Conjugated bile acid-driven CD14+CD16+ monocyte infiltration promotes cholestatic liver injury by enhancing hepatocyte necroptosis

Zhixian Zhu; Ziqian Xu; Xinyu Cao; Nan Zhao; Jiaxin Lei; Ling Li; Xi Li; Yan Li; Mingqiao Li; Tian Ren; Li Liu; James L Boyer; Shi-ying Cai; Qiong Pan; Xiaoxun Zhang; Jin Chai

doi:10.1016/j.jhepr.2025.101517

. 2025 Jul 11;7(10):101517. doi: 10.1016/j.jhepr.2025.101517

Conjugated bile acid-driven CD14⁺CD16⁺ monocyte infiltration promotes cholestatic liver injury by enhancing hepatocyte necroptosis

Zhixian Zhu ^1,^2,^3,^4,^†, Ziqian Xu ^1,^2,^3,^4,^†, Xinyu Cao ^1,^2,^3,^4,^†, Nan Zhao ^1,^2,^3,^4,^†, Jiaxin Lei ^1,^2,^3,^4,^†, Ling Li ^1,^2,^3,⁴, Xi Li ^1,^2,^3,⁴, Yan Li ^1,^2,^3,⁴, Mingqiao Li ^1,^2,^3,⁴, Tian Ren ^1,², Li Liu ^1,², James L Boyer ⁵, Shi-ying Cai ^5,^⁎, Qiong Pan ^1,^2,^3,^4,^⁎, Xiaoxun Zhang ^1,^2,^3,^4,^⁎, Jin Chai ^1,^2,^3,^4,^⁎

PMCID: PMC12448010 PMID: 40980164

Abstract

Background & Aims

Cell death and inflammation cause liver injury during cholestasis. Monocyte infiltration is crucial for the inflammatory response in tissues and organs. Thus, to study the role of monocytes in cholestatic liver disease, we used single-cell RNA sequencing (scRNA-seq) to analyze liver non-parenchymal cells (NPCs) from patients with obstructive cholestasis (OC).

Methods

Hepatic monocyte infiltration in liver was assessed using scRNA-seq and multiplex immunofluorescence. In vivo functional validation was conducted through serum biochemistry, liver histopathology, RT-qPCR, and Western blotting after intermediate monocyte infusion and elimination in mice. In vitro, CD14⁺CD16⁺ monocytes were isolated by flow cytometry for analysis of tumor necrosis factor-alpha (TNFα) and factor-related apoptosis ligand (FASL) expression.

Results

scRNA-seq and multiplex immunofluorescence revealed that CD14⁺CD16⁺ monocyte infiltration was increased in OC livers (n = 5–20; p <0.0001). The number of CD14⁺CD16⁺ monocytes was positively correlated with liver injury severity (alanine aminotransferase (ALT), n = 20, p = 0.00002; aspartate aminotransferase (AST), n = 20, p = 0.002). Infusion of human CD14⁺CD16⁺ monocytes (i.e. human intermediate monocytes) exacerbated cholestatic liver injury in mice fed 1% lithocholic acid (ALT, n = 5, p <0.01; AST, n = 5, p <0.01). Meanwhile, mice that received mouse intermediate monocytes also exhibited higher cholestatic liver injury (ALT, n = 5, p <0.01; AST, n = 5, p <0.01). Mechanistically, CD14⁺CD16⁺ monocyte infiltration increased hepatic levels of TNFα (n = 5, p <0.01) and FASL (n = 5, p <0.01), activating the receptor-interacting serine/threonine-protein kinase (RIPK)-1–RIPK3–mixed-lineage kinase domain-like protein (MLKL) axis to promote hepatocyte necroptosis. CD14⁺CD16⁺ monocytes isolated from peripheral blood mononuclear cells (PBMCs) from patients with OC expressed higher levels of TNFα (n = 3, p <0.05) and FASL (n = 3, p <0.05) compared with other PBMC populations. Bile acid-stressed hepatocytes drove CD14⁺CD16⁺ monocyte infiltration via JNK/c-Jun-mediated CCL2 (n = 5-20, p <0.0001) and CCL3 (n = 5-20, p <0.05) upregulation.

Conclusion

Infiltrated CD14⁺CD16⁺ monocytes promote hepatocyte necroptosis and exacerbate cholestatic liver injury. Thus, targeting their chemotaxis represents a promising therapeutic strategy against cholestasis.

Impact and implications

This study revealed that CD14⁺CD16⁺ monocyte-derived tumor necrosis factor-alpha (TNFα) and factor-related apoptosis ligand (FASL) drive hepatocyte necroptosis via the receptor-interacting serine/threonine-protein kinase (RIPK)-1–RIPK3–mixed-lineage kinase domain-like protein (MLKL) axis, establishing these infiltrating monocytes as crucial mediators of cholestatic liver injury. Bile acid-stressed hepatocytes promoted CD14⁺CD16⁺ monocyte infiltration by upregulating CCL2 and CCL3 via JNK/c-Jun signaling. These findings highlight the therapeutic potential of modulating monocyte infiltration or necroptosis pathways to alleviate disease progression, offering novel strategies for treating OC and related liver pathologies.

Keywords: Cholestasis, CD14⁺CD16⁺ monocyte, Hepatocyte necroptosis

Graphical abstract

Highlights

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CD14⁺CD16⁺ monocyte infiltration correlated with severity of cholestatic injury.
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Transplanted human/mouse intermediate monocytes exacerbated hepatocyte necroptosis via RIPK1–RIPK3–MLKL axis activation.
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Depletion of mouse intermediate monocytes ameliorated lithocholic acid-induced cholestatic injury.
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Conjugated bile acids upregulated hepatocyte CCL2/CCL3 through JNK/c-Jun signaling, recruiting CD14⁺CD16⁺ monocytes.

