Research ArticleHepatologyImmunologyInfectious disease Open Access | 10.1172/JCI175241

Juan Diego Sanchez Vasquez,1,2 Shirin Nkongolo,3,4 Daniel Traum,5,6 Valentin Sotov,7 Samuel C. Kim,8 Deeqa Mahamed,9 Aman Mehrotra,1 Anjali Patel,10 Diana Y. Chen,8 Scott Fung,1 Anuj Gaggar,8 Jordan J. Feld,1 Kyong-Mi Chang,5,6 Jeffrey J. Wallin,8 Ben X. Wang,7 Harry L.A. Janssen,11 and Adam J. Gehring1,2,12

1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

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1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

Find articles by Wallin, J. in: JCI | PubMed | Google Scholar

1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

Find articles by Wang, B. in: JCI | PubMed | Google Scholar

1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

Find articles by Janssen, H. in: JCI | PubMed | Google Scholar

1Toronto Centre for Liver Disease, Toronto General Hospital Research Institute, and

2Schwartz Reisman Liver Research Centre, University Health Network, Toronto, Ontario, Canada.

3Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany.

4German Center for Infection Research (DZIF), partner site Heidelberg, Germany.

5Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

6Medical Research, The Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania, USA.

7Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

8Gilead Sciences, Foster City, California, USA.

9Centre for Advanced Single Cell Analysis, The Hospital for Sick Children, Toronto, Canada.

10St. Michael’s Hospital, Toronto, Canada.

11Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands.

12Department of Immunology, University of Toronto, Toronto, Canada.

Address correspondence to: Adam J. Gehring, University Health Network, PMCRT, Room 2-306, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. Phone: 416.634.7095; Email: Adam.gehring@uhn.ca.

Find articles by Gehring, A. in: JCI | PubMed | Google Scholar |

Published April 15, 2025 - More info

Published in Volume 135, Issue 8 on April 15, 2025
J Clin Invest. 2025;135(8):e175241. https://doi.org/10.1172/JCI175241.
© 2025 Vasquez et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published April 15, 2025 - Version history
Received: August 29, 2023; Accepted: February 17, 2025 View PDF Abstract

Chronic liver injury triggers the activation and recruitment of immune cells, causing antigen-independent tissue damage and liver disease progression. Tissue inflammation can reshape macrophage composition through monocyte replacement. Replacement of tissue macrophages with monocytes differentiating in an inflammatory environment can potentially imprint a phenotype that switches the liver from an immune-tolerant organ to one predisposed to tissue damage. We longitudinally sampled the liver of patients with chronic hepatitis B who had active liver inflammation and were starting antiviral therapy. Antiviral therapy suppressed viral replication and liver inflammation, which coincided with decreased myeloid activation markers. Single-cell RNA-Seq mapped peripheral inflammatory markers to a monocyte-derived macrophage population, distinct from Kupffer cells, with an inflammatory transcriptional profile. The inflammatory macrophages (iMacs) differentiated from blood monocytes and were unique from macrophage found in healthy or cirrhotic liver. iMacs retained their core transcriptional signature after inflammation resolved, indicating inflammation-mediated remodeling of the macrophage population in the human liver that may affect progressive liver disease and immunotherapy.

Graphical Abstract Introduction

Macrophages play a pivotal role in tissue immunity, representing one of the first lines of defense against pathogens. These cells regulate tissue damage via pathogen clearance and secretion of cytokines, chemokines, and growth factors to avoid unchecked inflammation (1). The macrophage pool is highly heterogeneous, consisting mainly of self-renewing, embryo-derived macrophages that seed the organs during fetal development and monocyte-derived macrophages recruited from the blood upon tissue damage or infection (2, 3). Mouse studies have revealed that monocyte recruitment and imprinting are tissue specific, allowing these cells to differentiate into either long-lived or short-lived macrophages (4–6).

During acute infections, reshaping the macrophage population through an influx of monocyte-derived macrophages improves pathogen clearance and the return to baseline (7). However, more than 800 million people live with chronic liver disease (CLD), which is characterized by persistent inflammation, leading to over 2 million deaths per year from cirrhosis and hepatocellular carcinoma (HCC) (8, 9). In mouse models of liver inflammation,macrophages contribute to disease progression by producing inflammatory cytokines that activate surrounding intrahepatic cells, driving inflammation and tissue injury and causing progressive fibrosis that impairs liver regeneration (8). These inflammatory events can be ameliorated by macrophage depletion in experimental models, underscoring their significant role in tissue regulation (3, 10).

