dioctadecyl-3,3,3'3'-tetramethylindotricarbocyanine-iodide (DiR) fluorescent dye-labeled nanoparticles, including grapefruit nanovectors (GNV) and standard liposomes, were I.V. injected in the mice. The efficiency of redirecting GNVs from liver to other organs of injected mice was further analyzed with in vivo imaging. The concentration of chemo drugs delivered by GNV was measured by HPLC and the anti-lung metastasis therapeutic effects of chemo drugs delivered by GNVs in mouse breast cancer and melanoma cancer models were evaluated. Results: We show that tail vein-injected exosomes isolated from mouse peripheral blood were predominately taken up by liver Kupffer cells. Injection of peripheral blood-derived exosomes before I.V. injection of grapefruit-derived nanovector (GNV) decreased the deposition of GNV in the liver and redirected the GNV to the lung and to the tumor in breast and melanoma tumor-bearing mouse models. Enhanced therapeutic efficiency of doxorubicin (Dox) or paclitaxel (PTX) carried by GNVs for lung metastases was demonstrated when there was an I.V. injection of exosomes before therapeutic treatment. Furthermore, we found that CD36 and IGFR1 receptor-mediated pathways played a critical role in the exosome-mediated inhibitory effect of GNV entry into liver macrophages. Conclusions: Collectively, our findings provide a foundation for using autologous exosomes to enhance therapeutic vector targeted delivery to the lung.