Introduction

Cholestasis is characterized by the accumulation of bile acids (BAs) in the liver and systemic circulation, leading to liver injury.¹ Cholestasis can be caused by bile-duct obstruction (obstructive cholestasis, OC), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and others.²^,³ Accumulated BAs in the liver initiate an inflammatory response and promote hepatocyte death, driven, in part, by chemokine production and inflammatory cell infiltration.⁴^,⁵

Conjugated BAs induce the chemokines Ccl2 and Cxcl2 in mouse hepatocytes, attracting neutrophils and exacerbating hepatocyte necrosis in bile-duct ligation (BDL) cholestatic mice, whereas Ccl2 knockout significantly alleviates cholestatic liver injury.⁶ These chemokines can also attract monocytes to promote inflammation in liver fibrosis, metabolic dysfunction-associated steatotic liver disease (MASLD), and other conditions.⁷^,⁸ Monocytes are a type of mononuclear cell produced in the bone marrow. Human peripheral blood monocytes are divided into three subtypes: ‘classical’ CD14⁺CD16⁻ monocytes; ‘intermediate’ CD14⁺CD16⁺ monocytes; and ‘non-classical’ CD14^lowCD16⁺ monocytes.⁹ In mice, monocyte subsets are classified based on Ly6C and CD43 expression as: ‘classical’ Ly6c⁺CD43^low monocytes; ‘intermediate’ Ly6c⁺CD43⁺ monocytes; and ‘non-classical’ Ly6c^lowCD43⁺ monocytes. Intermediate monocytes have unique surface molecular characteristics and distinct biological functions.¹⁰ They contribute to the pathogenesis of inflammatory diseases, such as inflammatory bowel disease and neuromyelitis optica spectrum disorder, by producing high levels of proinflammatory cytokines, including tumor necrosis factor-alpha (TNFα), IL-1β, and IL-6.¹¹^,¹² However, the role of CD14⁺CD16⁺ monocytes and whether BAs induce hepatic chemokines to attract them into the liver in cholestasis remain unknown.

Hepatocyte death, including pyroptosis, efferocytosis, and apoptosis, drives cholestatic liver injury pathogenesis.[13], [14], [15] Interestingly, necroptosis has recently emerged as a new mechanism for hepatocyte death.¹⁶ Mechanistically, necroptosis is initiated downstream of many cellular stressors, including the signaling events activated by death receptor ligands, such as TNF-α, factor-related apoptosis ligand (FASL), or TRAIL. Once activated, receptor-interacting serine/threonine-protein kinase (RIPK)-3 interacts with RIPK1 to assemble the necrosome complex, ultimately leading to activation of mixed-lineage kinase domain-like protein (MLKL) and pore formation in the cell membrane, followed by cell death.¹⁷ A recent study established that necroptosis is commonly initiated by TNFα and FASL.¹⁸ Previous studies have observed hepatocyte necroptosis and elevated levels of hepatic TNFα in patients with OC.¹⁹ However, how cholestasis induces hepatocyte necroptosis remains unclear.

In this study, using single-cell RNA sequencing (sc-RNA seq) and multiplex immunofluorescence (mIF), we observed increased CD14⁺CD16⁺ monocytes in cholestatic livers from patients with OC and PBC. We then investigated the functional role and regulatory mechanisms involved both in vivo and in vitro. Our findings are the first to show that significantly increased CD14⁺CD16⁺ monocyte infiltrates in the cholestatic liver promote hepatocyte necroptosis and inflammation. Thus, targeting the CD14⁺CD16⁺ monocyte infiltration could be a novel therapeutic strategy for treating cholestatic liver injury.

Materials and methods

Human serum and liver samples

The study was approved by the Ethics Committees of the First Affiliated Hospital of Third Military Medical University (approval number KY2020313), conducted as per the Declaration of Helsinki (2024) with participants' written consent. Surgical liver samples were obtained from four control patients and four patients with OC for sc-RNA seq (Table S1). Liver samples from five control patients, five patients with PBC, and 20 patients with OC were analyzed by mIF, RNA extraction, and RT-qPCR (Table S2). Peripheral blood samples from control patients and patients with OC were used for fluorescence-activated cell sorting (FACS) of CD14⁺CD16⁺ monocytes (Table S2). For detailed Materials and methods, see the supplementary data and supplementary CTAT table.

Statistical analysis

Data are expressed as mean ± SD. Comparisons between the two groups were analyzed using the unpaired two-tailed Student’s t test, Welch's t test, or one-tailed Mann-Whitney U test. Multigroup comparison was determined using ordinary one-way ANOVA. p values were calculated using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA); p <0.05 was regarded as statistically significant.

Results

Single-cell transcriptomic analysis of non-parenchymal cells in human livers from patients with OC and control patients

We performed 5′/3′-sc-RNA seq analyses in liver tissues from patients with OC and their controls (n = 4 per group) (Fig. 1A; Table S1). 5′-scRNA-seq identified 18,516 and 16,946 liver non-parenchymal cells (NPCs) in control individuals and patients with OC, respectively. Uniform manifold approximation and projection (UMAP) unveiled 25 populations of NPCs in these specimens (Fig. 1B) from control and OC livers (Fig. 1C) spanning 10 cell lineages (Fig. 1D). Individual cell lineages had specific gene markers (Fig. 1E–G; Table S4). Interestingly, the distribution of these cell lineages differed between control and OC livers (Fig. 1H; Table S5). 3′-scRNA-seq data confirmed similar compositional alterations (Fig. S1). Differentially expressed genes (DEGs) in each liver cell subpopulation from the two groups were also analyzed (Fig. S2).

Numbers of hepatic CD14⁺CD16⁺ monocytes are positively correlated with severity of liver injury in patients with cholestasis

To further explore the potential pathophysiological role of NPCs in cholestasis, we performed advanced subset analysis (Fig. S3–S7). Although T and B cells exhibited changes at the major lineage level, no significant alterations were observed in subset compositions. Notably, the mononuclear phagocyte system demonstrated substantial restructuring of the proportions of different subsets. This population-specific reorganization prompted us to perform an in-depth investigation. sc-RNA seq data analysis displayed seven clusters of mononuclear phagocyte systems, based on their unique differentiation status and function (Fig. 2A–C; Fig. S8 and Table S6).²⁰^,²¹ These cell clusters exhibited unique gene expression profiles and biomarkers (Fig. 2D and E).

