Canine colostrum exosomes: characterization, anti-oxidative capacity and influence on canine mesenchymal stem cell

Background: Colostrum is a specific secretion of the mammary gland fundamental for the survival of the puppy during the first weeks after birth. It contains important bioactive molecules involved in the passive immunity and the maturation of various organs, highlighting small vesicles named exosomes. Exosomes have not been described yet in canine colostrum milk, where its role is crucial in dam-newborn communication for the development of the neonate. Results: Exosomes were abundant in canine colostrum milk and appeared with a characteristic cup-shaped morphology and well-defined round vesicles. Their size distribution was between 37−140 nm and western blot analysis showed an expression of specific exosomal markers. Proteomic analysis revealed a total of 826 proteins in exosomes cargo. We also found that exosomes modified proliferation and secretory profile in canine mesenchymal stem cells derived from bone marrow and adipose tissue, differently according to their origin. Besides, the exosomes of canine colostrum demonstrated a potent antioxidant effect. Conclusions: We described for the first time the isolation and characterization of milk exosomes from canine colostrum. Our findings highlight the abundant presence of exosomes in the colostrum of the canine species and contribute to explain their important antioxidant capacity after parturition and their role in the modulation of cell development and tissue differentiation in the newborn, where mesenchymal stem cells seem to play a role. bicinchoninic acid;

4 promoting the growth of intestinal epithelium, the proliferation of different cells and the signal transduction [16][17][18].
Our group described for the first time the characterization of exosomes from canine mesenchymal stem cells (MSCs), which showed differences according to their tissue source [29]. MSCs play a strategic role in the development, homeostasis and repair of different organs and tissues [30,31]. Its clinical use has shown promising results for different pathologies in canine species [32,33]. Although colostrum exosomes' effect has been evaluated in different cell types [34], it has never been assessed on MSCs.
Likewise, recent findings point to the importance of oxidative stress during the early postnatal period in the newborn [35], which may be responsible for serious alterations very well described in the human neonate [36,37] and in some domestic species [38].
Among the components of colostrum, there are different essential antioxidants against oxidative damage [38,39]. However, the antioxidant role of colostrum exosomes has not been described enough. As far as we know, this is the first time that exosomes from canine colostrum have been isolated and characterized. In addition, their effects on canine MSCs have been evaluated in vitro, as well as their antioxidant effect.
The purpose of our study was to characterize canine colostrum exosomes and evaluate their anti-oxidative capacity and influence on canine mesenchymal stem cell proliferation capacity and modification of its secretory profile.

Animals and colostrum sample collection
Eight client-owned healthy bitches of different breeds with a mean age of 3,87 ± 1,25 years and body weight of 16,5 +/-10,97 kg were selected as colostrum donors. The average litter size was 4,75 ± 1,65 puppies. Animals were up to date with vaccinations and deworming, and fed with a dry balanced diet for growing dogs ad libitum.
All animals were clinically examined previously, submitted to hematological and biochemical tests, and they did not manifest symptoms of infectious or parasitic diseases.
No medication was administered during pregnancy. Immediately after delivery, the mammary glands were disinfected, a previous massage was performed, and 3 mL of colostrum were collected using a manual milk extraction syringe. None of the animals required any type of anesthesia or sedation, and they were not sacrificed to obtain colostrum. The samples were stored at 4°C until analysis. Written consent was obtained from all dogs' owners.

Refractive index
The colostrum refractive index was measured in thawed colostrum at room temperature (21°C) with a handheld refractometer on samples diluted 1:2 in distilled water (Atago, Japan; refractive scale from 1.333 to 1.360) as previously described [4,40]. All samples were analyzed in the same session. 6 Colostrum exosomes isolation and characterization.
Pull of CCM exosomes from eight bitches were used. Isolation and characterization protocols by transmission electron microscopy (TEM) (Morgagni 268D electron microscope, Philips), Western Blot (WB) and proteomics analysis, were performed as previously described [29].
Size determination of purified exosomes was carried out using a Zetasizer Nano ZS (Malvern Instruments). Exosomes were diluted in 1mL of 1X phosphate buffered saline (PBS) and the parameters of Z potential (electronegativity) and size distribution were Canine MSCs culture and CCM exosomes proliferation effects Canine bone marrow (cBM-MSCs) and adipose tissue (cAd-MSCs) mesenchymal stem cells from the same donor were isolated and characterized as previously described [29,32,33].
Cultures were carried out in standard culture conditions: Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) exosomes free, 2.5mM Lglutamine, 100U/mL penicillin, 100 µg/mL streptomycin, and 1.25 µg/mL fungizone (all from Sigma-Aldrich). Cells were trypsinized at confluence and cryopreserved in liquid nitrogen. The experiments were carried out on culture passage 3. FBS exosome-free serum was obtained by ultra-centrifugation at 100.000 g for 60 min at 4 °C, using 70 Ti rotor in an Optima LE-80 K ultracentrifuge (Beckman Coulter). Supernatant was collected and precipitate (exosomes) was eliminated.
Cell proliferation was measured using MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega) according to the manufacturer's instructions. cBM-MSCs and cAd-MSCs were seeded at a concentration of 3 × 10 3 cells per well in a 96 well plate. Two doses of CCM exosomes (25µg/mL) were administered on days 1 and 6, and supernatants' absorbance optical density was measured at 490 nm at 1, 2, 5, 7, 9, 12 days using a microplate reader (ELx800, BioTek instruments).
Canine fibroblasts viability assay MTS assay was used to determine canine fibroblasts (Cellider Biotech) cell viability.

