Genome analysis.

<p>The genetic purity of IRAK-M−/− B6 mice was analyzed with genomic DNA from IRAK-M−/− B6 mice (breeders). WT B6 mice from the Jackson Laboratory were used as controls. Genomic SNP analysis of one WT control (upper) and one IRAK-M−/− mouse (lower) is shown in the figure.</p

Altered gut permeability and composition of gut bacteria in the intestine after alcohol treatment.

<p>(A) FITC-dextran concentration in blood after gut permeability test in wild type B6 mice (blue) and IRAK-M−/− mice (red). (B) LPS content in the blood of B6 mice (open bar) and IRAK-M−/− mice (solid bar). (C) Number of culturable bacteria in the intestine before (blue) and after (red) binge alcohol treatment (ALC) in wild type B6 and IRAK-M−/− mice. (D) G⁺/G⁻ gut bacteria ratio from mouse feces tested by Q-PCR before (blue) and after (red) binge alcohol treatment in wild type B6 and IRAK-M−/− mice. Experiments were performed 3 times for A and twice for B, C and D. The data presented in A, C and D were from one of the experiments, and those shown in B were from pooled 2 experiments. n = 2–3 in each group of each experiment. Error bars represent the <i>SD</i> of samples within a group. *<i>P</i><0.05, **<i>P</i><0.01 (Student’s t-test).</p

Inflammatory cytokine in LMNCs.

<p><i>Ex vivo</i> LMNCs were stained with intracellular cytokines and different surface markers as described in <i>Materials and Methods.</i> (A) Representative FACS plots showing IFNγ⁺ cells after gating CD8⁺ T cells in alcohol treated mice. (B) Summary of percentage of IFNγ producing CD8 T cells in LMNCs of control (CTL) and alcohol treated (ALC) B6 (blue) and IRAK-M−/− mice (red). (C) Representative FACS plots showing IL-6 producing (CD11b⁺Kupffer cells after gating CD11b⁺ LMNCs in alcohol treated mice. (D) Summary of percentage of IL-6 producing CD11b⁺ Kupffer cells in LMNCs of control (CTL) and alcohol treated (ALC) B6 (blue) and IRAK-M−/− mice (red). Error bars represent the <i>SD</i> of samples within a group. Experiments were performed 4 times and n = 2−4 in each group of each experiment. The data presented are from two pooled experiments. *<i>P</i><0.05, (Two-way ANOVA test).</p

Flow cytometric analysis of immune cell composition in liver after alcohol treatment.

<p>(A) Representative histograms of liver mononuclear cells (LMNCs) in wild type B6 mice (upper panel) and IRAK-M−/− mice (lower panel). (B) Summary of immune cell composition of LMNC in wild type B6 (blue) and IRAK-M−/− mice (red) in ALC groups. (C) Summary of immune cell composition of LMNC in wild type B6 (blue) and IRAK-M−/− mice (red) in CTL groups. (D) Representative dot plots of Treg (CD4⁺Foxp3⁺) cells in total LMNCs of control (CTL, left panel) and binge alcohol treated (ALC, right panel) WT B6 (upper panel) and IRAK-M−/− mice (lower panel). (E) Summary of percentage of Treg cells in total LMNCs of CTL and ALC treated WT B6 (blue) and IRAK-M−/− B6 (red) mice. (F) Representative dot plots of Treg (CD4⁺Foxp3⁺) cells in total splenocytes of control (CTL, left panel) and alcohol treated (ALC, right panel) WT B6 (upper panel) and IRAK-M−/− mice (lower panel). (G) Summary of percentage of Treg cells in total splenocytes of CTL and ALC treated WT B6 (blue) and IRAK-M−/− B6 (red) mice. Error bars represent the <i>SD</i> of samples within a group. The experiment was performed 3 times and the data presented are from one of the 3 experiments. **<i>P</i><0.01, Two way ANOVA analysis. <i>N.S.</i> not statistically significant.</p

Phagocytic activity of Kupffer cells in liver after alcohol treatment.

