Exosomes.

Exosomes are small, single-membrane, secreted organelles of ∼30 to ∼200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes contain an array of membrane-associated, high-order oligomeric protein complexes, display pronounced molecular heterogeneity, and are created by budding at both plasma and endosome membranes. Exosome biogenesis is a mechanism of protein quality control, and once released, exosomes have activities as diverse as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This pathway of intercellular vesicle traffic plays important roles in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses co-opt exosome biogenesis pathways both for assembling infectious particles and for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models.

Tumor-derived exosomes antagonize innate antiviral immunity.

Malignancies can compromise innate immunity, but the mechanisms of this are largely unknown. Here we found that, via tumor-derived exosomes (TEXs), cancers were able to transfer activated epidermal growth factor receptor (EGFR) to host macrophages and thereby suppress innate antiviral immunity. Screening of the human kinome identified the kinase MEKK2 in macrophages as an effector of TEX-delivered EGFR that negatively regulated the antiviral immune response. In the context of experimental tumor implantation, MEKK2-deficient mice were more resistant to viral infection than were wild-type mice. Injection of TEXs into mice reduced innate immunity, increased viral load and increased morbidity in an EGFR- and MEKK2-dependent manner. MEKK2 phosphorylated IRF3, a transcription factor crucial for the production of type I interferons; this triggered poly-ubiquitination of IRF3 and blocked its dimerization, translocation to the nucleus and transcriptional activity after viral infection. These findings identify a mechanism by which cancer cells can dampen host innate immunity and potentially cause patients with cancer to become immunocompromised.

Dysregulated Expression of the Nuclear Exosome Targeting Complex Component Rbm7 in Nonhematopoietic Cells Licenses the Development of Fibrosis.

Fibrosis is an incurable disorder of unknown etiology. Segregated-nucleus-containing atypical monocytes (SatMs) are critical for the development of fibrosis. Here we examined the mechanisms that recruit SatMs to pre-fibrotic areas. A screen based on cytokine expression in the fibrotic lung revealed that the chemokine Cxcl12, which is produced by apoptotic nonhematopoietic cells, was essential for SatM recruitment. Analyses of lung tissues at fibrosis onset showed increased expression of Rbm7, a component of the nuclear exosome targeting complex. Rbm7 deletion suppressed bleomycin-induced fibrosis and at a cellular level, suppressed apoptosis of nonhematopoietic cells. Mechanistically, Rbm7 bound to noncoding (nc)RNAs that form subnuclear bodies, including Neat1 speckles. Dysregulated expression of Rbm7 resulted in the nuclear degradation of Neat1 speckles, the dispersion of the DNA repair protein BRCA1, and the triggering of apoptosis. Thus, Rbm7 in epithelial cells plays a critical role in the development of fibrosis by regulating ncRNA decay and thereby the production of chemokines that recruit SatMs.

Exosomes mediate the cell-to-cell transmission of IFN-α-induced antiviral activity.

The cell-to-cell transmission of viral resistance is a potential mechanism for amplifying the interferon-induced antiviral response. In this study, we report that interferon-α (IFN-α) induced the transfer of resistance to hepatitis B virus (HBV) from nonpermissive liver nonparenchymal cells (LNPCs) to permissive hepatocytes via exosomes. Exosomes from IFN-α-treated LNPCs were rich in molecules with antiviral activity. Moreover, exosomes from LNPCs were internalized by hepatocytes, which mediated the intercellular transfer of antiviral molecules. Finally, we found that exosomes also contributed to the antiviral response of IFN-α to mouse hepatitis virus A59 and adenovirus in mice. Thus, we propose an antiviral mechanism of IFN-α activity that involves the induction and intercellular transfer of antiviral molecules via exosomes.

Size-Selective Capturing of Exosomes Using DNA Tripods.

