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. 2020 Aug;73(2):349–360. doi: 10.1016/j.jhep.2020.02.031

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© 2020 European Association for the Study of the Liver. Published by Elsevier B.V.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Fig. 5 — Injection of hAAMs reduces necrosis in APAP-ALI immunocompetent mice.

(A) hMDMs were differentiated from CD14+ cells isolated from healthy volunteer buffy coats, before incubation with hCSF-1 for 7 days. hAAMs were generated by stimulating hMDMs overnight with hCSF1, hIL-4, and hIL-13. (B) Representative flow cytometry plots demonstrating CD14-enrichment using CliniMACS® beads (C) Representative flow cytometry plots showing macrophage maturity markers (25F9, left panels; CD206, right panels) in CD14+ cells (top) and hMDMs (bottom) (D) Flow cytometry quantification of hMDMs (white), hMDMs stimulated with CSF-1 alone (light blue), and fully-stimulated hAAMs (dark blue), spots represent individual donors. (E) Panels show representative anti-FITC immunohistochemical stains in liver (left), spleen (centre), and lung (right) from APAP-ALI mice treated with PBS (top row) or hAAMs (bottom row). (F) Representative histological staining (left) and necrosis segmentation map (centre) in APAP-ALI mice with indicated treatments. Necrosis quantification in right panel (n ≥7 mice per group). (G) Percentage weight loss (left panel) and liver/body weight ratio (right panel) of APAP-ALI mice with indicated treatments (n ≥8 per group) (H). Serum injury/inflammatory markers in APAP-ALI mice receiving indicated treatments (n ≥6 per group; serum ALT activity, left panel; serum IL-6, centre panel; serum IL-10/-12p70 ratio, right panel, some sera has undetectable IL-12p70 levels). (I) Representative dual immunofluorescence images of HNF4α (yellow) and BrdU (magenta) against DAPI (counterstain, blue) in liver tissue from APAP-ALI mice treated with PBS (top row) or hAAMs (bottom row). White arrowheads indicate BrdU-positive cells, red arrowheads indicate dual-positive BrdU-positive HNF4α-positive cells (quantification right panel; n≥ 8). Scale bars 100 μm, unless otherwise indicated. p values indicated in panels, n.s. not significant. One-way ANOVA test in F, G (weight loss %), H (ALT), or Kruskal-Wallis test in G (liver/body ratio), H (IL-6, IL-10/-12 ratio), and I. ALI, acute liver injury; APAP, acetaminophen; APAP-ALI, APAP-induced ALI; hAAMs, alternatively activated macrophages; hMDMs, human monocyte-derived macrophages.