<p>(A) Representative histogram of FITC-dextran intake LMNCs in wild type B6 mice (blue line) and IRAK-M−/− mice (red line, 2%). (B) FITC-dextran uptake by LMNCs in wild type B6 (blue) and IRAK-M−/− mice (red, 23%). (C) FITC-dextran uptake by CD11b⁺ Kupffer cells in wild type B6 (blue) and IRAK-M−/− mice (red). (D) FITC-dextran uptake by CD68⁺ Kupffer cells in wild type B6 (blue) and IRAK-M−/− mice (red). Experiments were performed 3 times. N = 3–4 in each group of each experiment. The data presented are from one of the 3 experiments. Error bars represent the <i>SD</i> of samples within a group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, Two way ANOVA test.</p

CD107a expression in liver isolated CD8+ T-cells.

<p>(A) In vitro CD107a expression on liver (DBD and DCD) CD8+ T-cells was analysed after a co-culture with K562 cell line. Mann-Whitney U or Spearman correlation test was used (B) Gating of liver mononuclear cells to analyse CD107a expression on CD8+ T-cells. ns = not significant, * p<0.05.</p

The dominant T-cell subset of the liver is memory CD8 T-cells.

<p>Hepatic mononuclear cells (HMC) isolated from liver perfusate of living donors (LD, n = 11) were compared to matched PBMC. (A) Dot plots of CD4 and CD8 T-cells isolated from blood, through collagenase digestion and perfusion of the same liver. (B) Analysis of the combined expression of CD3, CD4, CD8, CD45RO and CD62L; on CD4+ (left) and CD8+ (right) T-cells (Mann-Whitney U test); on (C, D) CD4 (left) and CD8 (right) CD45RA+CD62L+ naïve (C) and CD45RO+CD62L- memory (D) CD69+ T-cells (Mann-Whitney U test); (E) CD127 expression on memory CD45RO+CD62L- CD4 (left) and CD8 (right) T-cells (Student t-test). ns = non-significant, *** p<0.001, ** p<0.01.</p

Intrahepatic T-cells respond to HMGB1 <i>in vitro</i>.

<p>(A, B) Biopsies obtained from liver allografts before (left) and one-hour after (right) reperfusion. (A) Haematoxylin and eosin staining of a representative biopsy from DBD or DCD livers showing hepatocellular necrosis (plain arrow) and neutrophil aggregation (arrowhead) in post-reperfusion samples; (B) Immuno-histochemical staining for HMGB1 of the biopsies shown in (A) reveals an increased proportion of hepatocytes showing nuclear expression of HMGB1 in post-reperfusion samples compared to the pre-reperfusion counterparts but no cytoplasmic expression; (C) Production of IFN-γ from T-cells of a representative donor co-cultured with syngeneic DC at the ratio of 20:1 for 7 days. Percentage of IFN-γ-producing cells gated on CD8+CD3+ T-cells in unstimulated cultures (negative control), in presence of anti-CD3/CD28 (positive control) or in presence of HMGB1 at 100ng/ml; (D) Proliferative response of T-cells in unstimulated cultures (negative control), in presence of anti-CD3/CD28 (positive control) or in presence of HMGB1 at 100ng/ml. 1 = negative, 3 = positive control and 2 = HMGB1; (E) Quantitative analysis of IFN-γ concentration in supernatants of cultures. NT = negative control, S = aCD3/CD28 stimulation, HMGB1 = cultures stimulated in presence of 100 or 10ng/ml of HMGB1. Depiction of IFN-γ concentration in pg/ml (left) and ratio of increase reported to the negative control (right). Kruskal Wallis test was used.</p

Clinical parameters of recipients.

<p>Clinical parameters of recipients.</p

The donation status of the liver allograft influences the cytokine production in culture.

<p>Cytokines in the supernatants of day-seven HMC culture were measured. Left column: levels of IL-2 (A), TNF-α (B), IFN-γ(B), IFN-in the supernatants of day-seven HMC culture were measured. Left column: levels of IL-2 (A), TNF-ells. Numbers indicate epatocellular injury iα (B), IFN-γ(B), IFN-in the supernatants of day-seven HMC un-stimulated and stimulated HMC. Mann Whitney U or Kruskal Wallis test was performed. ns = non-significant, * p<0.05, **p<0.01, ***p<0.001.</p