Hepatobiliary injury, incidence of biliary atresia and intraluminal rotavirus replication in the bile ducts.

<p>(A) Hematoxylin and eosin staining of livers. Ballooning degeneration and mononuclear cell infiltration were the basic pathologic changes in the liver of rotavirus infected mice in the NC group, but none suffered from hepatic cirrhosis. siNSP4 and siVP4 transfection decreased hepatic injury, but siVP7 transfected mice still suffered from significant hepatic injury. (B) Grading of extrahepatic bile duct injury. Grade 0: No obstruction, stenosis, necrotic epithelia or inflammatory cell infiltration. Grade 1: Mild stenosis and several inflammatory cells. Grade 2: Stenosis or obstruction caused by necrotic cells or inflammatory cells in bile duct lumens. Grade 3: Complete lumen obstruction. Black arrow pointed at the site of injury. (C) Summary of distribution of biliary injury grading. Biliary injury was significantly inhibited by siNSP4 (0/5), (<i>P</i><0.05) compared to NC (5/5). Three of siVP7 transfected mice suffered mild biliary injury. Half of siVP4 transfected mice suffered BA. (D) Measurement of inner and outer diameters of bile ducts. The dashed line and black line respectively indicated the inner and outer diameters. (E) Summary of distribution of inner/outer diameter index (I/O DI). siRNA transfected mice had relatively higher index value (all <i>P</i><0.05, compared to NC). siNSP4 had the highest I/O DI which was not significantly different from the blank controls (<i>P</i>>0.05). (F) Quantitative analysis of viral messenger RNA (mRNA) in bile ducts on 7 dpi using real-time reverse transcription polymerase chain reaction. siNSP4 decreased the level of all viral mRNAs (^*▴•<i>P</i><0.05, compared to NC). siVP7 inhibited the transcription of VP7 and VP4 mRNAs (^♦★<i>P</i><0.05, compared to NC). siVP4 only inhibited the mRNA expression of VP4 (^▪<i>P</i><0.05, compared to NC).</p

siRNA targets and sequences against NSP4, VP7 and VP4.

<p>siRNA targets and sequences against NSP4, VP7 and VP4.</p

Rotavirus replication, maturity and cytopathic effect of cultured extrahepatic biliary epithelia (EHBE).

<p>(A) Cytopathic effect (CPE) grading of cultured EHBE. The severity of EHBE injury was graded from 0 to 4 with 4 being the most severe. (B) Ultrastructural CPE. siNSP4 transfected EHBE had normal structure. The other 2 siRNAs also protected the shape of EHBE, but nuclear degeneration was noted (pointed by black arrows). (C) Rotavirus particles. Significantly less double-layered particles (DLPs, pointed by black arrows) and triple-layered particles (TLPs, pointed by black arrow heads with dash lines) existed in siNSP4 protected EHBE. siVP7 caused more synthesis of DLPs. (D) Viral particle quantification. TLPs in EHBE significantly reduced in all siRNA trasfected groups (^*•♦<i>P</i><0.05, compared to NC). DLPs in siVP7 group was increased significantly (^▴<i>P</i><0.05, compared to NC). (E) Representative gel images of reverse transcription polymerase chain reaction (RT-PCR). RT-PCR revealed the presence of 3 viral messenger RNAs (mRNAs) in NC group, but siNSP4 transfection reduced the level of NSP4 mRNA and completely inhibited the transcription of VP7 and VP4 mRNA. siVP7 caused absence of VP4 and VP7 mRNA. siVP4 reduced mRNA transcription. (F) Quantitative analysis using Gel Pro Analyzer. siNSP4 decreased the relative level of all viral mRNAs (^*▴•<i>P</i><0.05, compared to NC). siVP7 inhibited the transcription of VP7 and VP4 mRNAs (^♦★<i>P</i><0.05, compared to NC). siVP4 only inhibited the mRNA expression of VP4 (^▪<i>P</i><0.05, compared to NC).</p

Mean CPE percentage of different groups.

a<p>Blank Control, cells in blank control group that were not infected with rotavirus but transfected with siRNA Lamin A/C.</p><p>b Negative Control, cells in negative control were infected with rotavirus and transfected with siRNA Lamin A/C.</p>c, d, e<p>Cells in these groups were transfected with corresponding siRNAs.</p

Immunofluorescent assay for viral protein expression in biliary epithelia of extrahepatic bile ducts on 7 dpi.

