Characterisation of macrophages in healthy and diseased livers in mice: identification of necrotic lesion-associated macrophages

Dechun Feng; Yukun Guan; Yang Wang; Luca Maccioni; Bryan Mackowiak; Bin Gao

doi:10.1136/egastro-2025-100189

. 2025 Apr 7;3(2):e100189. doi: 10.1136/egastro-2025-100189

Characterisation of macrophages in healthy and diseased livers in mice: identification of necrotic lesion-associated macrophages

Dechun Feng ^1,^*, Yukun Guan ¹, Yang Wang ¹, Luca Maccioni ^1,², Bryan Mackowiak ¹, Bin Gao ^1,^✉

PMCID: PMC11979608 PMID: 40212045

Abstract

Background

Healthy livers contain a large number of resident macrophages named Kupffer cells (KCs), which are partially replaced by infiltrating monocyte-derived macrophages (MoMFs) during acute or chronic liver injury. Despite extensive research, understanding macrophage heterogeneity, spatial distribution and interactions with other cells within the liver remains challenging.

Methods

This study employs sequential multiplex immunofluorescence staining, advanced image analysis and single-cell RNA sequencing (scRNA-seq) analysis to characterise macrophages in both healthy and diseased livers in mice.

Results

Our data revealed that liver KCs made up more than 80% of total immune cells in healthy mouse livers, while massive amounts of MoMFs infiltrated into the livers after acute and chronic liver injury. KCs were more abundant and larger in Zones 1 and 2 compared with Zone 3 in healthy livers. Zone 1 KCs exhibited higher phagocytic activity than Zone 2/3 KCs and MoMFs. We simultaneously evaluated cell proliferation and apoptosis on one slide and found that proliferation and apoptosis of KCs and MoMFs significantly increased in acutely injured livers. We also performed scRNA-seq to investigate liver macrophage gene expression in naïve and concanavalin A (ConA)-treated mice. MoMF clusters expanded following ConA treatment, while KCs remained stable. Macrophages were divided into distinct subtypes, including C1q⁺MoMFs, with differential expression of genes like Trem2, Spp1, Fabp5 and Gpnmb. Newly recruited C1q⁻ MoMFs expressed high levels of Lyz and Ccr2, while Itgax (Cd11c)⁺ MoMFs expressed endothelin converting enzyme 1 (Ece1), a gene encoding ECE1 enzyme that activates endothelin to promote hepatic stellate cell contraction and necrotic lesion resolution. By immunostaining analysis of the proteins encoded by these signature genes, we identified several populations of MoMFs that were mainly located surrounding the necrotic lesion area and expressed various proteins that are involved in dead cell debris clearance.

Conclusion

We developed a robust framework for studying liver macrophages in vivo, offering insights into their roles in host defence and liver injury/repair. We identified several populations of MoMFs that surround necrotic lesion areas and express proteins that promote dead cell debris clearance. These necrotic lesion-associated macrophages likely play key roles in promoting necrotic lesion resolution.

Keywords: Liver Diseases, Kupffer Cells, Monocyte-derived Macrophages, Phagocytosis

WHAT IS ALREADY KNOWN ON THIS TOPIC

The liver is an immunological organ enriched with innate immune cells, particularly macrophages.
Liver macrophages play crucial roles in host defence, debris clearance and liver repair after injury.
Advances in single-cell RNA sequencing and spatial transcriptomics have identified several distinct populations of liver macrophages in both healthy and diseased livers.

WHAT THIS STUDY ADDS

Characterised the spatial distribution of Kupffer cells (KCs) and monocyte-derived macrophages (MoMFs) in both acute and chronic liver injury models.
Mapped KC and MoMF distribution and phagocytic ability in different liver zones of healthy livers.
Characterised necrotic lesion-associated macrophages in a concanavalin A-induced acute liver injury model via single-cell RNA-sequencing and immunostaining.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

This study provides a methodological framework for characterising liver macrophages in vivo.
Our findings offer insights into the role of liver macrophages in host defence and the resolution of liver damage, which may help design future research and therapeutic strategies.

Introduction

As the largest solid organ in the body, the liver consists of multiple cell types including hepatocytes, hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), bile duct cells and immune cells.¹ In addition to its critical role in metabolism, storage of nutrients and detoxification, the liver is also considered an immunological organ with predominant innate immunity.² The liver is enriched with innate immune cells, such as macrophages, natural killer (NK) cells, NKT cells and γδ T cells.^{1 2} These immune cells work in coordination with hepatocytes, HSCs and LSECs to maintain liver homeostasis, combat infections and support liver repair after acute and chronic injury.

Macrophages are the most abundant immune cells in healthy livers. Liver macrophages, which include liver resident Kupffer cells (KCs) and monocyte-derived macrophages (MoMFs), play major roles in clearing micro-organisms and cellular debris.³ KCs in particular act as the first line of defence against pathogens and pathogen-derived endotoxins from the gastrointestinal tract.⁴ They are also responsible for removing dead hepatocytes and damaged red blood cells.^{5 6} On liver injury, circulating monocytes rapidly migrate into the liver and differentiate into MoMFs. Together with KCs, MoMFs are critical for resolving liver damage and promoting liver regeneration by orchestrating multiple cell types including hepatocytes and HSCs.⁷ During infection, liver macrophages are also a key source of cytokines and chemokines that help recruit more immune cells into the liver for effective host defence against invading pathogens.^{8 9}

Although liver macrophages have been extensively studied over the past decades, many emerging questions need to be answered about liver macrophages’ function, plasticity and the interactions of macrophages with their microenvironment in both healthy and diseased livers. To effectively address these questions, the development of in vivo assays to characterise macrophages is urgently needed. The rapid advancements of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics approaches^{5 10 11} have enabled the identification of numerous subpopulations of macrophages and their potential crosstalk with neighbouring cells.^{11 12} However, validation of these findings in vivo remains a significant challenge.

