Enzootic bovine leukosis is a disease widespread in dairy herd livestock in many countries and is currently one of the most commonly reported neoplastic diseases in cattle. It is a cause of serious economic losses in cattle herds and is listed in the World Organisation for Animal Health’s Terrestrial Animal Health Code (26). The causative agent responsible for the disease is bovine leukaemia virus (BLV), a member of the
The viraemic state is rather short and can be detected during the first days after viral infection. The detection of viral antigens is difficult because viral protein production is suppressed by cytokines, especially by interleukin (IL)-6 and IL-10 (20, 23). These investigations indicated that BLV infection caused disease when changes occurred in host factors. There is no vaccine against or treatment for bovine leukosis.
Cells of eukaryotic and prokaryotic organisms release heterologous nanoparticles 40–5,000 nm in diameter called extracellular vesicles (EVs) into the extracellular space. The vesicles are spherical and enclosed by a phospholipid bilayer membrane (18, 19, 21). Under physiological conditions, the intact bilayer structures of EVs have the capability to protect their biomolecules against degradation by ribonucleases and digestive enzymes and maintain their integrity and biological activity (11). Extracellular vesicles were first discovered in human blood by Peter Wolf in 1967 (25) as “platelet dust”, and were subsequently also detected in reticulocytes and determined to be novel transfer vesicles. Extracellular vesicles can be categorised into three main groups: microvesicles, exosomes and apoptotic bodies (18, 19). They contain bioactive molecules comprising various proteins, microRNA (miRNA), messenger RNA (mRNA), and lipid compounds similar to those of the parent cells, and can affect other cells by the transfer of receptors and genetic cargo. Many authors have indicated their role in immune responses, angiogenesis, thrombosis, homeostasis, tumour development and metastasis (22, 30). The level of EVs is elevated in the course of cardiovascular diseases, viral infections, various types of neoplasm, and auto-immune, metabolic and parasitic diseases (2, 29, 30). Additionally, EVs have some cell-specific compounds and their content may be used for early diagnosis of these diseases.
Exosomes are small, lipid bilayer membranous vesicles of endocytic origin. These extracellular nanovesicles present in various body fluids are from 30 to 150 nm in diameter and are produced by the inward budding of the limiting membrane of multivesicular bodies. Exosomes carry surface and luminal proteins which are exchanged between cells (2, 3, 22). Exosomes’ protein compositions and functions depend on the organs and cell types that they were excreted from. They transfer information to the target cells
One of the most abundant protein families that are found in exosomes is the tetraspanins. Several members of this family, namely CD9, CD63, CD81 and CD82, are heavily present in exosomes of any cell type. Tetraspanins interact with many protein partners, including MHC molecules and integrins, which indicates that they are included in the organisation of large molecular complexes and membrane subdomains and take part in cell penetration, invasion and fusion events.
Many other compounds are present in exosomes, flotillin proteins among them. These proteins play important roles in many biological processes such as cell proliferation, apoptosis, adhesion and invasion. It was shown that flotillins are located on lipid microdomains and that the two proteins are involved in the retraction of plasma membrane vesicles. Flotillin-1 is widely expressed in the body and may have different action in different tissues and cells.
Exosomes contain proteins and nucleic acids with involvement in infection processes, because they bear mRNA and miRNA (7, 8, 9, 24, 29). It was reported that by cell-to-cell contact, exosomes can transport infectious agents and disseminate them to healthy cells, so infection is transmitted to all organs (28, 29). Neither vaccination against enzootic bovine leukosis being possible nor treatment of it being effective or economical, countering the disease must rely on detection of markers of infection with the virus to facilitate elimination of carrier animals. The aim of the study was isolation and molecular determination of exosomes in blood and supernatant fluids of bovine DC culture infected with BLV.
