No abstract

Intestinal mucus protects epithelial and immune cells from the gut resident microorganisms, and provides growth-promoting factors as mucus-derived O-glycans for beneficial bacteria. A lack of intestinal protective mucus results in changes in the commensal microflora composition, mucosal immune system reprogramming, and inflammation. Previous work has shown that fucose, the terminal glycan chain component of the intestinal glycoprotein Mucin2, and fucoidan polysaccharides have an anti-inflammatory effect in some mouse models of colitis. This study evaluates the effect of fucose on reproductive performance in heterozygous mutant Muc2 female mice. We found that even though Muc2+/− females are physiologically indistinguishable from C57Bl/6 mice, they have a significantly reduced reproductive performance upon dietary fucose supplementation. Metagenomic analysis reveals that the otherwise healthy wild-type siblings of Muc2−/− animals have reduced numbers of some of the intestinal commensal bacterial species, compared to C57BL/6 mice. We propose that the changes in beneficial microflora affect the immune status in Muc2+/− mice, which causes implantation impairment. In accordance with this hypothesis, we find that macrophage polarization during pregnancy is impaired in Muc2+/− females upon addition of fucose. Metabolic profiling of peritoneal macrophages from Muc2+/− females reveals their predisposition towards anaerobic glycolysis in favor of oxidative phosphorylation, compared to C57BL/6-derived cells. In vitro experiments on phagocytosis activity and mitochondrial respiration suggest that fucose affects oxidative phosphorylation in a genotype-specific manner, which might interfere with implantation depending on the initial status of macrophages. This hypothesis is further confirmed in BALB/c female mice, where fucose caused pregnancy loss and opposed implantation-associated M2 macrophage polarization. Taken together, these data suggest that intestinal microflora affects host immunity and pregnancy outcome. At the same time, dietary fucose might act as a differential regulator of macrophage polarization during implantation, depending on the immune status of the host.

Fucose, the terminal glycan of the intestinal glycoprotein Mucin2, was shown to have an anti-inflammatory effect in mouse colitis models and modulate immune response due to macrophage polarization changes. In this study we evaluated the effect of 0.05% L-fucose supplementation of drinking water on immune parameters in the intestine of homozygous mutant Muc2−/−, compared to Muc2+/+ mice. To get into innate and adaptive immunity mechanisms of gut inflammation, we tested PrkdcSCIDMuc2−/− strain, Muc2 knockout on SCID background, that is characterized by lack of lymphocytes, in comparison with PrkdcSCID mice. We evaluated intestinal cytokine profiling, macrophage and eosinophil infiltration, and expression of Nos2 and Arg1 markers of macrophage activation in all strains. Markers of Th1, Treg and Th17 cells (Tbx21, Foxp3, and Rorc expression) were evaluated in Muc2−/− and Muc2+/+ mice. Both Muc2−/− and PrkdcSCIDMuc2−/− mice demonstrated increased numbers of macrophages, eosinophils, elevated levels of TNFa, GM-CSF, and IL-10 cytokines. In Muc2−/− mice we observed a wide range of pro-inflammatory cytokines elevated, such as IFN-gamma, IL-1b, IL-12p70, IL-6, M-CSF, G-CSF, IL-17, MCP-1, RANTES, MIP1b, MIP2. Muc2−/− mice demonstrated increase of Nos2, Tbx21 and Foxp3 genes mRNA, while in PrkdcSCIDMuc2−/− mice Arg1 expression was increased. We found that in Muc2−/− mice L-fucose reduced macrophage infiltration and IL-1a, TNFa, IFNgamma, IL-6, MCP-1, RANTES, MIP1b levels, decreased Nos2 expression, and induced the expression of Treg marker Foxp3 gene. On the contrary, in PrkdcSCIDMuc2−/− mice L-fucose had no effect on macrophage and eosinophil numbers, but increased TNFa, GM-CSF, IL-12p70, IL-6, IL-15, IL-10, MCP1, G-CSF, IL-3 levels and Nos2 gene expression, and decreased Arg1 gene expression. We demonstrated that anti-inflammatory effect of L-fucose observed in Muc2−/− mice is not reproduced in PrkdcSCIDMuc2−/−, which lack lymphocytes. We conclude that activation of Treg cells is a key event that leads to resolution of inflammation upon L-fucose supplementation in Muc2−/− mice.

Genome editing of farm animals has undeniable practical applications. It helps to improve production traits, enhances the economic value of livestock, and increases disease resistance. Gene-modified animals are also used for biomedical research and drug production and demonstrate the potential to be used as xenograft donors for humans. The recent discovery of site-specific nucleases that allow precision genome editing of a single-cell embryo (or embryonic stem cells) and the development of new embryological delivery manipulations have revolutionized the transgenesis field. These relatively new approaches have already proven to be efficient and reliable for genome engineering and have wide potential for use in agriculture. A number of advanced methodologies have been tested in laboratory models and might be considered for application in livestock animals. At the same time, these methods must meet the requirements of safety, efficiency and availability of their application for a wide range of farm animals. This review aims at covering a brief history of livestock animal genome engineering and outlines possible future directions to design optimal and cost-effective tools for transgenesis in farm species.