Bovine FcRn-Mediated Human Immunoglobulin G Transfer across the Milk-Blood Barrier in Transgenic Mice

<div><p>Maternal-fetal IgGs transport occurs either prenatally or postnatally, which confers the newborns with passive immunity before their own immune system has matured. However, little is known about the mechanisms of postnatal IgGs passage in the mammary gland. To investigate how FcRn mediates the IgGs transport in the mammary gland, we first generated bFcRn and anti-HAV mAb transgenic mice, and then obtained HF transgenic mice expressing both transgenes by mating the above two strains. Transgene expression of bFcRn in the four lines was determined by qRT-PCR and western blot. We then localized the expression of bFcRn to the acinar epithelial cells in the mammary gland, and anti-HAV mAb was mainly detected in the acini with weak staining in the acinar epithelial cells. Human IgGs could be detected in both milk and serum of HF transgenic mice by western blot and ELISA. A significantly lower milk to serum ratio of human IgGs in HF mice compared with that of anti-HAV mAb mice, indicating that bFcRn could transport human IgGs across the milk-blood barrier from milk to serum during lactation in HF mice. While, there were no transport of murine IgGs, IgAs, or IgMs. These results provide understandings about the mechanisms of maternal-fetal immunity transfer in the mammary gland.</p></div

bFcRn cannot transport murine IgAs and IgMs across the mammary gland.

<p>bFcRn could neither increase the concentration of IgAs and IgMs (A and C) nor transport them across the milk-blood barrier in the mammary gland (B and D). The ratio of serum/milk indicates the transfer of IgAs or IgMs from serum to milk. The data were combined from three independent experiments; the bars represent the means ± SD (n = 7–8 mice per group).</p

Mammary gland expression of the bFcRn transgenes in transgenic mice.

<p>(A) <i>bFcRn α</i> mRNA levels in the mammary gland of transgenic mice. The expression levels were determined by the expression related to <i>GAPDH</i> (an internal control). The data were combined from three independent experiments; the bars represent the means ± SD (n = 7–8 mice per group); Neg, wild-type mice as a control; <i>**P</i><0.01. (B) Western blot of bFcRn in the four lines of transgenic mice. HRP-conjugated K6 antibody against a polypeptide (AGLAQPLTVE) representing amino acids 279–288 was used. Tubulin was used as a loading control. Neg-41, protein from a wild-type mouse as a control; P, bovine mammary gland. (C) Immunofluorescent analysis of the mammary gland during lactation. Tissue sections prepared from the mammary gland were stained with the FITC-conjugated anti-bFcRn α antibody (K6) (Green). DAPI (blue) indicated nuclear staining. The data are representative of at least three sections. Images were obtained with a 40X water objective lens.</p

ELISA analysis of human IgGs in the mammary gland of transgenic mice.

<p>Human IgGs were detected in the serum of HF mice (A–B) and the milk/serum ratio was significant lower in HF transgenic mice (C–D). The milk/serum ratio is used as an index for the transfer of human IgGs from milk to serum in the mammary gland. The concentrations of murine IgGs (E–F) and the ratio of serum/milk (G–H) indicated that there was no transfer of murine IgGs by bFcRn. The ratio of serum/milk is used as an index for the transfer of murine IgGs from serum to milk. D3-serum, serum obtained on day 3 of lactation; C, colostrum collected on day 3 of lactation; D10-serum, serum obtained on day 10 of lactation; M, milk collected on day 10 of lactation. The data were combined from three independent experiments; the bars represent the means ± SD (n = 7–8 mice per group); <i>*P</i><0.05; <i>**P</i><0.01.</p

Analysis of the distribution of human IgGs in anti-HAV mAb and HF transgenic mice.

<p>Human IgGs distribution in the anti-HAV mAb transgenic mice (A) and co-localization of bFcRn and human IgGs in the HF transgenic mice (B). Tissue sections prepared from the mammary gland of anti-HAV mAb mice were incubated with Cy3-conjugated (red) goat anti-human IgG antibody. For the HF mice, the sections were hybridized with a combination of Cy3-conjugated (red) goat anti-human IgG antibody and FITC-conjugated anti-bFcRn α antibody (K6). The nucleus was stained with DAPI (blue). The data obtained are representative of at least three sections. Images were obtained with a 40X water objective lens in (A) and a 100X oil objective lens in (B). (C) Human IgGs were transferred from milk to serum in the HF transgenic mice. Milk and serum (day3) were performed western blot using goat anti-human IgG antibody in four different genotypes of mice from line-1. S, serum; M, milk; P, positive control, human IgG.</p

Generation of the bFcRn and anti-HAV mAb transgenic mice.

<p>(A–B) Schematic representation of the bFcRn and anti-HAV mAb constructs for microinjection. Chicken β-globin insulator (2x), un-translated exons E1, E8, E9, parts of E2 and E7, and also β-casein 3′ genomic DNA are indicated by bars. Goat β-casein promoter is indicated by solid arrow. The restriction sites of <i>Sal</i>I, <i>Xho</i>I, <i>Not</i>I, <i>Eco</i>RI, <i>Noc</i>I are shown. The hatched boxes represent insertion of sequences of <i>bFcRn α, bFcRn β, human IgG HC,</i> and <i>human IgG LC</i> to <i>Xho</i>I restriction site, respectively. Double arrows indicate the probes used for the southern blot for <i>bFcRn</i> and <i>anti-HAV mAb</i> transgenes. Arrows in (A) represent the quantitative real-time PCR primers used for the detection of bFcRn α. (C–D) Identify transgenic founders using PCR. M, 1 kb DNA ladder in (C) or 100 bp DNA ladder in (D); P1, P2, positive plasmids control for <i>bFcRn α</i>, <i>bFcRn β,</i> respectively; P, positive plasmids control for <i>human IgG HC</i> or <i>human IgG LC</i>; Neg, genomic DNA from wild-type mice as a negative control. (E–F) Southern blot analysis of transgenes. P10, plasmid control of ten copies of <i>bFcRn α</i> or <i>bFcRn β</i>; P1, P5, P10, plasmids control equivalents of 1, 5, 10 copies of <i>human IgG HC</i> or <i>human IgG LC</i>; Neg, genomic DNA from an age matched wild-type mouse as a negative control.</p

Susceptibility analysis of PRRSV receptor transgenic cell lines.