Investigations were performed on a group of 12 Polish black and white lowland cows at age 4–6 years naturally infected with BLV. Four healthy cows at the same age as the experimental group were enrolled as negative controls. Blood samples from animals were taken in an abattoir, and for this reason the approval of the local ethics committee was not required. The supernatants from HeLa cell cultures and cultures of foetal lamb kidney permanently infected with BLV (FLK-BLV) were used as positive controls. Blood samples were drawn from the jugular vein and collected into tubes with or without ethylenediaminetetraacetic acid (EDTA)-K2 anticoagulant. White blood cells and lymphocytes were counted. A part of the blood samples with anticoagulant was centrifuged at 2,000 ×
The methods of DC generation and cell culture were adapted from those described by Szczotka
Sera, plasma, FLK-BLV lysates, and the supernatant of DC cultures were centrifuged at 4°C at low speed (1,500 ×
The presence of BLV glycoprotein gp51 in dendritic cells and the positive control FLK-BLV cell line was determined in an immunofluorescence (IF) reaction. Monoclonal anti-BLV gp51 fluorescein isothiocyanate–conjugated antibodies (VMRD, Pullman, WA, USA) were used and cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclei visualisation, then the pictures were merged.
The pellet containing exosomes was resuspended in a dedicated buffer (Total Exosome RNA and Protein Isolation Kit; Invitrogen, Carlsbad, CA, USA). The protein level was measured with the NanoDrop spectrophotometer and 50 ng of protein of every sample was used for sodium dodecyl sulphate polyacrylamide gel electrophoresis, which was run at 100 V for 3 h. Electrotransfer to nitrocellulose (Protran 0.45 NC; GE HealthCare Life Sciences/Amersham, Chalfont St. Giles, UK) was performed overnight at 20 V in standard conditions. During immunodetection, nitrocellulose membranes were blocked in 2% bovine serum albumin in PBS. Next, blots were washed with PBS-Tween buffer and incubated at room temperature for 2 h with the mouse IgG primary antibody diluted to 1 : 2,000 according to the manufacturer’s instructions. Monoclonal antibodies for exosomal cellular markers were used as the primary antibody, namely anti-bovine CD9 (Kingfisher Biotech, St. Paul, MN, USA), anti-bovine CD63 (Bio-Rad, Hercules, CA, USA) and anti-flotillin-1 (BD Biosciences, Lexington, KY, USA). Markers of BLV were detected with the use of monoclonal anti-gp51 and anti-p24 antibodies (both from VMRD). After washing, all blots were incubated with the goat anti-mouse IgG horseradish peroxidase–conjugated secondary antibody diluted to 1 : 10,000 (Thermo Scientific/Pierce Biotechnology, Rockford, IL, USA) for 1 h at room temperature, then after final washing, chemiluminescence was detected with the use of Amersham ECL (enhanced chemiluminescence) reagent (Cytiva, Marlborough, MA, USA).
A scanning electron microscope (Zeiss, Jena, Germany) and Libra 120 transmission electron microscope (Zeiss) were used for morphological examination of DCs and exosomes, which was performed according to the method described by Hanaishi
Exosomal and viral marker analysis revealed that the morphology of monocytes took on dendritic cell characteristics after 24 h of culture
The presence of CD9, CD63 and flotillin-1 exosomal markers in exosomes isolated from the blood plasma of bovine leukaemia virus–infected cows as detected by Western blot
M – Marker ladder
The presence of glycoprotein (gp)51 and core protein (p)24 viral markers in exosomes isolated from the blood plasma of bovine leukaemia virus–infected cows as detected by Western blot
M – Marker ladder; K+ – exosomes in supernatant of permanently bovine leukaemia virus–infected foetal lamb kidney cell culture; 1–4 – exosomes isolated from the blood plasma of bovine leukaemia virus–infected cows
Expression of bovine leukaemia virus glycoprotein 51 in dendritic cells generated from blood samples of bovine leukaemia virus–infected cattle, as visualised by an immunofluorescence reaction
In exosomes isolated from the blood plasma and supernatant of
The expression of BLV gp51 in dendritic cells generated from monocytes of BLV-infected cattle is presented in Fig. 3. The green fluorescence is visible in the whole cell, both in the veil and the processes (dendrites), which indicates the presence of BLV gp51 protein and infection with BLV. The cellular nucleus stained with DAPI is blue.
The FLK-BLV cell line was used as a positive control for the IF reaction test. Visible green fluorescence indicated the expression of BLV gp51, and blue denoted nuclei staining with DAPI.