Fractionating and characterizing target samples are fundamental to the analysis of biomolecules. Extracellular vesicles (EVs), containing information regarding the cellular birthplace, are promising targets for biology and medicine. However, the requirement for multiple-step purification in conventional methods hinders analysis of small samples. Here, we apply a DNA origami tripod with a defined aperture of binders (e.g., antibodies against EV biomarkers), which allows us to capture the target molecule. Using exosomes as a model, we show that our tripod nanodevice can capture a specific size range of EVs with cognate biomarkers from a broad distribution of crude EV mixtures. We further demonstrate that the size of captured EVs can be controlled by changing the aperture of the tripods. This simultaneous selection with the size and biomarker approach should simplify the EV purification process and contribute to the precise analysis of target biomolecules from small samples.

Challenges and opportunities for cancer stem cell-targeted immunotherapies include immune checkpoint inhibitor, cancer stem cell-dendritic cell vaccine, chimeric antigen receptor immune cells, and modified exosomes.

Cancer stem cells (CSCs) are associated with the tumor microenvironment (TME). CSCs induce tumorigenesis, tumor recurrence and progression, and resistance to standard therapies. Indeed, CSCs pose an increasing challenge to current cancer therapy due to their stemness or self-renewal properties. The molecular and cellular interactions between heterogeneous CSCs and surrounding TME components and tumor-supporting immune cells show synergistic effects toward treatment failure. In the immunosuppressive TME, CSCs express various immunoregulatory proteins, growth factors, metabolites and cytokines, and also produce exosomes, a type of extracellular vesicles, to protect themselves from host immune surveillance. Among these, the identification and application of CSC-derived exosomes could be considered for the development of therapeutic approaches to eliminate CSCs or cancer, in addition to targeting the modulators that remodel the composition of the TME, as reviewed in this study. Here, we introduce the role of CSCs and how their interaction with TME complicates immunotherapies, and then present the CSC-based immunotherapy and the limitation of these therapies. We describe the biology and role of tumor/CSC-derived exosomes that induce immune suppression in the TME, and finally, introduce their potentials for the development of CSC-based targeted immunotherapy in the future.

Exosomes target HBV-host interactions to remodel the hepatic immune microenvironment.

Chronic hepatitis B poses a significant global burden, modulating immune cells, leading to chronic inflammation and long-term damage. Due to its hepatotropism, the hepatitis B virus (HBV) cannot infect other cells. The mechanisms underlying the intercellular communication among different liver cells in HBV-infected individuals and the immune microenvironment imbalance remain elusive. Exosomes, as important intercellular communication and cargo transportation tools between HBV-infected hepatocytes and immune cells, have been shown to assist in HBV cargo transportation and regulate the immune microenvironment. However, the role of exosomes in hepatitis B has only gradually received attention in recent years. Minimal literature has systematically elaborated on the role of exosomes in reshaping the immune microenvironment of the liver. This review unfolds sequentially based on the biological processes of exosomes: exosomes' biogenesis, release, transport, uptake by recipient cells, and their impact on recipient cells. We delineate how HBV influences the biogenesis of exosomes, utilizing exosomal covert transmission, and reshapes the hepatic immune microenvironment. And based on the characteristics and functions of exosomes, potential applications of exosomes in hepatitis B are summarized and predicted.

Regulatory T cells: exosomes deliver tolerance.

T regulatory (Treg) cells enforce peripheral tolerance through regulation of diverse immune responses in a context-specific manner. Okoye et al. show one way that Treg cells suppress Th1 cell responses is through nonautonomous gene silencing mediated by microRNA-containing exosomes.

MicroRNA-containing T-regulatory-cell-derived exosomes suppress pathogenic T helper 1 cells.

Foxp3(+) T regulatory (Treg) cells prevent inflammatory disease but the mechanistic basis of suppression is not understood completely. Gene silencing by RNA interference can act in a cell-autonomous and non-cell-autonomous manner, providing mechanisms of intercellular regulation. Here, we demonstrate that non-cell-autonomous gene silencing, mediated by miRNA-containing exosomes, is a mechanism employed by Treg cells to suppress T-cell-mediated disease. Treg cells transferred microRNAs (miRNA) to various immune cells, including T helper 1 (Th1) cells, suppressing Th1 cell proliferation and cytokine secretion. Use of Dicer-deficient or Rab27a and Rab27b double-deficient Treg cells to disrupt miRNA biogenesis or the exosomal pathway, respectively, established a requirement for miRNAs and exosomes for Treg-cell-mediated suppression. Transcriptional analysis and miRNA inhibitor studies showed that exosome-mediated transfer of Let-7d from Treg cell to Th1 cells contributed to suppression and prevention of systemic disease. These studies reveal a mechanism of Treg-cell-mediated suppression mediated by miRNA-containing exosomes.

