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Lung inflammation is a hallmark of coronavirus disease 2019 (COVID-19). In this study, we show that mice develop inflamed lung tissue after being administered exosomes released from the lung epithelial cells exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp12 and Nsp13 (exosomesNsp12Nsp13). Mechanistically, we show that exosomesNsp12Nsp13 are taken up by lung macrophages, leading to activation of nuclear factor κB (NF-κB) and the subsequent induction of an array of inflammatory cytokines. Induction of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β from exosomesNsp12Nsp13-activated lung macrophages contributes to inducing apoptosis in lung epithelial cells. Induction of exosomesNsp12Nsp13-mediated lung inflammation was abolished with ginger exosome-like nanoparticle (GELN) microRNA (miRNA aly-miR396a-5p. The role of GELNs in inhibition of the SARS-CoV-2-induced cytopathic effect (CPE) was further demonstrated via GELN aly-miR396a-5p- and rlcv-miR-rL1-28-3p-mediated inhibition of expression of Nsp12 and spike genes, respectively. Taken together, our results reveal exosomesNsp12Nsp13 as potentially important contributors to the development of lung inflammation, and GELNs are a potential therapeutic agent to treat COVID-19., Graphical Abstract, COVID-19 is a disease affecting the pulmonary system with devastating inflammation. Teng et al. have shown that exosomes released from SARS-CoV-2-infected lung epithelia cells are preferentially taken up by lung macrophages that subsequently induce lung inflammation. Ginger exosome-like nanoparticle miRNA inhibits SARS-CoV-2-induced lung inflammation and viral replication.

Macrophages have been linked to tumor initiation, progression, metastasis, and treatment resistance. However, the transcriptional regulation of macrophages driving the protumor function remains elusive. Here, we demonstrate that the transcription factor c-Maf is a critical controller for immunosuppressive macrophage polarization and function in cancer. c-Maf controls many M2-related genes and has direct binding sites within a conserved noncoding sequence of the Csf-1r gene and promotes M2-like macrophage–mediated T cell suppression and tumor progression. c-Maf also serves as a metabolic checkpoint regulating the TCA cycle and UDP-GlcNAc biosynthesis, thus promoting M2-like macrophage polarization and activation. Additionally, c-Maf is highly expressed in tumor-associated macrophages (TAMs) and regulates TAM immunosuppressive function. Deletion of c-Maf specifically in myeloid cells results in reduced tumor burden with enhanced antitumor T cell immunity. Inhibition of c-Maf partly overcomes resistance to anti–PD-1 therapy in a subcutaneous LLC tumor model. Similarly, c-Maf is expressed in human M2 and tumor-infiltrating macrophages/monocytes as well as circulating monocytes of human non–small cell lung carcinoma (NSCLC) patients and critically regulates their immunosuppressive activity. The natural compound β-glucan downregulates c-Maf expression on macrophages, leading to enhanced antitumor immunity in mice. These findings establish a paradigm for immunosuppressive macrophage polarization and transcriptional regulation by c-Maf and suggest that c-Maf is a potential target for effective tumor immunotherapy.

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.

SUMMARY Dermal γδT cells play critical roles in skin homeostasis and inflammation. However, the underlying molecular mechanisms by which these cells are activated have not been fully understood. Here, we show that the mechanistic or mammalian target of rapamycin (mTOR) and STAT3 pathways are activated in dermal γδT cells in response to innate stimuli such as interleukin-1β (IL-1β) and IL-23. Although both mTOR complex 1 (mTORC1) and mTORC2 are essential for dermal γδT cell proliferation, mTORC2 deficiency leads to decreased dermal γδT17 cells. It appears that mitochondria-mediated oxidative phosphorylation is critical in this process. Notably, although the STAT3 pathway is critical for dermal Vγ4T17 effector function, it is not required for γδ6T17 cells. Transcription factor IRF-4 activation promotes dermal γδT cell IL-17 production by linking IL-1β and IL-23 signaling. The absence of mTORC2 in dermal γδT cells, but not STAT3, ameliorates skin inflammation. Taken together, our results demonstrate that the mTOR-STAT3 signaling differentially regulates dermal γδT cell effector function in skin inflammation., In Brief Cai et al. demonstrate that the mTOR and STAT3 signaling pathways differentially regulate dermal Vγ4 and Vγ6 T cell effector function, leading to distinct outcomes in skin inflammation., Graphical Abstract

The importance of gut microbiota in human health and pathophysiology is undisputable. Despite the abundance of metagenomics data, the functional dynamics of gut microbiota in human health and disease remain elusive. Urolithin A (UroA), a major microbial metabolite derived from polyphenolics of berries and pomegranate fruits displays anti-inflammatory, anti-oxidative, and anti-ageing activities. Here, we show that UroA and its potent synthetic analogue (UAS03) significantly enhance gut barrier function and inhibit unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2–related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. Importantly, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction in addition to anti-inflammatory activities. Cumulatively, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing inflammation to protect from colonic diseases., Urolithins are microbial metabolites derived from food polyphenols. Here, Singh et al. show that urolithin A and a synthetic analogue enhance gut barrier function via Nrf2-dependent pathways and mitigate inflammation and colitis in mice, highlighting a potential application for inflammatory bowel diseases.

The IL-1 signaling pathway has been shown to play a critical role in the pathogenesis of chronic, autoinflammatory skin diseases such as psoriasis. However, the exact cellular and molecular mechanisms have not been fully understood. Here, we show that IL-1β is significantly elevated in psoriatic lesional skin and imiquimod-treated mouse skin. In addition, IL-1R signaling appears to correlate with psoriasis disease progression and treatment response. IL-1 signaling in both dermal γδ T cells and other cells such as keratinocytes is essential to an IMQ-induced skin inflammation. IL-1β induces dermal γδ T cell proliferation and IL-17 production in mice. In addition, IL-1β stimulates keratinocytes to secrete chemokines that preferentially chemoattract peripheral CD27– CCR6+IL-17 capable of producing γδ T cells (γδT17). Further studies showed that endogenous IL-1β secretion is regulated by skin commensals to maintain dermal γδT17 homeostasis in mice. Mouse skin associated with Corynebacterium species, bacteria enriched in human psoriatic lesional skin, has increased IL-1β and dermal γδT17 cell expansion. Thus, the IL-1β–IL-1R signaling pathway may contribute to skin inflammation and psoriasis pathogenesis via the direct regulation of dermal IL-17–producing cells and stimulation of keratinocytes for amplifying inflammatory cascade.