The results of the determination of exosomal and viral markers are presented in Table 1. When supernatants of
The viral and exosomal markers detected by Western blot in bovine leukaemia virus (BLV)-infected cows
Origin of exosomes | BLV infection | BLV markers | Cellular markers | ||||
---|---|---|---|---|---|---|---|
gp51 | p24 | CD63 | CD9 | flotillin-1 | |||
Positive control – supernatant of FLK-BLV culture | + | + | + | + | + | + | |
Supernatant of cultured |
1 | + | + | + | + | + | + |
2 | + | + | + | + | + | + | |
3 | + | + | + | + | + | + | |
4 | + | + | + | + | + | + | |
5 | + | + | + | + | + | + | |
Negative control – plasma BLV− | − | − | − | + | + | + | |
Positive control – lysate of FLK–BLV cells | + | + | + | + | + | + | |
Bovine BLV+ sera (1–7) and BLV− sera (8–11) | 1 | + | + | + | + | + | + |
2 | + | + | + | + | + | + | |
3 | + | + | + | + | + | + | |
4 | + | + | + | + | + | + | |
5 | + | + | + | + | + | + | |
6 | + | + | + | + | + | + | |
7 | + | + | + | + | + | + | |
8 | − | − | − | + | + | + | |
9 | − | − | − | + | + | + | |
10 | − | − | − | + | + | + | |
11 | − | − | − | + | + | + |
gp – glycoprotein; p – core protein; FLK – foetal lamb kidney; DC – dendritic cell
Scanning microscopy images are presented in Figs 5, 6, 7 and 9. There are small vesicles with diameters of 1 μm between cultured dendritic cells. The cells, 10–18.8 μm in diameter, have characteristic dendrites. Figure 9 presents extracellular vesicles in the
Extracellular vesicles in
Mag – magnification; EHT – electron high tension; WD – working distance; SE1 – secondary electron 1
Extracellular vesicles in
Mag – magnification; EHT – electron high tension; WD – working distance; SE1 – secondary electron 1
Extracellular vesicles in
Exosomes in
Mag – magnification; EHT – electron high tension; WD – working distance; SE1 – secondary electron 1
Transmission electron microscopy images are presented in Figs 8 and 10. The visible small vesicles differ in size. Variation in vesicle size is also evident in the scanning electron microscopy images of exosomes isolated from sera, plasma and cultured DCs.
Electron micrograph of exosomes in
Electron micrograph of exosomes in
In the supernatants of leukaemic dendritic cell cultures (Figs 5, 6 and 7), extracellular vesicles 1 μm in size were detected and the cells were 18.8 μm in diameter. Similar results were found in the HeLa cell cultures (Fig. 9). Analysis in the transmission electron microscope showed the presence of vesicles 49 to 65 μm in size (Figs 8 and 10).
Expression of bovine leukaemia virus glycoprotein 51 in a permanently bovine leukaemia virus–infected foetal lamb kidney cell line, as visualised by an immunofluorescence reaction
In the current study we investigated cellular and viral markers of exosomes isolated from the blood and supernatant of cultures of DCs from BLV-infected cattle. The CD9, CD63 and flotillin-1 cellular markers were detected in these exosomes. The presence of the gp51 and p24 BLV antigens was confirmed. Yamada
Exosomes have great potential as liquid biopsy specimens in the diagnosis of many diseases because of their presence in most body fluids and general stability (9, 12, 18, 24, 30 ). Cancer-derived exosomes carry cargo reflective of genetic or signalling alterations in cancer cells and can be used as biomarkers for the early detection of neoplasia (6, 12). Exosomes contain many different proteins: both their own and proteins derived from parent cells. They are very convenient biomarkers because they have cancer-related compounds such as proteins, lipids, RNA, miRNA and DNA (18, 19, 24). They are very small, so they can easily pass through the tissue barrier and are present in various body fluids (1, 6, 22). Their lipid bilayer membrane protects them and their contents from degradation by enzymes that are present in circulating blood. Another factor in exosomes’ diagnostic potential is specifically in the diagnosis of early-stage cancer: in humans, each millilitre of blood contains about 1×109 easily separated exosomes which can be analysed for indications of these diseases (6, 7, 8). Exosomes secreted by different cells are important mediators between tumour cells and stromal cells in their function of transforming information from the bone marrow microenvironment (3, 27, 28). Their action in cancer drug resistance and the possibility for their therapeutic application are still being investigated in many scientific centres (12). Exosomes derived from bone marrow mesenchymal stem cells play a significant role in cancer development (2, 3). Additionally, they promoted the metastatic potential of leukaemia cells (6, 13). Li
