Study On The Mechanism Of Lymphocytes From Mammary Gland Crossing Epithelium Into Milk Colostrum In Sows

The colostrum is rich in nutrients and contains a large number of immune cells including lymphocytes that play important roles in neonatal piglets withimmaturate immune systems.Recently,many studies have investigated the protective effects of lymphocytes in the colostrum on newborn piglets,however,little is known about the transport mechanism of colostrum lymphocytes across the mammary glands into the colostrum.In this study,we explored the mechanism of lymphocyte migration across the mammary epithelium in the colostrum.Firstly,we isolated colostral lymphocytes from milk colostrum in sows and functional analysis showed they play a role in the proliferation of lymphocytes and the secretion of cytokines.Secondly,a mammary epithelial cell(Ep H4-Ev)/T cell co-culture system was established to explore the mechanism of lymphocyte transmigration across mammary epithelial cells in vitro.The results suggested that prolactin could promote the migration of lymphocytes from the mammary epithelium to the colostrum by promoting the production of the CCL28 chemokine.Finally,we investigated the characteristics of the intestinal mucosa of newborn piglets and the results showed that the intestinal immune development of newborn piglets was defective,providing a theoretical basis for the necessity of timely access to colostrum in neonatal piglets.The content of this research article is divided into the following three parts:1.The separation of lymphocytes from the sow colostrum and the study of its morphology and function.In this experiment,the colostrum lymphocytes from sows were separated and the cell morphology was observed by Giemsa staining.Our results showed that the lymphocytes had a full body with a clear boundary between the cytoplasm and nucleus.The cells had an irregular plasma membrane with nuclear chromatin aggregated in a large area.The cytoplasm was very small and presented as a thin ring shape.Immunofluorescence staining was used to detect CD3~+T lymphocytes.Flow cytometry was used to determine the proportion of CD3~+T lymphocytes in the colostrum which was 8%and the ratio of CD4:CD8 cells was 0.4～0.5.We then explored the proliferative function and the secretion of cytokines in the lymphocytes.Immunofluorescence staining of nuclear proliferation antigen(PCNA)showed that CD3~+T lymphocytes in colostrum can proliferate.Compared to lymphocytes in the mesenteric lymph nodes(MLN),we found that colostrum lymphocytes had higher expression levels of inflammatory cytokines including interleukin-1β(IL-1β),tumor necrosis factorα(TNF-α),Interleukin-4(IL-4)and Interleukin-8(IL-8).In this study,we initially explored the morphology and function of lymphocytes in the colostrum to provide a theoretical basis for exploration of the immune effects of colostrum lymphocytes in newborn piglets.2.The distribution of lymphocytes in the mammary glands of sows and the mechanism of lymphocyte migration in the colostrum.The lymphocytes in the colostrum home to the mammary gland through the circulation after antigen-sensitized activation.The lymphocytes then migrate to the colostrum through the epithelium of the mammary gland in the breast stroma.Many previous studies have investigated the homing of lymphocytes,however,the mechanism of lymphocytes migration across the mammary epithelium to colostrum in the breast remains unclear.To explore the source of lymphocytes in the colostrum and the mechanism of transmammary epithelial migration,we first detected the distribution of T lymphocytes in the mammary glands of sows by HE and immunohistochemical staining.The results revealed that lymphocytes in the breast were mainly distributed in the breast acinar or between the mammary epithelium.CD3~+T lymphocytes were mostly located at the base of the breast.Next,we successfully constructed a breast epithelial barrier model using breast epithelial cells.In the co-culture model of breast epithelial cells(Ep H4-Ev cells)/T cells,cell counts and immunofluorescence assays showed that prolactin could significantly promote the migration of lymphocytes across the mammary epithelium.To further investigate the migration mechanism,we demonstrated the expression of CCL28 in mammary epithelial cells was significantly increased in the presence of prolactin and facilitates the transmigration of lymphocytes by q PCR and ELISA.Our data indicated that prolactin promotes the migration of lymphocytes across the mammary epithelium by increasing the production of chemokines in mammary epithelial cells.3.Characterization of the small intestinal mucosa of newborn piglets and the necessity to acquire colostrum.The intestinal mucosa of newborn piglets is not well developed and can be easily attacked by pathogenic bacteria after birth.Therefore,the timely supplementation of the colostrum is particularly important for for enhancing the immunity and the survival of piglets.In this study,morphological examination of the small intestine mucosa showed that the development of immune function was significantly lower than that of weaned piglets compared with weaned piglets.Also,the expression of Claudin3 and Occludin was significantly lower in the intestine of newborn piglets compared to their weaned counterparts.Finally,q PCR testing revealed that most pattern recognition receptor genes were expressed at lower levels in both duodenum and jejunum of newborn piglets compared to weaned piglets.However,the patterns of expression in the ileum were reversed.The expression levels of IL-1βand TNF-αin the mucosa of the jejunum and the ileum of newborn piglets were lower than in weaned piglets.These data indicated that the intestinal mucosa of newborn piglets was immature and the immune function was inadequate.Our results provided preliminary data to support the further study of intestinal mucosa development in newborn piglets.