Effects Of The FcRn Cytoplasmic Tail On IgG Metabolism And Transfer In Mice

As the immune system has not been fully developed, passive acquirement of IgG from the mother is essential for protecting vulnerable neonates from infections in their early life. The neonatal Fc receptor,namely, FcRn, has been clearly shown to mediate IgG transfer across the placenta, as well as IgG absorption in the neonatal small intestine, although whether this receptor is involved in IgG transfer from maternal serum to milk remains controversial. When examining FcRn heavy chain sequences in various mammals,a surprising length variation in the cytoplasmic tail (CYT) was found to be consistently related to the method of IgG transfer that animals adopt. Specifically, mammals with FcRn a chain that has a shorter CYT in the C-terminus (10 amino acids truncated) tend to rely largely on postnatal IgG transfer through milk (cow, pig, sheep, dog, cat, etc), whereas other mammals with a longer FcRn CYT acquire maternal IgG mostly through the placenta prenatally (human, rabbit, etc) or to a lesser degree,through milk (mouse,rat,etc). Therefore, this consistent and remarkable length variation allows us to speculate that CYT sequence might be positively correlated with IgG transport processes.Firstly, I isolated MDCK cell lines after the transfection of two plasmids expressing either full-length or truncated mouse FcRn. Both types of FcRn were shown to be highly expressed in their respective clones and be able to bind IgG under mildly acidic pH. The subcellular localization of FcRn was detected by immunofluorescence, which showed that the distribution patterns of the two clones were similar. Then a Transwell system was established to compare the IgG trans-cellular capacity of the two kinds of FcRn. In vitro assay using this system clearly suggested that the FcRn CYT truncation significantly strengthened basolateral to apical and weakened apical to basolateral directed transcytosis(p = 0.037).To further address this hypothesis, I generated FcRn-Tr mice using CRISPR/Cas9 mediated homology recombination, resulting in no translation of the last 10 amino acids of FcRn heavy chain.Overall, FcRn truncation-homozygous mice were indistinguishable with the wild type mice in FcRn expressional patterns. A series of experiments were then conducted to investigate the effect of FcRn CYT truncation on IgG metabolism and transfer. ELISA assay measuring the serum IgG concentrations or antigen-specific IgG titers between FcRn-Tr and wild type mice suggested that FcRn truncation did not influence IgG metabolism in either steady or immunized state. Serum IgG concentrations in different genotypes of fetal mice (collected 1 to 2 days preparturition) were examined, and the results suggested that the FcRn truncation did not influence IgG transfer across the placenta. Then I examined the external IgG in the neonatal circulation of newborns after absorpted by the enterocytes and found that the FcRn truncation did not influence IgG transfer across the small intestine. The IgG transfer across the mammary gland in FcRn-Tr mice was not altered as shown by measuring the IgG concentrations in the serum and milk. These results suggested that the 10 amino acids truncation of FcRn CYT did not influence the passive transfer of mouse IgG.In summary, our study demonstrated that the truncation of 10 amino acids in the murine FcRn CYT conferred more apically transcytosed IgG in vitro in a Transwell assay but did not influence IgG metabolism and passive transfer in vivo in FcRn-Tr mice. These results denied the correlation of the FcRn CYT length and IgG transport process, and further suggested that the IgG transfer from maternal serum to milk may have a distinct mechanism from those utilized by IgG transfer across the placenta or small intestinal epithelial cells.