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.
As far as we know, this is the first study that describes and characterizes the presence of exosomes from CCM and evaluates some in vitro functions in addition to demonstrate their interaction on canine MSCs and fibroblast. Our results reveal the existence of abundant exosomes in CCM, which significantly promote the proliferation and viability of canine MSCs according to their origin, modifying their secretory profile and showing antioxidant potential on canine fibroblast.
Ultracentrifugation techniques allowed isolating abundant CCM exosomes, in a similar manner to that already described in the isolation of other origins [27, 29, 33]. The presence of CCM exosomes were confirmed by TEM, size determination and western blot analysis, expressing ALIX, Hsp70 and TSG101 exosomal markers, according to the recommendation of the International Society for Extracellular Vesicles [45, 46].
Exosomes play a key role in cell-to-cell communication and contain different specific proteins depending on their cellular origin. Nevertheless, they share a subset of essential proteins for vesicular biogenesis, structure and distribution [33, 47, 48]. Through proteomic analysis, we identified 892 proteins mainly related to functions such as transport, metabolism, regulation of different biological functions, cell differentiation, organization and biogenesis. These results coincide with the colostrum milk exosomes of other species [7, 48], which suggest the evolutionary importance of these vesicles to regulate different cellular functions in the newborn [6, 13, 14, 25, 49], and it is shared between different species of mammals [10].
Newborns are more prone to suffer oxidative stress than adults [35, 50–52], bringing on an increase in the risk factors that trigger inflammation, infection and ischemia, resulting in multiple organs damage, which plays a fundamental role in the pathogenesis of several pregnancy and perinatal diseases [53–55].
Colostrum is essential in the antioxidant mechanism of the newborn [56–59] and our results demonstrate, the antioxidant potential of CCM exosomes on canine fibroblast.
Fibroblasts are a very abundant cell type in the body and suffer the effect of free radicals in very important stages of maturation of certain vital organs such as the lung [60, 61]. This has justified the use of this cell type to evaluate the antioxidant capacity of CCM exosomes.
Evaluating MSCs as the target of colostrum exosomes, we found interesting results, which depend on the cellular source. CCM Exosomes co-culture with MSCs determined a significant 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, 62]. 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 [63].
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 [64,65]. They are exerted through a large diversity of adipokines secretions with complex autocrine and paracrine effects [66]. MSCs are postnatal multipotential progenitors and can be found in adipose tissue, bone marrow and other connective tissues [29, 67]. MSCs fat residents are generally the principal source for adipocytes during postnatal growth and maintenance of adipose tissue [68].
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 [69]. 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 [70], necessary for the development and growth process.
cBM-MSCs stimulated with CCM 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 [71]. IL–12p40 has an important role in the development of T cells and enhance the production of immune factors [72]. NO is a highly immunosuppressive soluble factor that decreases the proliferation and modulation of T cells and promotes apoptosis of immune cells [73].
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 [74]. IFN-γ intervenes in the macrophage’s 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 [75, 76].
In addition, we found that colostrum exosomes increased the secretion of factors related to neurogenesis (NGF-β) by cAd-MSCS in a very intense way. NGF plays a crucial role in the peripheral and central nervous systems that regulate the growth, differentiation and survival of neurocytes, improves cognitive functions and shows potential to induce angiogenesis in physiological and pathological conditions [77, 78].
Although both types demonstrate a secretory similarity in the functions related to angiogenesis, migration and chemotaxis of immune cells, the different behavior of each cell type would confirm the importance of the cellular niche in the different biological functions of individuals. Thus, while adipose tissue MSC show an important endocrine and metabolic potential in adipose tissue development and neurogenesis, the response of BM-MSCs is more consistent with immunity, cell mobilization, angiogenesis and hematopoiesis.
Therefore, the results presented here open new ways for improving knowledge of colostrum functions and for a possible future use of these vesicles in the immune system modulation, the antioxidant response and the growth of puppies [8, 16, 47].
Although our study obviously had limitations due to the small sample size of colostrum donors and the restriction posed by the lack of specific reagents available for the canine species, we believe that our work is a first step in this direction. However, a more in-depth investigation of exosomes’ functions, the focus on miRNA cargos, gene regulation, immunity and metabolism may be an interesting line of research.