SARS coronavirus 2 (SARS-CoV-2) is a novel viral pathogen that causes a clinical disease called coronavirus disease 2019 (COVID-19). Although most COVID-19 cases are asymptomatic or involve mild upper respiratory tract symptoms, a significant number of patients develop severe or critical disease. Patients with severe COVID-19 commonly present with viral pneumonia that may progress to life-threatening acute respiratory distress syndrome (ARDS). Patients with COVID-19 are also predisposed to venous and arterial thromboses that are associated with a poorer prognosis. The present study identified the emergence of a low-density inflammatory neutrophil (LDN) population expressing intermediate levels of CD16 (CD16Int) in patients with COVID-19. These cells demonstrated proinflammatory gene signatures, activated platelets, spontaneously formed neutrophil extracellular traps, and enhanced phagocytic capacity and cytokine production. Strikingly, CD16Int neutrophils were also the major immune cells within the bronchoalveolar lavage fluid, exhibiting increased CXCR3 but loss of CD44 and CD38 expression. The percentage of circulating CD16Int LDNs was associated with D-dimer, ferritin, and systemic IL-6 and TNF-α levels and changed over time with altered disease status. Our data suggest that the CD16Int LDN subset contributes to COVID-19–associated coagulopathy, systemic inflammation, and ARDS. The frequency of that LDN subset in the circulation could serve as an adjunct clinical marker to monitor disease status and progression.

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., High-fat diet plays a role in development of insulin resistance. Here, the authors report a mechanism that underlies the development of diet induced insulin resistance through the activation of an aryl hydrocarbon receptor mediated signalling pathway in the liver by faecal exosomes derived from intestinal cells.

Within the past decades, extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in both prokaryotes and higher eukaryotes to regulate a diverse range of biological processes. Besides EVs, exosome-like nanoparticles (ELNs) derived from plants were also emerging. Comparing to EVs, ELNs are source-widespread, cost-effective and easy to obtain. Their definite activities can be utilized for potential prevention/treatment of an abundance of diseases, including metabolic syndrome, cancer, colitis, alcoholic hepatitis and infectious diseases, which highlights ELNs as promising biotherapeutics. In addition, the potential of ELNs as natural or engineered drug carriers is also attractive. In this review, we tease out the timeline of plant EVs and ELNs, introduce the arising separation, purification and characterization techniques, state the stability and transport manner, discuss the therapeutic opportunities as well as the potential as novel drug carriers. Finally, the challenges and the direction of efforts to realize the clinical transformation of ELNs are also discussed.

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.

One of the defining characteristics of a pre-metastatic niche, a fundamental requirement for primary tumor metastasis, is infiltration of immunosuppressive macrophages. How these macrophages acquire their phenotype remains largely unexplored. Here, we demonstrate that tumor-derived exosomes (TDEs) polarize macrophages toward an immunosuppressive phenotype characterized by increased PD-L1 expression through NF-kB-dependent, glycolytic-dominant metabolic reprogramming. TDE signaling through TLR2 and NF-κB leads to increased glucose uptake. TDEs also stimulate elevated NOS2, which inhibits mitochondrial oxidative phosphorylation resulting in increased conversion of pyruvate to lactate. Lactate feeds back on NF-κB, further increasing PD-L1. Analysis of metastasis-negative lymph nodes of non-small-cell lung cancer patients revealed that macrophage PD-L1 positively correlates with levels of GLUT-1 and vesicle release gene YKT6 from primary tumors. Collectively, our study provides a novel mechanism by which macrophages within a pre-metastatic niche acquire their immunosuppressive phenotype and identifies an important link among exosomes, metabolism, and metastasis.

© 2019 Elsevier Inc. The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data. The Extracellular RNA Communication Consortium (ERCC) presents progress toward understanding the biology of extracellular RNAs and their use as biomarkers and therapeutics.

Acquired resistance to chemotherapy remains a major stumbling block in cancer treatment. Chronic inflammation has a crucial role in induction of chemoresistance and results, in part, from the induction and expansion of inflammatory cells that include myeloid-derived suppressor cells (MDSCs) and IL-13+ Th2 cells. The mechanisms that lead to induction of activated MDSCs and IL-13+ Th2 cells have not yet been identified. Here we demonstrated that doxorubicin (DOX) treatment of 4T1 breast tumor-bearing mice led to the induction of IL-13R+miR-126a+ MDSCs (DOX-MDSC). DOX-MDSC promote breast tumor lung metastasis through MDSC miR-126a+ exosomal-mediated induction of IL-13+ Th2 cells and tumor angiogenesis. The induction of DOX-MDSC is regulated in a paracrine manner. DOX treatment not only increases interleukin (IL)-33 released from breast tumor cells, which is crucial for the induction of IL-13+ Th2 cells, but it also participates in the induction of IL-13 receptors and miR-126a expressed on/in the MDSCs. IL-13 released from IL-13+Th2 cells then promotes the production of DOX-MDSC and MDSC miR-126a+ exosomes via MDSC IL-13R. MDSC miR-126a+ exosomes further induce IL13+ Th2 cells in a positive feed-back loop manner. We also showed that MDSC miR-126a rescues DOX-induced MDSC death in a S100A8/A9-dependent manner and promotes tumor angiogenesis. Our findings provide insight into the MDSC exosomal-mediated chemoresistance mechanism, which will be useful for the design of inhibitors targeting the blocking of induction of miR-126a+ MDSCs.

