MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure

Abstract

Objective: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response.

Design: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer^-/-) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice.

Results: We demonstrate a significant expansion of resolution-like MerTK+HLA-DR^high cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCII^high macrophages during the resolution phase in ALF, APAP-treated Mer^-/- mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DR^high phenotype which promotes neutrophil apoptosis and their subsequent clearance.

Conclusions: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury.

Keywords: ACUTE LIVER FAILURE; IMMUNOLOGY; INFLAMMATION; MACROPHAGES.

Figure 1

Characterisation of Mer tyrosine kinase (MerTK)+ circulating monocytes and liver-derived hepatic macrophages in patients with acute liver failure (ALF). (A) Flow cytometry analysis and gating strategy used to determine MerTK expression in circulating monocytes and their subsets. (B) Data show MerTK expression levels of (1) monocytes in patients with ALF (n=15), chronic liver disease (CLD) (n=10) and healthy controls (HC) (n=15), (2) liver-derived macrophages isolated from ALF (n=8), CLD (n=10) and normal liver (n=6) tissue. (C) Representative histograms and expression levels of monocyte surface markers in MerTK+HLA-DR± cells. (D) MerTK+HLA-DR+ and MerTK+HLA-DR- subsets as proportion (%) of circulating monocytes in HC (n=15), patients with CLD (n=10) and ALF on admission (n=15) and days 3–5 following their admission (n=8). (E) Inflammatory cytokine secretion in HC and ALF peripheral blood mononuclear cells (PBMC) supernatants (n=5 each) after microbial challenge (LPS 100 ng/mL, 6 hours), as determined by ELISA. (F) Proportion of MerTK+HLA-DR± monocytes that efferocytosed apoptotic neutrophils, phagocytosed E. coli bioparticles and produced tumour necrosis factor (TNF)-α after microbial challenge (n=5 each). Non-parametric (Mann-Whitney) statistical analysis was used. Data presented as median values with IQR. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. SSC, side scatter; FSC, forward scatter.

Figure 2

Gene expression pattern of Mer tyrosine kinase (MerTK)+ monocytes in heatlhy controls (HC) and acute liver failure (ALF). (A) Human MerTK+ and MerTK− monocytes were FACS-sorted in HC and patients with ALF (n=3 each), with a gating strategy displayed by representative flow cytometry plots. (B–E) Highly pure isolates of MerTK± subsets were subjected to quantitative microarray gene expression analysis (nCounter GX Human Immunology V2 kit, profiling 594 immunology-related human genes; NanoString Technologies, Seattle, Washington, USA). Data show the log2 fold-change of expression and agglomerative cluster (heatmap, z-score; green=min and red=max magnitude of expression) of 50 chosen differentially expressed genes, comparing MerTK+ versus MerTK− monocytes in HC (B and C), and MerTK+ monocytes in ALF versus MerTK+ monocytes in HC (D and E). *p<0.05, **p<0.01, ***p<0.001. SSC, side scatter; FSC, forward scatter.

Figure 3

Mer tyrosine kinase (MerTK)+ hepatic macrophages are expanded in human and experimental APAP-induced acute liver injury. (A) Representative confocal images for MerTK (green), CD163 (red), HLA-DR (red), DAPI (blue) and colocalisation (yellow) in pathological control (n=4) and acute liver failure (ALF) (n=6) human liver tissue (100×, inset 200×). Data show enumeration of CD163+MerTK+ and MerTK+HLA-DR+ cells in centrilobular areas of pathological control (PC, n=4) and ALF (n=6) liver. (B–E) Wild-type (WT) mice dosed with APAP were studied at 8, 24 and 48 hours, while untreated mice served as baseline controls (n=4/group). (B) Schematic of experimental dosing, representative flow cytometry analysis and gating strategy used to identify F4/80+ hepatic macrophages and determine their MerTK expression levels. (C) Ly6G+ neutrophils and F4/80+ macrophages as percentage (%) of total liver CD45+ leucocytes, as determined by flow cytometry. (D) Representative flow cytometry gating strategy used to identify (CD11b_lowF4/80^high)-resident Kupffer cells (KC) and (CD11b^highF4/80_low) monocyte-derived macrophages (MoMF) to determine their MerTK expression. Further subanalysis examined the MHC class II and Ly6C expression levels of MerTK+ macrophages in both MoMF (grey bars) and KC (black bars) subpopulations. (E) Data show MerTK+MHCII+ macrophages as proportion of total F4/80+ cells and the relative contribution of MoMF (grey) or KC (black). Non-parametric (Mann-Whitney) statistical analysis was used. Data are presented as median values with IQR. * or ^#p<0.05, **p<0.01, ****p<0.0001.

