Microbes and Infection 16 (2014) 269e272 www.elsevier.com/locate/micinf
Mycobacteria, meet your messenger*
Biology textbooks contain detailed descriptions about cellular communication involving hormones, the interactions of immune cells, and ion gradients across neuronal membranes. However, space in more recent editions will need to be accorded to a new system of cellular communication involving microscopic particles called exosomes. These mini-messengers are attracting a great deal of attention from academia and industry alike, notably for their ability to manipulate immune responses. In this issue of Microbes and Infection, Tang and colleagues examine how exosomes may shape immune responses during the course of infectious disease . Exosomes are small (30e120 nm) membrane vesicles that are probably secreted by all types of cell. These vesicles are formed inside the cell by the inward budding of cellular compartments called multivesicular endosomes (MVE). Exosomes internalised in the MVE are released into the extracellular environment when the MVE fuses with the plasma membrane. They are then taken up by target cells either via the target cell’s endocytic pathway or by fusing directly with the plasma membrane and releasing their content directly into the cytoplasm. However, their function in intercellular communication took a long time to be recognised. Their first reported cargo was the transferrin receptor [2,3] which was shedded by reticulocytes during their differentiation. This led to the initial belief that exosomes were the garbage bags of a cellular trash disposal system, and ten years passed before the discovery of some of the more exotic activities of these microvesicles. Raposo and colleagues found that exosomes secreted by EpsteineBarr virus-transformed B cells contained MHC class II dimers bound to antigenic peptides that could stimulate T cell responses . The study of exosomes subsequently exploded and their cargo became meticulously catalogued. Besides membrane-associated proteins, mass spectrometry has shown that over 4400 various protein components can be transported by these microvesicles . One big surprise came with the discovery of nucleic acid content in the form of
Article highlight of “Proteomic analysis and immune properties of exosomes released by macrophages infected with Mycobacterium avium” by J.J. Wang et al. .
mRNA and microRNA raising the possibility of genetic transfer between cells . As a result of this extensive cargo manifest, exosomes have been implicated in a wide variety of processes, including apoptosis, angiogenesis, inflammation, coagulation, and the creation of polarity during development . Their role in the immune system has been extensively studied. Besides direct antigen presentation, exosomes can also carry antigens from the cells from which they originate. These exosomes can be taken up by dendritic cells which present degraded antigens coupled with MHC molecules to T cells. Exosomes may also contain microbial components that stimulate innate immune responses. Beatty and colleagues were the first to show that the cell wall components of mycobacteria were trafficked inside the endocytic compartment of infected macrophages . These pathogen-associated molecular patterns (PAMPs) were somehow passed on to uninfected bystander cells. Not long after, this relay mechanism was shown to involve exosomes . Exosomes released from mouse macrophages infected with Mycobacterium tuberculosis were capable of inducing a pro-inflammatory response in uninfected macrophages. Thus, exosomes function in a system of pathogen surveillance, increasing the exposure of intracellular pathogens to the immune cells and showing these microbial agents that there is nowhere to hide inside the infected cell. Tang and co-workers now report similar findings in a human cell system with Mycobacterium avium, which is a close relative of M. tuberculosis. M. avium is less virulent than M. tuberculosis, which makes it much safer to handle. Tang treated uninfected macrophages either with exosomes derived from macrophages infected with M. avium or exosomes derived from uninfected macrophages and examined the response of these resting macrophages. Uninfected macrophages that were treated with exosomes derived from M. avium infected macrophages secreted higher concentrations of the pro-inflammatory cytokines TNF-a and IFN-g than uninfected macrophages treated with control exosomes. In fact, the response effectively mimicked that of infection of macrophages with M. avium itself, albeit with somewhat delayed kinetics. Exosomes derived from M. avium infected macrophages also promoted the expression of CD80 and CD86 in uninfected cells. These cell surface molecules work in tandem
http://dx.doi.org/10.1016/j.micinf.2014.03.004 1286-4579/Ó 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Highlight / Microbes and Infection 16 (2014) 269e272
to activate T cells, suggesting that exosomes may also stimulate the adaptive arm of immunity upon infection with M. avium. Exosome isolation is a delicate process, and obtaining enough material to examine their potential microbial contents is no trivial matter. Although the authors were unable to definitively say that these exosomes contained M. avium components, previous reports that exosomes secreted by macrophages carry microbial antigens and PAMPs [9,10] support the hypothesis that exosomes introduce immunogenic proteins to uninfected macrophages. Thus, exosomes function as relay warning signals that alert resting macrophages to the presence of infection. Although in this instance exosomes appeared to boost immune responses, they may be a double edged sword, notably during immune responses to cancer. Exosomes secreted by tumour cells can transport tumour antigens to dendritic cells . However, exosomes secreted by some tumours may also contain immunosuppressive molecules which can (at least in vitro) impair the proliferation of immune cells [12,13]. Cancerous cells may even impart the malignant gift of oncogenic gene products to their unwitting neighbours . Nonetheless, the applications of exosomes are bountiful.
trials . It appears that exosomes may just be the next small thing.