In this study, we employed sequential multiplex immunofluorescence staining of cell markers together with whole-slide scanning to obtain and analyse large-scale data on cell location, size, proliferation and apoptosis with spatial context in both healthy and diseased livers in mice. Additionally, we developed methods to evaluate the phagocytic ability of liver macrophages localised in different liver zones in vivo. Furthermore, we performed scRNA-seq and bulk RNA-seq, and immunohistochemistry staining to extensively characterise and validate the phenotypes of macrophages in a model of concanavalin A (ConA)-induced acute liver injury.

Materials and methods

Animal study: C57BL/6J (Cat.No:000664) was obtained from The Jackson Laboratory. For the ConA-induced liver injury model, ConA (Sigma-Aldrich) was dissolved in phosphate-buffered saline (PBS) and injected through the tail vein at a dose of 12 mg/kg. For the acetaminophen (APAP)-induced liver injury model, APAP (Sigma-Aldrich) was dissolved in PBS and given to mice by intraperitoneal injection at a dose of 350 mg/kg. For the high-fat diet (HFD) feeding model, mice were fed on diet with 60 kcal% fat (Research Diets, D12492) for 3 months. For the metabolic dysfunction-associated steatohepatitis (MASH) model, mice were fed on Gubra-amylin (GAN) diet (Research Diets, D09100310) for 6 months. For the ConA and APAP models, both male and female mice were used. For the HFD and MASH models, male mice were used because they are more sensitive to these models.¹³ Animals were randomly assigned to each group. All animals were cared for in accordance with National Institutes of Health guidelines.

Other materials and methods used in this study can be found in online supplemental materials.

Results

Composition of cell types in healthy adult mouse livers by sequential multiplex immunostaining

To quantify the proportion of different cell types in healthy mouse liver, we used sequential multiplex immunostaining of markers for the five types of cells in the liver: Hepatocyte nuclear factor (HNF)4α for hepatocytes, CK19 for bile duct cells (BDCs), Desmin for HSCs, CD31 for LSECs and CD45 for immune cells. In addition, we included four immune cell markers: Ionized calcium-binding adaptor molecule 1 (IBA1) for macrophages, CD3 for T, NKT and mucosal-associated invariant T (MAIT) cells, CD20 for B cells and S100A9 for neutrophils. Representative staining results are shown in figure 1A,B. After whole-slide scanning, approximately 10 000 cells were segmented per sample using ilastik and analysed with CellProfiler (figure 1A,B). Our data indicate that hepatocytes account for approximately 60% of total cells in healthy mouse livers. LSECs and immune cells represent 17% and 13%, respectively, while HSCs and BDCs contribute around 3% and 1%, respectively, of the total cells (figure 1C). Among the immune cells, IBA1⁺ macrophages are the predominant cell type, accounting for>80% of immune cells in healthy mouse livers. Other immune cell types, including T, NKT, MAIT, B cells and neutrophils, collectively constitute<15% of the total immune cell population (figure 1C). Thus, macrophages comprise roughly 10% of the total cells in healthy mouse livers. Notably, we lacked a reliable marker for NK cells in our immunostaining panel, and 1.4% of unidentified immune cells include NK cells.

IBA1 and CLEC4F staining effectively distinguish resident KCs and MoMFs

Two distinct populations of macrophages, liver resident KCs and MoMFs, have been identified in both healthy and diseased livers. Recent studies highlighted differences between the two types of macrophages in various aspects, including their origin, gene expression profiles and functions.⁵ F4/80 has been widely used as a marker for KCs over the past decades. Practically, flow cytometry can separate the two populations using CD11b and F4/80 staining: KCs are characterised by high F4/80 expression and low CD11b levels, whereas MoMFs exhibit low F4/80 expression and high CD11b levels.¹⁴ However, in immunohistochemical or immunofluorescence staining, F4/80 cannot effectively distinguish between these two populations, as both KCs and MoMFs are stained positively with F4/80 (figure 2A). In recent years, we and others have used IBA1/C-Type Lectin Domain Family 4 Member F (CLEC4F) co-staining to effectively distinguish KCs from MoMFs in healthy liver and injured liver on formalin-fixed, paraffin-embedded (FFPE) sections.^{7 15 16} KCs express both IBA1 and CLEC4F while MoMFs only express IBA1 but not CLEC4F. Here, we stained healthy liver and livers from four different liver disease models with IBA1 and CLEC4F, and our data clearly revealed that healthy control mouse livers mainly contained IBA1⁺CLEC4F⁺ KCs, while IBA1⁺ CLEC4F⁻ MoMFs were markedly increased in injured livers (figure 2B).

