Effects Of Transgenic Cow’s Milk Containing Recombinant Human Lactoferrin On Immune Responses And Bone Formation In Piglets

Lactoferrin (LF), an80kDa iron binding glycoprotein, is a member of the transferrin family. It has many biological effects such as immune-modulating, enhancement of iron transport and absorption, promotion of balanced intestinal flora, bone formation, broad-spectrum antibacterial, antioxidant, anti-inflammatory, antiviral, and anticancer properties etc. As a functional ingredient in some fields such as food and medicine, LF has wide applying and developing prospects. LF is found in neutrophilic granulocytes and epithelial cells of the exocrine glands such as colostrum and milk. Compared with human milk (1-5mg/mL), infant formulas provide little LF (0.02-0.2mg/mL), suggesting a special role of LF. Our laboratory has produced transgenic cows harboring a BAC of hLF gene, which secrete rhLF at the concentration of3.4g/L in the milk. The aim of the present study was to evaluate the effect of rhLF milk supplementation on immune responses and bone metabolism in vitro and in vivo. We chose neonatal piglets, a well-established animal model for studying infant nutrition, for this study.Eighteen Landrace piglets at7days of age were fed ordinary milk (OM),1:1mix of ordinary and rhLF milk (MM), or rhLF milk (LFM) for30days. The incidence of diarrhea in piglets in natural condition was observed. After feeding experiment, the animals were sacrificed and blood and tissue samples were taken for analysis, including food allergies, immune responses and bone status.We found that LFM-fed significantly reduced incidence of diarrhea (P<0.05). The production of IgE and histamine were measured by enzyme-linked immunosorbent assay (ELISA). The concentration of IgE in the plasma, jejunum, ileum or colon was not significant higher than that in OM group (P>0.05). There was no significant difference in the concentration of histamine in the jejunum, ileum or colon among the three groups (P>0.05). The MM diets decreased the expression of FCER1B mRNA by76%in the ileum (P<0.05). These results showed that rhLF milk did not elicit food allergies in neonatal piglets. The concentration of total IgG in the plasma (P<0.05) and colon (P<0.01) and IgA (P<0.01) in the plasma of LFM piglets was significantly higher than that of the OM group, which suggested the humoral immunity may be enhanced. In the spleen, the production of IL-2was higher (P <0.01) in LFM piglets than that in OM piglets which biased systemic T-cell responses towards T helper (Th)1-type. However, Th2-type responses were also promoted, as evidenced by the increased secretion of IL-5(P<0.05). In the plasma, the production of IL-12in LFM piglets was significantly higher than that in OM piglets (P<0.01), showing that the Thl-type response was increased. However, production of the anti-inflammatory cytokine IL-10in LFM piglets was significantly higher than that in OM piglets (P<0.01), showing that the Th2-type response was also increased. Intestinal morphological analysis showed that villus heights and villus height:crypt depth ratio were increased and crypt depths were decreased in the ileum of LFM group piglets (P<0.01). The mRNA levels of immune-related genes were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). rhLF milk increased mRNA levels of toll-like receptor (TLR)2in the ileum and nuclear factor-κB (NF-κB) p65in the colon (P<0.05). Concentration of serum calcium, bone mineral density (BMD), bone mineral content (BMC), and bone strength of tibia were increased by20.33%,14.81%,28.57%, and38.39%, respectivly, by rhLF milk supplementation compared with the control. In addition, osteoblast cell proliferation and differentiation were significantly promoted in vitro, when the culture medium supplemented with rhLF at concentration of200and400μg/mL. In addition, we examined the underlying signal mechanism. Treatment of human osteoblast cells with rhLF rapidly induced phosphorylation of p44/p42mitogen-activated protein kinase (p44/p42MAPK, ERK1/2). In conclusion, these in vitro and in invo findings suggested that rhLF supplementation could have a beneficial effect on neonate systematic and intestinal immune responses and bone health.