Macrophage-mediated inflammation presents a stark contrast to the steady-state environment of the liver, which is one of tolerance and immune suppression. The tolerogenic environment is maintained via IL-10 production from Kupffer cells (KCs), which are embryonically derived macrophages of the liver (11, 12). This suggests that during CLD, the KC niche is being reshaped by monocyte-derived macrophages that are more inflammatory. This could change the dynamics of immune regulation in the liver and contribute to chronic liver damage, as has been observed in mouse models (10, 13–15).

Understanding how inflammation reshapes liver macrophage composition in humans has been restricted by the ability to collect longitudinal tissue samples within a time frame relevant to inflammation dynamics. However, patients with chronic HBV infection who present to the clinic with active liver damage are started on antiviral therapy. Antiviral therapy suppresses HBV replication and stops liver damage within 6 months. Once started, antiviral treatment can be life-long to maintain suppression of viral replication. Withdrawal of therapy can lead to HBV reactivation and potentially life-threatening liver inflammation that is characterized by a macrophage signature (16–19). Using longitudinal liver fine-needle aspirates (FNAs), we captured dynamic changes in cellular composition and activation during the first 6 months of therapy (20). By combining liver FNA sampling in patients starting antiviral therapy with single-cell RNA-Seq (scRNA-Seq), we achieved a resolution necessary to define heterogeneous macrophage populations and their activation status in the liver of patients with chronic hepatitis B (CHB). This led to the identification of an inflammatory, monocyte-derived macrophage population unique to inflamed livers. These monocyte-derived macrophages were imprinted with a transcriptional profile that was distinguishable even after 4 years of antiviral therapy.

Results

Serum markers of myeloid activation map to liver macrophages. To investigate the cellular dynamics of liver inflammation and damage in CLD, we recruited 15 patients with CHB who had ongoing liver damage, as evidenced by elevated levels of serum alanine aminotransferase (ALT) represented as a fold increase over normal values (upper limit of normal [ULN) >1). Patients started antiviral treatment with tenofovir alafenamide (TAF) (25 mg daily), which reduced liver damage and hepatitis B virus (HBV) replication (21) (Figure 1A). We collected liver FNAs at study entry and 12 and 24 weeks after TAF initiation and performed scRNA-Seq in 5 patients (red lines) to link inflammatory biomarkers in the serum to activation of immune cells in the liver (Figure 1B). This allowed us to compare intrahepatic immune profiles during liver injury (baseline) and as inflammation resolved (weeks 12 and 24).

Figure 1

Study design and identification of hepatic cell populations at single-cell resolution from liver FNAs. (A) HBV DNA and ALT (displayed as fold change [FC] of the ULN) in blood over time, with patients whose samples were sequenced highlighted in red (n = 5). (B) Eleven CHB patients with elevated ALT levels started NUC therapy with TAF (25 mg/d). At baseline and after 12 and 24 weeks of therapy, blood and liver FNAs were collected. Longitudinal FNAs from 5 patients were subjected to scRNA-Seq. (C) Clustering and annotation of 41,829 cells from human livers for 5 patients across 3 time points (baseline, week 12, and week 24). Uniform manifold approximation and projection (UMAP) dimensionality reduction identified 30 clusters. (D) Luminex data from plasma immune profiles of all patients across time. (E) Feature plots depicting single-cell gene expression of individual genes detected by the Luminex assay. (F) Feature plots depicting single-cell gene expression of liver myeloid cells. P values were determined by repeated-measures 1-way ANOVA (*P < 0.05, **P < 0.005, and ***P < 0.001).