Reactive oxygen species measurement
Reactive oxygen species (ROS) detection was performed using DCFDA / H2DCFDA -Cellular ROS Assay Kit (Abcam) according to the manufacturer's instructions. Canine fibroblasts were co-cultured with exosomes (25μg/ml) for 24h after being exposed to H 2 O 2 (500 μM) 9 for 3 h. Standard culture conditions were used after H 2 O 2 treatment for the control group.

Statistical analysis
Data analysis was performed by SigmaPlot 11.0 software and each test was repeated three times. The data are presented as mean ± standard deviation (SD). Student's t test was used for MSCs proliferation, canine fibroblast viability and ELISA assay results, and the P-value was adjusted using the Bonferroni method for multiple comparisons. The degree of significance was established in the following ranges: P<0.05 (*), P<0.01 (**) and P<0.001(***).

Colostrum refractive index
The values obtained from all colostrum samples were within the standard values described for this species. The average refractive index value was 1.343 ± 0.0014 (Table 1).

Canine colostrum exosomes characterization
The mean of exosome concentrations obtained in the eight CCM samples were 305.60 ± 46.7 µg/mL. CCM exosomes were visualized by TEM (Fig. 1a), and their size distribution was between 37−140 nm with a zeta potential of -11.40 ± 0.53 mV (Fig. 1c). The measurement of size is based on the Dynamic Light Scattering (DLS) technique.

Proteomic analysis
The total number of peptides was performed by mass spectrometry and analyzed using Canis lupus familiaris protein database. We found 826 proteins in CCM exosomes.
Biological processes of characterized exosomes proteins were determined by Gene Ontology parameters (Fig. 2).