Figure 4

Mer tyrosine kinase (MerTK)-deficient mice are characterised by increased hepatic inflammation and reduced macrophages following APAP-induced liver injury. Wild-type (WT) (black bars) and Mer^−/− (grey bars) mice dosed with APAP were studied at 8, 24 and 48 hours, and untreated mice served as baseline controls (n=4/group). (A) Representative images of H&E stained liver tissue and quantification of necrotic area (%). (B) F4/80+ hepatic macrophages, Kupffer cells (KC) and monocyte-derived macrophages (MoMF) as percentage of total liver CD45+ leucocytes, as determined by flow cytometry. (C) Representative flow cytometric analysis from liver CD45+ leucocytes showing detection of (CD11b^highF4/80_low) MoMF and (CD11b_lowF4/80^high) KC. (D) Ly6G+ hepatic neutrophils as percentage of total liver CD45+ leucocytes and enumeration of MPO+ neutrophils using flow cytometry and immunohistochemistry, respectively. (E) Representative images of liver tissue stained for MPO+ (purple) cells (200×). Non-parametric (Mann-Whitney) statistical analysis was used. Data are presented as median values with IQR. * or ^#p<0.05, ***p<0.001, ****p<0.0001.

Figure 5

Secretory leucocyte protease inhibitor (SLPI) induces a Mer tyrosine kinase (MerTK)^highHLA-DR^high phenotype in monocytes and liver-derived macrophages. (A–B) Effects of recombinant human (rh)-SLPI (0 and 0.5 µg/mL) on monocyte migration across hepatic endothelium were determined (n=3 independent experiments). (A) Schematic of migration assay: CD14-isolated monocytes are added on top of a preformed hepatic endothelium monolayer; non-migrated monocytes are harvested 1.5 hours after, while subendothelial monocytes are obtained 24 hours later. Phenotypic characterisation of non-migrated and subendothelial monocytes was determined by flow cytometry. (B) Data show HLA-DR, CD163 and MerTK expression levels and representative histograms (CD14++CD16+ subset) for (top panel) non-migrated and (lower panel) subendothelial monocytes. Results expressed as mean fluorescence intensity (MFI). (C and D) Effects of (rh)-SLPI (0 and 0.5 µg/mL) on hepatic macrophages isolated from normal liver explant tissue were assessed (n=5). (C) Data show representative histograms and surface marker expression in the CD14++CD16+ subset and intracellular cytokine levels in total monocytes following microbial challenge (LPS 100 ng/mL). (D) LPS-stimulated inflammatory cytokine levels (pg/mL) in hepatic mononuclear cell culture supernatants, as determined by ELISA. Non-parametric (Mann-Whitney) statistical analysis was used. Data presented as median values with IQR. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. IFN, interferon; IL, interleukin; iso, isotype control antibody; ns, non-significant; TNF, tumour necrosis factor.

