Morbidity and mortality from diarrheal illnesses remains a burden worldwide. While not able to prevent diarrhea, oral rehydration solution can reverse the fluid loss and dehydration responsible for a majority of the deaths due to diarrhea and remains the main treatment for this condition [29
]. More recent work has demonstrated that supplementation with micronutrients such as zinc, glutamine and alanyl-glutamine can improve outcomes [30
]. However, the mechanisms involved in these biologic effects remain poorly defined. As a potential alternative or addition to oral rehydration therapy, we report here the impact of goat milk with and without increased concentrations of the human milk antimicrobial protein lysozyme on intestinal barrier function in vitro.
We conducted several assays which demonstrated the mitigation of the effects of EAEC with both goat milk and goat milk containing increased concentrations of lysozyme. In the absence of injury, both HLZ transgenic and control goat milk stimulated cellular proliferation in IEC-6 cells compared to cells with no milk supplementation. Milk is known to be beneficial to growth and these data demonstrate that goat milk in general can be beneficial to intestinal cell proliferation. Both protein and fat are required nutrients for proper cellular functioning and it is likely that the presence of these ‘extra’ nutrients contributed to the proliferation of the intestinal cells in culture. These data also indicate that the presence of lysozyme in milk does not directly enhance proliferation in its own. It should be noted that these assays conducted with milk without fat resulted in no differences (data not shown) pointing to the presence of the fat as an important component.
When cell injury was induced by the scraping of the cell monolayer, the presence of milk did not significantly improve migration in the absence of bacteria, however, in the presence of EAEC-042 milk significantly improved cell migration with HLZ transgenic milk performing better than control goat milk. Nutrients in the milk, particularly the fat, could be acting to prevent the EAEC from associating with the intestinal cells thus allowing them to maintain migration. Goat's milk has a high content of short chain fatty acids (caprylic, capric and caproic) that are important in reversing poor dietary intake and intestinal disorders [34
] and have been found to act as antimicrobial agents [35
]. The enhanced effect by the lysozyme-containing milk from transgenic animals over non-transgenic control milk may be due to the antimicrobial activity of lysozyme. Previous work has demonstrated that there were no significant differences in the overall amount of fat in HLZ milk compared to control milk [18
] and that the protein composition of the two milks differ only in the presence of lysozyme [37
]. The HLZ milk has been shown to have antimicrobial activity against several microorganisms including E. coli
both in vitro [19
] and at the level of the intestine [20
]. However, purified lysozyme alone or added to control milk was not as efficacious an antimicrobial agent as the HLZ milk itself [19
], indicating that the in vivo-produced HLZ is more potent. It is therefore likely that lysozyme is exerting antimicrobial activity to reduce the number of EAEC present, although this could not be quantified with this assay. Upon intestinal infection, certain bacteria can adhere to the intestinal wall and cause damage to the intestinal epithelium. Thus, the maintenance of the epithelial barrier is essential for proper functioning and prevention of the entry of pathogenic bacteria leading to an inflammatory response [38
]. Both control and transgenic goat milk significantly reduced bacterial adhesion with HLZ milk offering no advantage over control milk. In this assay, cells were incubated with milk for only 1.5
hours, likely too little time for lysozyme to act in a significant antimicrobial fashion. The mechanism of pathogenesis of EAEC infection involves the adherence of EAEC-042 to the mucosal surface of the intestine via aggregative adherence fimbriae (AAF), secretion of toxins and mucosal inflammation by the induction of interleukin 8 (IL-8) release [39
]. IL-8 plays an important role in the pathogenesis of EAEC, as it recruits neutrophils to the intestinal epithelial mucosa causing destruction of the epithelium and promoting fluid secretion [40
]. This may be related to atrophy of the villi with reduced digestion and absorption of nutrients, one of the mechanisms that could explain weight loss in affected malnourished children. While HLZ milk offered no distinct advantage over control milk in this assay, previous work in vivo has demonstrated increased villi height and expression of the anti-inflammatory cytokine TGF-β upon consumption of HLZ milk indicating that the milk has a protective effect on the intestinal epithelium [21
] and combined with this data suggests a likely beneficial clinical impact on EAEC infection.
Evidence of altered intestinal barrier function in the presence of EAEC-042 was seen as there was a reduction in IEC-6 viability accompanied by an increase in apoptosis (2 times greater than control) and necrosis (5 times greater than control). When supplemented with milk from HLZ transgenic and non-transgenic control goats, these effects were mitigated. Again, the nutrients in the milk may be protecting the cells from the effects of the bacteria and enhancing their viability. Further studies are required to elucidate the mechanisms involved. Cell viability of IEC-6 cells with induced oxidative damage was increased by the addition of human colostrum, but not cow milk or infant formulas, and the increase was attributed to the polyamine spermine reducing the oxidative stress [41
]. Polyamines are small cationic molecules that are found in human milk and are required for intestinal growth and development [42
]. Lysozyme also carries a net positive charge and is a relatively small protein (14
kDa) and it is likely that properties of lysozyme other than its antimicrobial function are contributing to the effect of lysozyme-rich milk. Further work is required to dissect out the role of lysozyme on these various aspects of gut barrier function.