Yuelong Liu

A Novel Nanoparticle Drug Delivery System: The Anti-inflammatory Activity of Curcumin Is Enhanced When Encapsulated in Exosomes

Monocyte-derived myeloid cells play vital roles in inflammation-related autoimmune/inflammatory diseases and cancers. Here, we report that exosomes can deliver anti-inflammatory agents, such as curcumin, to activated myeloid cells in vivo. This technology provides a means for anti-inflammatory drugs, such as curcumin, to target the inflammatory cells as well as to overcome unwanted off-target effects that limit their utility. Using exosomes as a delivery vehicle, we provide evidence that curcumin delivered by exosomes is more stable and more highly concentrated in the blood. We show that the target specificity is determined by exosomes, and the improvement of curcumin activity is achieved by directing curcumin to inflammatory cells associated with therapeutic, but not toxic, effects. Furthermore, we validate the therapeutic relevance of this technique in a lipopolysaccharide (LPS)-induced septic shock mouse model. We further show that exosomes, but not lipid alone, are required for the enhanced anti-inflammatory activity of curcumin. The specificity of using exosomes as a drug carrier creates opportunities for treatments of many inflammation-related diseases without significant side effects due to innocent bystander or off-target effects.

Induction of myeloid-derived suppressor cells by tumor exosomes.

Myeloid‐derived suppressor cells (MDSCs) promote tumor progression. The mechanisms of MDSC development during tumor growth remain unknown. Tumor exosomes (T‐exosomes) have been implicated to play a role in immune regulation, however the role of exosomes in the induction of MDSCs is unclear. Our previous work demonstrated that exosomes isolated from tumor cells are taken up by bone marrow myeloid cells. Here, we extend those findings showing that exosomes isolated from T‐exosomes switch the differentiation pathway of these myeloid cells to the MDSC pathway (CD11b+Gr‐1+). The resulting cells exhibit MDSC phenotypic and functional characteristics including promotion of tumor growth. Furthermore, we demonstrated that in vivo MDSC mediated promotion of tumor progression is dependent on T‐exosome prostaglandin E2 (PGE2) and TGF‐β molecules. T‐exosomes can induce the accumulation of MDSCs expressing Cox2, IL‐6, VEGF, and arginase‐1. Antibodies against exosomal PGE2 and TGF‐β block the activity of these exosomes on MDSC induction and therefore attenuate MDSC‐mediated tumor‐promoting ability. Exosomal PGE2 and TGF‐β are enriched in T‐exosomes when compared with exosomes isolated from the supernatants of cultured tumor cells (C‐exosomes). The tumor microenvironment has an effect on the potency of T‐exosome mediated induction of MDSCs by regulating the sorting and the amount of exosomal PGE2 and TGF‐β available. Together, these findings lend themselves to developing specific targetable therapeutic strategies to reduce or eliminate MDSC‐induced immunosuppression and hence enhance host antitumor immunotherapy efficacy.

Tumor Exosomes Inhibit Differentiation of Bone Marrow Dendritic Cells

The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. In this study, we show that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b+ myeloid precursors in the bone marrow (BM) in vivo, and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that TS/A exosomes block the differentiation of murine myeloid precursor cells into dendritic cells (DC) in vitro. Addition of tumor exosomes at day 0 led to a significant block of differentiation into DC, whereas addition at later time points was less effective. Similarly, exosomes produced by human breast tumor cells inhibited the differentiation of human monocytes in vitro. The levels of IL-6 and phosphorylated Stat3 were elevated 12 h after the tumor exosome stimulation of murine myeloid precursors, and tumor exosomes were less effective in inhibiting differentiation of BM cells isolated from IL-6 knockout mice. Addition of a rIL-6 to the IL-6 knockout BM cell culture restored the tumor exosome-mediated inhibition of DC differentiation. These data suggest that tumor exosome-mediated induction of IL-6 plays a role in blocking BM DC differentiation.