// Huang-Ge Zhang 1, 2 , Pengxiao Cao 2 , Yun Teng 2 , Xin Hu 4, 5 , Qilong Wang 2, 8 , Ashish S. Yeri 6 , Xiaoying Zhuang 2 , Abhilash Samykutty 2 , Jingyao Mu 2 , Zhong-Bin Deng 3 , Lifeng Zhang 2 , James A. Mobley 7 , Jun Yan 3 , Kendall Van Keuren-Jensen 6 , Donald Miller 3 1 Louisville Veterans Administration Medical Center, Louisville, KY 40206, USA 2 James Brown Cancer Center, Department of Microbiology and Immunology, University of Louisville, KY 40202, USA 3 Department of Medicine, University of Louisville, KY 40202, USA 4 Program in Biostatistics, Bioinformatics and Systems Biology, The University of Texas Graduate School of Biomedical Sciences at Houston, TX 77030, USA 5 Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 6 Translational Genomics Research Institute, Phoenix, AZ 85004, USA 7 Mass Spectrometry/Proteomics Shared Facility, University of Alabama at Birmingham, Birmingham, AL 35294, USA 8 Department of Clinical Oncology, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, 223300, China Correspondence to: Huang-Ge Zhang, email: H0Zhan17@louisville.edu Keywords: isolation and identification extracellular microvesicles, in vivo predominately population, HG-NV, exosomes Received: November 12, 2015 Accepted: April 16, 2016 Published: May 12, 2016 ABSTRACT Extracellular microvesicles (EVs) have been recognized for many potential clinical applications including biomarkers for disease diagnosis. In this study, we identified a major population of EVs by simply screening fluid samples with a nanosizer. Unlike other EVs, this extracellular nanovesicle (named HG-NV, HG-NV stands for HomoGenous nanovesicle as well as for Huang-Ge- nanovesicle) can be detected with a nanosizer with minimal in vitro manipulation and are much more homogenous in size (8–12 nm) than other EVs. A simple filtration platform is capable of separating HG-NVs from peripheral blood or cell culture supernatants. In comparison with corresponding exosome profiles, HG-NVs released from both mouse and human breast tumor cells are enriched with RNAs. Tumor derived HG-NVs are more potent in promoting tumor progression than exosomes. In summary, we identified a major subset of EVs as a previously unrecognized nanovesicle. Tumor cell derived HG-NVs promote tumor progression. Molecules predominantly present in breast tumor HG-NVs have been identified and characterized. This discovery may have implications in advancing both microvesicle biology research and clinical management including potential used as a biomarker.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that promote tumor progression. In this study, we demonstrated that activation of a C-type lectin receptor, dectin-1, in MDSC differentially modulates the function of different MDSC subsets. Yeast-derived whole β-glucan particles (WGP; a ligand to engage and activate dectin-1, oral treatment in vivo) significantly decreased tumor weight and splenomegaly in tumor-bearing mice with reduced accumulation of polymorphonuclear MDSC but not monocytic MDSC (M-MDSC), and decreased polymorphonuclear MDSC suppression in vitro through the induction of respiratory burst and apoptosis. On a different axis, WGP-treated M-MDSC differentiated into F4/80+CD11c+ cells in vitro that served as potent APC to induce Ag-specific CD4+ and CD8+ T cell responses in a dectin-1–dependent manner. Additionally, Erk1/2 phosphorylation was required for the acquisition of APC properties in M-MDSC. Moreover, WGP-treated M-MDSC differentiated into CD11c+ cells in vivo with high MHC class II expression and induced decreased tumor burden when inoculated s.c. with Lewis lung carcinoma cells. This effect was dependent on the dectin-1 receptor. Strikingly, patients with non–small cell lung carcinoma that had received WGP treatment for 10–14 d prior to any other treatment had a decreased frequency of CD14−HLA-DR−CD11b+CD33+ MDSC in the peripheral blood. Overall, these data indicate that WGP may be a potent immune modulator of MDSC suppressive function and differentiation in cancer.

Exosomes have shown increasing potential as delivery vesicles for therapy, but challenges like cost/yield, drug payload, and targeting specificity still exist. Plant derived exosome-like nanoparticles have been reported as a promising substitution and exhibit biocompatibility through oral, intranasal administration; however, systemic delivery of siRNA by exosome-like nanoparticles directly isolated from plants has not been reported. Recently, we reported the control of RNA orientation to decorate human derived exosome with cell targeting ligands for specific delivery of siRNA to tumors. Here, we expand to the application of arrowtail RNA nanoparticles for displaying ligands on ginger derived exosome-like nanovesicles (GDENs) for siRNA delivery and tumor inhibition through IV administration. Cushion ultracentrifugation coupled with equilibrium density gradient ultracentrifugation were used for purifying GDENs that displayed size, density, and morphology similar to human derived exosomes. Folic acid (FA), as a ligand, was displayed on the surface of GDENs for targeted delivery of survivin siRNA to KB cancer models. In vitro gene knockdown efficacy by FA-3WJ/GDENs/siRNA complex was comparable to transfection. We observed inhibition of tumor growth on a xenograft model by intravenous administration, which reveals the potential of GDENs as an economic delivery system for siRNA.

Exosomes are emerging mediators of intercellular communication; whether the release of exosomes has an effect on the exosome donor cells in addition to the recipient cells has not been investigated to any extent. Here, we examine different exosomal miRNA expression profiles in primary mouse colon tumour, liver metastasis of colon cancer and naive colon tissues. In more advanced disease, higher levels of tumour suppressor miRNAs are encapsulated in the exosomes. miR-193a interacts with major vault protein (MVP). Knockout of MVP leads to miR-193a accumulation in the exosomal donor cells instead of exosomes, inhibiting tumour progression. Furthermore, miR-193a causes cell cycle G1 arrest and cell proliferation repression through targeting of Caprin1, which upregulates Ccnd2 and c-Myc. Human colon cancer patients with more advanced disease show higher levels of circulating exosomal miR-193a. In summary, our data demonstrate that MVP-mediated selective sorting of tumour suppressor miRNA into exosomes promotes tumour progression., Exosomes are involved in the development of metastasis but how their composition is regulated is not well known. Here the authors propose that major vault protein-dependent loading of miR-193a into exosomes could be a general mechanism by which cancer cells get rid of oncosuppressor miRNAs.

The miRNA cluster miR-17∼92 targets mRNAs involved in distinct pathways that either promote or inhibit tumor progression. However, the cellular and molecular mechanisms underlying miR-17∼92 cluster-mediated protumorigenic or anti-tumorigenic effects have not been studied. Herein, we determined that inhibition of colon cancer progression is dictated by quantitatively controlling expression of the miR-17∼92 cluster. miR-19 in the context of the miR-17∼92 cluster at medium levels promoted tumor metastasis through induction of Wnt/β-catenin–mediated epithelial-mesenchymal transition by targeting to the tumor-suppressor gene, PTEN . However, higher levels of the miR-17∼92 cluster switched from PTEN to oncogenes, including Ctnnb1 (β-catenin) via miR-18a, which resulted in inhibition of tumor growth and metastasis. However, overexpression of Ctnnb1 in tumor cells with high-level miR-17∼92 did not lead to an increase in the levels of β-catenin protein, suggesting that other factors regulated by higher levels of miR-17∼92 might also contribute to inhibition of tumor growth and metastasis. Those unidentified factors may negatively regulate the production of β-catenin protein. Collectively, the data presented in this study revealed that higher levels of miR-17∼92 were a critical negative regulator for activation of the Wnt/β-catenin pathway and could have a potential therapeutic application.

