1. Volume 64, Issue 5, Pages 1147-1157 (May 2016)
IL-23 prevents IL-13-dependent tissue repair associated with Ly6Clo monocytes in Entamoeba histolytica-induced liver damage 
Jill Noll, Elena Helk, Helena Fehling, Hannah Bernin, Claudia Marggraff, Thomas Jacobs, Samuel Huber, Penelope Pelczar, Thomas Ernst, Harald Ittrich, Benjamin Otto, Hans-Willi Mittrücker, Christoph Hölscher, Frank Tacke, Iris Bruchhaus, Egbert Tannich, Hannelore Lotter 
Journal of Hepatology 
Volume 64, Issue 5, Pages (May 2016)
DOI: /j.jhep
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
Role of IL-23 in E. histolytica-induced liver damage. WT and IL-23p19−/− mice were intra-hepatically infected with E. histolytica trophozoites, and liver mRNA accumulation was determined by qPCR at indicated time points post-infection (p.i.). (A) IL-23p19; (B) IL-17; (C) CCL2 mRNA accumulation in sham treated and infected mice; (D) serum levels of CCL2; (n=3–6). (E) Representative T2-weighted MRI images of mouse liver tissue showing the size of E. histolytica-induced liver abscesses on days 3, 5, and 7p.i. and (F) calculated abscess volumes (three experiments; n=4–5). (G) mRNA accumulation of E. histolytica-specific actin and cysteine peptidase A1 in WT and IL-23p19−/− mice during the course of infection. (H) Gating strategy used to identify CD11b+Ly6Chi/lo monocytes and CD11b+Ly6G+ neutrophils by flow cytometry in the liver of WT and IL-23p19−/− mice. (I) Frequencies of the respective cell populations at the indicated time points p.i. (three independent experiments, n=3–4). Data are expressed as the mean±SEM. p values were determined using the Mann-Whitney U test (∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Lack of IL-17 and CCR2/CCL2 results in a similar disease course and monocyte recruitment pattern as those observed in IL-23p19−/− mice. (A) FACS-gating strategy to determine IL-17A+ cells in WT and IL-23p19−/− mice through the course of E. histolytica infection. (B) MRI-determined abscess volumes in IL-17A/F−/− mice and WT mice i.p.-injected with Rat IgG or anti-IL-17A. (C) Gating strategy for γδTCR+IL-17A+ cells. (D) Course of γδTCR+IL-17A+ frequencies during ALA development in the livers of WT and IL-23p19−/− mice. (E) Expression of surface markers on IL-17+ cells in infected WT and IL-23p19−/− mice compared with an unstained control. (F) CCL2 mRNA accumulation during the first 24hp.i. in the livers of WT and IL-17A/F−/− mice as determined by qPCR (n=3–6). (G) Percentage of Ly6Chi/lo monocytes within the CD11b+ liver leukocyte population in WT and IL-17A/F−/− mice, and mice depleted for IL-17, on Day 3p.i. (H) CD11b+Ly6Chi/lo monocyte frequencies in the liver of WT and CCR2−/− mice at indicated time points p.i. (B) and (E) show data from three independent experiments. Data are expressed as the mean±SEM. p values were determined using the Mann-Whitney U test (∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Definition of biological functional groups and kinetics of selected chemokines/cytokines based on mRNA accumulation in the liver of E. histolytica-infected WT and IL-23p19−/− mice. (A) Differentially expressed mRNAs in the liver of WT and IL-23p19−/− mice at 6hp.i. as determined by microarray analysis and categorized into Ingenuity’s biological functional groups. The selection of biological functions relevant to the phenotype of ALA is highlighted by annotation (n=3). (B) Kinetics of mRNA accumulation for selected chemokines and cytokines in infected WT and IL-23p19−/− mice, as determined by qPCR (n=4–5). Data are expressed as the mean±SEM. p values were determined using the Mann-Whitney U test (∗p<0.05).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Characterization of IL-13 producers by FACS analysis. Gating strategy, histograms and quantitative analysis of liver-specific (A) IL-13+ leukocytes, (B) IL-13+ monocytic cells, and (C) IL-13+ lymphocytic cells in naïve and E. histolytica-infected WT and IL-23p19−/− mice relative to the IL-13 isotype control at 12hp.i. as determined by mean fluorescence intensity (MFI). (D) Gating strategy and histograms to determine IL-13+CD11b+, IL-13+Ly6Chi, IL-13+Ly6Clo monocytes, IL-13+Ly6G+ neutrophils, IL-13+SiglecF+ eosinophils, IL-13+CD3+CD4+, and IL-13+F4/80+ macrophages in the livers of naïve and infected WT and IL-23p19−/− mice relative to the IL-13 isotype control by MFI. Quantitative MFI based analysis of (E) IL-13+CD11b+ and (F) IL-13+Ly6Clo monocytes in naïve and infected WT and IL-23p19−/− mice at 12h and 1dayp.i. (A–F) show the data from two independent experiments. Data are expressed as the mean±SEM. p values were determined using the Mann-Whitney U test (∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
NOS2, Arg1 and Ki67 expression in E. histolytica-infected WT and IL-23p19−/− mice. WT and IL-23p19−/− mice were intra-hepatically infected with E. histolytica trophozoites. Accumulation of (A) NOS2 and (B) Arg1 mRNA in the liver over time. (C) Ratio of Arg1/NOS2 mRNA accumulation in IL-23p19−/− mice compared with that in WT mice. (D) Increase in the MFI of liver-specific Arg1+Ly6Clo and (E) F4/80+Ly6Clo monocytes. Data represent the mean±SEM (n=4-5). (F) Sequential slices of paraffin-embedded liver sections from WT and IL-23p19−/− mice at Day 3p.i. were stained with a rabbit serum pool against different surface and intracellular E. histolytica molecules, H&E as well as mAbs against Ki67 (upper detail), neutrophils (7/4), macrophages (F4/80), CD11b, NOS2, and Arg1. A higher magnification of the same liver area is shown. (G) Quantification of Ki67+ cells from the abscess center (layer 3), abscess margin (layer 2), and adjacent liver tissue (layer 1). Positive cells were manually counted in representative sections (2–3) from each slide of infected livers from IL-23p19−/− (n=3) and WT (n=4) mice and analyzed using Image J software. p values were determined using the unpaired Student’s t test or the Mann-Whitney U test (∗p<0.05; ∗∗p<0.01).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

7. Fig. 6
Immuno-depletion and reconstitution of IL-13. (A) Schematic showing the treatment scheme of anti-IL-13-mAb and abscess volumes, as determined by MRI, in E. histolytica-infected IL-23p19−/− and WT mice following administration of anti-IL-13 or a goat IgG ctrl mAb (100μg/animal). (B) Schematic showing the administration of the IL-13/αIL-13 complex in WT mice and abscess volumes in mice receiving either rIL-13/anti-IL-13 (1.2μg rIL-13/6.4μg anti-IL13-mAb/animal) or goat IgG ctrl mAb (100μg/animal). Data from two independent experiments are shown and expressed as the mean±SEM. p values were determined using the Mann-Whitney U test (∗p<0.05; ∗∗∗p<0.001; ∗∗∗∗p<0.0001).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions