Margareta Sjöstrand

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called “exosomal shuttle RNA” (esRNA).

Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages

BackgroundExosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages.MethodExosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy.ResultsRNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages.ConclusionsExosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.

Endogenous IL-17 as a mediator of neutrophil recruitment caused by endotoxin exposure in mouse airways

We have previously demonstrated that administration of the recently described cytokine IL-17 in rat airways in vivo recruits and activates neutrophils locally. In the current study, we examined whether endogenous IL-17 is involved in mediating neutrophil recruitment caused by endotoxin exposure in mouse airways. Our in vivo data show that local endotoxin exposure causes the release of free, soluble IL-17 protein 6 h later. Systemic pretreatment with a neutralizing anti-IL-17 Ab almost completely inhibits neutrophil recruitment 24 h, but not 6 h, after endotoxin exposure in the airways. Pretreatment with neutralizing anti-IL-6 and anti-macrophage inflammatory protein (MIP)-2 Abs inhibits neutrophil recruitment caused by local endotoxin exposure and IL-17, respectively. Our in vitro data show that endotoxin exposure stimulates the release of soluble IL-17 protein in T lymphocytes harvested from lung and spleen, respectively, and that this cytokine release requires coculture with airway macrophages. Intracellular IL-17 protein is detected in T lymphocytes from spleen but not in airway macrophages after coculture and stimulation of these two cell types. Finally, anti-IL-17 does not alter endotoxin-induced release of IL-6 and MIP-2 from T lymphocytes and airway macrophages in coculture. In conclusion, our results indicate that endotoxin exposure causes the release of IL-17 from T lymphocytes and that this cytokine release requires the presence of macrophages. Once released, endogenous IL-17 acts in part by inducing local release of neutrophil-mobilizing cytokines such as IL-6 and MIP-2, from nonlymphocyte, nonmacrophage cells, and this contributes to recruitment of neutrophils in the airways. These IL-17-related mechanisms constitute potential targets for pharmacotherapy against exaggerated neutrophil recruitment in airway disease.

Exosomes Communicate Protective Messages during Oxidative Stress; Possible Role of Exosomal Shuttle RNA

Background Exosomes are small extracellular nanovesicles of endocytic origin that mediate different signals between cells, by surface interactions and by shuttling functional RNA from one cell to another. Exosomes are released by many cells including mast cells, dendritic cells, macrophages, epithelial cells and tumour cells. Exosomes differ compared to their donor cells, not only in size, but also in their RNA, protein and lipid composition. Methodology/Principal Findings In this study, we show that exosomes, released by mouse mast cells exposed to oxidative stress, differ in their mRNA content. Also, we show that these exosomes can influence the response of other cells to oxidative stress by providing recipient cells with a resistance against oxidative stress, observed as an attenuated loss of cell viability. Furthermore, Affymetrix microarray analysis revealed that the exosomal mRNA content not only differs between exosomes and donor cells, but also between exosomes derived from cells grown under different conditions; oxidative stress and normal conditions. Finally, we also show that exposure to UV-light affects the biological functions associated with exosomes released under oxidative stress. Conclusions/Significance These results argue that the exosomal shuttle of RNA is involved in cell-to-cell communication, by influencing the response of recipient cells to an external stress stimulus.

Eosinophilopoiesis in A Murine Model of Allergic Airway Eosinophilia: Involvement of Bone Marrow IL-5 and IL-5 Receptor α

The airway inflammation in asthma is dominated by eosinophils. The aim of this study was to elucidate the contribution of newly produced eosinophils in airway allergic inflammation and to determine mechanisms of any enhanced eosinophilopoiesis. OVA-sensitized BALB/c mice were repeatedly exposed to allergen via airway route. Newly produced cells were identified using a thymidine analog, 5-bromo-2′-deoxyuridine, which is incorporated into DNA during mitosis. Identification of IL-5-producing cells in the bone marrow was performed using FACS. Bone marrow CD3+ cells were enriched to evaluate IL-5-protein release in vitro. Anti-IL-5-treatment (TRFK-5) was given either systemically or directly to the airways. IL-5R-bearing cells were localized by immunocytochemistry. Repeated airway allergen exposure caused prominent airway eosinophilia after three to five exposures, and increased the number of immature eosinophils in the bone marrow. Up to 78% of bronchoalveolar lavage (BAL) granulocytes were 5-bromo-2′-deoxyuridine positive. After three allergen exposures, both CD3+ and non-CD3 cells acquired from the bone marrow expressed and released IL-5-protein. Anti-IL-5 given i.p. inhibited both bone marrow and airway eosinophilia. Intranasal administration of anti-IL-5 also reduced BAL eosinophilia, partly via local effects in the airways. Bone marrow cells, but not BAL eosinophils, displayed stainable amounts of the IL-5R α-chain. We conclude that the bone marrow is activated by airway allergen exposure, and that newly produced eosinophils contribute to a substantial degree to the airway eosinophilia induced by allergen. Airway allergen exposure increases the number of cells expressing IL-5-protein in the bone marrow. The bone marrow, as well as the lung, are possible targets for anti-IL-5-treatment.