To quantify the number of KCs and MoMFs, we extracted the spatial distribution information of IBA1⁺CLEC4F⁺ KCs and IBA1⁺CLEC4F⁻ MoMFs by using CellProfiler and plotted them in figure 3A. In healthy liver, most IBA1⁺ macrophages also express high levels of CLEC4F, indicating they are KCs, while IBA1⁺ cells adjacent to BDCs do not have CLEC4F staining, suggesting that they are MoMFs. These observations are in agreement with a previous study by Guilliams et al.¹⁰ In two models of acute liver injury induced by ConA and APAP, massive MoMFs were found in necrotic areas while KCs were predominantly located in non-damaged regions (figure 3A). Interestingly, in the ConA-induced immune-mediated liver injury model, more than 95% of macrophages in necrotic regions are MoMFs. In contrast, in the APAP model, MoMFs only account for about 60% of all macrophages and~40% of macrophages are KCs in necrotic regions (figure 3A–D). Mice with 3-month HFD feeding (HFD model) showed slightly increased percentages of both KCs and MoMFs. MoMFs were detected in inflammatory foci and crown-like structures in the HFD model. In mice fed with a 6-month GAN diet (MASH model), the number of IBA1⁺CLEC4F⁺ KCs/KC-like cells and MoMFs were significantly increased compared with naïve and HFD mice, with much more inflammatory foci and crown-like structures. Similar to the HFD model, MoMFs were mainly detected in these inflammatory foci and crown-like structures. Interestingly, we also found that some crown-like structures consist of IBA1⁺Clec4F⁺ KCs (figure 3A–D). We speculate that these cells originate from MoMFs but acquire KC phenotypes within the liver, as MASH represents a chronic disease model.

Zonal distribution of KCs in healthy mouse livers

We next evaluated the zonal distribution of KCs in healthy livers using sequential multiplex immunostaining (figure 4A). After the staining of IBA1 and CLEC4F as mentioned above, liver sections were subsequently stained with zonation markers glutamine synthetase (GS) and E-cadherin (ECAD) after antibody stripping. The three zonation masks, Zone 1 (GS⁺ECAD⁻), Zone 2 (GS⁻ECAD⁻) and Zone 3 (GS⁻ECAD⁺), were generated based on GS and ECAD staining patterns. The number, location and size of KCs in three zones were extracted and analysed by CellProfiler. The location of KCs in three zones was plotted in figure 4A. We analysed the proportion of KCs among total cells in all three zones and found comparable KC percentages in Zone 1 and Zone 2. However, the KC percentage was significantly lower in Zone 3 (figure 4B), which is consistent with previous findings.¹⁷ Further analysis revealed that the size of the KCs, along with their major and minor axis lengths, were significantly smaller in Zone 3 compared with Zones 1 and 2 (figure 4C).

Evaluation of phagocytosis of liver macrophages in vivo

One of the most important roles of liver macrophages, particularly KCs, is to sense and engulf invading pathogens like bacteria and dead cells. Currently, widely used assays to evaluate the phagocytic ability of macrophages typically involve isolating macrophages and incubating them with fluorescent-labelled particles.¹⁸ These macrophages engulfed with fluorescent-labelled particles are then analysed by flow cytometry. However, isolation of KCs requires in situ perfusion and enzyme digestion, which takes hours to prepare samples, and enzyme digestion may affect KCs’ ability to phagocytose. Here, we developed a robust assay to evaluate the phagocytosis of liver macrophages in vivo using a combination of sequential multiplex immunostaining and subsequent data extraction/analysis. Naïve mice were injected with pHrodo Red Escherichia coli (E. coli) BioParticles via tail vein. These E. coli particles are non-fluorescent outside the cells, but fluoresce bright red in phagosomes with low pH environments. 1 hour later, liver tissues were collected and paraffin-embedded for further analysis. Liver sections were first scanned for E. coli particle fluorescence. Then, these sections were subjected to IBA1 and CLEC4F staining to visualise MoMFs and KCs, followed by GS and ECAD staining to map liver zonation. E. coli particles were exclusively detected inside IBA1⁺ liver macrophages (figure 5A). Similar to KC zonal distribution analysis mentioned above, KCs in all three zones and MoMFs were identified by CellProfiler, and the mean fluorescence intensity was quantified for each KC and MoMF. KCs in Zone 1 exhibited significantly greater ability to take up E. coli particles compared with KCs in Zones 2 and 3, both of which had similar phagocytic capabilities (figure 5B). In contrast, MoMFs exhibited a much lower phagocytic ability compared with KCs across all three zones. Representative images of KCs in all zones and MoMFs engulfing E. coli particles are provided in figure 5C.

Simultaneous evaluation of proliferation and apoptosis in KCs and MoMFs

In healthy livers, very few KCs and MoMFs undergo proliferation or apoptosis. However, we observed notable proliferation and apoptosis of liver macrophages in several diseased liver models.^{19 20} Here, we developed an assay to simultaneously quantify the proliferation and apoptosis of KCs and MoMFs. Sequential multiplex immunostaining was performed on liver sections 72 hours after ConA-induced liver injury. We selected the 72-hour time point because this time point is associated with a significant amount of MoMF accumulation in the border region of the necrotic area.⁷ The staining targeted KC/MoMF markers IBA1/CLEC4F, proliferation marker KI67 and apoptosis marker Cleaved-Caspase3 (Cl-Cas3). As shown in figure 6A, we identified proliferating MoMFs (IBA1⁺CLEC4F⁻KI67⁺), apoptotic MoMFs (IBA1⁺CLEC4F⁻Cl-CAS3⁺), proliferating KCs (IBA1⁺CLEC4F⁺KI67⁺) and apoptotic KCs (IBA1⁺CLEC4F⁺Cl-CAS3⁺) on the image. Then, we employed CellProfiler to quantify the percentage of these four types of cells based on the expression of these markers. Compared with healthy livers, both apoptosis and proliferation of KC/MoMF increased in injured livers (figure 6B). More MoMFs underwent proliferation than KCs in the recovery stage of ConA-induced liver injury (figure 6B). Interestingly, there is no significant difference in the percentage of apoptotic KCs and MoMFs in this model (figure 6B).