Figure 6

Secretory leucocyte protease inhibitor (SLPI) suppresses neutrophils through Mer tyrosine kinase (MerTK)^highHLA-DR^high monocytes in a paracrine manner. (A) Representative immunohistochemistry (IHC) (left) and confocal (right) micrographs and enumeration of (MPO+) hepatic neutrophils and (MPO+TUNEL+) apoptotic neutrophils in centrilobular areas of pathological control (PC, n=4) and acute liver failure (ALF) (n=6) human liver tissue. IHC images (400×) show MPO+ (purple) cells. Confocal images show MPO (green), TUNEL (red), DAPI (blue) and coexpression (yellow) (400×). (B–E) Autocrine and paracrine effects of recombinant human (rh)-SLPI (0 and 0.5 μg/mL) on neutrophils were examined (n=5). (B) Neutrophil extracellular trap formation (NETosis) was determined fluorometrically (DNA, ng/mL) in culture following stimulation with PMA (25nM) or LPS (100 ng/mL). (Left) Representative images of NETs using SYTOX Green Dye (1 μM). (Right) Data show NETosis (DNA, ng/mL) quantified in culture (1) with/without SLPI and (2) with ±SLPI-treated monocyte culture supernatants (n=3). (C) Paracrine experimental approach for neutrophils and data showing monocyte MerTK expression levels after culture with/without SLPI (48 hours). (D) Representative Annexin-V/7-AAD staining and percentage of apoptotic neutrophils in culture with ±SLPI-treated monocyte supernatants (n=5). (E) SLPI paracrine effects were assessed by blocking SLPI's activity on monocytes in ALF plasma (α-SLPI, 5 µg/mL) (n=5). (Left) Representative Annexin-V/7-AAD staining and percentage of apoptotic neutrophils; (middle) NET formation (DNA, ng/mL) and (right) LPS-stimulated (100 ng/mL) intracellular cytokine levels of neutrophils (n=5 each). Data presented as median values with IQR. *p<0.05, **p<0.01, ****p<0.0001. HC, healthy controls; IL, interleukin; ns, non-significant; TNF, tumour necrosis factor.

Figure 7

Secretory leucocyte protease inhibitor (SLPI) enhances the monocyte clearance of apoptotic neutrophils. (A–D) CD14-isolated monocytes were cultured with medium or (rh)-SLPI (0.5 μg/mL) or dexamethasone (100 nM) for 48 hours and then coincubated (4 hours) with apoptotic neutrophils (n=3 independent experiments). (A) Representative confocal microscopy images of CMTPX-labelled monocyte engulfment of apoptotic CMFDA-labelled neutrophils (original magnification ×63); merge/z-stack images: arrows showing colocalised/engulfed cells. (B) Data show Mer tyrosine kinase (MerTK) expression in monocyte subsets after culture (48 hours) with different treatments. (C and D) Representative flow cytometry plots and percentage of monocytes that phagocytosed CMFDA-labelled neutrophils. (E and F) CD14-isolated monocytes were cultured with medium or (rh)-SLPI (0.5 μg/mL) for 48 hours and then coincubated (4 hours) with apoptotic Huh-7 hepatoma cells (n=3 independent experiments). (E) (Upper) Representative confocal microscopy images of CMFDA-labelled Huh-7 cells and (lower) representative Annexin-V/7-AAD staining of Huh-7 cells treated with/without 20 μM STS (50 μm, scale bars). (F) Representative flow cytometry analysis and percentage of monocytes that phagocytosed CMFDA-labelled (STS-treated) apoptotic Huh-7 cells. Non-parametric (Mann-Whitney) statistical analysis was used. Data are expressed as median values with IQR. *p<0.05, **p<0.01, ****p<0.0001. ns, non-significant.

Figure 8

Secretory Leucocyte protease inhibitor (SLPI) administration in wild-type (WT) mice induces Mer tyrosine kinase (MerTK)+ macrophages and promotes hepatic resolution following APAP-induced acute liver injury. WT mice were dosed with SLPI (−/+) or APAP (+/−) or APAP plus SLPI (+/+) while untreated mice (−/−) served as baseline controls (n=6/group). Mice were studied at baseline (white bars), 24 hours (black bars) and 48 hours (grey bars). (A) Schematic describes the experimental dosing and representative images of H&E-stained livers (baseline and 48 hours). (B) Quantification of necrosis and plasma alanine transaminase (ALT) levels. (C) Representative flow cytometry analysis and data show MerTK expression of F4/80+ macrophages at baseline and following SLPI administration. (D) Data show MerTK expression levels of F4/80+ macrophages, subanalysed into (CD11b_lowF4/80^high)-resident Kupffer cells (KC) and (CD11b^highF4/80_low) monocyte-derived macrophages (MoMF). (E) Representative liver immunohistochemistry images at baseline and 24 hours (n=5 each) and enumeration of MPO+ (purple) hepatic neutrophils (200×). (F) Representative liver confocal micrographs at baseline and 24 hours (n=5 each) stained for MPO (green), TUNEL (red), DAPI (blue); coexpression (yellow) (400×). Data show the percentage of (MPO+TUNEL+) apoptotic neutrophils. Non-parametric (Mann-Whitney) statistical analysis was used. Data are presented as median values with IQR. * or #p<0.05, **p<0.01, **** p<0.0001. SSC, side scatter.