Exosomes may serve as diagnostic markers for various diseases. These microvesicles represent a neat little packing system because all the protein and RNA cargo is encased in a membrane so they are impervious to circulating nucleases and proteases in the blood. They have a very long half-life in the bloodstream, permitting their transport between anatomically distant locations. As a testament to their stability, they have been found in many bodily fluids including blood, saliva, urine, epididymal fluid, amniotic liquid, synovial fluid, and breast milk. Custom made exosomes may be used to deliver vaccines or therapeutic agents. In the last decade, several therapeutic approaches involving exosomes have been devised and tested and some have even made it to Phase II clinical
2. Interview with Li-Jung Tang
1. Biosketch e Li-Jung Tang Dr. Li-Jung Tang completed his Ph.D at XiangYa School of Medicine, Central South University, China during which he cloned several novel genes related to human multiple myeloma and carried out preliminary functional analyses of these genes. He then carried out postdoctoral training with Dr. Garcia Rodolfo in the International Center for Genetic Engineering and Biotechnology (ICGEB) in Trieste, Italy between 2005 and 2007. As a post-doctorate he used 2DE MALDI TOF/TOF MS to examine protein patterns in human body fluids upon infection. He was also involved in research of the macrophage responses to mycobacterium via the Toll-like receptor pathway. He returned to China with his family in 2007 and became an associate professor at the School of Biological Science and Technology, Central South University where he studied macrophage defence mechanisms against mycobacterium that involve exosomes secreted from cells. In 2013 he became a professor at Central South University.
1. What triggered your interest in the immune functions of exosomes? I studied at the International Centre for Genetic and Engineering Biotechnology (ICGEB) in Trieste, Italy where I first started working with exosomes with my PostDoc supervisor. I realised that exosomes are mysterious and wonderful biological products that deserved to be researched. 2. What is the take-home message of the article?
Highlight / Microbes and Infection 16 (2014) 269e272
Exosomes are involved in the immune system’s defence mechanisms against mycobacteria. Background 3. Do you have a personal motto, quote or leading sentence? Persist and you will succeed. 4. What advice would you give to the young next-generation scientists? The most important thing is to be passionate about what you are doing, and then of course you need to work hard. 5. What is your favourite hang-out method after a tough day at the lab? Jogging and swimming. 6. In your opinion, what is the most important (scientific) discovery of the last fifty years? The finding that cells can communicate through exosomes.
Exosomes are small (30e120 nm) membrane vesicles that are probably secreted by all types of cell. Although they were initially believed to be a by-product of the in vitro manipulation of cells, and subsequently as a vehicle for cells to shed unwanted proteins, their important role as molecular messengers enabling cell communication between distant sites is becoming increasingly studied. Exosomes are a stable packaging device for the transport of proteins and nucleic acid between cells and are found in a large number of bodily fluids. They have been implicated in a wide variety of biological processes. Notably, their contents can modulate both adaptive and innate immune responses during the course of infection. There is hope that one day custom made exosomes may be used to deliver therapeutic agents in the fight against disease.
7. If you could travel back in time what scientific discovery would like to assist to? In a Nutshell The discovery of the structure of DNA by Watson, Crick, and Franklin. 8. If you could travel forward in time e what eventual invention would you like to check out? The use of exosomes to treat diseases. Exosomes are very important molecular carriers. But we still have a lot of work to do!
Uninfected macrophages that were exposed to exosomes derived from macrophages infected with M. avium expressed higher levels of molecules involved in T cell activation, CD80 and CD86, than macrophages treated with exosomes derived from noninfected macrophages. Exosomes derived from macrophages infected with M. avium induced a pro-inflammatory response in uninfected macrophages, involving the higher production of TNFa and IFNg than uninfected macrophages treated with control exosomes. Thus, exosomes released by infected cells mimic the effect of infection. The abundance of twelve proteins was different between exosomes derived from macrophages infected with M. avium and those derived from noninfected macrophages. Five of these proteins were identified by mass spectrometry. These were cytoskeletal proteins, a protein involved in protein processing, and a protein involved in signal transduction. The presence of bacterial proteins in exosomes was not investigated due to technical limitations.
Drawing by Sophia Ha¨fner.
 Wang JJ, Chen C, Xie PF, Pan Y, Tan YH, et al. Proteomic analysis and immune properties of exosomes released by macrophages infected with Mycobacterium avium. Microbes Infect 2014;16:283e91.
Highlight / Microbes and Infection 16 (2014) 269e272
 Harding C, Heuser J, Stahl P. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol 1983;97:329e39.  Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol 1985;101:942e8.  Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med 1996;183:1161e72.  Mathivanan S, Simpson RJ. ExoCarta: a compendium of exosomal proteins and RNA. Proteomics 2009;9:4997e5000.  Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, et al. Exosomemediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654e9.  Vlassov AV, Magdaleno S, Setterquist R, Conrad R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 2012;1820:940e8.  Beatty WL, Rhoades ER, Ullrich HJ, Chatterjee D, Heuser JE, et al. Trafficking and release of mycobacterial lipids from infected macrophages. Traffic 2000;1:235e47.  Bhatnagar S, Shinagawa K, Castellino FJ, Schorey JS. Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo. Blood 2007;110:3234e44.  Giri PK, Kruh NA, Dobos KM, Schorey JS. Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and
culture filtrate protein-treated macrophages. Proteomics 2010;10: 3190e202. Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, et al. Tumorderived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 2001;7:297e303. Andreola G, Rivoltini L, Castelli C, Huber V, Perego P, et al. Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles. J Exp Med 2002;195:1303e16. Liu C, Yu S, Zinn K, Wang J, Zhang L, et al. Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function. J Immunol 2006;176:1375e85. Al-Nedawi K, Meehan B, Micallef J, Lhotak V, May L, et al. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 2008;10:619e24. Viaud S, Thery C, Ploix S, Tursz T, Lapierre V, et al. Dendritic cellderived exosomes for cancer immunotherapy: what’s next? Cancer Res 2010;70:1281e5.
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11 March 2014 Available online 21 March 2014