Signature gene expression pattern of macrophages in healthy and acutely injured livers

In our previous study,⁷ scRNA-seq analysis of purified MoMFs from ConA-induced injured liver identified a C1q⁺ macrophage population. To further characterise liver macrophages in both healthy and diseased states, we performed scRNA-seq sequencing on all cell types isolated from the livers of naïve mice and mice treated with ConA for 72 and 96 hours. Across these samples, we identified the expression of 32 285 genes in 52 005 cells (figure 7A). Unsupervised clustering revealed 15 distinct cell populations, including hepatocytes, NKT/T cells, LSECs, erythroid cells, neutrophils, B cells, dendritic cells, BDCs, HSCs, mast cells, macrophages and KCs (figure 7B). Notably, two MoMF clusters (clusters 2 and 9) expanded post-ConA treatment, while KCs (cluster 5) remained unchanged (figure 7A,B). Additionally, neutrophil and NKT/T cell populations increased, while differences in other cell subsets were less pronounced.

We focused on the MoMF and KC clusters and identified 16 macrophage subclusters (figure 7C,D). Among macrophage signature genes (online supplemental figure S2), we observed distinct expression patterns across these subclusters: (1) KCs were divided into high C1q (clusters 2 and 12) and low C1q (cluster 9) groups. (2) C1q⁺ MoMFs were categorised into two groups: those with high expression of Trem2, Spp1, Fabp5 and Gpnmb (clusters 1 and 8) and those with low expression of these genes (clusters 3 and 4). (3) C1q⁻ MoMFs exhibited high expression of Lyz and Ccr2, indicating they are newly recruited macrophages (clusters 0, 11 and 14). (4) The Ece1, a gene encoding ECE1 that converts endothelin precursors into active endothelin to promote HSC contraction,⁷ was highly expressed by Cd11c⁺ MoMFs (clusters 5, 6 and 7). (5) S100a8⁺MoMFs expressed high levels of Mmp9, Cd9, S100a8 and S100a9.

To validate these findings, we performed bulk RNA-seq on MoMFs purified from livers at different time points post ConA treatment. All signature genes mentioned above were upregulated following liver injury (online supplemental figure S3). We further verified the protein levels of these signature genes by using immunohistochemical staining (figure 8A,B). We first examined the expression of the M1/M2 macrophage markers CD86 and CD206 (encoded by Mrc1). CD86 was detected in KCs in naïve mice and significantly increased in both KCs and MoMFs following liver injury. Notably, a subset of MoMFs in the necrotic region exhibited particularly high levels of CD86. In contrast, CD206 was consistently expressed at high levels in nearly all liver macrophages, regardless of liver health or injury status.

Next, we analysed proteins associated with C1q⁺ MoMFs, including TREM2, SPP1, FABP5 and GPNMB.^{7 21 22} These proteins were predominantly detected in IBA1⁺MoMFs located in necrotic lesion regions of the injured livers but not in KCs. The dendritic cell marker CD11c, expressed by a subset of MoMFs, was enriched in necrotic lesion regions. The ECE1 enzyme protein, expressed by Cd11c⁺MoMFs, was detected in MoMFs surrounding necrotic lesion areas.

Interestingly, MMP9 and CD9 were primarily expressed by S100a8⁺MoMFs located inside the necrotic area, rather than at its border. Proteinases involved in protein degradation, such as Legumain (LGMN), cathepsin B (CTSB) and cathepsin D (CTSD), were not exclusive to macrophages but were significantly enriched in MoMFs within the necrotic regions. Macrophage scavenger receptor 1 (MSR1), which was important in the development of MASH,^{23 24} was expressed in both KCs and MoMFs in necrotic areas.

As summarised in figure 8C, immunohistochemical staining validated the signature genes identified by scRNA-seq in various MoMF groups. Among these, two populations, Trem2^highC1q⁺MoMFs and Cd11c⁺MoMFs, were predominantly located at the border of necrotic areas, while S100a8⁺MoMFs were primarily found inside the necrotic regions. Trem2^highC1q⁺MoMFs exhibited high expression of TREM2, SPP1, FABP5 and GPNMB, whereas Cd11c⁺MoMFs expressed elevated levels of ECE1. Proteinase genes such as LGMN, CTSB and CTSD were expressed in both KCs and MoMFs, but their expression was notably higher in MoMFs within necrotic regions.

Discussion

In this study, we used a combination of sequential multiplex immunofluorescence staining, whole-slide scanning and in-depth data analysis to characterise liver macrophages. We also demonstrated how to distinguish KCs and MoMFs in healthy and diseased mouse liver sections. Furthermore, we developed tools to quantify the number, size, percentage and location of KCs and MoMFs, respectively, in various liver injury models. By adding zonation markers, we determined the distribution and phagocytic ability of KCs across different liver zones. Additionally, we simultaneously evaluated the proliferation and apoptosis of KCs and MoMFs on a single slide. Finally, we demonstrated the staining results for a set of signature genes identified by scRNA-seq in a model of acute liver injury induced by ConA.

In this study, we stained markers for nearly all major cell types in the healthy liver, including hepatocytes, BDCs, HSCs, LSECs, macrophages, T/NKT cells, B cells and neutrophils, on each sample. Each cell type was segmented based on cell-type specific markers using ilastik, a machine learning algorithm to efficiently segment cells.²⁵ We analysed six healthy liver samples from 8 to 10-week-old mice, with more than 10 000 cells counted for each sample. We found the percentage of hepatocytes accounts for 60%~65% of total cells in the adult mouse livers, which is different from the traditional notion that hepatocytes constitute about 60%~80% of total cells in the liver.¹ We found a higher percentage of HSCs (~3%) in the current study compared with <0.5% reported in the previous traditional estimation.¹ Macrophages represent~85% of total immune cells and~10% of total cells in healthy mouse livers, consistent with their critical roles in sensing and engulfing pathogens from the intestinal blood supply. Finally, we were unable to reliably visualise NK cells due to the lack of reliable staining antibodies. The antibodies for labelling NK cells for flow cytometry analysis, such as anti-NK1.1 and anti-NKp46 did not produce satisfactory staining results in our experiments.

