GPR120 expressions by Macrophages in pediatric NAFLD patients.

<p>A) Immunohistochemistry for GPR120 in sections of pediatric NAFLD biopsies. B) Double immunofluorescence for CD68 (red) and GPR120 (green) in NAFLD biopsies. Nuclei are shown in blue. CD68-positive macrophages express GPR120 (yellow arrows). Original magnification: 40×. C) The histogram shows the number of GPR120-positive macrophages per high power field (HPF) at the baseline and after DHA treatment. A significant reduction of GPR120-positive macrophages was seen after DHA treatment. * = p<0.05.</p

Nuclear expression of phosphorylated (p) NF-κB by Macrophages in pediatric NAFLD patients.

<p>A) Immunohistochemistry for serine-311-phosphorylated NF-κB (pNF-κB) in pediatric NAFLD biopsies before and after DHA treatment. In parallel with GPR120 expression, the DHA treatment determined a reduction of pNF-κB expression and its nuclear translocation in macrophages (A, yellow arrows). Original magnification: 40×. B) Histogram shows the significant reduction of pNF-κB nuclear translocation in macrophages after DHA treatment. Data are shown as Means ± Standard Deviation. * = p<0.05. C–D) The number of GPR120-positive macrophages (C) and the pNF-κB nuclear expression in macrophages. (D) were correlated with serum level of inflammatory cytokines.</p

Immunohistochemistry for Cytokeratin(CK) 7 and EpCAM in liver biopsies of pediatric NAFLD patients.

<p>A) at the beasline, pediatric NAFLD biopsies were characterized by a prominent expansion of hepatic progenitor cell (HPC) pool and the presence of reactive ductules at the periphery of portal spaces. After DHA treatment, a minimal involvement of the HPC compartment was present. Original magnification 20×. B) EpCAM-positive HPCs (arrows) were significant reduced by DHA treatment in comparison with the biopsies at the baseline. Original magnification 40×. C) Histograms show the reduction of HPC expansion (ductular reaction extension) and activation (number of EpCAM+ cells) after DHA treatment. Data are shown as Means ± Standard Deviation. * = p<0.05.</p

Anthropometric and laboratory parameters of the study-population.

<p>Data are reported as Means ± SD * = p<0.05.</p

Nuclear expression of phosphorylated (p) NF-κB by hepatocytes in pediatric NAFLD patients.

<p>A) Immunohistochemistry for pNF-κB in pediatric NAFLD biopsies demonstrates the reduction of pNF-κB in hepatocyte nuclei (red arrows) after DHA treatment. B) DHA treatment determined a significant reduction of pNF-κB expression and its nuclear translocation in hepatocytes. Data are shown as Means ± Standard Deviation. * = p<0.05.</p

GPR120 expression by HPC and Hepatocytes in pediatric NAFLD patients.

<p>A) Immunohistochemistry for EpCAM and GPR120 in serial sections of pediatric NAFLD biopsies. CK-7-positive HPCs highly express GPR120 (arrows). B) Double immunofluorescence for EpCAM (green) and GPR120 (red) in NAFLD biopsies. Nuclei are shown in blue. Numerous EpCAM-positive progenitor cells co-express GPR120 (yellow cells: yellow arrows) confirming the findings obtained through immunohistochemistry in serial sections. C) Immunohistochemistry for GPR120 in sections of pediatric NAFLD biopsies before and after DHA treatment. A significant increase of GPR120-positive hepatocytes was clearly seen after DHA treatment. Original magnification: 40×.</p

Correlations between total macrophage number and histo-pathological parameters in all pediatric NAFLD biopsies at baseline (N = 32).

<p>Correlations between total macrophage number and histo-pathological parameters in all pediatric NAFLD biopsies at baseline (N = 32).</p

Modification of phosphorylated (p) β-catenin expression in ductular reaction and associated hepatic progenitor cells (HPCs) in pediatric NAFLD after docosahexaenoic acid (DHA) treatment.

<p>A) Immunofluorescence for phosphorylated (p) β-catenin and Cytokeratin(CK)7 in pediatric NAFLD biopsies. After DHA treatment, the number of pβ-catenin positive cells within reactive ductules is increased (yellow arrows); green arrows indicate CK7+ ductular cells not expressing pβ-catenin. Original Magnification (OM) = 20x. B) Immunohistochemistry for pβ-catenin in pediatric NAFLD biopsies after DHA treatment confirms the expression of pβ-catenin by ductular reaction (arrows). OM = 10x. C) Immunofluorescence for phosphorylated (p) β-catenin and SOX9 in pediatric NAFLD biopsies. pβ-Catenin+ cells within reactive ductules were positive for the progenitor cell marker SOX9 (arrows). OM = 40x.</p

Comparison of anthropometrics and laboratory data between NAFLD patients who did not receive DHA supplementation (T1—NAFLD) and NAFLD patients who received DHA supplementation (T1—DHA).

<p>Comparison of anthropometrics and laboratory data between NAFLD patients who did not receive DHA supplementation (T1—NAFLD) and NAFLD patients who received DHA supplementation (T1—DHA).</p

Docosahexaenoic acid (DHA) treatment modifies macrophage subsets in pediatric NAFLD.

<p>A) Immunohistochemistry for CD68 in pediatric NAFLD biopsies. The number of portal (red arrowheads) CD68+ macrophages (MΦs) is reduced after DHA treatment (T1) in comparison with baseline (T0) biopsies. No modifications in lobular CD68+ MΦ number (yellow arrows) are observed at T1. Original Magnification (OM) = 10x. B) Immunohistochemistry for S100A9 in pediatric NAFLD biopsies. The number of S100A9+ macrophages is reduced after DHA treatment (T1) in comparison with baseline (T0) biopsies. C) Double immunofluorescence for CD206 and CD68 in pediatric NAFLD biopsies. CD206+ macrophages are increased (yellow arrows) after DHA treatment (T1) in comparison with baseline (T0) biopsies. Original Magnification (OM) = 10x.</p