Gallbladder wall abnormality in biliary atresia of mouse Sox17+/- neonates and human infants

Abstract

Biliary atresia (BA) is characterized by the inflammation and obstruction of the extrahepatic bile ducts (EHBDs) in newborn infants. SOX17 is a master regulator of fetal EHBD formation. In mouse Sox17^+/- BA models, SOX17 reduction causes cell-autonomous epithelial shedding together with the ectopic appearance of SOX9-positive cystic duct-like epithelia in the gallbladder walls, resulting in BA-like symptoms during the perinatal period. However, the similarities with human BA gallbladders are still unclear. In the present study, we conducted phenotypic analysis of Sox17^+/- BA neonate mice, in order to compare with the gallbladder wall phenotype of human BA infants. The most characteristic phenotype of the Sox17^+/- BA gallbladders is the ectopic appearance of SOX9-positive peribiliary glands (PBGs), so-called pseudopyloric glands (PPGs). Next, we examined SOX17/SOX9 expression profiles of human gallbladders in 13 BA infants. Among them, five BA cases showed a loss or drastic reduction of SOX17-positive signals throughout the whole region of gallbladder epithelia (SOX17-low group). Even in the remaining eight gallbladders (SOX17-high group), the epithelial cells near the decidual sites were frequently reduced in the SOX17-positive signal intensity. Most interestingly, the most characteristic phenotype of human BA gallbladders is the increased density of PBG/PPG-like glands in the gallbladder body, especially near the epithelial decidual site, indicating that PBG/PPG formation is a common phenotype between human BA and mouse Sox17^+/- BA gallbladders. These findings provide the first evidence of the potential contribution of SOX17 reduction and PBG/PPG formation to the early pathogenesis of human BA gallbladders.This article has an associated First Person interview with the joint first authors of the paper.

Keywords: Biliary atresia; Cholecystitis; Human; Mouse; PBG; PPG; Peribiliary gland; Pseudopyloric gland; SOX17.

Fig. 1.

Increased density of PBG-like glands in mouse Sox17^+/− gallbladders. (A) DBA-stained EHBDs of wild-type neonates (from 18.5 dpc to 7 dpp). The lower panels display higher magnification views of the cystic duct (boxed region) in the upper panels. The arrowheads indicate the first sign of PBG formation in the DBA-positive cystic duct wall at 0 dpp. (B,C) Confocal microscope images of whole-mount DBA/SOX9 double-stained EHBD samples of cystic duct (B) and gallbladder (C) from Sox17^+/− (left) and wild-type (right) mice at 3 and 7 dpp. Dotted white boxes indicate areas shown at higher magnification in the upper right insets; lower right insets show the SOX9 expression patterns in these regions. Arrowheads indicate SOX9-positive cells. Asterisks in the luminal space of the Sox17^+/− bile ducts (B′,B‴,C′) indicate the aberrant accumulation of DBA-positive cell debris. (D) H&E, PAS and Alcian blue staining of the sagittal EHBD sections including gallbladder (gb) and cystic duct (cd) in mouse Sox17^+/− and wild-type littermates at 21 dpp. The PBG-like glands (also known as PPG) are indicated by arrowheads. Higher magnification is shown in the lower insets. (E) Morphometric analyses using whole-mount DBA-stained samples (box plots), showing PBG/PPG count in the cystic duct (upper) and gallbladder (lower) of Sox17^+/− (white bar) and wild-type (black bar) littermates. PBG/PPG density in the gallbladder was significantly higher in Sox17^+/− pups than in wild-type littermates. The upper number in each box is the sample number. The horizontal line within each box is the median value (50th percentile), box indicates first to third interquartile ranges and whiskers indicate the highest/lowest values. Each PBG/PPG density in Sox17^+/− pups versus that in wild-type pups (mean±s.e.m.) are as follows: 1.4±0.2 versus 1.9±0.1 at 3 dpp, 1.6±0.3 versus 1.6±0.4 at 7 dpp in the cystic duct region; 0.6±0.2 versus 0.1±0.0 at 3 dpp, 0.5±0.1 versus 0.2±0.1 at 7 dpp in the gallbladder region. *P<0.05, Student's two-tailed t-test. a.cy, cystic artery; cd, cystic duct; gb, gallbladder; LML, left medial lobe of liver; Ms, mouse; hd, hepatic duct; RML, right medial lobe of liver. Scale bars: 100 µm.

Fig. 2.

The expression profiles of SOX17 and SOX9 in human non-BA and control (cont) gallbladder walls. (A,B) Lower-magnification images (Alcian blue staining) of the gallbladder walls (gallbladder body) in non-BA and control patients (14-day∼7-year old). (C-E) Anti-SOX17 (upper) and SOX9 (lower) immunostaining of two serial sections of the fundus, body and neck regions of the gallbladders in human non-BA (C) and control (D,E) infants, in addition to the SOX17 and SOX9 expression profiles in the gallbladder body of three control patients at 4 months, 3 years and 7 years old (D). Insets show higher-magnification images of SOX17/SOX9-positive epithelial cells. Hm, human. Scale bars: 100 µm.

Fig. 3.

The expression profiles of SOX17 and SOX9 in human BA gallbladder walls. (A) Anti-SOX17 (upper) and anti-SOX9 (lower) immunostaining of two serial sections of three BA gallbladders [gallbladder body; two-SOX17-high (BA-high) and one SOX17-low (BA-low)]. Insets show higher-magnification images of the gallbladder epithelia in each sample. A non-BA sample (non-BA#6) is also shown. Insets labeled ‘bv’ display the SOX17-positive endothelial cells observed in the same stained section. Arrowheads show PBG/PPG-like glands. (B) Morphometric analyses using the SOX17/SOX9-stained sections (box plots), displaying the SOX17/SOX9 indices (left) and relative numbers of SOX17-positive (center) or SOX9-positive (right) epithelial cells in the gallbladder body of each group. The horizontal line within each box is the median value, box indicates first to third interquartile ranges and whiskers indicate the highest/lowest values. *P<0.05, Student's two-tailed unpaired t-test. bv, blood vessel; cont, control. Scale bars: 100 µm.

Fig. 4.

Appearance of PBG/PPG structures, especially near the decidual sites, in human BA gallbladders. (A,B) Alcian blue and anti-SOX17 staining of gallbladders from SOX17-low (BA#2; A) and SOX17-high (BA#13; B) groups, showing the epithelial decidual sites (dashed lines) and PBG/PPG-like glands (arrowheads) of human BA gallbladders. Insets show higher-magnification images of the SOX17-negative epithelial site or glands, indicated by a white or black arrow, respectively. In the lower inset of A, the SOX9-stained image of the same gland is also shown. (C) Alcian blue and anti-SOX9 staining, showing PBG/PPG-like glands (arrowheads) in the gallbladder walls (BA#11 in SOX17-high). Images of non-BA (non-BA#2) and control (cont#6) gallbladder walls are also shown. (D) Morphometric analyses (box plots) showing a significant increase in the abundance of PBG/PPG-like glandular structures (per 100 µm length) in BA compared with non-BA and control gallbladders. The horizontal line within each box is the median value, box indicates first to third interquartile ranges and whiskers indicate the highest/lowest values. *P<0.05, Student's two-tailed, unpaired t-test. (E) Schematic showing the gallbladder wall phenotypes shared between human BA patients and mouse Sox17^+/− neonates (red) (Uemura et al., 2013; Higashiyama et al., 2017). The potential causes and progression of human gallbladder pathogenesis (Bezerra et al., 2018) are also indicated (gray). gb, gallbladder; Hm, human; Ms, mouse. Scale bars: 100 µm.