Circulating Pro-Inflammatory Exosomes Worsen Stroke Outcomes in Aging.

[Figure: see text].

Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer-BioNTech) Vaccination prior to Development of Antibodies: A Novel Mechanism for Immune Activation by mRNA Vaccines.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with exosomes carrying spike protein.

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.

The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic KrasG12D, a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.

Plasmacytoid dendritic cells cross-prime naive CD8 T cells by transferring antigen to conventional dendritic cells through exosomes.

Although plasmacytoid dendritic cells (pDCs) have been shown to play a critical role in generating viral immunity and promoting tolerance to suppress antitumor immunity, whether and how pDCs cross-prime CD8 T cells in vivo remain controversial. Using a pDC-targeted vaccine model to deliver antigens specifically to pDCs, we have demonstrated that pDC-targeted vaccination led to strong cross-priming and durable CD8 T cell immunity. Surprisingly, cross-presenting pDCs required conventional DCs (cDCs) to achieve cross-priming in vivo by transferring antigens to cDCs. Taking advantage of an in vitro system where only pDCs had access to antigens, we further demonstrated that cross-presenting pDCs were unable to efficiently prime CD8 T cells by themselves, but conferred antigen-naive cDCs the capability of cross-priming CD8 T cells by transferring antigens to cDCs. Although both cDC1s and cDC2s exhibited similar efficiency in acquiring antigens from pDCs, cDC1s but not cDC2s were required for cross-priming upon pDC-targeted vaccination, suggesting that cDC1s played a critical role in pDC-mediated cross-priming independent of their function in antigen presentation. Antigen transfer from pDCs to cDCs was mediated by previously unreported pDC-derived exosomes (pDCexos), that were also produced by pDCs under various conditions. Importantly, all these pDCexos primed naive antigen-specific CD8 T cells only in the presence of bystander cDCs, similarly to cross-presenting pDCs, thus identifying pDCexo-mediated antigen transfer to cDCs as a mechanism for pDCs to achieve cross-priming. In summary, our data suggest that pDCs employ a unique mechanism of pDCexo-mediated antigen transfer to cDCs for cross-priming.

Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity.

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.

Associations Among Adipose Tissue Immunology, Inflammation, Exosomes and Insulin Sensitivity in People With Obesity and Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIMS: Insulin resistance is a key factor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We evaluated the importance of subcutaneous abdominal adipose tissue (SAAT) inflammation and both plasma and SAAT-derived exosomes in regulating insulin sensitivity in people with obesity and NAFLD. METHODS: Adipose tissue inflammation (macrophage and T-cell content and expression of proinflammatory cytokines), liver and whole-body insulin sensitivity (assessed using a hyperinsulinemic-euglycemic clamp and glucose tracer infusion), and 24-hour serial plasma cytokine concentrations were evaluated in 3 groups stratified by adiposity and intrahepatic triglyceride (IHTG) content: (1) lean with normal IHTG content (LEAN; N = 14); (2) obese with normal IHTG content (OB-NL; N = 28); and (3) obese with NAFLD (OB-NAFLD; N = 28). The effect of plasma and SAAT-derived exosomes on insulin-stimulated Akt phosphorylation in human skeletal muscle myotubes and mouse primary hepatocytes was assessed in a subset of participants. RESULTS: Proinflammatory macrophages, proinflammatory CD4 and CD8 T-cell populations, and gene expression of several cytokines in SAAT were greater in the OB-NAFLD than the OB-NL and LEAN groups. However, with the exception of PAI-1, which was greater in the OB-NAFLD than the LEAN and OB-NL groups, 24-hour plasma cytokine concentration areas-under-the-curve were not different between groups. The percentage of proinflammatory macrophages and plasma PAI-1 concentration areas-under-the-curve were inversely correlated with both hepatic and whole-body insulin sensitivity. Compared with exosomes from OB-NL participants, plasma and SAAT-derived exosomes from the OB-NAFLD group decreased insulin signaling in myotubes and hepatocytes. CONCLUSIONS: Systemic insulin resistance in people with obesity and NAFLD is associated with increased plasma PAI-1 concentrations and both plasma and SAAT-derived exosomes. ClinicalTrials.gov number: NCT02706262 (https://clinicaltrials.gov/ct2/show/NCT02706262).