The intestinal immune system is continuously exposed to massive amounts of nanoparticles derived from food. Whether nanoparticles from plants we eat daily have a role in maintaining intestinal immune homeostasis is poorly defined. Here, we present evidence supporting our hypothesis that edible nanoparticles regulate intestinal immune homeostasis by targeting dendritic cells (DCs). Using three mouse colitis models, our data show that orally given nanoparticles isolated from broccoli extracts protect mice against colitis. Broccoli-derived nanoparticle (BDN)-mediated activation of adenosine monophosphate-activated protein kinase (AMPK) in DCs plays a role in not only prevention of DC activation but also induction of tolerant DCs. Adoptively transferring DCs pre-pulsed with total BDN lipids, but not sulforaphane (SFN)-depleted BDN lipids, prevented DSS-induced colitis in C57BL/6 (B6) mice, supporting the role of BDN SFN in the induction of DC tolerance. Adoptively transferring AMPK +/+ , but not AMPK −/− , DCs pre-pulsed with SFN prevented DSS-induced colitis in B6 mice, further supporting the DC AMPK role in SFN-mediated prevention of DSS-induced colitis. This finding could open new preventive or therapeutic avenues to address intestinal-related inflammatory diseases via activating AMPK.

Exosomes are small (30 to 100 nm) membrane-bound particles that are released from normal, diseased, and neoplastic cells and are present in blood and other bodily fluids. Exosomes contain a variety of molecules including signal peptides, mRNA, microRNA, and lipids. Exosomes can function to export from cells unneeded endogenous molecules and therapeutic drugs. When exosomes are taken up by specific cells, they may act locally to provide autocrine or paracrine signals or, at a distance, as a newly described nanoparticle-based endocrine system. Specifically, mRNA transferred to cells by exosomes can result in the production of new proteins. In cancer, signals via exosomes affect the immune system by inhibiting the functions of T cells and normal killer (NK) cells and by inhibiting the differentiation of precursors to mature antigen-presenting cells. Also, exosomes increase the number and/or activity of immune suppressor cells, including myeloid-derived suppressor cells, T-regulatory cells, and CD14+, HLA-DR−/low cells. The effects of exosomes on the development and progression of cancers, with an emphasis on suppression of immune surveillance, is described. Also discussed are potential uses of exosomes clinically, in the development of vaccines, in targeting tumors, and in diagnosis and/or early detection. Clin Cancer Res; 17(5); 959–64. ©2011 AACR.

An important characteristic of tumors is that they at some point in their development overcome the surveillance of the immune system. Tumors secrete exosomes, multivesicular bodies containing a distinct set of proteins that can fuse with cells of the circulating immune system. Purified exosomes from TS/A breast cancer cells, but not non-exosomal fractions, inhibit (at concentrations of nanograms per ml protein) IL-2-induced natural killer (NK) cell cytotoxicity. The dietary polyphenol, curcumin (diferuloylmethane), partially reverses tumor exosome-mediated inhibition of natural killer cell activation, which is mediated through the impairment of the ubiquitin–proteasome system. Exposure of mouse breast tumor cells to curcumin causes a dose-dependent increase in ubiquitinated exosomal proteins compared to those in untreated TS/A breast tumor cells. Furthermore, exosomes isolated from tumor cells pretreated with curcumin have a much attenuated inhibition of IL-2 stimulated NK cell activation. Jak3-mediated activation of Stat5 is required for tumor cytotoxicity of IL-2 stimulated NK cells. TS/A tumor exosomes strongly inhibit activation of Stat5, whereas the tumor exosomes isolated from curcumin-pretreated tumor cells have a lowered potency for inhibition of IL-2 stimulated NK cell cytotoxicity. These data suggest that partial reversal of tumor exosome-mediated inhibition of NK cell tumor cytotoxicity may account for the anti-cancer properties of curcumin.

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.

Inflammation is a hallmark of cancer. Activated immune cells are intrinsically capable of homing to inflammatory sites. Using three inflammatory-driven disease mouse models, we show that grapefruit-derived nanovectors (GNV) coated with inflammatory-related receptor enriched membranes of activated leukocytes (IGNVs) are enhanced for homing to inflammatory tumor tissues. Blocking LFA-1 or CXCR1 and CXCR2 on the IGNVs significantly inhibits IGNV homing to the inflammatory tissue. The therapeutic potential of IGNVs was further demonstrated by enhancing the chemotherapeutic effect as shown by inhibition of tumor growth in two tumor models and inhibiting the inflammatory effects of dextran sulfate sodium-induced mouse colitis. The fact that IGNVs are capable of homing to inflammatory tissue and that chemokines are overexpressed in diseased human tissue provides the rationale for using IGNVs to more directly deliver therapeutic agents to inflammatory tumor sites and the rationale for the use of IGNVs as treatment for certain cancers in personalized medicine. Cancer Res; 75(12); 2520–9. ©2015 AACR.

In common with many other cell types, synovial fibroblasts produce exosomes. In this study, we show that the exosomes produced by synovial fibroblasts obtained from individuals with rheumatoid arthritis (RASF), but not exosomes produced by synovial fibroblasts obtained from individuals with osteoarthritis, contain a membrane bound form of TNF-α as demonstrated by colloidal gold immunostaining of TNF-α and confirmed by both Western blot and mass spectrometry. The RASF-derived exosomes, but not exosomes derived from fibroblasts obtained from individuals with osteoarthritis, are cytotoxic for the L929 cell, a TNF-α-sensitive cell line, and stimulate activation of NF-κB and induction of collagenase-1 in RASF. These effects are blocked by addition of soluble TNFR1 (sTNFbp), suggesting that a TNF-α-signaling pathway mediates these biological activities. sTNFbp also reduced the production of exosomes by RASF, suggesting the interruption of a positive amplification loop. Exosomes can transmit signals between cells, and RASF exosomes, effectively taken up by anti-CD3-activated T cells, activated AKT and NF-κB and rendered these activated T cells resistant to apoptosis. Neutralization of exosomal membrane TNF-α by sTNFbp partially reversed this resistance, suggesting that not only TNF-α but also additional exosomal proteins may contribute to the development of apoptosis resistance.