Fig. 2 — **Number of CD14⁺CD16⁺ monocyte infiltrates in the liver is high and correlated with the degree of liver injury in patients with cholestasis.** (A) UMAP of 6,892 DCs and MPs from four CTR and four OC livers identified seven distinct clusters of the mononuclear phagocyte system. (B) Canonical marker gene expression across mononuclear phagocyte clusters. (C) Cell distribution in CTR (blue) and OC (orange) livers. (D) Heatmap of the relative expression levels of marker genes in different clusters of the MP system (top, color-coded by cluster and condition), with exemplar genes labeled (right). Columns denote cells; rows denote genes. (E) Violin plots of the expression levels of enriched marker genes across distinct subpopulations of the MP system. (F) Proportions of MP system subpopulations in control and OC livers; n = 4 per group. (G) Proportions of MP system subpopulations in CTR and PBC livers; n = 4–5 per group. (H) mIF analysis of CD68⁺CD14⁺CD16⁺ cells in livers using antibodies against CD14 (red), CD16 (green), and CD68 (purple). CTR, n = 5; PBC, n = 5; OC, n = 20. (I) Quantitative analysis of CD14⁺CD16⁺ monocytes showing increased infiltration in OC and PBC livers. CTR, n = 5; PBC, n = 5; OC, n = 20. (J) Correlation between CD14⁺CD16⁺ monocyte counts and serum levels of ALT, AST, ALP, and GGT in patients with OC (n = 20). Data are mean ± SD, analyzed using the unpaired two-tailed Student’s t test or Mann-Whitney U test. Correlation analyses performed using Spearman’s rank test. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CTR, control; DC, dendritic cell; GGT, gamma-glutamyl transferase; mIF, multiplex immunofluorescence; OC, obstructive cholestasis; MP, mononuclear phagocyte; PBC, primary biliary cholangitis; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.

Within these seven clusters, monocytes comprised three subsets: CD14⁺ monocytes (human classical monocytes); CD16⁺ monocytes (human non-classical monocytes); and CD14⁺CD16⁺ monocytes (human intermediate monocytes). Notably, compared with control individuals, the frequency of CD14⁺CD16⁺ monocytes increased in the livers of patients with OC (Fig. 2F). Similarly, a higher percentage of CD14⁺CD16⁺ monocytes was also observed in the livers of patients with PBC (Fig. 2G).²² The expression of genes encoding CD14 and CD16 is shown in Fig. S9A and B. The frequency of other types of NPCs in the liver also varied. To verify the uniqueness of CD14⁺CD16⁺ monocytes, we analyzed the DEGs and relevant pathways by Gene Ontology (GO; Fig. S10A and B). The results revealed that CD14⁺CD16⁺ monocytes exhibited unique DEG profiles distinct from other monocyte subsets, indicating a mechanistically divergent transcriptional landscape within the mononuclear phagocyte system (Fig. S10E). Complementing our findings with Monocle2-based trajectory analysis, we found that CD14⁺CD16⁺ monocytes preferentially clustered at specific differentiation stages, revealing hepatic microenvironment-driven divergence in monocyte differentiation pathways (Fig. S10C and D).

To verify our observations, mIF staining was performed in human liver tissues. The results revealed significantly higher numbers of CD14⁺CD16⁺ monocytes infiltrating the livers of patients with OC or PBC compared with controls (Fig. 2H and I). Interestingly, there was a significant positive correlation between the numbers of hepatic CD14⁺CD16⁺ monocytes and levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Fig. 2J; Table S2), suggesting that CD14⁺CD16⁺ monocytes aggravate liver injury in cholestasis.

CD14⁺CD16⁺ monocyte infiltration significantly enhances cholestatic liver injury in a mouse model of cholestasis induced by an LCA diet

To further investigate the functional role of infiltrated intermediate monocytes in cholestasis, two animal models were established. In the first model, we purified and injected human peripheral blood CD14⁺CD16⁺ monocytes into immunodeficient NOD-Prkdc^scidIl2rg^em1/Smoc (M-NSG) mice. These mice were then fed a 1% lithocholic acid (hereafter referred to as LCA) diet to induce cholestasis (Fig. 3A and B). In the second model, clodronate liposomes were used to deplete endogenous mononuclear phagocytes in C57BL/6 mice. Two days after injection of clodronate liposome, we isolated primary mouse intermediate monocytes alongside non-intermediate monocytes from the bone marrow of the mice. Then, one group of mice received injections of intermediate monocytes, while the other was injected with non-intermediate monocytes (Fig. 3C and D). Human CD14⁺CD16⁺ monocyte infusions significantly elevated serum ALT and AST in cholestatic M-NSG mice compared with those not receiving an infusion (Fig. 3E). Similarly, compared with the group injected with non-intermediate monocytes, mice that received intermediate monocytes showed higher serum ALT and AST levels (Fig. 3F). We also constructed an intermediate monocyte-DTR knockout system. We found that knockdown of intermediate monocytes with diphtheria toxin attenuated LCA-induced injury (Figs. S11 and S12). Furthermore, histology confirmed that the ratio of hepatic necrosis and the scores of inflammation were also significantly increased in cholestatic mice that received an intermediate monocyte (human and mouse) injection (Fig. 3G–J). However, there was no significant difference in scores of liver fibrosis or bile-duct proliferation between these two groups (Fig. S13). Taken together, these findings provide solid evidence that infiltration of intermediate monocytes significantly aggravates cholestatic liver injury.