Contribution of MyD88 to the Tumor Exosome-Mediated Induction of Myeloid Derived Suppressor Cells

In this study we observed that mice pretreated with tumor exosomes had a significant acceleration of tumor metastasis in the lung. Tumor metastasis correlated significantly with an increase in recruitment of more Myeloid-derived suppressor cells (MDSCs) in the lung of C57BL/6j (B6) mice pretreated with tumor exosomes. These effects were blunted when MyD88 knockout (KO) mice were pretreated with tumor exosomes. MDSCs induced by tumor exosomes and isolated from wild-type B6 mice also more potently inhibited T cell activation and induction of interleukin-6 and tumor necrosis factor-alpha than MDSCs isolated from the lung of MyD88 KO mice. In vitro, addition of tumor exosomes to bone marrow-derived CD11b(+)Gr-1(+) cells isolated from wild-type B6 mice resulted in more cytokine production, including tumor necrosis factor-alpha, interleukin-6, and the chemokine CCL2, than CD11b(+)Gr-1(+) cells isolated from MyD88 KO mice. Moreover, lower levels of CCL2 were observed in the lungs in MyD88 KO mice pretreated with tumor exosomes than that in wild-type mice. Together these data demonstrate a pivotal role for MyD88 in tumor exosome-mediated expansion of MDSCs and tumor metastasis.

Adipose Tissue Exosome-Like Vesicles Mediate Activation of Macrophage-Induced Insulin Resistance

OBJECTIVE We sought to determine whether exosome-like vesicles (ELVs) released from adipose tissue play a role in activation of macrophages and subsequent development of insulin resistance in a mouse model. RESEARCH DESIGN AND METHODS ELVs released from adipose tissue were purified by sucrose gradient centrifugation and labeled with green fluorescent dye and then intravenously injected into B6 ob/ob mice (obese model) or B6 mice fed a high-fat diet. The effects of injected ELVs on the activation of macrophages were determined through analysis of activation markers by fluorescence-activated cell sorter and induction of inflammatory cytokines using an ELISA. Glucose tolerance and insulin tolerance were also evaluated. Similarly, B6 mice with different gene knockouts including TLR2, TLR4, MyD88, and Toll-interleukin-1 receptor (TIR) domain–containing adaptor protein inducing interferon-β (TRIF) were also used for testing their responses to the injected ELVs. RESULTS ELVs are taken up by peripheral blood monocytes, which then differentiate into activated macrophages with increased secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Injection of obELVs into wild-type C57BL/6 mice results in the development of insulin resistance. When the obELVs were intravenously injected into TLR4 knockout B6 mice, the levels of glucose intolerance and insulin resistance were much lower. RBP4 is enriched in the obELVs. Bone marrow–derived macrophages preincubated with recombinant RBP4 led to attenuation of obELV-mediated induction of IL-6 and TNF-α. CONCLUSIONS ELVs released by adipose tissue can act as a mode of communication between adipose tissues and macrophages. The obELV-mediated induction of TNF-α and IL-6 in macrophages and insulin resistance requires the TLR4/TRIF pathway.

Thymus Exosomes-Like Particles Induce Regulatory T Cells

Exosomes released from different types of cells have been proposed to contribute to intercellular communication. We report that thymic exosome-like particles (ELPs) released from cells of the thymus can induce the development of Foxp3+ regulatory T (Treg) cells in the lung and liver. Thymic ELPs also induce the conversion of thymic CD4+CD25− T cells into Tregs. Tregs induced by thymic ELPs suppress the proliferation of CD4+CD25− T cells in vitro and in vivo. We further show that neutralization of TGF-β in ELPs partially reverses thymic ELP-mediated induction of CD4+Foxp3+ T cells in the lung and liver. This study demonstrates that thymic ELPs participate in the induction of Foxp3+ Tregs. Also, TGF-β of thymic ELPs might be required for the generation of Tregs in the peripheral tissues.

Immature myeloid cells induced by a high-fat diet contribute to liver inflammation.