Ubiquitin inhibitors act at many levels to enhance apoptosis signaling. For TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis signaling, there are at least five mechanisms by which apoptosis are regulated by the ubiquitin-proteasome pathway. First, proteasome inhibitors can decrease Fas-like inhibitor protein (FLIP) protein levels in tumors, resulting in increased apoptosis signaling due to increased caspase-8 activation. This appears to involve the ubiquitin ligase TNF receptor activation factor-2 (TRAF2) and acts indirectly by causing cell-cycle arrest at a stage where there is high degradation of the FLIP-TRAF2 complex. Second, the regulation of the proapoptotic Bcl-2 family member BAX occurs indirectly. Apoptosis signaling and caspase activation results in a confirmation change in the normally monomeric BAX, which exposes the BH3 domain of BAX, leading to dimerization and resistance to ubiquitin degradation. BAX then translocates into the mitochondria, resulting in the release of proapoptotic mitochondrial factors such as cytochrome c and second mitochondria-derived activator of caspase (SMAC). This results in the activation of caspase-9 and formation of the apoptosome and efficient apoptosis signaling. A third mechanism of the regulation of TRAIL signaling in the ubiquitin-proteasome pathway is mediated by the inhibitor of apoptosis proteins (IAP) E3 ligases. These IAPs can directly bind to caspases but also can act as ubiquitin ligases for caspases, resulting in the degradation of these caspases. IAP binding to caspases can be inhibited by SMAC, which exhibits a caspase-9 homology domain. The fourth mechanism for apoptosis activation by proteasome inhibitors is through the stabilization of the inhibitor of the kappaB (IkappaB)/NF-kappaB complex and prevention of nuclear translocation of the antiapoptosis transcription factor NF-kappaB. During TRAIL-DR4, DR5 signaling, this pathway is activated by interactions of activated Fas-associated death domain with activated receptor-interacting protein (RIP), which in turn activates NF-kappaB-inducing kinase and phosphorylates IkappaB. Therefore, the inhibition of IkappaB degradation blocks this RIP-mediated antiapoptosis signaling event. Last, p53 protein levels, and susceptibility to apoptosis, can be deregulated by the human homolog Hdm2 (Mdm2) E3 ligase. This process is inhibited by p53 phosphorylation and by sequestration of Mdm2 by ARF. Better mechanisms to inhibit the ubiquitin-proteasome pathway targeted at the ubiquitin-proteasome degradation process itself, or more specifically at the E3 ligases known to modulate and downregulate proapoptosis pathways will lead to the enhancement of TRAIL apoptosis signaling and better cancer therapeutic outcomes act through this pathway.

Summary Dendritic cells (DC) genetically engineered to express high levels of Fas ligand (FasL/CD95L) have been demonstrated to delete T cells in an antigen specific manner in several different animal models in vivo. However, the immunomodulatory capacity of primary human FasL-expressing Killer-DC has not been determined. Therefore, human Killer-DC were generated from mature monocyte-derived DC using the inducible CRE/LoxP adenoviral vector system, and the immunoregulatory capacity of these cells was analyzed in cocultures with primary human T cells in vitro. Combined transductions of DC by AdloxPFasL and AxCANCre resulted in FasL expression in >70% of DC without affecting the mature phenotype. Proliferation of activated primary human T cells was inhibited up to 80% in cocultures with FasL-expressing DC but not EGFP-transduced DC, which was due to induction of apoptosis in activated but not resting CD4+ and CD8+ T cells. Apoptosis induced by Killer-DC could be blocked by an anti-FasL-antibody in a dose dependent fashion. The present results demonstrate that FasL-expressing Killer-DC eliminate activated but not resting primary human CD4+ and CD8+ T cells by induction of Fas-mediated apoptosis supporting the concept to apply Killer-DC as a novel strategy for the treatment of T cell-dependent autoimmune disease and allograft rejection in humans.

During his long research career in the field of aging, Dr Bernard Strehler developed a series of theories concerning the identity of genes that can promote longevity and their role in natural selection. As a tribute to Dr Strehler, we have taken this opportunity to summarize a selection of these theories and to illustrate how these insights have influenced our search for longevity genes within the immune system. The identification of longevity genes has proven difficult. We believe that, at least in part, this reflects the emphasis on the concept of survival of the ‘physically’ fittest. We have used the immune system as a model to demonstrate that, over and above the self-evident advantage of those genes that contribute the attributes commonly associated with survival of the ‘physically’ fittest, those genes that lead to a predisposition to cooperate also confer a competitive survival advantage. As the acquisition of cooperativity in a society is linked to support mechanisms provided by older individuals, the search for longevity genes should not be limited to those genes that are associated with extended expression of a youthful phenotype. Rather these studies should be expanded to include identification of those genes that regulate physiologic parameters that affect individual longevity, even if they do not correspond with the traditional view of reproductive competitiveness. At the societal level, longevity genes may encode attributes that regulate sociologic or psychological parameters that may contribute to a tendency to non-aggressive or cooperative behavior that leads to achievement of common goals necessary for the survival of the species. This view of the selection for longevity impacts the analysis of longevity genes and aging at the organismal level. Dr Strehler viewed organismal aging as an integrated functional state, in which he conceived the outcome as reflecting the net balance of functional decrementers and evolved compensatory features. We propose that, in more evolved species, the longevity genes will be those genes, or sets of genes, that counterbalance of age-related functional decrementers with the age-related manifestation of evolved compensatory features. Thus, as illustrated here through analysis of the immune system, the longevity genes may well be those genes that promote overall systemic cooperation and compensation within the immune system and associated systems, rather than the genes that prevent age-related alterations in only one or a limited number of pathways.

Molecular imaging can reveal in vivo analysis and quantification of biochemical reactions. To enable cell-surface imaging of receptors, novel ligands have been developed which can be radiolabeled or imaged by bioluminescence. Specific examples include somatostatin receptors, estrogen and progesterone receptors, receptors involved in adhesion and externalization of phosphatidyl serine as an indicator of apoptosis. Central nervous system imaging can be carried out using ligands for receptors including dopamine, serotonin and Gamma amino butyric acid (GABA). In addition, tumor and metabolic imaging can be carried out with the Na-K ATPase pump using the tracer thallium-201 for SPECT or F-18 FDG for PET imaging. Finally, novel receptors or endogenous metabolic pathways can be analyzed combining cell-gene therapy to create specific tracer targets in cells that can be studied by molecular imaging. The challenge of molecular imaging is to first identify key pathways that are unique for a specific disease processes, such as atherosclerosis, cancer, CNS disorders, immunologic and arthritis disorders and next to devise a high-affinity specific small molecular ligand that can be adapted to be a radiolabeled tracer to study this pathway. Advances in genomics and proteomics combine with new peptide-chemistry approaches should provide a large number of targets and tracers in the near future to achieve these imaging objectives.