Fig. 3 — **Infusion with human or mouse-derived intermediate monocytes aggravates cholestatic liver injury in murine models of 1% LCA-induced cholestasis.** (A) Immunodeficient M-NSG mice. (B) Experimental procedure for investigating the potential roles of CD14⁺CD16⁺ monocytes in mice with cholestasis. (C) Clodronate liposome mechanism for depleting endogenous monocytes. (D) Model for investigating the effects of mouse-derived intermediate monocytes in mice with cholestasis. (E) Serum ALT and AST levels among different groups: ∗p <0.05 *vs.* CTR; #p <0.05 *vs.* LCA group; n = 4–5 per group. (F) Serum ALT and AST levels among different groups: ∗p <0.05 *vs.* CTR; #p <0.05 *vs.* LCA + non-intermediate monocyte group; n = 5 per group. (G,H) Histological analyses of liver sections after H&E, Sirius Red, and Ck19 immunofluorescence staining; n = 4–5 per group. (I) Quantitative analyses of necrosis and inflammation; ∗p <0.05 *vs.* CTR; #p <0.05 *vs.* LCA group; n = 4–5 per group. (J) Quantitative analyses of necrosis and inflammation; ∗p <0.05 *vs.* CTR; #p <0.05 *vs.* LCA + non-intermediate monocyte group; n = 5 per group. Data are mean ± SD, analyzed using the unpaired two-tailed Student’s t test or Mann-Whitney U test. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CTR, control; LCA, lithocholic acid; M-NSG, NOD-*Prkdc*^scidIl2rg^em1/Smoc.

Hepatocyte necroptosis is significantly increased and positively correlated with CD14⁺CD16⁺ monocyte numbers in LCA-induced cholestatic mice or patients with cholestasis

The marked elevation of serum ALT and AST indicates substantial hepatocyte death. Through dual assessment of HNF4α (a hepatocyte-specific nuclear marker) and propidium iodide (PI) nuclear staining, we observed that LCA-fed M-NSG mice that did not receive an intermediate monocyte transfusion exhibited HNF4α-positive nuclei predominantly retaining intact nuclear morphology (i.e. round/oval shape with uniform fluorescence intensity), reflecting baseline cell death under cholestatic stress. By contrast, LCA-fed mice that received a CD14⁺CD16⁺ monocyte transfusion demonstrated profoundly aberrant HNF4α-positive nuclear architectures, characterized by chromatin condensation, karyorrhexis, and irregular nuclear contours (Fig. 4A).

Fig. 4 — **Infusion with human CD14⁺CD16⁺ monocytes deteriorates LCA-induced liver necroptosis in mice.** (A) Hepatic nuclear morphology by immunofluorescence analysis of HNF4α (green) and PI staining (red). (B) Real-time qPCR analysis of hepatic *Ripk1, Ripk3*, and *Mlkl* mRNA levels; ∗p <0.05 *vs.* LCA group; n = 5 per group. (C) Immunofluorescence analysis of hepatic Ripk1 (red) and Ripk3 (red) expression in mice. (D) Western blot analyses of the relative Ripk1, Ripk3, and Mlkl protein expression levels, and their phosphorylation in mouse liver; ∗p <0.05 *vs.* LCA group; n = 5 per group. (E) Western blot analyses of the relative Mlkl membrane protein expression levels in mouse liver; ∗p <0.05 *vs.* LCA group; n = 5 per group. (F) Real-time qPCR analysis of hepatic *TNFα* and *FASL* mRNA levels in mouse liver; ∗p <0.05 *vs.* LCA group; n = 5 per group. (G) Real-time qPCR analysis of hepatic *RIPK1, RIPK3*, and *MLKL* mRNA levels; ∗p <0.05 *vs.* CTR patient group; CTR, n = 5; OC, n = 20. (H) Hepatic *RIPK1, RIPK3*, and *MLKL* mRNA transcript levels; ∗p <0.05 *vs.* CTR patient group; CTR, n = 5; PBC, n = 5. (I) Real-time qPCR analysis of relative *TNFα* and *FASL* mRNA transcript levels in CD14⁺CD16⁺ monocytes isolated from PBMCs and the remaining PBMC populations; ∗p <0.05 *vs.* remaining PBMC populations; n = 3 per group. (J) Correlation analysis between CD14⁺CD16⁺ monocyte counts and hepatic *RIPK1, MLKL*, and *RIPK3* mRNA transcripts in patients with OC (n = 20). Data are mean ± SD, analyzed using the unpaired two-tailed Student’s t test, Welch's t test, or one-tailed Mann-Whitney U test. Correlation analyses performed using Spearman’s rank tests. CTR, control; FASL, Factor-related apoptosis ligand; MLKL, mixed-lineage kinase domain-like protein; OC, obstructive cholestasis; PBC, primary biliary cholangitis; PBMC, peripheral blood mononuclear cell; PI, propidium iodide; RIPK, receptor-interacting serine/threonine-protein kinase; TNFα, tumor necrosis factor-alpha.

To investigate how CD14⁺CD16⁺ monocytes aggravated hepatocyte-specific death, we analyzed the different forms of cell death, by using Caspase3 and Bax for apoptosis, Gsdmd for pyroptosis, and Ripk1, Ripk3, and Mlkl for necroptosis. Levels of Caspase3 and Bax (apoptosis) and Gsdmd (pyroptosis) expression did not change significantly in cholestatic mice with and without human CD14⁺CD16⁺ monocyte injection (Figs. S14 and S15). By contrast, the relative levels of hepatic Ripk1, Ripk3, and Mlkl (necroptosis) mRNA transcripts significantly increased in LCA-induced cholestatic M-NSG mice infused with human CD14⁺CD16⁺ monocytes (Fig. 4B). Immunofluorescence (IF) analysis confirmed the increased expression of Ripk1 and Ripk3 proteins in these human CD14⁺CD16⁺ monocyte-infused mouse livers (Fig. 4C).

Furthermore, Western blotting revealed significantly increased hepatic levels of Ripk1, Ripk3, and Mlkl phosphorylation and their total proteins, but decreased Caspase8 in LCA-induced cholestatic M-NSG mice administered human CD14⁺CD16⁺ monocyte injections (Fig. 4D; Fig. S14C). Elevated Mlkl membrane translocation in these mice confirmed necroptosis activation (Fig. 4E). In another model, which received mouse intermediate monocyte and non-intermediate monocyte transplantation, activation of the necroptotic signaling axis was also confirmed by Western blot analysis (Fig. S16A and B).