<p>Extrahepatic bile ducts infected by rotavirus expressed cytoplasmic viral proteins in the epithelial layer. siNSP4 transfection decreased the expression of NSP4, VP7 and VP4. In serial sections, NSP4 was positive but VP4 and VP7 were both negative in siVP7 transfected mice. siVP4 transfected mice had positive expression of VP7 and NSP4.</p

HMGB1-Promoted and TLR2/4-Dependent NK Cell Maturation and Activation Take Part in Rotavirus-Induced Murine Biliary Atresia

<div><p>Recent studies show that NK cells play important roles in murine biliary atresia (BA), and a temporary immunological gap exists in this disease. In this study, we found high-mobility group box-1 (HMGB1) and TLRs were overexpressed in human and rotavirus-induced murine BA. The overexpressed HMGB1 released from the nuclei of rotavirus-infected cholangiocytes, as well as macrophages, activated hepatic NK cells via HMGB1-TLRs-MAPK signaling pathways. Immature NK cells had low cytotoxicity on rotavirus-injured cholangiocytes due to low expression of TLRs, which caused persistent rotavirus infection in bile ducts. HMGB1 up-regulated the levels of TLRs of NK cells and promoted NK cell activation in an age-dependent fashion. As NK cells gained increasing activation as mice aged, they gained increasing cytotoxicity on rotavirus-infected cholangiocytes, which finally caused BA. Adult NK cells eliminated rotavirus-infected cholangiocytes shortly after infection, which prevented persistent rotavirus infection in bile ducts. Moreover, adoptive transfer of mature NK cells prior to rotavirus infection decreased the incidence of BA in newborn mice. Thus, the dysfunction of newborn NK cells may, in part, participate in the immunological gap in the development of rotavirus induced murine BA.</p></div

Expression of HMGB1, TLR2 and TLR4 in livers of infants with biliary atresia (BA) and in bile ducts of mice challenged with RRV.

<p>(<b>A</b>) Paraffin sections of liver tissues from infants at the time of operation for congenital dilation of the bile duct (CDB) (N = 5) or BA (N = 9) immunostained with anti-HMGB1, anti-TLR2 or anti-TLR4 antibody. Brown staining represents positive signals. The scale bar = 20 µm. (<b>B</b>) Protein levels of HMGB1, TLR2 and TLR4 detected by western blotting in liver tissues from patients with CDB or BA. The protein levels are normalized to β-actin. (<b>C</b>) mRNA levels of HMGB1, TLR2 and TLR4 detected by realtime RT-PCR. The data are normalized to <i>GAPDH</i>. (<b>D</b>) All mice were injected with 50 µl vehicle medium or 50 µl RRV supernatant intraperitoneally within 12 hours after birth and were euthanized 7 days later. Paraffin sections of livers were immunostained with anti-HMGB1, anti-TLR2 or anti-TLR4 antibody. The scale bar = 15 µm. (<b>E, F</b> and <b>G</b>) mRNA levels of HMGB1, TLR2 and TLR4 in livers were detected by realtime RT-PCR. The data are normalized to <i>GAPDH</i>. *<i>p</i><0.05, **<i>p</i><0.01; N = 7–16 mice per group. The values were expressed as mean ± SD.</p

Age affects the activation of NK cells <i>in vitro</i>.