Myeloid-Cell-Specific IL-6 Signaling Promotes MicroRNA-223-Enriched Exosome Production to Attenuate NAFLD-Associated Fibrosis.

BACKGROUND ANDS AIMS: NAFLD is associated with elevation of many cytokines, particularly IL-6; however, the role of IL-6 in NAFLD remains obscure. The aim of this study was to examine how myeloid-specific IL-6 signaling affects NAFLD by the regulation of antifibrotic microRNA-223 (miR-223) in myeloid cells. APPROACH AND RESULTS: Patients with NAFLD or NASH and healthy controls were recruited, and serum IL-6 and soluble IL-6 receptor α (sIL-6Rα) were measured. Compared to controls, serum IL-6 and sIL-6Rα levels were elevated in NAFLD/NASH patients. IL-6 levels correlated positively with the number of circulating leukocytes and monocytes. The role of IL-6 in NAFLD was investigated in Il6 knockout (KO) and Il6 receptor A (Il6ra) conditional KO mice after high-fat diet (HFD) feeding. HFD-fed Il6 KO mice had worse liver injury and fibrosis, but less inflammation, compared to wild-type mice. Hepatocyte-specific Il6ra KO mice had more steatosis and liver injury, whereas myeloid-specific Il6ra KO mice had a lower number of hepatic infiltrating macrophages (IMs) and neutrophils with increased cell death of these cells, but greater liver fibrosis (LF), than WT mice. Mechanistically, the increased LF in HFD-fed, myeloid-specific Il6ra KO mice was attributable to the reduction of antifibrotic miR-223 and subsequent up-regulation of the miR-223 target gene, transcriptional activator with PDZ-binding motif (TAZ), a well-known factor to promote NASH fibrosis. In vitro, IL-6 treatment up-regulated exosome biogenesis-related genes and subsequently promoted macrophages to release miR-223-enriched exosomes that were able to reduce profibrotic TAZ expression in hepatocytes by exosomal transfer. Finally, serum IL-6 and miR-223 levels were elevated and correlated with each other in NAFLD patients. CONCLUSIONS: Myeloid-specific IL-6 signaling inhibits LF through exosomal transfer of antifibrotic miR-223 into hepatocytes, providing therapeutic targets for NAFLD therapy.

Exosome-educated macrophages and exosomes differentially improve ligament healing.

Recently, our group used exosomes from mesenchymal stromal/stem cells (MSCs) to simulate an M2 macrophage phenotype, that is, exosome-educated macrophages (EEMs). These EEMs, when delivered in vivo, accelerated healing in a mouse Achilles tendon injury model. For the current study, we first tested the ability of EEMs to reproduce the beneficial healing effects in a different rodent model, that is, a rat medial collateral ligament (MCL) injury model. We hypothesized that treatment with EEMs would reduce inflammation and accelerate ligament healing, similar to our previous tendon results. Second, because of the translational advantages of a cell-free therapy, exosomes alone were also examined to promote MCL healing. We hypothesized that MSC-derived exosomes could also alter ligament healing to reduce scar formation. Similar to our previous Achilles tendon results, EEMs improved mechanical properties in the healing ligament and reduced inflammation, as indicated via a decreased endogenous M1/M2 macrophage ratio. We also showed that exosomes improved ligament remodeling as indicated by changes in collagen production and organization, and reduced scar formation but without improved mechanical behavior in healing tissue. Overall, our findings suggest EEMs and MSC-derived exosomes improve healing but via different mechanisms. EEMs and exosomes each have attractive characteristics as therapeutics. EEMs as a cell therapy are terminally differentiated and will not proliferate or differentiate. Alternatively, exosome therapy can be used as a cell free, shelf-stable therapeutic to deliver biologically active components. Results herein further support using EEMs and/or exosomes to improve ligament healing by modulating inflammation and promoting more advantageous tissue remodeling.