Aberrant microRNA (miRNA) expression has been identified in various human solid cancers. However, whether the levels of miRNA expression in tumor cells have any effect on tumor progression has not been determined. In this proof-of-concept study, the restoration of high-level expression of the miR17–92 cluster of miRNAs reveals its function as a tumor suppressor in murine solid cancer cells. Specifically, genetically engineered expression of higher levels of miR17/20a in the miR17–92 cluster in both murine breast cancer and colon cancer cells triggered natural killer (NK)–cell recognition by inhibiting the expression of MHC class I (H-2D) through the Mekk2–Mek5–Erk5 pathway. Results from the mouse tumor studies were recapitulated using samples of human solid tumors. Together, these data indicate that miR17/20a miRNAs function as tumor suppressors by reprogramming tumor cells for NK cell-mediated cytotoxicity. Cancer Immunol Res; 2(8); 789–99. ©2014 AACR.

Exosomes are tiny membrane-bound vesicles that are over produced by most proliferating cell types during normal and pathological states. Their levels are up-regulated during pregnancy and disease states such as cancer. Exosomes contain a wide variety of proteins, lipids, RNAs, non-transcribed RNAs, microRNAs and small RNAs that are representative to their cellular origin and shuttle from a donor cell to a recipient cell. From intercellular communication to tumor proliferation, exosomes carry out a diverse range of functions, both helpful and harmful. Useful as biomarkers, exosomes may be applicable in diagnostic assessments as well as cell-free anti-tumor vaccines. Exosomes of ovarian cancer contain different set of proteins and miRNAs compared to exosomes of normal, cancer-free individuals. These molecules may be used as multiple "barcode" for the development of a diagnostic tool for early detection of ovarian cancer.

A novel anti-human DR5 monoclonal antibody, TRA-8, induces apoptosis of most tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive tumor cells both in vitro and in vivo. In contrast to both the membrane-bound form of human TRAIL, which induced severe hepatitis in mice, and the soluble form of human TRAIL, which induced apoptosis of normal human hepatocytes in vitro, TRA-8 did not induce significant cell death of normal human hepatocytes. However, both primary hepatocellular carcinoma cells and an established liver cancer cell line were highly susceptible to the killing mediated by TRA-8. We show here that elevated levels of cell-surface expression of DR5 and increased susceptibility to DR5-mediated apoptosis are characteristics of malignant tumor cells. In contrast, DR5 alone is not sufficient to trigger apoptosis of normal hepatocytes. Therefore, selective, specific targeting of DR5 with an agonistic antibody might be a safe and effective strategy for cancer therapy.

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap.

The current studies of apoptosis in rheumatoid arthritis (RA) suggest that molecules (Fas-related or TNF-related), pathways (activation of pro-apoptosis or anti-apoptosis pathway), cell types (lymphocytes or synovial fibroblast), and the mechanism that triggers apoptosis (tolerance induction-related, down-modulation of inflammation-related, or DNA damage-related) all play a fundamental role to determine the induction or prevention of RA. These series of defects at different levels and in different cells lead to hyperproliferation, defective apoptosis, or hyperapoptosis. This review summarizes the available knowledge of apoptosis and RA to help identify candidate target cells and target molecules for delivery of gene constructs or modified biological or chemical reagents to the target site for effective modification of these cells.

An adenovirus vector encoding the human Bcl-2 gene (hBcl-2) was derived. In vivo expression of hBcl-2 in murine livers enhanced and prolonged adenovirus-mediated gene expression. Furthermore, in the hBcl-2-treated group a significant reduction in the apoptosis induced by the adenovirus vector was observed. Thus, the cytoprotection of the vector-infected cells with antiapoptotic genes appears promising for successful in vivo gene therapy.

An adenovirus vector encoding murine Fas ligand (mFasL) under an inducible control was derived. In vivo ectopic expression of mFasL in murine livers induced an inflammatory cellular infiltration. Furthermore, ectopic expression of mFasL by myocytes did not allow prolonged vector-mediated transgene expression. Thus, ectopic expression of functional mFasL in vector-transduced cells does not appear to confer, by itself, an immunoprivileged site sufficient to mitigate adenovirus vector immunogenicity.

A chimeric Adenovirus-Simian Virus 40 (AdSV40) containing the large T antigen was used to transform rheumatoid synovial fibroblasts. A rheumatoid synovial fibroblast cell line was established by infection of primary rheumatoid arthritis (RA) synovial fibroblasts at Passage 10 with AdSV40 recombinants followed by selection in semisoft agarose cultures. The transformed cells grew anchor independent, exhibited continuous proliferation (65 passages) in monolayer culture, and formed multiple visible foci. The transformed synovial fibroblasts showed expression of the simian virus 40 large T antigen in the nucleus as determined by immunofluorescence staining. In addition, indirect immunofluorescence staining demonstrated that the transformed cells stained specifically with a fibroblast-specific antibody 1B10. Studies involving expression of metalloproteinases showed that collagenase and stromelysin were induced by phorbal 12-myristate 13-acetate (PMA), and such an induction was repressed by dexamethasone typical of primary RA fibroblasts. Levels of mRNAs for IL-1 beta, TNF-alpha, and c-jun were increased by PMA, and the mRNA transcripts of these genes were also repressed by addition of dexamethasone to the culture media. Our results indicate that transformed RA synovial fibroblasts display a similar gene expression pattern in response to PMA and dexamethasone as observed for untransformed primary RA synovial fibroblasts. These transformed rheumatoid arthritis synovial fibroblast cells provide an ideal cell culture model in which to test the efficacy of novel arthritis gene therapy reagents.

Normal and diseased cells release bilayered membrane-bound nanovesicles into interstitial spaces and into bodily fluids. A subgroup of such microvesicles is called exosomes and is described in blood as 30 to 100 nm in diameter and as spherical to cup-shaped nanoparticles with specific surface molecular characteristics (eg, expression of the tetraspanins CD9, CD81, and CD63). Extracellular microvesicles provide local signals (eg, autocrine and paracrine) and distant endocrine signals to cells via the transfer of their contents, which include signal proteins, lipids, miRNAs, and functional mRNAs. Exosomes and related microvesicles also aid cells in exporting less-needed molecules and potentially harmful molecules, including drugs; in the case of neoplasia, the export of chemotherapeutic drugs may facilitate cellular chemoresistance. Cancers have adapted the exosome and related microvesicles as a pathway by which neoplastic cells communicate with each other (autocrine) and with nonneoplastic cells (paracrine and endocrine); via this pathway, cancer suppresses the immune system and establishes a fertile local and distant environment to support neoplastic growth, invasion, and metastases. Because exosomes mirror and bind to the cells from which they arise, they can be used for delivery of drugs, vaccines, and gene therapy, as biomarkers and targets. We review how exosomes and related extracellular microvesicles facilitate the progression and metastases of cancers and describe how these microvesicles may affect clinical care.