A role of GM-CSF in the accumulation of neutrophils in the airways caused by IL-17 and TNF‐α

The T‐cell cytokine interleukin (IL)-17 selectively accumulates neutrophils in murine airways in vivo and may thus constitute a link between activation of T‐lymphocytes and accumulation of neutrophils. In this study, the authors evaluated the role of granulocyte macrophage-colony stimulating factor (GM-CSF) in accumulation of neutrophils in the airways caused by IL-17 and tumour necrosis factor (TNF)‐α. In vitro, human (h) IL-17 concentration-dependently stimulated the release of GM-CSF protein (enzyme-linked immunosorbent assay) in human bronchial epithelial cells (16HBE). IL-17 also time-dependently stimulated the release of GM-CSF protein in venous endothelial (human umbilical vein endothelial cells) cells in vitro. Co-stimulation with IL-17 plus the pro-inflammatory cytokine TNF‐α potentiated the release of GM-CSF protein in 16HBE cells. hIL-17 also enhanced the expression of GM-CSF messenger ribonucleic acid in 16HBE cells (reverse transcriptase polymerase chain reaction), with a similar order of magnitude as TNF‐α. Conditioned cell medium from bronchial epithelial cells co-stimulated with hIL-17 plus TNF‐α prolonged survival (trypan blue exclusion) of human neutrophils in vitro and this effect was blocked by an anti-GM-CSF antibody. In vivo, local co-stimulation with mouse IL-17 plus TNF‐α caused an additive potentiation of the accumulation of neutrophils in bronchoalveolar lavage fluid from mouse airways and this effect was blocked by an anti‐GM-CSF antibody given systemically. In conclusion, granulocyte macrophage-colony stimulating factor is involved in the accumulation of neutrophils in the airways caused by interleukin-17 and tumour necrosis factor‐α, probably via effects on both recruitment and survival of neutrophils.

Characterization of mRNA and microRNA in human mast cell-derived exosomes and their transfer to other mast cells and blood CD34 progenitor cells

Background: Exosomes are nanosized vesicles of endocytic origin that are released into the extracellular environment by many different cells. It has been shown that exosomes from various cellular origins contain a substantial amount of RNA (mainly mRNA and microRNA). More importantly, exosomes are capable of delivering their RNA content to target cells, which is a novel way of cell-to-cell communication. The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34+ progenitor cells. Methods: The mRNA and microRNA content of exosomes from a human mast cell line, HMC-1, was analysed by using microarray technology. Co-culture experiments followed by flow cytometry analysis and confocal microscopy as well as radioactive labeling experiments were performed to examine the uptake of these exosomes and the shuttle of the RNA to other mast cells and CD34+ progenitor cells. Results: In this study, we show that human mast cells release RNA-containing exosomes, with the capacity to shuttle RNA between cells. Interestingly, by using microRNA microarray analysis, 116 microRNAs could be identified in the exosomes and 134 microRNAs in the donor mast cells. Furthermore, DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. In addition, transfer experiments revealed that exosomes can shuttle RNA between human mast cells and to CD34+ hematopoietic progenitor cells. Conclusion: These findings suggest that exosomal shuttle RNA (esRNA) can play a role in the communication between cells, including mast cells and CD34+ progenitor cells, implying a role in cells maturation process. To access the supplementary material to this article: Tables S1-S6, please see Supplementary files under Article Tools online.

MicroRNA-155 is essential for TH2-mediated allergen-induced eosinophilic inflammation in the lung