<p>(A) Quantitative RT-PCR (qPCR) analysis of pCD163, pCD169, and sCD151 mRNA expression in transgenic BHK-21 cells: the respective expressions of the target genes were calculated and normalized to GAPDH using the 2^-Δct method. (B) qPCR analysis of pCD163, pCD169, and sCD151 mRNA expression in transgenic BHK-21 cells at the 5^th,15^th, and 25^th passage. Cells were collected and total RNA was extracted, reverse transcribed, and quantitated by qPCR. (C) Immunofluorescence assay (IFA) analysis of PRRSV N protein expression in transgenic BHK-21 cells infected with PRRSV. Transgenic cells were infected with PRRSV CH-1a or JXwn06 (MOI = 1), and expression of the N protein was examined at 36 hpi using the monoclonal antibody SDOW17 and a secondary antibody conjugated to Alexa Fluor 594. Bar = 200 μm. (D) qPCR analysis of viral ORF7 RNA expression in BHK-21 transgenic cells at 12 hpi and 24 hpi. The four transgenic BHK-21 cells were infected with PRRSV CH-1a or JXwn06 (MOI = 0.5) and viral ORF7 in the cells was analyzed by qPCR at 12 hpi and 24 hpi. (E) The supernatant containing PRRSV RNA was analyzed based on absolute quantitative RT-PCR at the indicated time points. Transgenic cells were infected with PRRSV CH-1a or JXwn06 (MOI = 0.5), and the supernatant was collected and used for RNA extraction and absolute qPCR of the virions at 12 hpi and 24 hpi. The data were representative of the results of three independent experiments (mean ± SD). Statistical significances were analyzed using Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001; NS, not significant.</p

Comparison of PRRSV infection efficiency between BHK-21-TTG and MARC-145 cells.

<p>(A) Schematic of the experimental design for the detection of PRRSV infection in different transgenic BHK-21 cells at the indicated time points. The cells were incubated with CH-1a or JXwn06 (MOI = 1) for 2 h, and medium containing virus was replaced with fresh medium at 2 hpi after washing the cells. The viruses were then detected using the indicated methods. (B) Viral ORF7 RNA of the two PRRSV strains, CH-1a and JXwn06, was determined at the indicated times by qPCR. Three wells of each cell line at each time point were used. Data were presented as means ± SD of at least three independent experiments. (C) Representative IFA of the PRRSV N protein at 36 hpi. BHK-21, BHK-21-TTG, and MARC-145 cells were inoculated with JXwno6 or CH-1a (MOI = 1). PRRSV was detected using the monoclonal anti-N protein antibody (SDOW17) followed by a secondary antibody conjugated to Alexa Fluor 594. BHK-21 cells were used as the negative control. (Bar = 200 μm). (D) Supernatant containing PRRSV RNA was analyzed by absolute quantitative RT-PCR at the indicated time points. (E) IFA of the PRRSV N protein in MARC-145 cells after being inoculated with supernatant from BHK-21, MARC-145, and BHK-21-TTG cells. The supernatant (BHK-21-S, MARC-145-S, and BHK-21-TTG-S) of infected cells was collected at 36 hpi, and inoculated into MARC-145 cells by 100 μL/well plus 100 μL medium in 48 well plates, after which IFAs for N protein in MARC-145 were performed at 36 hpi (Bar = 200 μm). (F) Titer determination of the PRRSV strains in MARC-145 and BHK-21-TTG cells. MARC-145 and BHK-21-TTG cells were infected with JXwn06 PRRSV at an MOI of 1. At 48 hpi, viruses were extracted by freezing and thawing three times and the virus titers (TCID₅₀/ml) in MARC-145 cells were measured. Each data point represents the mean ± SD from three independent experiments. Statistical significances were analyzed by Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001.</p

ELISA analysis of MSTN protein with a functional C-terminal domain in cloned cattle.

<p>We also produced a healthy cloned calf with monoallelic <i>MSTN</i> mutation, which consisted of a 55-bp insertion in intron 1, and caused the premature termination of translation. This allele is represented by “55 bp inserted” in the chart, and “6 bp/117 bp deleted” represents the double-muscled cloned bovine previously discussed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095225#pone-0095225-g004" target="_blank">Figure 4</a>. The level of MSTN protein with a functional C-terminal domain was reduced by approximately 50% in both mutant calves, compared to that of the WT calf. The ELISA data were analyzed using paired Student’s <i>t</i>-tests. The error bars represent the standard deviations of three experiments (*<i>P</i><0.05 indicates a statistically significant difference compared to the WT calf).</p

Comparison of gene mutations induced using MSTN-ZFN mRNA (set 1) and plasmid DNA in single-cell colonies.

<p>*the mutant efficiency was calculated by mutant colonies/total single cell colonies.</p