F4/80 has long been used as a marker for KCs in the liver.⁵ However, F4/80 staining alone cannot effectively distinguish KCs from MoMFs in both healthy and diseased livers. In contrast, IBA1 and CLEC4F double staining is a reliable and straightforward method for visualising KCs and MoMFs on liver sections. In healthy liver and acute liver injury models, IBA1 and CLEC4F can be used to clearly distinguish KCs from MoMFs. In chronic liver disease models, IBA1⁺CLECF4⁻ MoMFs were identifiable in most crown-like structures and inflammatory foci. However, we observed a subset of macrophages in crown-like structures expressing both CLEC4F and IBA1, resembling KCs. We speculate that these cells are MoMFs that acquire KC phenotype in the liver, a process that usually takes about 1~2 weeks after MoMFs migrate into the liver.²⁶ The spatial distribution of KCs and MoMFs was analysed within defined regions. This approach revealed distinct patterns of KC and MoMF distribution in the necrotic regions of the ConA-induced and APAP-induced liver injury models. In the ConA model, the necrotic region was almost exclusively populated by MoMFs, whereas in the APAP model, MoMFs accounted for approximately 60% of total macrophages in the necrotic region. In the ConA-induced liver injury model, hepatocytes, LSECs and KCs were all damaged by activated T/NKT cells in the necrotic areas. In contrast, hepatocytes are the major cell types that are damaged in the APAP model while the majority of KCs are not affected because toxic APAP adducts are mainly formed by cytochrome P450 2E1 (CYP2E1), which is highly expressed in hepatocytes but not KCs. These findings suggest different mechanisms of dead cell clearance and liver repair following injury in the two models.^{7 27 28}

The liver is organised into three zones along the portal-central axis of liver lobules, which are influenced by gradients of oxygen, nutrients, hormones and other factors.²⁹ Hepatocytes in different zones are specialised for distinct tasks.^{30 31} However, the functional differences of non-parenchymal cells in different zones remain poorly understood due to technical challenges. Here, we used sequential multiplex immunostaining, whole-slide scanning and data extraction/analysis to characterise the distribution, cell size and phagocytic ability of liver macrophages across the three zones. Zone 1 KCs had a higher density and greater phagocytic activity than KCs in Zones 2 and 3, which are in agreement with previous findings.^{4 17} The gut microbiota has been identified as a major driving force for the increased number and heightened phagocytic activity of Zone 1 KCs.^{4 17} Interestingly, we also observed that KCs in Zone 1 are larger than those in other zones. The relationship between KC size and functions such as phagocytosis, as well as the mechanisms regulating KC size, remains unclear and warrants further investigation.

Very few (1%~2%) KCs and MoMFs undergo proliferation in healthy liver. In response to liver injury, numerous monocytes migrate into the liver and differentiate into MoMFs. However, it is unclear whether these MoMFs can efficiently proliferate within the liver or what their fate is in injured liver tissue. Here, we simultaneously evaluate the rates of proliferation and apoptosis in both KCs and MoMFs in injured liver. The proliferation of both KCs and MoMFs significantly increased in injured liver. Notably, the percentage of proliferating MoMFs (~12%) was significantly higher than that of KCs (~6%) in injured liver. However, the percentage of apoptotic MoMFs is similar to KCs in both healthy and acutely injured livers. These data demonstrated that the proliferation of liver macrophages, particularly MoMFs, plays a critical role in maintaining sufficient macrophage populations during the recovery process following acute liver injury.

In our previous study,⁷ scRNA-seq analysis of purified MoMFs from ConA-induced injured liver identified a C1q⁺ macrophage population, which expresses a variety of genes related to phagocytosis and clearance. Although we had difficulties in getting satisfactory staining for C1q on FFPE liver samples, our immunohistochemical analyses revealed that proteins encoded by those signature genes are predominantly located in macrophages encircling or inside necrotic lesions. Some of these proteins can be used as markers of C1q⁺ macrophages.⁷ In the current study, we further conducted scRNA-seq on all cell types obtained from the livers of naïve mice and mice treated with ConA for 72 and 96 hours. Two MoMF clusters (clusters 2 and 9) expanded significantly following ConA treatment, while KCs (cluster 5) remained largely unchanged. We identified 16 distinct subclusters for liver macrophages, each displaying unique gene expression patterns. KCs were further divided into two groups: those with high C1q expression (clusters 2 and 12) and those with low C1q expression (cluster 9). Moreover, C1q⁺ MoMFs were categorised into two groups with high expression (clusters 1 and 8) and low expression (clusters 3 and 4) of Trem2, Spp1 and Gpnmb. In contrast, C1q⁻ MoMFs expressed high levels of Lyz and Ccr2, indicating that they are likely newly recruited macrophages. Moreover, our data also identified Cd11c⁺MoMFs that express high levels of Ece1, a gene encoding ECE1 enzyme that converts endothelin precursors into active endothelin to promote HSC contraction.⁷ Finally, we verified the location and expression of proteins encoded by these signature genes using immunohistochemical staining. We successfully obtained satisfactory staining of 15 proteins. Among them, TREM2, SPP1, FABP5 and GPNMB were exclusively expressed on MoMFs around necrotic areas but not KCs. In addition, ECE1 expressed by Cd11c⁺MoMFs was also predominantly found in necrotic areas. MMP9 and CD9 were identified as markers for visualising MoMFs inside the necrotic area rather than at their borders. Genes associated with protein degradation, including LGMN, CTSB and CTSD, were expressed at higher levels in MoMFs surrounding necrotic regions, though they were also expressed at relatively lower levels in KCs. The function of some signature genes mentioned in this study is not fully understood. Moreover, the roles of these populations of macrophages located in the necrotic area in the resolution of liver injury will be interesting topics for future studies.