We also measured the hepatic levels of TNFα and FASL given that they are important inducers of cell necroptosis.²³ RT-qPCR showed that TNFα and FASL mRNAs were significantly higher in LCA-induced M-NSG mice infused with human CD14⁺CD16⁺ monocytes compared with control mice (Fig. 4F). Similar results were seen in the LCA-induced cholestatic mouse model when injected with mouse intermediate monocytes (Fig. S16C). To determine whether hepatocyte necroptosis arises from BA-mediated direct cytotoxicity, we stimulated primary mouse hepatocytes with taurocholic acid (TCA). However, BA treatment did not significantly alter Ripk1, Ripk3, or Mlkl protein levels (Fig. S17). Similarly, necroptotic markers (RIPK1, RIPK3, MLKL) were significantly increased in patients with OC or PBC (Fig. 4G and H; Fig. S18), indicating activated hepatic necroptosis in human cholestasis. The intermediate monocytes not only caused hepatocyte necroptosis, but also contributed to T-cell infiltration. We observed an elevation of T cells in a model infused with intermediate monocytes through immunofluorescence for T cell markers (Fig. S19). Interestingly, RT-qPCR analysis revealed that human CD14⁺CD16⁺ monocytes expressed higher levels of necroptotic inducers (TNFα and FASL) compared with other peripheral blood mononuclear cells (PBMCs) (Fig. 4I). Moreover, CD14⁺CD16⁺ monocyte numbers were positively correlated with levels of RIPK1 or MLKL mRNA transcripts in human cholestatic livers (Fig. 4J). Together, these results suggest that accumulation of CD14⁺CD16⁺ monocytes in the liver triggers secretion of TNFα and FASL to induce hepatocyte necroptosis in cholestatic liver.

Conjugated BAs induce chemokine CCL2 and CCL3 production to attract CD14⁺CD16⁺ monocyte infiltration in the liver during cholestasis

Chemokines regulate inflammatory cell migration via their receptors.²⁴ Currently, 49 chemokines are known to regulate this biological process, of which 12 facilitate monocyte chemotaxis (Fig. 5A).²⁵ To identify those regulating CD14⁺CD16⁺ monocyte migration in cholestasis, we analyzed their expression in patients with OC. RT-qPCR detected significant increases in CCL2, CCL3, CCL22, and CXCL17 mRNA transcripts but not in CCL1, CCL4, CCL5, CCL8, CCL14, CCL25, CXCL12, and CX3CL1 mRNA transcripts in the livers of patients with OC, compared with controls (Fig 5B; Fig. S20). Similarly, we also observed significant increases in Ccl2, Ccl3, and Cxcl17 mRNA transcripts but not Ccl22 mRNA transcripts in the livers of mouse models of cholestasis, including BDL-, 1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-, and LCA-induced cholestasis (Fig. 5C–E). Furthermore, RT-qPCR analysis revealed that treatment with conjugated BAs, including TCA, taurochenodeoxycholic acid (TCDCA), and taurodeoxycholic acid (TDCA), significantly enhanced the expression of Ccl2 and Ccl3 but not Cxcl17 in primary mouse hepatocytes (Fig. 5F).

Fig. 5 — **BAs stimulate CCL2 and CCL3 expression in hepatocytes to recruit the migration and accumulation of CD14⁺CD16⁺ monocytes in the liver.** (A) Chemokine and monocyte chemotaxis. (B) Real-time qPCR analysis of hepatic *CCL2, CCL3, CCL22,* and *CXCL1*7 mRNA levels; ∗p <0.05 *vs.* CTR patient group; CTR, n = 5; OC, n = 20. (C) Real-time qPCR analysis of hepatic *Ccl2, Ccl3, Ccl22*, and *Cxcl1*7 mRNA levels in bile-duct-ligated mouse models; ∗p <0.05 *vs.* Sham-operated mouse group; n = 4–5 per group. (D) Real-time qPCR analysis of hepatic *Ccl2, Ccl3, Ccl22*, and *Cxcl1*7 mRNA levels in 0.1% DDC diet mice; ∗p <0.05 *vs.* normal diet group; n = 4–6 per group. (E) Real-time qPCR analysis of hepatic *Ccl2, Ccl3, Ccl22*, and *Cxcl1*7 mRNA levels in LCA diet cholestatic mice; ∗p <0.05 *vs.* normal diet group; n = 4–6 per group. (F) Real-time qPCR analysis of hepatic *Ccl2, Ccl3*, and *Cxcl1*7 mRNA levels in primary mouse hepatocytes treated with 50 μM TCA, TCDCA, or TDCA; ∗p <0.05 *vs.* DMSO group; n = 3 per group. (G) Experimental procedures for Transwell migration assays with the co-culture of CD14⁺CD16⁺ cells and TCA (50 μM)-stimulated primary mouse hepatocytes *in vitro*; n = 3 per group. (H) Transwell assay analysis of the migratory ability of CD14⁺CD16⁺ cells co-cultured with primary mouse hepatocytes under treatment with DMSO, TCA, and TCA + anti-Ccl2/Ccl3 neutralizing antibodies; n = 3 per group. Data are mean ± SD, analyzed using the unpaired two-tailed Student’s t test, Welch's t test, or Mann-Whitney U test. BA, bile acid; CTR, control; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; LCA, lithocholic acid; OC, obstructive cholestasis; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid.

Next, we used a Transwell co-culture system to determine whether conjugated BAs induce Ccl2 and Ccl3 to attract CD14⁺CD16⁺ monocytes. TCA treatment significantly increased the number of migrated CD14⁺CD16⁺ monocytes that transmigrated across Transwell membranes toward primary mouse hepatocytes (Fig. 5G). However, this increase was significantly impaired in the presence of anti-Ccl2 or anti-Ccl3 neutralizing antibodies (Fig. 5H). Collectively, these findings indicate that conjugated BAs induce hepatocyte CCL2 and CCL3 expression to attract the infiltration of CD14⁺CD16⁺ monocytes in the liver.

