Fig 1.
Impact of 0.25x106 ffu (low-dose) RRV on disease phenotype and outcome.
(A) Weight gain, jaundice and survival following different doses of RRV in newborn mice shows that 48% of mice that received the low dose survived through day of life 21 (N = 12–25 per group). (B) Sections of extrahepatic bile ducts (top panels) from low-dose challenged mice show an inflammatory obstruction of the duct lumen on day 7 with subsequent clearance by day 14 and persistent focal epithelial and submucosal injury on day 21. Accompanying liver sections (middle and lower panels) show persistent periportal inflammatory infiltrate from days 7–21, with the development of fibrosis on day 21. Tissue sections stained with H&E or Sirius Red (where noted); liver magnification x40 for day 7 and 14 H&E livers, and x20 for day 21 H&E and Sirius Red; N = 4–5 per group at each time point.
Fig 2.
Hepatic inflammation after low-dose RRV.
Flow-cytometry based quantification of hepatic mononuclear cells shows similar high populations of CD8+ T- and NK cells (A and B) and over-expression of Ifnγ, Cxcl9 and Cxcl10 (C) between low-dose (0.25x106 ffu) and high-dose (1.5x106 ffu) RRV (* = P<0.05, ns = not significant, N = 3–9 per group).
Table 1.
Efficacy of NK cell depletion following RRV challenge.
Fig 3.
Improved outcome after NK cell depletion in low-dose RRV mice.
Depletion of NK cells after the development jaundice (day 6) results in improved growth, earlier resolution of phenotypic jaundice and improved mortality (Kaplan-Meier survival analysis, N = 14–30 per group) (A), lower plasma ALT and total bilirubin (B), and decreased portal inflammation/expansion (C). Panel D contains a dot plot representation of the relative surface area (left graph) of cytokeratin-positive stained portal profiles (representative immunostaining panels on the right) showing a decrease in cholangiocyte profiles in mice undergoing NK cell depletion where each dot represents an individual portal triad (cholangiocytes; green; N = 3–4 mice per group).
Fig 4.
Gene expression, histology and fibrosis after NK cell depletion.
Depletion of NK cells after the development of jaundice (day 6) results in significantly decreased hepatic markers of (A) NK cell cytotoxicity and inflammation (Klrk1/Nkg2d, Klrc1/Nkg2a, Klrd1/CD94, Ncr1/Nkp46, FasL, and Tnfa) and (B) fibrosis (Col1a1, Timp1, Sma, Mmp8, and Mmp9) on days 7, 14 and 21 following low-dose injection. * = P<0.05, ** = P<0.001 and *** = P≤0.0001 (A and B). Panel C contains corresponding liver histology demonstrating persistent periportal expansion and inflammation (arrow heads on H&E) and the development of fibrosis (Sirius Red) on days 7, 14 and 21 that are suppressed by the depletion of NK cells.
Fig 5.
Molecular signature of extrahepatic bile ducts.
Condition tree of the 82 genes, showing the level of expression 14 days after RRV or normal saline (NS) control. Levels of expression are depicted as a range in color, in which red represents up-regulation, blue represents down-regulation, and yellow represents baseline levels.
Fig 6.
Expression and biological relatedness of genes differentially expressed in extrahepatic bile ducts on day 14 following RRV challenge.
(A) A functional enrichment analysis by CIMminer classifies 82 genes into 3 functional categories: inflammation/immunity (blue), metabolic [24] and other (pink). The red areas in the heatmap indicate closely related biological processes that are shared by the subgroup of genes shown on the horizontal axis. (B) Ranking of the top 24 inflammation/immunity based on fold change compared to normal saline controls.
Fig 7.
Suppression of hepatic gene expression by NK cell depletion.
Decreased mRNA expression of Igj, Ltf, Mmp7, LCcn2 and S100a9 following intraperitoneal injections of 30 μL of rabbit anti-asialo GM1 antiserum beginning on day 6 following low-dose RRV infection (N = 3–4 per group).