In summary, we present a set of methods for characterising the features of macrophages in healthy and diseased livers in vivo. Combined with the analysis of omics data, we believe that image analysis of multiplex immunofluorescence data can offer a more reliable and insightful understanding of target cells. Furthermore, by integrating scRNA-seq analysis with immunohistochemical staining verification, we identified several populations of necrotic lesion-associated macrophages that have different signature genes and surround necrotic liver lesions, playing an important role in promoting necrotic lesion resolution during ConA-induced acute liver injury.

Supplementary material

online supplemental figure 1

egastro-3-2-s001.pdf^{(2.1MB, pdf)}

DOI: 10.1136/egastro-2025-100189

online supplemental file 1

egastro-3-2-s002.docx^{(37.8KB, docx)}

DOI: 10.1136/egastro-2025-100189

The funder did not influence the results/outcomes of the study despite author affiliations with the funder.

Footnotes

Funding: The work was supported by the intramural programme of NIAAA and NIH (AA000369 and AA000368) (BG).

Prepub: Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (https://doi.org/10.1136/egastro-2025-100189).

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient consent for publication: Not applicable.

Ethics approval: Animal experiments were approved by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) Animal Care and Use Committee (Animal Protocol LLD-BG-01).

Data availability free text: The data can be found from the Gene Expression Omnibus (GEO) under accession GSE287737.

Patient and public involvement: Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

Data availability statement

Data are available in a public, open access repository.

References

1.Racanelli V, Rehermann B. The liver as an immunological organ. Hepatology. 2006;43:S54–62. doi: 10.1002/hep.21060. [DOI] [PubMed] [Google Scholar]
2.Gao B, Jeong WI, Tian Z. Liver: An organ with predominant innate immunity. Hepatology. 2008;47:729–36. doi: 10.1002/hep.22034. [DOI] [PubMed] [Google Scholar]
3.Guillot A, Tacke F. Spatial dimension of macrophage heterogeneity in liver diseases. eGastroenterology. 2023;1:e000003. doi: 10.1136/egastro-2023-000003. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Miyamoto Y, Kikuta J, Matsui T, et al. Periportal macrophages protect against commensal-driven liver inflammation. Nature New Biol. 2024;629:901–9. doi: 10.1038/s41586-024-07372-6. [DOI] [PubMed] [Google Scholar]
5.Guilliams M, Scott CL. Liver macrophages in health and disease. Immunity. 2022;55:1515–29. doi: 10.1016/j.immuni.2022.08.002. [DOI] [PubMed] [Google Scholar]
6.Wen Y, Lambrecht J, Ju C, et al. Hepatic macrophages in liver homeostasis and diseases-diversity, plasticity and therapeutic opportunities. Cell Mol Immunol. 2021;18:45–56. doi: 10.1038/s41423-020-00558-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Feng D, Xiang X, Guan Y, et al. Monocyte-derived macrophages orchestrate multiple cell-type interactions to repair necrotic liver lesions in disease models. J Clin Invest. 2023;133:e166954. doi: 10.1172/JCI166954. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Tacke F. Targeting hepatic macrophages to treat liver diseases. J Hepatol. 2017;66:1300–12. doi: 10.1016/j.jhep.2017.02.026. [DOI] [PubMed] [Google Scholar]
9.Wong CHY, Jenne CN, Petri B, et al. Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance. Nat Immunol. 2013;14:785–92. doi: 10.1038/ni.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Guilliams M, Bonnardel J, Haest B, et al. Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches. Cell. 2022;185:379–96. doi: 10.1016/j.cell.2021.12.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Xiong X, Kuang H, Ansari S, et al. Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis. Mol Cell. 2019;75:644–60. doi: 10.1016/j.molcel.2019.07.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Liang Y, Kaneko K, Xin B, et al. Temporal analyses of postnatal liver development and maturation by single-cell transcriptomics. Dev Cell. 2022;57:398–414. doi: 10.1016/j.devcel.2022.01.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Hwang L-L, Wang C-H, Li T-L, et al. Sex differences in high-fat diet-induced obesity, metabolic alterations and learning, and synaptic plasticity deficits in mice. Obesity (Silver Spring) 2010;18:463–9. doi: 10.1038/oby.2009.273. [DOI] [PubMed] [Google Scholar]
14.Wang M, You Q, Lor K, et al. Chronic alcohol ingestion modulates hepatic macrophage populations and functions in mice. J Leukoc Biol. 2014;96:657–65. doi: 10.1189/jlb.6A0114-004RR. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Guillot A, Buch C, Jourdan T. Kupffer Cell and Monocyte-Derived Macrophage Identification by Immunofluorescence on Formalin-Fixed, Paraffin-Embedded (FFPE) Mouse Liver Sections. Methods Mol Biol. 2020;2164:45–53. doi: 10.1007/978-1-0716-0704-6_6. [DOI] [PubMed] [Google Scholar]
16.Guillot A, Guerri L, Feng D, et al. Bile acid-activated macrophages promote biliary epithelial cell proliferation through integrin αvβ6 upregulation following liver injury. J Clin Invest. 2021;131:e132305. doi: 10.1172/JCI132305. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Gola A, Dorrington MG, Speranza E, et al. Commensal-driven immune zonation of the liver promotes host defence. Nature New Biol. 2021;589:131–6. doi: 10.1038/s41586-020-2977-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Montaño F, Grinstein S, Levin R. Quantitative Phagocytosis Assays in Primary and Cultured Macrophages. Methods Mol Biol. 2018;1784:151–63. doi: 10.1007/978-1-4939-7837-3_15. [DOI] [PubMed] [Google Scholar]
19.Li M, He Y, Zhou Z, et al. MicroRNA-223 ameliorates alcoholic liver injury by inhibiting the IL-6-p47phox-oxidative stress pathway in neutrophils. Gut. 2017;66:705–15. doi: 10.1136/gutjnl-2016-311861. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Ait Ahmed Y, Fu Y, Rodrigues RM, et al. Kupffer cell restoration after partial hepatectomy is mainly driven by local cell proliferation in IL-6-dependent autocrine and paracrine manners. Cell Mol Immunol. 2021;18:2165–76. doi: 10.1038/s41423-021-00731-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Fabre T, Barron AMS, Christensen SM, et al. Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation. Sci Immunol. 2023;8:eadd8945. doi: 10.1126/sciimmunol.add8945. [DOI] [PubMed] [Google Scholar]
22.Ramachandran P, Dobie R, Wilson-Kanamori JR, et al. Resolving the fibrotic niche of human liver cirrhosis at single-cell level. Nature New Biol. 2019;575:512–8. doi: 10.1038/s41586-019-1631-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Bieghs V, Wouters K, van Gorp PJ, et al. Role of scavenger receptor A and CD36 in diet-induced nonalcoholic steatohepatitis in hyperlipidemic mice. Gastroenterology. 2010;138:2477–86. doi: 10.1053/j.gastro.2010.02.051. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Govaere O, Petersen SK, Martinez-Lopez N, et al. Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease. J Hepatol. 2022;76:1001–12. doi: 10.1016/j.jhep.2021.12.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Berg S, Kutra D, Kroeger T, et al. ilastik: interactive machine learning for (bio)image analysis. Nat Methods. 2019;16:1226–32. doi: 10.1038/s41592-019-0582-9. [DOI] [PubMed] [Google Scholar]
26.Scott CL, Zheng F, De Baetselier P, et al. Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells. Nat Commun. 2016;7:10321. doi: 10.1038/ncomms10321. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Matchett KP, Wilson-Kanamori JR, Portman JR, et al. Multimodal decoding of human liver regeneration. Nature New Biol. 2024;630:158–65. doi: 10.1038/s41586-024-07376-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Ben-Moshe S, Veg T, Manco R, et al. The spatiotemporal program of zonal liver regeneration following acute injury. Cell Stem Cell. 2022;29:973–89. doi: 10.1016/j.stem.2022.04.008. [DOI] [PubMed] [Google Scholar]
29.Paris J, Henderson NC. Liver zonation, revisited. Hepatology. 2022;76:1219–30. doi: 10.1002/hep.32408. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Wei Y, Wang YG, Jia Y, et al. Liver homeostasis is maintained by midlobular zone 2 hepatocytes. Science. 2021;371:eabb1625. doi: 10.1126/science.abb1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.He L, Pu W, Liu X, et al. Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair. Science. 2021;371:eabc4346. doi: 10.1126/science.abc4346. [DOI] [PubMed] [Google Scholar]