Ab therapy against surface Ags on tumor cells has demonstrated significant efficacy for some cancers. However, it is costly and patients frequently develop acquired resistance over time. In cases of Ab therapy resistance, T cell responses have been shown to be essential in controlling disease progression. Thus, vaccination that generates a sustained Ab response as well as a T cell response may be more effective and economical. In this article, we have developed a vaccination strategy by targeting protein Ags to B cells via a CD19 single-chain variable fragment miniAb. Using the tumor-associated Ag her-2/neu extracellular domain, we showed that the coengagement of CD19 and BCR induced full B cell activation to produce a high titer of Abs and enhanced CD4 Th2 response and CD8 T cell activation and differentiation. These Abs competitively inhibited humanized her-2/neu Ab binding and were capable of activating the complement and inhibiting human breast cancer growth in vitro. Therapeutic efficacy was demonstrated in vivo using murine mammary carcinoma models. Furthermore, four different extracellular domains of her-2/neu could be targeted to B cells to generate Abs against particular domains with different antitumor properties. This approach may offer a new avenue for vaccine development with significantly lower cost, which may be of use not only for cancer therapy but also for infectious agents.

The gut mucosal immune system is considered to play an important role in counteracting potential adverse effects of food-derived antigens including nanovesicles. Whether nanovesicles naturally released from edible fruit work in a coordinated manner with gut immune cells to maintain the gut in a noninflammatory status is not known. Here, as proof of concept, we demonstrate that grapefruit-derived nanovesicles (GDNs) are selectively taken up by intestinal macrophages and ameliorate dextran sulfate sodium (DSS)-induced mouse colitis. These effects were mediated by upregulating the expression of heme oxygenase-1 (HO-1) and inhibiting the production of IL-1β and TNF-α in intestinal macrophages. The inherent biocompatibility and biodegradability, stability at wide ranges of pH values, and targeting of intestinal macrophages led us to further develop a novel GDN-based oral delivery system. Incorporating methotrexate (MTX), an anti-inflammatory drug, into GDNs and delivering the MTX-GDNs to mice significantly lowered the MTX toxicity when compared with free MTX, and remarkably increased its therapeutic effects in DSS-induced mouse colitis. These findings demonstrate that GDNs can serve as immune modulators in the intestine, maintain intestinal macrophage homeostasis, and can be developed for oral delivery of small molecule drugs to attenuate inflammatory responses in human disease.

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.

Promoting complement (C) activation may enhance immunological mechanisms of anti-tumor Abs for tumor destruction. However, C activation components, such as C5a, trigger inflammation, which can promote tumor growth. We addressed the role of C5a on tumor growth by transfecting both human carcinoma and murine lymphoma with mouse C5a. In vitro growth kinetics of C5a, control vector, or parental cells revealed no significant differences. Tumor-bearing mice with C5a-transfected xenografted tumor cells had significantly less tumor burden as compared with control vector tumors. NK cells and macrophages infiltrated C5a-expressing tumors with significantly greater frequency, whereas vascular endothelial growth factor, arginase, and TNF-α production were significantly less. Tumor-bearing mice with high C5a-producing syngeneic lymphoma cells had significantly accelerated tumor progression with more Gr-1+CD11b+ myeloid cells in the spleen and overall decreased CD4+ and CD8+ T cells in the tumor, tumor-draining lymph nodes, and the spleen. In contrast, tumor-bearing mice with low C5a-producing lymphoma cells had a significantly reduced tumor burden with increased IFN-γ–producing CD4+ and CD8+ T cells in the spleen and tumor-draining lymph nodes. These studies suggest concentration of local C5a within the tumor microenvironment is critical in determining its role in tumor progression.

Exosomal particular size of 30-100 nm matches the size criterion for nanoparticles, and opens up the possibility of using exosomes as a nanoparticle drug carrier. More importantly, exosomes released from different types of host cells have different biological effects and targeting specificities. Therefore, depending on the therapeutic goal, different types of exosomes can be combined with specific drugs and serve as carriers so that personalized medicine needs are met. In addition, exosomes do not appear to have cytotoxicity. Based on the perceived advantages of exosomes, they may well serve as a next generation drug delivery mechanism that combines nanoparticle size with a non-cytotoxic effect, target specificity, and a high drug carrying capacity, to make them useful in the treatment of a variety of diseases. This review will focus on exosomes as a biological nanoparticle drug carrier with emphasis on their immune-regulatory activities.

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.

Until now the essential transcription factor that determines the epithelial phenotype of breast cancer has not been identified and its role in epithelial-to-mesenchymal transition (EMT) and tumor progression remain unclear. Here, by analyzing large expression profiles of human breast cancer cells, we found an extraordinary correlation between the expression of Grainyhead transcription factor Grhl2 and epithelial marker E-cadherin. Knockdown of Grhl2 expression by shRNA in human mammary epithelial cell MCF10A leads to down-regulation of E-cadherin and EMT. Grhl2 is down-regulated in disseminated cancer cells that have undergone EMT, and over-expression of Grhl2 is sufficient to induce epithelial gene expression. Large clinical datasets reveal that expression of Grhl2 is significantly associated with poor relapse free survival and increased risk of metastasis in breast cancer patients. In mouse models, over-expression of Grhl2 significantly promotes tumor growth and metastasis. Further testing of several Grhl2 regulated genes leads to the same conclusions that the tumorigenic and metastatic potentials of tumor cells are linked to epithelial phenotype but not mesenchymal phenotype. In conclusion, our findings indicate that Grhl2 plays an essential role in the determination of epithelial phenotype of breast cancers, EMT and tumor progression.

Summary Interleukin-23 (IL-23) and CD4 + T helper 17 (Th17) cells are thought to be critical in psoriasis pathogenesis. Here, we report that IL-23 predominantly stimulated dermal γδ T cells to produce IL-17 that led to disease progression. Dermal γδ T cells constitutively expressed the IL-23 receptor (IL-23R) and transcriptional factor RORγt. IL-17 production from dermal γδ T cells was independent of αβ T cells. The epidermal hyperplasia and inflammation induced by IL-23 were significantly decreased in T cell receptor δ-deficient ( Tcrd −/− ) and IL-17 receptor-deficient ( Il17ra −/− ) mice but occurred normally in Tcra −/− mice. Imiquimod-induced skin pathology was also significantly decreased in Tcrd −/− mice. Perhaps further promoting disease progression, IL-23 stimulated dermal γδ T cell expansion. In psoriasis patients, γδ T cells were greatly increased in affected skin and produced large amounts of IL-17. Thus, IL-23-responsive dermal γδ T cells are the major IL-17 producers in the skin and may represent a novel target for the treatment of psoriasis.