Tetraspanins are involved in the process of exosome production. In antigen-presenting cells, the functions of MHC-II molecules are regulated by their integration into the cytoplasmic membrane regions, which are enriched in the tetraspanin CD9 (3). Tetraspanins may be analysed for diagnosis of various tumours and infectious diseases. It was evident that CD63+ exosomes were significantly increased in patients with melanoma and other cancers (21, 30), and CD63 has been suggested as a protein marker of cancer. Similarly, the CD81 protein, another member of the tetraspanin family, plays an important role in cell entry by the hepatitis C virus and was demonstrated to be significantly increased in the serum of patients with chronic hepatitis C, indicating that CD81 may be used as a marker for the diagnosis of hepatitis C viral infection (13, 22). Exosomes released by virus-infected cells contain viral components as well as components of cellular origin. It means that viruses not only transport their own products in exosomes but also exert some determining effect on the type of cellular products transported within the excretory vesicles. It was shown that exosomes released from HIV-1-infected and uninfected cells differ in their densities, which indicated that exosomes from infected cells are different from exosomes of uninfected cells (2, 21). Released exosomes can bind to neighbouring cells and travel passively through the bloodstream to very distant parts of the carrier’s body, where they can induce biological changes depending on the kind of products they carry. For example, Nef is one of the HIV-1 proteins that is released within exosomes. Nef plays an important role in the activation of CD4+ T cells, and when these cells are activated, they are susceptible to HIV infection and viral replication. Some authors indicated that HIV-1 may facilitate its spread to neighbouring cells by secreting viral chemokine co-receptors CCR5 and CXCR4 in exosomes. Exosomes from HIV-1 infected cells carry several viral miRNAs (2). Another human retrovirus, HTLV-1, exports viral components with the use of an exosomal transport system (1, 12).
Exosomes serve as transporters that deliver virus receptors to target cells that make them susceptible to virus entry. Tumour-derived exosomes have been shown to promote cancer development and metastasis (11, 13). It was found that bovine milk-derived exosomes can be orally administered with the cancer chemotherapeutic paclitaxel, altering the drug’s stability and toxicity (1). The important role of exosomes in cancer cell survival and proliferation was identified. On the other hand, evidence suggests that exosomes are useful in cancer treatment and may also be an effective approach in leukaemia diagnosis (6, 8). A critical attribute of tumour cells is that these cells can evade host immune surveillance. It has been reported that exosomes derived from leukaemic cells have a suppressive effect on the immune system and that as a consequence, it is possible for them to escape the immune response. The sera of patients with acute myeloblastic leukaemia were shown to contain a higher level of exosomes than the sera of healthy people (6, 18).
Since exosomes are present in most biological fluids, they are apt to be used clinically in the early diagnosis and prognosis of viral infections and diseases caused by infective microorganisms. Conventional diagnostic procedures for leukaemia have some limitations. The method based on exosome isolation from body fluids and the supernatant of dendritic cell culture is non-invasive and can be useful for early detection of haematological malignancies (8, 12). Plasma is an important source of cancer markers; its mRNA, miRNA and protein contents can be used for the early diagnosis of the disease. Research revealed that cancer cells’ exosomes had different miRNA profiles to normal cells, which is important in light of the heavy involvement of miRNAs in tumorigenesis (9, 10). Exosomes contain miRNA and other genetic information and can be exploited as biomarkers (24).
The results of recent studies indicate the potential role of exosomal miRNAs as biomarkers. In the future, they may become effective therapeutic tools (1, 6). A significant role of exosomes is the