<p>(<b>A</b>, <b>C</b> and <b>E</b>) Flow cytometric analyses of activation markers of CD69, TNF-α and IFN-γ on CD49b⁺ NK cells stimulated by HMGB1 in different age groups. All NK cells were harvested from livers of mice of various ages. NK cells were either stimulated by HMGB1 or un-stimulated. Values in the right-upper quadrant of dot plots represent percent cells positive for CD49b and activation markers (CD69, TNF-α or IFN-γ) of NK cells, and the data are shown as representative dot plots. The average percentages of activation marker positive NK cells are shown in <b>B</b>, <b>D</b> and <b>F</b>. The change of percentages of CD69⁺ (<b>G</b>), TNF-α⁺ (<b>H</b>) and IFN-γ⁺ (<b>I</b>) NK cells in 1 day old, 7 day old and adult mice were illustrated in line charts. *<i>p</i><0.05 and **<i>p</i><0.01; N = 5 mice per group. The values are expressed as mean ± SD.</p

Roles of TLR2, TLR4 and MAPK families in HMGB1-induced activation of NK cells.

<p>(<b>A</b> and <b>C</b>) Flow cytometric analyses of activation markers on <i>Tlr2</i>^−/− and <i>Tlr4</i>^−/− NK cells (CD49b⁺) stimulated by HMGB1. NK cells were derived from livers of adult <i>Tlr2</i>^−/− mice, <i>Tlr4</i>^−/− mice and their wild-type controls. All NK cells in this experiment were stimulated by HMGB1. Data are shown as representative dot plots and the values in the right-upper quadrant represent percent cells positive for CD49b and activation markers of NK cells, and the average percentages of activation marker positive NK cells are shown in <b>B</b> and <b>D</b>. (<b>E</b>) Flow cytometric analyses of activation markers on HMGB1 stimulated CD49b⁺ NK cells under blockade of p38, JNK or ERK. All NK cells were derived from adult wild-type B6 mice. Values in the right-upper quadrant represent percent cells positive for CD49b and activation markers of NK cells and the average percentages of activation-marker positive NK cells are shown in <b>F</b>. *<i>p</i><0.05, **<i>p</i><0.01; N = 5 mice per group. The values are expressed as mean ± SD.</p

Adoptive transfer of mature NK cells decreases the incidence of BA and improves survival, and the level of VP4 in cholangiocytes and the incidence of BA are decreased as the age of mice increases.

<p>(<b>A</b>) The expression of CD69, TNF-α and IFN-γ of NK cells derived from the livers of B6 mice in different age groups was detected by flow cytometry (N = 5) at 24 hours after RRV challenge and the analysis of incidence of BA (N = 11–16). (<b>B</b> and <b>C</b>) RRV challenge was performed in mice of different age groups (N = 5). The protein and mRNA levels of VP4 in the bile ducts were detected by ELISA and realtime RT-PCR at different time points. The values of protein were expressed as mean ng/mg per bile duct weight ± SD. (<b>D</b>) The mRNA levels of HMGB1 in the bile ducts (N = 5 per group) were detected by realtime RT-PCR and are normalized to <i>GAPDH</i>. (<b>E</b>) Histopathological changes of bile ducts in different age groups on different days post-infection. The arrows indicate infiltrated inflammatory cells. The arrow heads indicate injured cholangiocytes. The scale bar = 50 µm. (<b>F</b>) Transferred EGFP-NK cells (white arrows) were found in the liver of recipient newborn B6 mice infected by RRV. The scale bar = 30 µm. (<b>G</b>) The upper panels show gel images representing EGFP mRNA levels in the livers by RT-PCR. The lower panel shows quantitative analysis of EGFP mRNA by realtime RT-PCR. **<i>p</i><0.01. The data of mRNA levels are normalized to <i>β-actin</i>. (<b>H</b>) The summary of distribution of biliary injury grading. Each dot represents an individual mouse pup. The scale bar = 50 µm. (<b>I</b>) Survival analysis of mice subjected to different treatment (N = 8–16). **<i>p</i><0.01.</p