Discussion
Colostrum plays a fundamental role for survival in the neonatal period of the puppy, as well as for its future development as adult [1,3]. To date, despite knowledge of the nutritional and immunological components of colostrum in dogs [4,5], there is no study on biological nanostructures, such as exosomes, their composition and biological functions in canine colostrum related to the modulation and maturation of the immune system of the newborn.
As far as we know, this is the first study that describes and characterizes the presence of Ultracentrifugation techniques allowed isolating abundant exosomes from all canine colostrum samples, in a similar manner to that already described in the isolation of other origins [27,29].
The exosomes quantity obtained was like that described in the breast milk of other species [25,45]. This concentration was between 40 and 350 times higher compared to the exosomes isolated by our group from canine MSCs [33], but very similar to the dog peripheral blood exosomes quantity [27].
According to the recommendation of the International Society for Extracellular Vesicles [46,47], the identities of canine colostrum exosomes were confirmed by TEM, size determination and western blot analysis, expressing ALIX, Hsp70 and TSG101 exosomal markers.
Exosomes contain different specific proteins depending on their cellular origin.
Nevertheless, they share a subset of essential proteins for vesicular biogenesis, structure and distribution [33,48,49]. Through proteomic analysis, our group identified 892 proteins mainly related to functions such as transport, metabolism and regulation of 13 different biological functions, cell differentiation, organization and biogenesis. These results coincide with the colostrum milk exosomes of other species [7,50], which suggest the evolutionary importance of these particles to regulate different cellular functions in the newborn, and it is shared between different species of mammals [10].
Exosomes play a key role in cell-to-cell communication. Despite the few studies of breast milk exosomes, they have been reported to act to elicit multiple regulatory functions in the recipient cells of the newborn [6]. Among them stand out those associated with the modulation of the immune response, promoting thymic regulatory T cell maturation [25], proliferation of intestinal cell and digestive tract development [13,14], as well as contributing to the development of microbiota [51], crucial for the growth and health of newborns.
Oxidative stress is due to an imbalance between the formation of ROS and the capacity of the organism to eliminate reactive intermediates or to repair their resulting damage [52].
Newborns are more prone to suffer oxidative stress than adults [35,53]. Inadequate balances determine an increase in the risk factors that trigger inflammation, infection and ischemia, resulting in multiple organs damage promoting ROS production, which plays a fundamental role in the pathogenesis of several pregnancy and perinatal diseases [54][55][56].
Colostrum is essential in the antioxidant mechanism of the newborn [57,58]. Some factors such as their conservation or mother diseases can affect their ROS potential [59,60].
Our results demonstrate for the first time the antioxidant potential of canine colostrum exosomes. We believe that the administration of antioxidant supplements in puppies fed with colostrum substitutes is important to counteract the generation of postpartum ROS [38].
Evaluating MSCs as the target of colostrum exosomes, we found interesting results, which depend on the cellular source. Exosomes co-culture with MSCs determined a significant 14 increase in cAD-MSCs proliferation, whereas this effect was not observed in cBM-MSCs.
The energy supply during the first days of life through colostrum plays a very important role in the growth rate of the puppy and affect the risk of neonatal mortality [1,61]. Fat content was low in newborns and increased rapidly during the first month of life, this does not appear to be related to breed size [62].
Adipose tissue, besides being an energy reservoir, represents a natural defense against hypothermia and fulfills metabolic, endocrine and regulatory functions, both with systemic and local effects [63,64]. They are exerted through a large diversity of adipokines secretions with complex autocrine and paracrine effects [65]. MSCs are postnatal multipotential progenitors and can be found in adipose tissue, bone marrow and other connective tissues [29,66]. MSCs fat residents are generally the principal source for adipocytes during postnatal growth and maintenance of adipose tissue [67].
In this study we demonstrated that canine colostrum exosomes determine changes in the secretory profile of both types of canine MSCs studied, but in a very different way. Of the 13 analytes evaluated, we found a significant increase in the production of 5 of them in cAD-MSCS (IL-8, MCP-1, IFN-γ, TNF-α and NGF-β), and 6 in cBM-MSCs (IL-12p40, IL-6, IL-8,

MCP-1, SCF and NO).
Both cell types showed an increase in the secretion of IL-8 and MCP-1, factors related to migration, chemotaxis and angiogenesis.
IL-8, also known as CXCL8, has been shown to have potent pro-angiogenic properties, promoting vein endothelial cell proliferation, migration, tube-formation and the ability to attract and activate neutrophils [68]. MCP-1, is one of the factors associated with the immunomodulatory effects of MSCs, reduces apoptosis and plays a direct mediating role for angiogenesis, which is manifested by the formation of new blood vessels [69], necessary for the development and growth process.
cBM-MSCs stimulated with colostrum exosomes specifically increase the production of factors related to immunity (IL-6, IL-12p40, NO) and regulation and mobilization of hematopoiesis (SCF). IL-6is a pleiotropic cytokine with a key role in different biological processes, such as regulation of the immune response, inflammation, hematopoiesis, apoptosis, cell survival and cell proliferation [70]. IL-12p40 has an important role in the development of T cells and enhance the production of immune factors [71]. NO is a highly immunosuppressive soluble factor that decreases the proliferation and modulation of T cells and promotes apoptosis of immune cells [43].
In contrast to BM-MSCs, colostrum exosomes in cAd-MSCs, in addition to stimulating their proliferation, demonstrated a change in their secretory profile by increasing the release of proinflammatory cytokines (TNF-α and IFN-γ). TNF-α is a pleiotropic cytokine with important but sometimes contradictory functions in numerous physiological processes related to immunity and inflammation [72]. IFN-γ intervenes in the macrophages activation, induces the expression of MHC class II molecules, increases the cytotoxic potential and favors, together with TNF-α, the development of the fundamental Th1 cell responses to control viral infections [73].
MSCs with a predominantly pro-inflammatory profile have been associated with early stage infections and infection processes, which stimulate the response of the innate immune system, fundamental in these early stages of life [74].
In addition, we found that colostrum exosomes increased the secretion of factors related  Availability of data and materials 18 All data generated or analysed during this study are included in this published article (and its supplementary information files).

Competing interests
The authors declare that they have no competing interests.   Data are expressed as mean ± SD.

Supplementary Files
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Additional file 1_Exosome proteomic profile.pdf