Ubiquitinated endosomal proteins that are deposited into the lumens of multivesicular bodies are either sorted for lysosomal-mediated degradation or secreted as exosomes into the extracellular milieu. The mechanisms that underlie the sorting of cellular cargo proteins are currently unknown. In this study, we show that the COP9 signalosome (CSN)-associated protein CSN5 quantitatively regulated proteins that were sorted into exosomes. Western blot analysis of exosomal proteins indicated that small interfering (si)RNA knockdown of CSN5 results in increased levels of both ubiquitinated and non-ubiquitinated exosomal proteins, including heat shock protein 70, in comparison with exosomes isolated from the supernatants of 293 cells transfected with scrambled siRNA. Furthermore, 293 cells transfected with JAB1/MPN/Mov34 metalloenzyme domain-deleted CSN5 produced exosomes with higher levels of ubiquitinated heat shock protein 70, which did not affect non-ubiquitinated heat shock protein 70 levels. The loss of COP9-associated deubiquitin activity of CSN5 also led to the enhancement of HIV Gag that was sorted into exosomes as well as the promotion of HIV-1 release, suggesting that COP9-associated CSN5 regulates the sorting of a number of exosomal proteins in both a CSN5 JAB1/MPN/Mov34 metalloenzyme domain-dependent and -independent manner. We propose that COP9-associated CSN5 regulates exosomal protein sorting in both a deubiquitinating activity-dependent and -independent manner, which is contrary to the current idea of ubiquitin-dependent sorting of proteins to exosomes.

Fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis (RA FLSs) exhibit prosurvival, rather than apoptotic, response to tumor necrosis factor (TNF)-alpha stimulation. Here, we show that JAB1 is a critical regulator of the TNF-alpha-mediated anti-apo-ptosis pathways in RA FLSs. We found that knockdown of JAB1 using small interfering (si)RNA led to restoration of the TNF-alpha-induced apoptosis response, reduction of nuclear factor-kappaB activity, delayed degradation of IkappaB-alpha, and inhibited phosphorylation of JNK. Analysis of the interactions of JAB1 by reciprocal co-immunoprecipitations and confocal microscopy revealed that JAB1 interacts with TNF receptor-associated-factor 2 (TRAF2). The generation of the anti-apoptotic signal on binding of TNF-alpha to the TNF receptor (TNFR)1 has been shown to be associated with the recruitment of TRAF2 to the TNFR1 in a process that requires ubiquitination of TRAF2 with lysine-63-linked polyubiquitin chains. We found that TNF-alpha stimulation of JAB1 siRNA-transfected RA FLSs failed to stimulate ubiquitination of TRAF2. Thus, we conclude that JAB1-regulated ubiquitination of TRAF2 is a novel mechanism whereby TNF-alpha can induce anti-apoptosis signaling and production of matrix metalloproteinases through activation of nuclear factor-kappaB and JNK in RA FLSs.

Background Methionine, an essential amino acid, is important for normal growth and development, as it is required for both protein and polyamine synthesis as well as in methylation reactions. It has been reported that high concentrations of methionine inhibit cellular growth and gene expression in the human breast tumor-derived MCF-7 cells. These effects are thought to be mediated by the modulation of p53. However, the generalizability of this observation and the precise role of p53 in methionine-induced growth suppression needs to be determined. Methods To determine if the inhibition of cell growth by methionine applies to other cell lines and to characterize further the role of p53 in methionine-induced growth suppression, we have assessed the effects of methionine on cellular growth and proliferation and p53 expression in cells expressing native p53, eg, breast cancer MCF-7 cells and prostate cancer LNCaP cells, and also in cells expressing a mutated (point) form of p53, eg, prostate cancer DU-145 cells. These cell lines were treated with varying concentrations of l -methionine. The effects of l -methionine on cell growth were assyed by using cell viability assays and immunostaining for Ki-67, a cell proliferation marker. The effects of methionine on p53 expression were assessed by using reverse transcriptase–polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. The role of p53 in l -methionine–mediated growth suppression was evaluated using short-interference RNA for p53 (siRNA-p53), immunoprecipitation, and direct DNA sequencing. Results We demonstrated that methionine at a concentration of 1 to 5 mg/mL inhibited the growth of both MCF-7 and LNCaP cells. In association with the inhibition of growth, methionine also inhibited native p53 expression at the mRNA and protein levels, respectively. Furthermore, transfection with siRNA-p53, to knock down p53 expression, increased cell growth and proliferation of the LNCaP cells even when they were exposed to methionine. In contrast, the same treatment did not diminish growth or proliferation of the DU-145 cells. Also, the expression of mutated p53 at the mRNA or protein levels was not altered. Conclusion Our results extend a prior observation to other cell lines and demonstrate that high concentrations of methionine suppress the expression of native but not mutated p53. These inhibitory effects on cellular growth are, in part, due to inhibition of cellular proliferation probably via a p53-dependent pathway.

International audience; This white paper by eighty members of the Complex Trait Consortium presents a community's view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?

Several in vitro and animal studies have been performed to modulate the interaction of APCs and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells in an Ag-specific manner. However, due to the difficulties in vector generation and low transduction frequencies, similar studies with primary human APC are still lacking. To evaluate whether Fas ligand (FasL/CD95L) expressing killer APC could be generated from primary human APC, monocyte-derived dendritic cells (DC) were transduced using the inducible Cre/Loxp adenovirus vector system. Combined transduction of DC by AdLoxpFasL and AxCANCre, but not single transduction with these vectors, resulted in dose- and time-dependent expression of FasL in >70% of mature DC (mDC), whereas 80% of iDC, whereas FasL-expressing mDC were protected from FasL/Fas (CD95/Apo-1)-mediated apoptosis despite coexpression of Fas. FasL-expressing mDC eliminated Fas+ Jurkat T cells as well as activated primary T cells by apoptosis, whereas nonactivated primary T cells were not deleted. Induction of apoptosis in Fas+ target cells required expression of FasL in DC and cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. Induction of apoptosis in Fas+ target cells required expression of FasL in DC, cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. The present results demonstrate that FasL-expressing killer APC can be generated from human monocyte-derived mDC using adenoviral gene transfer. Our results support the strategy to use killer APCs as immunomodulatory cells for the treatment of autoimmune disease and allograft rejection.