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

Expression of total and phosphorylated p38, ERK and JNK.

<p>(<b>A</b>) Specific bands of p38, p-p38, ERK, p-ERK, JNK and p-JNK of NK cells derived from adult wild-type B10 mice and <i>Tlr4</i>^−/− mice were detected. NK cells were stimulated with HMGB1 or un-stimulated. (<b>B</b>–<b>G</b>) Fold changes of total and phosphorylated p38, ERK and JNK of NK cells derived from wild-type B10 mice and <i>Tlr4</i>^−/− mice were quantified. (<b>H</b>) Specific bands of p38, p-p38, ERK, p-ERK, JNK and p-JNK of NK cells derived from adult wild-type B6 mice and <i>Tlr2</i>^−/− mice were detected. (<b>I</b>–<b>N</b>) Relative protein levels of total and phosphorylated p38, ERK and JNK of NK cells derived from wild-type B6 mice and <i>Tlr2</i>^−/− mice were quantified. *<i>p</i><0.05, **<i>p</i><0.01; N = 5 mice per group. The protein levels are normalized to internal β-Tubulin controls and 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

Synthesis and release of HMGB1 induced by RRV infection on cultured cholangiocytes.

<p>(<b>A</b>) Release of HMGB1 from the nuclei of cholangiocytes was detected after rhesus-rotavirus (RRV) infection at different time points by immunocytofluorescent staining. Representative images are showing the location relationship between HMGB1 (red) and nuclei (blue) at 0, 12, 24 and 36 hours after RRV infection. White arrows are showing the location of HMGB1 during release. The scale bar = 20 µm. (<b>B</b>) Immunocytofluorescent staining of HMGB1 in the nuclei of cultured cholangiocytes at 12, 24 and 36 hours after RRV infection. Cells in the control group were incubated with vehicle medium. The upper panels represent nuclei (blue), the middle panels represent HMGB1 staining (red), the lower panels represent overlays. The scale bar = 50 µm. (<b>C</b>) Quantification of the mRNA levels of HMGB1 in cultured cholangiocytes at 12, 24 and 36 hours after RRV infection. The values were normalized to <i>GAPDH</i>. (<b>D</b>) Concentration of released HMGB1 in the culture medium at 12, 24 and 36 hours after RRV infection. Four independent samples are tested in each group and each sample is run in triplicate. **<i>p</i><0.01. The values were 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

Expression of TLR2/TLR4 and NK cell cytotoxicity increases as mice age.

<p>(<b>A</b>) Specific bands of TLR2 of NK cells derived from <i>Tlr4</i>^−/− mice and (<b>B</b>) specific bands of TLR4 of NK cells derived from <i>Tlr2</i>^−/− mice with different ages (1 day, 7 days and 10 weeks). NK cells were stimulated by HMGB1 or un-stimulated. (<b>C</b>) The relative protein level of TLR2 of NK cells derived from <i>Tlr4</i>^−/− mice in different age groups. **<i>p</i><0.01 compared to the control group; N = 5 mice per group. The values are normalized to the expression of β-Tublin and expressed as mean ± SD. (<b>D</b>) The relative protein level of TLR2 of NK cells derived from <i>Tlr4</i>^−/− mice in different age groups. **<i>p</i><0.01 compared to the control group; N = 5 mice per group. The values are normalized to the expression of β-Tublin and expressed as mean ± SD. (<b>E</b>) Cytotoxicity is measured by percentage of cholangiocyte death. NK cells were derived from <i>Tlr2</i>^−/− mice, <i>Tlr4</i>^−/− mice and their wild-type controls. Livers of mice with different ages (1 day, 7 days and 10 weeks) were used as the source of NK cells. From the left to the right, the treatment was (1) non-RRV infected cholangiocytes + non-HMGB1 stimulated NK cells, (2) non-RRV infected cholangiocytes + non-HMGB1 stimulated NK cells, (3) RRV-infected cholangiocytes + non-HMGB1 stimulated NK cells or (4) RRV-infected cholangiocytes + HMGB1 stimulated NK cells. N = 5 mice per group. The values represent the percentages of cholangiocyte death and are 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