Chronic inflammation plays a critical role in promoting obesity‐related disorders, such as fatty liver disease. The inflammatory cells that mediate these effects remain unknown. This study investigated the accumulation of immature myeloid cells in the liver and their role in liver inflammation. We found that the accumulation of immature myeloid cells, i.e., CD11b+Ly6ChiLy6G− cells, in the liver of B6 mice fed a high‐fat diet contribute to liver inflammation. Adoptive transfer of CD11b+Ly6ChiLy6G− cells isolated from the liver of obese B6 mice, but not from lean B6 mice, resulted in liver damage that was evident by an increase in the activity of liver transferases in serum. CD11b+Ly6ChiLy6G− cells isolated from the liver of obese mice are more easily activated by way of Toll‐like receptor (TLR) stimulation resulting in interleukin 12 and other inflammatory cytokine expression in an MyD88‐dependent fashion. TLR7‐activated CD11b+Ly6ChiLy6G− cells also enhance liver natural killer T cell (NKT) death in an Fas‐dependent manner. Experiments using mice depleted of Gr‐1+ immature myeloid cells demonstrated the important role of CD11b+Ly6ChiLy6G− in liver inflammation. Repeated injection of exosome‐like particles causes CD11b+ cell activation and subsequent homing to and accumulation of the cells in the liver. Conclusion: Consumption of a high‐fat diet by B6 mice triggers an accumulation of immature myeloid cells in the liver. The immature myeloid cells release proinflammatory cytokines and induce NKT cell apoptosis. Activation‐induced NKT apoptosis further promotes excessive production of Th‐1 cytokines. This diet‐induced accumulation of immature myeloid cells may contribute to obesity‐related liver disease. (HEPATOLOGY 2009.)

TLR2-Mediated Expansion of MDSCs Is Dependent on the Source of Tumor Exosomes

Exosomes released from tumor cells having been shown to induce interleukin-6 release from myeloid-derived suppressor cells in a Toll-like receptor 2/Stat3-dependent manner. In this study, we show that exosomes released from tumor cells re-isolated from syngeneic mice are capable of inducing interleukin-6 in a Toll-like receptor 2-independent manner, whereas the data generated from exosomes of tumor cells having undergone numerous in vitro passages induce interleukin-6 in a Toll-like receptor 2-dependent manner. This discrepancy may be due to the source of tumor cells used to generate the exosomes for this study. These results suggest that exosomes released from tumor cells that are not within a tumor microenvironment may not realistically represent the role of tumor exosomes in vivo. This is an important consideration since frequently passing tumor cells in vivo is an accepted practice for studying tumor exosome-mediated inflammatory responses.

Exosome-like Nanoparticles from Intestinal Mucosal Cells Carry Prostaglandin E2 and Suppress Activation of Liver NKT Cells

Regulation and induction of anergy in NKT cells of the liver can inhibit autoimmune and antitumor responses by mechanisms that are poorly understood. We investigated the effects of PGE2, delivered by intestinal, mucus-derived, exosome-like nanoparticles (IDENs), on NKT cells in mice. In this study, 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-galactosylceramide (α-GalCer)–stimulated liver NKT cells in a PGE2 E-type prostanoid 2/E-type prostanoid 4 receptor–mediated manner. Proinflammatory 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.

Exosomes and immune surveillance of neoplastic lesions: a review

Abstract The immune system has been reported to suppress the development and progression of neoplastic lesions; however, the exact mechanisms by which neoplastic lesions and the immune system interact are not well understood. Within the last decade, tiny membrane bound particles, approximately 30–100 nm in diameter, have been observed in the blood and other body fluids. These particles, currently called exosomes, are released from many types of tissues including tumors, and they contain and carry many proteins, and mRNAs and microRNA species. We review here how tumors suppress the immune system, especially by the formation of exosomes. Exosomes released from tumors are carried in part by the vascular system to distant cells, which phagocytose them. Depending on the proteins, mRNAs or microRNAs in the exosomes and the cell type, phagocytosis of exosomes may provide a modulating signal to the cell. In the case of exosomes from tumors, uptake of the exosomes by cells of the immune system has been reported to have three main effects: 1) suppression of the number and activity of natural killer cells, 2) suppression of the activity of T cells and 3) suppression of the number and maturation of mature dendritic cells.