Among the 41,829 cells across the 3 longitudinal time points, we identified 30 distinct cell clusters that had a similar distribution among the different donors (Figure 1C and Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI175241DS1). We previously demonstrated that the myeloid activation marker soluble CD163 (sCD163) was significantly increased during liver damage across multiple stages of HBV infection (19). Analysis of serum immune markers in our patient cohort confirmed elevated levels of sCD163 in the serum of patients with hepatitis that significantly declined after 12 weeks of TAF therapy. Consistent with the decline in sCD163, additional myeloid activation markers — IL-18 and galectin-9 — also significantly decreased after starting therapy (Figure 1D). Using the transcriptome data, we mapped these markers to the monocyte and macrophage clusters, defined by the expression of CD68 (monocyte and macrophage marker), C1QA/B/C (macrophage marker), VCAN (classical monocyte marker), and FCGR3A (nonclassical monocyte marker) (Figure 1, E and F) (22–24). These data validate our previous observations and implicate monocytes and macrophages as potential key players in the pathogenesis of liver injury in CHB (19).

Inflammatory macrophages are phenotypically distinct from KCs. Because serum markers suggested myeloid involvement, CD68+ cells were subclustered for further analysis. This led to the identification of 2 classical monocyte clusters (expression of VCAN, LYZ, and CD14), 1 nonclassical monocyte cluster (CD16+ monocytes; FCGR3A [CD16a], SIGLEC10, and PECAM1); and 2 macrophage clusters (Liver Mac 1 and Liver Mac 2; C1QA [complement component]; SLC40A1 [ferroportin]; and MARCO) (Figure 2, A and B) (4, 5, 22–25).

Figure 2

Identification and characterization of myeloid cells during liver inflammation. (A) CD68+ clusters were reclustered using UMAP dimensionality reduction for 5 patients across 3 time points (baseline, week 12, and week 24). (B) Feature plots depicting single-cell gene expression of individual myeloid genes (scale: log-transformed gene expression). (C) Violin plots of macrophage-defining genes. All selected genes have an adjusted P value of less than 0.05. (D) IMC depicting liver macrophages and CD8+ T cell colocalization in inflamed livers of patients with CHB. The portal region is outlined by yellow dotted lines. (E) Enrichment of liver macrophages depicted by the ratio of the portal divided by the lobular cell count/mm2 between inflamed (n = 28) and noninflamed (n = 6) liver tissue sections. Lobular enrichment values were multiplied by –100. P values were determined by a nonparametric Mann-Whitney U test (*P < 0.05, **P < 0.005, and ***P < 0.001). (F and G) Simple correlation analysis between portal CD8+ T cells/mm2 and (F) serum ALT levels and (G) iMacs/mm2 in patients with CHB. (H) Proximity analysis of CD8+ T cells and liver macrophages within 15 μm between inflamed (n = 28) and noninflamed (n = 6) liver tissue sections. P values were determined by nonparametric Wilcoxon test (*P < 0.05, **P < 0.005, and ***P < 0.001). (I) Multiplex IF images showing liver macrophages in inflamed liver of patients with CHB at inflammatory foci and noninflamed regions (n = 4). Original magnification, ×20. (J) Quantification of liver macrophages in inflamed liver of patients with CHB at inflammatory foci and noninflammatory regions. P values were determined by nonparametric Wilcoxon test (*P < 0.05, **P < 0.005, and ***P < 0.001).

Both macrophage clusters expressed CD68 and FCGR3A (CD16a), but Liver Mac 1 could be distinguished by the lack of CD14 expression (Figure 2C, first column). Liver Mac 2 expressed markers of embryonically derived macrophages such as TIMD4 (26, 27), CETP, and CD163 (Figure 2C, second panel). In contrast, Liver Mac 1 was enriched for markers of recent monocyte-to-macrophage differentiation (ZFP36L1, IRF8, and MAFB) (4, 5, 28, 29) (Figure 2C, third panel), immune activation (IL18, galectin-9 [LGALS9], and BAFF [TNFSF13B]) (30–32) (Figure 2C, fourth panel), and IFN signaling (IFI27, IFIT3, and GBP5) (33, 34) (Figure 2C, fifth panel). These data demonstrate the coexistence of multiple macrophage populations during active liver damage in patients with CHB. Based on the transcriptional profiles, Liver Mac 1 and Liver Mac 2 are referred to hereafter as inflammatory macrophages (iMacs) and KCs, respectively.

Consistent with the transcriptional differences for each macrophage population, their predicted functional profiles differed by gene set enrichment analysis (GSEA) (35). We performed GSEA between baseline and week 24. We found that processes related to inflammation, IFN signaling, and differentiation were enriched in the iMacs at baseline (Supplemental Figure 2A). KCs were also enriched in IFN signaling and antigen presentation but differed by pathways