Conjugated BAs enhance the activity of c-Jun binding to CCL2 and CCL3 promoters to induce their expression by JNK/MAPK signaling in cholestasis

To reveal the mechanisms underlying conjugated BA-induced CCL2 and CCL3 expression in cholestasis, we analyzed potential transcription factor-binding sites in the CCL2 and CCL3 promoters by conducting in silico analysis (of hTFtarget, ENCODE, CHEA, KnockTF, GTRD, and ChIP-Atlas) (Fig. 6A). RT-qPCR showed elevated hepatic c-Jun mRNA levels positively correlating with CCL2 and CCL3 in patients with OC (Fig. 6B and C), suggesting that c-Jun upregulates the expression of these chemokines. Interestingly, TCA induced the expression of c-Jun at the mRNA and protein levels as well as phosphorylation levels of JNK in a dose-dependent manner in primary mouse hepatocytes (Fig. 6D and E). Furthermore, ChIP assays evidenced that TCA significantly increased the binding of c-Jun to the Ccl2 or Ccl3 promoter (Fig. 6F and G). However, these increases were diminished in the presence of SP600125, a specific JNK inhibitor (Fig. 6H). Therefore, these data indicated that conjugated BAs induce CCL2 and CCL3 expression in hepatocytes during cholestasis by enhancing c-Jun expression and activity.

Fig. 6 — **c-Jun regulates CCL2 and CCL3 expression in hepatocytes during the pathogenic process of cholestasis.** (A) Strategies for predicting transcription factor-binding sites in the promoter region of *CCL2* and *CCL3* and for selection of the transcription factor c-Jun. (B) Real-time qPCR analysis of the relative levels of hepatic *c-Jun* mRNA transcripts; ∗p <0.05 *vs.* CTR patient group; CTR, n = 5; OC, n = 20. (C) Correlation analysis of the hepatic levels of c-Jun and *CCL2* and *CCL3* mRNA transcripts in patients with OC (n = 20). (D) Real-time qPCR analysis of hepatic *c-Jun* mRNA levels in primary mouse hepatocytes; ∗p <0.05 *vs.* DMSO group; n = 3 per group. (E) Relative protein levels for c-Jun and upstream JNK and p-JNK, in primary mouse hepatocytes exposed to DMSO or TCA (25 μM or 50 μM). (F,G) Assessment of chromatin immunoprecipitation assays for c-Jun binding to *Ccl2* and *Ccl3* promoters in primary mouse hepatocytes; ∗p <0.05 *vs.* DMSO group; n = 3 per group. (H) Treatment with the JNK inhibitor SP600125 (25 μM) attenuates BA-stimulated Ccl2 and Ccl3 expression in primary mouse hepatocytes; ∗p <0.05 *vs.* DMSO group; #p <0.05 *vs.* TCA (50 μM) group; n = 3 per group. Data are mean ± SD, analyzed using the unpaired two-tailed Student’s t test, Welch's t test, or one-tailed Mann-Whitney U test. Correlation analyses performed using Spearman’s rank tests. BA, bile acid; CTR, control; OC, obstructive cholestasis; TCA, taurocholic acid.

Discussion

The current study investigated the functional role and regulatory mechanisms of CD14⁺CD16⁺ monocytes in cholestatic liver injury. This study highlights four major findings: (1) CD14⁺CD16⁺ monocytes were significantly increased in the liver of patients with cholestasis (OC and PBC; Fig. 2F–I), and correlated positively with liver injury (Fig. 2J); (2) injection of mice with human and mouse intermediate monocytes aggravated cholestatic liver injury by promoting hepatocyte necroptosis via the Ripk1–Ripk3–Mlkl signaling pathway (Fig. 4); (3) depletion of mouse intermediate monocytes significantly alleviated LCA-induced injury; (4) conjugated BAs stimulated Ccl2 and Ccl3 expression in hepatocytes by enhancing c-Jun binding to their promoters, which triggered CD14⁺CD16⁺ monocyte chemotaxis (Fig. 5, Fig. 6). These findings indicated that conjugated BAs induced expression of Ccl2 and Ccl3 in hepatocytes by increasing the activity of c-Jun binding to their promoters, thereby promoting CD14⁺CD16⁺ monocyte infiltration and cholestatic liver injury aggravation (Fig. 7). Thus, targeting CD14⁺CD16⁺ monocyte infiltration into the liver could be a strategy for therapeutic intervention.

Fig. 7 — **Potential mechanisms by which conjugated BAs stimulate CCL2 and CCL3 expression, promoting CD14⁺CD16⁺ monocyte infiltration and triggering hepatocyte necroptosis in cholestasis disease.** In brief, BAs accumulated during cholestasis upregulate c-Jun, enhancing hepatocyte CCL2 and CCL3 expression. Subsequently, these chemokines recruit CD14⁺CD16⁺ monocyte infiltration. CD14⁺CD16⁺ monocytes secrete high levels of TNFα and FASL, activating the RIPK1–RIPK3–MLKL axis to induce hepatocyte necroptosis. BA, bile acid; FASL, factor-related apoptosis ligand; MLKL, mixed-lineage kinase domain-like protein; RIPK, receptor-interacting serine/threonine-protein kinase; TNFα, Tumor necrosis factor-alpha.