BACKGROUND Allergic asthma is a chronic disease of the conducting airways characterized by T(H)2 inflammation and tissue remodeling after exposure to inhaled allergens. Although the T(H)2 profile is undisputed, the underlying molecular mechanisms leading to this abnormal T(H)2 profile remain largely unclear. MicroRNAs (miRNAs) are short noncoding RNAs that are important regulators of gene expression in the immune system. However, the role of miRNAs, specifically miR-155, in the regulation of allergic airway inflammation is unexplored. OBJECTIVES We sought to assess the contribution of miR-155 in a mouse model of allergic airway inflammation. METHODS To investigate a role for miR-155 in the regulation of allergic inflammation in vivo, we used miR-155 knockout (KO) and wild-type (WT) mice sensitized and exposed to ovalbumin. RESULTS miR-155 deficiency resulted in diminished eosinophilic inflammation and mucus hypersecretion in the lungs of allergen-sensitized and allergen-challenged mice compared with WT control animals. This was supported by a reduction in T(H)2 cell numbers and airway T(H)2 cytokine levels and complete abrogation of allergen-induced airway eotaxin-2/CCL24 and periostin levels in miR-155 KO mice. Intranasal instillation of eotaxin-2/CCL24 before allergen challenge partially restored airway eosinophilia in miR-155 KO mice, and adoptive transfer of CD4(+) T cells resulted in a similar degree of airway eosinophilia in miR-155 KO and WT mice. Furthermore, the transcription factor PU.1, a negative regulator of T(H)2 cytokine production, was upregulated in the airways of allergen-challenged miR-155 KO mice compared with WT mice. CONCLUSIONS Our data provides evidence that miR-155 contributes to the regulation of allergic airway inflammation by modulating T(H)2 responses through the transcription factor PU.1.

RNA-containing exosomes in human nasal secretions.

Background Exosomes are nanovesicles of endocytic origin released by cells and present in human body fluids such as plasma, breast milk, and bronchoalveolar lavage fluid. These vesicles take part in communication between cells. Recently, it was shown that exosomes contain both mRNA and microRNA. This RNA can be shuttled between cells (exosomal shuttle RNA), which is a new route of communication between cells. The aim of this study was to determine whether nasal secretions harbor exosomes and furthermore, whether these exosomes contain RNA. Methods Human nasal lavage fluid (NLF) underwent centrifugation and filtration to discard cells and debris, followed by a final ultracentrifugation at 120,000 × g to pellet the exosomes. Exosomes were detected using electron microscopy (EM), flow cytometry, and Western blot. RNA was extracted and analyzed using a Bioanalyzer. Results Exosomes were visualized as 40–80 nm, CD63+ vesicles using EM. Flow cytometry of exosomes using anti–major histocompatibility complex class II beads revealed exosomes positive for the tetraspanins CD9, CD63, and CD81. Western blot confirmed the presence of exosomal protein and absence of proteins from the endoplasmic reticulum (ER), because the exosomes were positive for Tsg101, but negative for the ER marker, calnexin. Bioanalyzer analysis revealed that, these exosomes contain RNA. Conclusion This study shows for the first time that NLF contains exosomes and that these exosomes contain RNA. Further characterization of the exosomal RNA and proteins may provide important information about communication in the nose and potentially provide a source of biomarkers for upper airway diseases.

Differential effects of fluticasone and montelukast on allergen-induced asthma

Early asthmatic responses (EAR) and late asthmatic responses (LAR) to allergen are induced by the local release of a series of bronchoconstrictor mediators, including leukotrienes and histamine. Both anti‐leukotrienes and other anti‐asthma drugs, such as inhaled glucocorticoids, have been shown to reduce both EAR and LAR. The aim of the present study was to directly compare the effects of regular treatment with an oral anti‐leukotriene, montelukast (Mont; 10 mg once daily, for 8 days), and an inhaled glucocorticoid [fluticasone propionate (FP) 250 μg twice daily for 8 days] on the EAR and LAR to an inhaled allergen challenge. Patients with a documented EAR and LAR at a screening visit were randomized to these treatments, or placebo, in a double‐blind, double‐dummy, crossover fashion. Allergen challenge at a dose causing both an EAR and LAR was given on the eighth day of treatment. The maximum fall in FEV1 during the EAR was 17.8% during placebo treatment, 8.3% during Mont and 16.3% during FP (P < 0.05 for Mont vs placebo). The maximum fall during the EAR was 13.8% during placebo treatment, 11.8% during Mont and 2% during FP treatment (P < 0.05 for FP vs placebo and FP vs Mont). PC20 methacholine was significantly higher 24 h after allergen challenge during FP‐treatment compared with Mont (P < 0.05). Both montelukast and fluticasone reduced the relative amount of sputum eosinophils after allergen compared with placebo treatment. This study shows that anti‐leukotrienes are effective to attenuate the EAR, whereas inhaled glucocorticoids are more effective than anti‐leukotrienes in attenuating the EARs and improves bronchial hyperresponsiveness to a greater extent. In conclusion, inhaled glucocorticoids have overall greater efficacy than oral anti‐leukotrienes to attenuate allergen‐induced airway responses in mild asthmatic patients.