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DOI: 10.1136/egastro-2025-100189

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DOI: 10.1136/egastro-2025-100189

Data Availability Statement

Data are available in a public, open access repository.

[R1] 1.Racanelli V, Rehermann B. The liver as an immunological organ. Hepatology. 2006;43:S54–62. doi: 10.1002/hep.21060. [DOI] [PubMed] [Google Scholar]

[R2] 2.Gao B, Jeong WI, Tian Z. Liver: An organ with predominant innate immunity. Hepatology. 2008;47:729–36. doi: 10.1002/hep.22034. [DOI] [PubMed] [Google Scholar]

[R3] 3.Guillot A, Tacke F. Spatial dimension of macrophage heterogeneity in liver diseases. eGastroenterology. 2023;1:e000003. doi: 10.1136/egastro-2023-000003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Miyamoto Y, Kikuta J, Matsui T, et al. Periportal macrophages protect against commensal-driven liver inflammation. Nature New Biol. 2024;629:901–9. doi: 10.1038/s41586-024-07372-6. [DOI] [PubMed] [Google Scholar]

[R5] 5.Guilliams M, Scott CL. Liver macrophages in health and disease. Immunity. 2022;55:1515–29. doi: 10.1016/j.immuni.2022.08.002. [DOI] [PubMed] [Google Scholar]

[R6] 6.Wen Y, Lambrecht J, Ju C, et al. Hepatic macrophages in liver homeostasis and diseases-diversity, plasticity and therapeutic opportunities. Cell Mol Immunol. 2021;18:45–56. doi: 10.1038/s41423-020-00558-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] 7.Feng D, Xiang X, Guan Y, et al. Monocyte-derived macrophages orchestrate multiple cell-type interactions to repair necrotic liver lesions in disease models. J Clin Invest. 2023;133:e166954. doi: 10.1172/JCI166954. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] 8.Tacke F. Targeting hepatic macrophages to treat liver diseases. J Hepatol. 2017;66:1300–12. doi: 10.1016/j.jhep.2017.02.026. [DOI] [PubMed] [Google Scholar]