M2 Macrophage-Derived Exosomes Promote Angiogenesis and Growth of Pancreatic Ductal Adenocarcinoma by Targeting E2F2.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive tumors all over the world, has a generally poor prognosis, and its progression is positively correlated with the density of blood vessels. Recently, tumor-associated macrophages (TAMs) were proven to be beneficial for angiogenesis, but their mechanism of action remains unclear. Our study indicated that M2 macrophages were positively correlated with the microvessel density (MVD) of PDAC tissues, and M2 macrophage-derived exosomes (MDEs) could promote the angiogenesis of mouse aortic endothelial cells (MAECs) in vitro. At the same time, the M2 MDEs could also promote the growth of subcutaneous tumors and increase the vascular density of mice. Moreover, we also found that miR-155-5p and miR-221-5p levels in the M2 MDEs were higher than those in M0 MDEs, and they could be transferred into MAECs, as demonstrated by RNA sequencing (RNA-seq) and qPCR analysis. Our data confirmed the interaction between TAMs and the angiogenesis of PDAC by exosomes. Additionally, targeting the exosomal miRNAs derived from TAMs might provide diagnostic and therapeutic strategies for PDAC.

What counts in the immunological synapse?

Molecular interactions at the interface between helper T cells and antigen-presenting B cells govern the ability to produce specific antibodies, which is a central event in protective immunity generated by natural infection or man-made vaccines. In order for a T cell to deliver effective help to a B cell and guide affinity maturation, it needs to provide feedback that is proportional to the amount of antigen the B cell collects with its surface antibody. This review focuses on mechanisms by which T and B cells manage to count the products of antigen capture and encourage B cells with the best receptors to dominate the response and make antibody-producing plasma cells. We discuss what is known about the proportionality of T cells responses to presented antigens and consider the mechanisms that B cells may use to keep count of positive feedback from T cells.

Exosomes with FOXP3 from gene-modified dendritic cells ameliorate the development of EAE by regulating the balance of Th/Treg.

Objective: To investigate the efficiency and potential mechanisms of exosomes from dendritic cells (DCs) transfected with Forkhead box protein P3 (FOXP3) in the development of experimental autoimmune encephalomyelitis (EAE). Method: Mouse bone marrow-derived immature DCs were loaded with adenovirus carrying FOXP3 gene, and exosomes were generated. Then the exosomes with FOXP3 (FOXP3-EXOs) were co-cultured with CD4+T cell in vitro to evaluate their potential on CD4+T cell proliferation and differentiation, and injected into EAE mice to assess their effects on the development of EAE. Result: FOXP3-EXOs were effective to inhibit the CD4+T cell proliferation and the production of Interferon gamma (IFN-γ), interleukin (IL)-6, and IL-17, while they promoted the production of IL-10 in vitro. Moreover, FOXP3-EXOs treatment significantly decreased the neurological scores, reduced the infiltration of inflammatory cells into the spinal cord, and decreased demyelination in comparison to saline and Con-EXOs treated EAE mice. Moreover, the FOXP3-EXOs treatment resulted in obvious increases in the levels of regulatory T (Treg) cells and IL-10, whereas levels of T helper 1 (Th1) cells, Th17 cells, IFN-γ, IL-6, and IL-17 decreased significantly in the splenocyte culture of EAE mice. Conclusion: The present study preliminarily investigated the effects and potential mechanisms of FOXP3-EXOs in EAE and revealed that the FOXP3-EXOs could inhibit the production of Th1 and Th17 cells and promote the production of Treg cells as well as ameliorate the development of EAE. The neuroprotective effects of FOXP3-EXOs on EAE are likely due to the regulation of Th/Treg balance.