Many studies have highlighted the role of inflammatory responses in cholestatic liver injury.²⁶^,²⁷ Infiltrated macrophages also mediate liver inflammation and fibrosis.²⁸ However, the precise role of each type of inflammatory infiltrate in cholestatic liver injury has not been clarified. Here, we first identified a subpopulation of monocytes, CD14⁺CD16⁺ monocytes, which increased in the livers of patients with OC and PBC, but their function and regulatory mechanisms in cholestasis remained unknown. A previous study reported that CD14⁺CD16⁺ monocytes produce high levels of proinflammatory cytokines, such as TNFα,²⁹ in agreement with our results that CD14⁺CD16⁺ monocytes expressed high levels of TNFα and FASL compared with blood PBMCs (Fig. 4I). TNFα and FASL are important inducers of cell necroptosis,³⁰ and genetic ablation of the gene encoding Tnf receptor 1 in mice significantly attenuates hepatic necrosis in cholestasis.³¹ Our findings suggest that CD14⁺CD16⁺ monocytes induce hepatocyte necroptosis. Indeed, in LCA-induced cholestatic M-NSG mice, infusion with human CD14⁺CD16⁺ monocytes significantly increased hepatic necroptosis with elevated TNFα and FASL expression (Fig. 4F), corroborated by murine intermediate monocytes (Fig. S16). Via their receptors, chemokines are crucial for inflammatory cell migration.³² Currently, 49 chemokines have been identified, 12 of which can facilitate monocyte chemotaxis. Among these 12 monocyte-related chemokines, CCL2, CCL3, and CXCL17 were significantly elevated in patients with cholestasis and in cholestatic mice (Fig. 5B–E). Conjugated BAs stimulated Ccl2 and Ccl3 expression in primary mouse hepatocytes (Fig. 5F) and transmigration of CD14⁺CD16⁺ monocytes across the Transwell membranes in a co-culture system (Fig. 5G and H). Infusion of intermediate monocytes also promoted the infiltration of T cells. Interestingly, previous research demonstrated that hepatic accumulation of conjugated BAs also stimulated the expression of CCL2, CXCL2, and CXCL10 to attract neutrophils and CD8⁺ T-cell migration, resulting in hepatic inflammation during cholestasis.⁶^,³³ Moreover, conjugated BAs also induced hepatocyte pyroptosis in cholestasis through activation of TWEAK–TNFRSF12A signaling.³⁴ Chen et al.¹⁴ reported that Arid3a promoted macrophage liver accumulation and cholestatic liver injury by impairing efferocytosis. These findings highlight that conjugated BAs initiate the increase of chemokines in the liver, subsequently leading to the infiltration of inflammatory cells that cause hepatocyte death, resulting in cholestatic liver injury.

Molecularly, BAs can activate various signaling pathways that regulate downstream genes with distinct functions. Here, we observed activation of the JNK/c-Jun signaling pathway in BA-stressed hepatocytes, which stimulated Ccl2 and Ccl3 expression (Fig. 5, Fig. 6). Moreover, BAs also activate NFAT to induce the expression of CXCL2 and CXCL10.³⁵ These chemokines then attract inflammatory cells to trigger cell death and liver inflammation, resulting in liver injury (Fig. 7).³⁶^,³⁷ By contrast, BAs can also activate certain signaling pathways that attenuate liver injury by repressing chemokine expression. Previous research showed that accumulation of liver BAs activated the JAK/STAT3 pathway, which inhibited CCL2 and CXCL2 production by mediating RUNX1 expression.⁴ Interestingly, this STAT3 activation also upregulated hepatic SLC35C1 expression and facilitated CEACAM1 fucosylation, suppressing CCL2 and CXCL2 expression.³⁸ Together, these findings suggest that accumulation of BAs activates a variety of signaling pathways that both induce hepatocyte injury and activate adaptive responses to maintain hepatic homeostasis during cholestasis.

In conclusion, we showed that elevated CD14⁺CD16⁺ monocyte infiltration in cholestatic livers aggravates liver injury by promoting hepatocyte necroptosis. This is mediated by the induction of CCL2 and CCL3 expression in hepatocytes by conjugated BAs through the JNK/c-Jun signaling pathway. Therefore, blocking CD14⁺CD16⁺ monocyte infiltration into the liver could be a promising therapeutic strategy for treating cholestatic liver injuries.

Abbreviations

ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BA, bile acid; BDL, Bile-duct ligation; CTR, control; DC, dendritic cell; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; DEG, differentially expressed gene; FACS, fluorescence-activated cell sorting; FASL, factor-related apoptosis ligand; GO, Gene Ontology; GGT, gamma-glutamyl transferase; IF, immunofluorescence; LCA, lithocholic acid; M-NSG, NOD-Prkdc^scidIl2rg^em1/Smoc; MASLD, metabolic dysfunction-associated steatotic liver disease; MLKL, mixed-lineage kinase domain-like protein; MP, mononuclear phagocyte; mIF, multiplex immunofluorescence; NPC, non-parenchymal cells; OC, obstructive cholestasis; PBC, primary biliary cholangitis; PBMC, peripheral blood mononuclear cell; PI, propidium iodide; PSC, primary sclerosing cholangitis; RIPK, receptor-interacting serine/threonine-protein kinase; scRNA-seq, single-cell RNA sequencing; TBIL, total bilirubin; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; TNFα, tumor necrosis factor-alpha; UMAP, uniform manifold approximation and projection.

Financial support

This work was supported by the National Natural Science Foundation of China (82325008, 82422014, and 92268110), the Outstanding Medical Research Group of Chongqing (414Z381), and Open Project of Chongqing Engineering Research Center of Organ Intelligent Bio-Manufacturing (CQERC-OIBM20241010). The funders had no role in the study design, data collection and analysis, publication decisions, or manuscript preparation.

Authors’ contributions

Designed the experiments: JC. Contributed to reagents/materials/analysis tools: NZ, LL, XL, YL, ML, TR, LLiu, QP, JLB, S-YC. Performed the experiments: ZX, XC, JL, ZZ, NZ. Analyzed the data: ZZ, ZX. Wrote the manuscript: ZZ, ZX, XC, XZ. Critically revised the manuscript: JC Jin Chai, XZ, QP, S-YC.