[R9] 9.Wong CHY, Jenne CN, Petri B, et al. Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance. Nat Immunol. 2013;14:785–92. doi: 10.1038/ni.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Guilliams M, Bonnardel J, Haest B, et al. Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches. Cell. 2022;185:379–96. doi: 10.1016/j.cell.2021.12.018. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Xiong X, Kuang H, Ansari S, et al. Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis. Mol Cell. 2019;75:644–60. doi: 10.1016/j.molcel.2019.07.028. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] 12.Liang Y, Kaneko K, Xin B, et al. Temporal analyses of postnatal liver development and maturation by single-cell transcriptomics. Dev Cell. 2022;57:398–414. doi: 10.1016/j.devcel.2022.01.004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Hwang L-L, Wang C-H, Li T-L, et al. Sex differences in high-fat diet-induced obesity, metabolic alterations and learning, and synaptic plasticity deficits in mice. Obesity (Silver Spring) 2010;18:463–9. doi: 10.1038/oby.2009.273. [DOI] [PubMed] [Google Scholar]

[R14] 14.Wang M, You Q, Lor K, et al. Chronic alcohol ingestion modulates hepatic macrophage populations and functions in mice. J Leukoc Biol. 2014;96:657–65. doi: 10.1189/jlb.6A0114-004RR. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Guillot A, Buch C, Jourdan T. Kupffer Cell and Monocyte-Derived Macrophage Identification by Immunofluorescence on Formalin-Fixed, Paraffin-Embedded (FFPE) Mouse Liver Sections. Methods Mol Biol. 2020;2164:45–53. doi: 10.1007/978-1-0716-0704-6_6. [DOI] [PubMed] [Google Scholar]

[R16] 16.Guillot A, Guerri L, Feng D, et al. Bile acid-activated macrophages promote biliary epithelial cell proliferation through integrin αvβ6 upregulation following liver injury. J Clin Invest. 2021;131:e132305. doi: 10.1172/JCI132305. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] 17.Gola A, Dorrington MG, Speranza E, et al. Commensal-driven immune zonation of the liver promotes host defence. Nature New Biol. 2021;589:131–6. doi: 10.1038/s41586-020-2977-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] 18.Montaño F, Grinstein S, Levin R. Quantitative Phagocytosis Assays in Primary and Cultured Macrophages. Methods Mol Biol. 2018;1784:151–63. doi: 10.1007/978-1-4939-7837-3_15. [DOI] [PubMed] [Google Scholar]

[R19] 19.Li M, He Y, Zhou Z, et al. MicroRNA-223 ameliorates alcoholic liver injury by inhibiting the IL-6-p47phox-oxidative stress pathway in neutrophils. Gut. 2017;66:705–15. doi: 10.1136/gutjnl-2016-311861. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Ait Ahmed Y, Fu Y, Rodrigues RM, et al. Kupffer cell restoration after partial hepatectomy is mainly driven by local cell proliferation in IL-6-dependent autocrine and paracrine manners. Cell Mol Immunol. 2021;18:2165–76. doi: 10.1038/s41423-021-00731-7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Fabre T, Barron AMS, Christensen SM, et al. Identification of a broadly fibrogenic macrophage subset induced by type 3 inflammation. Sci Immunol. 2023;8:eadd8945. doi: 10.1126/sciimmunol.add8945. [DOI] [PubMed] [Google Scholar]

[R22] 22.Ramachandran P, Dobie R, Wilson-Kanamori JR, et al. Resolving the fibrotic niche of human liver cirrhosis at single-cell level. Nature New Biol. 2019;575:512–8. doi: 10.1038/s41586-019-1631-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Bieghs V, Wouters K, van Gorp PJ, et al. Role of scavenger receptor A and CD36 in diet-induced nonalcoholic steatohepatitis in hyperlipidemic mice. Gastroenterology. 2010;138:2477–86. doi: 10.1053/j.gastro.2010.02.051. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Govaere O, Petersen SK, Martinez-Lopez N, et al. Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease. J Hepatol. 2022;76:1001–12. doi: 10.1016/j.jhep.2021.12.012. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Berg S, Kutra D, Kroeger T, et al. ilastik: interactive machine learning for (bio)image analysis. Nat Methods. 2019;16:1226–32. doi: 10.1038/s41592-019-0582-9. [DOI] [PubMed] [Google Scholar]

[R26] 26.Scott CL, Zheng F, De Baetselier P, et al. Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells. Nat Commun. 2016;7:10321. doi: 10.1038/ncomms10321. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] 27.Matchett KP, Wilson-Kanamori JR, Portman JR, et al. Multimodal decoding of human liver regeneration. Nature New Biol. 2024;630:158–65. doi: 10.1038/s41586-024-07376-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] 28.Ben-Moshe S, Veg T, Manco R, et al. The spatiotemporal program of zonal liver regeneration following acute injury. Cell Stem Cell. 2022;29:973–89. doi: 10.1016/j.stem.2022.04.008. [DOI] [PubMed] [Google Scholar]

[R29] 29.Paris J, Henderson NC. Liver zonation, revisited. Hepatology. 2022;76:1219–30. doi: 10.1002/hep.32408. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.Wei Y, Wang YG, Jia Y, et al. Liver homeostasis is maintained by midlobular zone 2 hepatocytes. Science. 2021;371:eabb1625. doi: 10.1126/science.abb1625. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.He L, Pu W, Liu X, et al. Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair. Science. 2021;371:eabc4346. doi: 10.1126/science.abc4346. [DOI] [PubMed] [Google Scholar]

PERMALINK

Characterisation of macrophages in healthy and diseased livers in mice: identification of necrotic lesion-associated macrophages

Dechun Feng

Yukun Guan

Yang Wang

Luca Maccioni

Bryan Mackowiak

Bin Gao