Table 1.
Anthropometrics and laboratory data of twelve patients affected by NAFLD and who underwent lifestyle intervention program and did not receive DHA administration.
Table 2.
Anthropometrics, laboratory and histological data of NAFLD patients who received DHA supplementation for 18 months.
Table 3.
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).
Fig 1.
Ductular Reaction (DR) and portal fibrosis in pediatric NAFLD.
A) Immunohistochemistry for cytokeratin (CK)7 in pediatric NAFLD biopsies. DR extension is increased in definite steatohepatitis (NASH) in comparison with not-SH (NAFL). Original Magnification (OM) = 10x. B-C) Immunohistochemistry for CK7 is counterstained with Sirius Red. DR extension is associated with portal fibrosis. OM = 10x (B) and 20x (C). (D) Box-and-Whisker Plots (median, quartile ranges, minimum—maximum) regarding DR and EpCAM+ hepatocytes.
Table 4.
Macrophage number and phenotype in liver biopsies obtained from normal subjects and in pediatric patients affected by NAFLD.
Fig 2.
Macrophage subsets in pediatric NAFLD.
A) Immunohistochemistry for CD68 in pediatric NAFLD biopsies. The number of CD68+ macrophages is increased (yellow arrows) in definite steatohepatitis (NASH) in comparison with not steatohepatitis (simple steatosis: NAFL). Original Magnification (OM) = 10x. B) Immunohistochemistry for CD68 in pediatric NAFLD biopsies. The number of portal CD68+ macrophages is increased (yellow arrows) in NASH in comparison with NAFL. OM = 10x. C) Immunohistochemistry for CD68 and Cytokeratin (CK) 7 in serial section shows that portal macrophages (arrows) are spatially associated with reactive ductules (dotted red line and arrowheads). These data were confirmed by immunofluorescence for Pan-CK and CD68. OM = 20x. D) Immunohistochemistry for S100A9 in pediatric NAFLD biopsies. The number of S100A9+ macrophages is increased (yellow arrows) in NASH in comparison with NAFL. Original Magnification (OM) = 10x. E) Immunohistochemistry for CD206 in pediatric NAFLD biopsies. The number of CD206+ macrophages is reduced (yellow arrows) in definite NASH in comparison with NAFL. Original Magnification (OM) = 10x.
Table 5.
Correlations between total macrophage number and histo-pathological parameters in all pediatric NAFLD biopsies at baseline (N = 32).
Table 6.
Macrophage number and phenotype in liver biopsies obtained from pediatric patients affected by NAFLD at the baseline (T0) and at the end of DHA administration (T1).
Fig 3.
Docosahexaenoic acid (DHA) treatment modifies macrophage subsets in pediatric NAFLD.
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.
Fig 4.
Phenotype, apoptosis and wnt3a expression in macrophages are modified by docosahexaenoic acid (DHA) treatment in pediatric NAFLD.
A) Immunofluorescence for Arginase-1 and CD163 confirms the increase of macrophages (MΦs) with an anti-inflammatory phenotype after DHA treatment (T1) in comparison with baseline biopsies (T0). Original Magnification (OM) = 20x. B) Immunofluorescence for Caspase-3 and CD68 shows the increased macrophage apoptosis at T1. OM = 20x. C) Immunohistochemistry for Wnt3a in pediatric NAFLD biopsies. The number of macrophages expressing Wnt3a is increased after DHA treatment (arrows) in comparison with baseline biopsies. OM = 10x. D) Immunofluorescence for Wnt3a and CD68 in biopsies after DHA confirms the Wnt3a expression in MΦs (arrows). OM = 10x.
Table 7.
Correlations between modifications of macrophage phenotype, histo-pathological features, ductular reaction and serum inflammatory cytokine levels after DHA treatment.
Table 8.
Correlations between modifications of macrophage phenotype, histo-pathological features, ductular reaction and serum inflammatory cytokine levels after DHA treatment.
Fig 5.
Modification of phosphorylated (p) β-catenin expression in ductular reaction and associated hepatic progenitor cells (HPCs) in pediatric NAFLD after docosahexaenoic acid (DHA) treatment.
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.