Data availability

The raw sequencing data from this research have been deposited in the National Genomics Data Center (https://ngdc.cncb.ac.cn/gsa-human/) with the accession number HRA008337. Other data required to evaluate conclusions of this study are provided in the manuscript or the supplementary data. Raw datasets are available from the corresponding authors upon reasonable request.

Conflicts of interest

The authors declare that there are no conflicts of interest regarding the publication of this article. Please refer to the accompanying ICMJE disclosure forms for further details.

Acknowledgements

The authors are grateful to the patients for providing biomaterials and informed consent for research. We thank our team members (Cholestatic Liver Diseases Center and Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) Medical Research Center, The First Affiliated Hospital (Southwest Hospital) of Third Military Medical University (Army Medical University)) for their generous help.

Footnotes

Author names in bold designate shared co-first authorship.

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhepr.2025.101517.

Contributor Information

Shi-ying Cai, Email: shi-ying.cai@yale.edu.

Qiong Pan, Email: qiong.pan@cldcsw.org.

Xiaoxun Zhang, Email: xiaoxun.zhang@cldcsw.org.

Jin Chai, Email: jin.chai@cldcsw.org.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

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mmc2.docx^{(64.2KB, docx)}

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mmc3.pdf^{(10.4MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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mmc2.docx^{(64.2KB, docx)}

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Data Availability Statement

[bib1] 1.Li Y., Tang R., Leung P.S.C., et al. Bile acids and intestinal microbiota in autoimmune cholestatic liver diseases. Autoimmun Rev. 2017;16(9):885–896. doi: 10.1016/j.autrev.2017.07.002. [DOI] [PubMed] [Google Scholar]

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[bib4] 4.Zhang L., Pan Q., Zhang L., et al. Runt-related transcription factor-1 ameliorates bile acid-induced hepatic inflammation in cholestasis through JAK/STAT3 signaling. Hepatology. 2023;77:1866–1881. doi: 10.1097/HEP.0000000000000041. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib6] 6.Cai S.Y., Ouyang X., Chen Y., et al. Bile acids initiate cholestatic liver injury by triggering a hepatocyte-specific inflammatory response. JCI Insight. 2017;2(5) doi: 10.1172/jci.insight.90780. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib11] 11.Hanai H., Iida T., Takeuchi K., et al. Adsorptive depletion of elevated proinflammatory CD14+CD16+DR++ monocytes in patients with inflammatory bowel disease. Am J Gastroenterol. 2008;103(5):1210–1216. doi: 10.1111/j.1572-0241.2007.01714.x. [DOI] [PubMed] [Google Scholar]

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[bib14] 14.Chen R., Huang B., Lian M., et al. A+T rich interaction domain protein 3a (Arid3a) impairs Mertk-mediated efferocytosis in cholestasis. J Hepatol. 2023;79(6):1478–1490. doi: 10.1016/j.jhep.2023.08.016. [DOI] [PubMed] [Google Scholar]

[bib15] 15.Ghanbarinejad V., Jamshidzadeh A., Khalvati B., et al. Apoptosis-inducing factor plays a role in the pathogenesis of hepatic and renal injury during cholestasis. Naunyn Schmiedebergs Arch Pharmacol. 2021;394(6):1191–1203. doi: 10.1007/s00210-020-02041-7. [DOI] [PubMed] [Google Scholar]

[bib16] 16.Schwabe R.F., Luedde T. Apoptosis and necroptosis in the liver: a matter of life and death. Nat Rev Gastroenterol Hepatol. 2018;15(12):738–752. doi: 10.1038/s41575-018-0065-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Conjugated bile acid-driven CD14+CD16+ monocyte infiltration promotes cholestatic liver injury by enhancing hepatocyte necroptosis

Zhixian Zhu

Ziqian Xu

Xinyu Cao

Nan Zhao

Jiaxin Lei

Ling Li

Xi Li

Yan Li

Mingqiao Li

Tian Ren

Li Liu

James L Boyer

Shi-ying Cai

Qiong Pan

Xiaoxun Zhang

Jin Chai

Abstract

Background & Aims

Methods

Results

Conclusion

Impact and implications

Graphical abstract

Highlights

Introduction

Materials and methods

Human serum and liver samples

Statistical analysis

Results

Single-cell transcriptomic analysis of non-parenchymal cells in human livers from patients with OC and control patients

Fig. 1.

Numbers of hepatic CD14+CD16+ monocytes are positively correlated with severity of liver injury in patients with cholestasis

Fig. 2.

CD14+CD16+ monocyte infiltration significantly enhances cholestatic liver injury in a mouse model of cholestasis induced by an LCA diet

Fig. 3.

Hepatocyte necroptosis is significantly increased and positively correlated with CD14+CD16+ monocyte numbers in LCA-induced cholestatic mice or patients with cholestasis

Fig. 4.

Conjugated BAs induce chemokine CCL2 and CCL3 production to attract CD14+CD16+ monocyte infiltration in the liver during cholestasis

Fig. 5.

Conjugated BAs enhance the activity of c-Jun binding to CCL2 and CCL3 promoters to induce their expression by JNK/MAPK signaling in cholestasis

Fig. 6.

Discussion

Fig. 7.

Abbreviations

Financial support

Authors’ contributions

Data availability

Conflicts of interest

Acknowledgements

Footnotes

Contributor Information

Appendix A. Supplementary data

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Conjugated bile acid-driven CD14⁺CD16⁺ monocyte infiltration promotes cholestatic liver injury by enhancing hepatocyte necroptosis

Numbers of hepatic CD14⁺CD16⁺ monocytes are positively correlated with severity of liver injury in patients with cholestasis

CD14⁺CD16⁺ monocyte infiltration significantly enhances cholestatic liver injury in a mouse model of cholestasis induced by an LCA diet

Hepatocyte necroptosis is significantly increased and positively correlated with CD14⁺CD16⁺ monocyte numbers in LCA-induced cholestatic mice or patients with cholestasis

Conjugated BAs induce chemokine CCL2 and CCL3 production to attract CD14⁺CD16⁺ monocyte infiltration in the liver during cholestasis