CCR8 orchestrates an immunosuppressive niche in the liver to promote Echinococcus multilocularis infection

Jiao Hou; Haining Fan

doi:10.1371/journal.pntd.0014018

Abstract

Echinococcus multilocularis (E. m) infection causes alveolar echinococcosis (AE), a serious zoonotic disease characterized by invasive larval growth in the liver. The parasite establishes a chronic infection, suggesting effective modulation of host immunity. Here, we investigated the role of the CCR8/CCL1 chemokine axis in shaping the hepatic immune microenvironment during E.m infection. In infected wild-type (WT) mice, chronic infection specifically activated the hepatic CCR8/CCL1 axis, which was associated with a marked accumulation of FOXP3⁺ regulatory T cells (Tregs). Notably, although CCR8⁺ T cells expanded numerically, their production of effector (IFN-γ, TNF-α, and perforin) was significantly impaired. In contrast, infected CCR8-knockout (KO) mice developed smaller hepatic lesions, exhibited a reduction in liver weight, and had significantly lower serum ALT levels. Mechanistically, CCR8 deficiency enhanced the effector functions of CD4⁺ and CD8⁺ T cells, skewing the immune response towards a Th1 phenotype, and partially reversed the immunosuppressive milieu. Our findings establish that the CCR8/CCL1 axis drives the formation of an immunosuppressive niche in the liver by recruiting both Tregs and functionally suppressed CCR8⁺ T cells, thereby facilitating parasite immune evasion. This study not only elucidates a pivotal mechanism of immune escape in AE but also identifies CCR8 as a promising novel immunotherapeutic target for this neglected tropical disease.

Author summary

The parasite Echinococcus multilocularis causes a devastating liver disease, forming tumor-like growths that are difficult to treat. A major reason for its success is its ability to “switch off” the body’s immune defenses in the liver. We investigated a specific communication signal in cells, known as the CCR8/CCL1 pathway, to understand how the parasite achieves this. We discovered that the parasite activates this pathway, which acts like a homing beacon for the liver. This beacon attracts two types of problematic cells: powerful immune-suppressing cells (Tregs) and, surprisingly, normal immune fighter cells (T cells) that become functionally impaired. While these fighter cells are present in large numbers, they lose their ability to attack the parasite. When we genetically removed the CCR8 signal in mice, the immune system fought back effectively. These mice had much healthier livers and smaller parasite lesions because their immune cells remained active and potent. Our work shows that blocking the CCR8 pathway can reverse the parasite’s immune-suppressing tricks, revealing a promising new strategy for treating this serious disease.

Citation: Hou J, Fan H (2026) CCR8 orchestrates an immunosuppressive niche in the liver to promote Echinococcus multilocularis infection. PLoS Negl Trop Dis 20(3): e0014018. https://doi.org/10.1371/journal.pntd.0014018

Editor: Chao Yan, Xuzhou Medical University, CHINA

Received: November 21, 2025; Accepted: February 9, 2026; Published: March 6, 2026

Copyright: © 2026 Hou, Fan. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data are in the manuscript and/or supporting information files.

Funding: This study was supported by National Key Clinical Specialty Discipline Construction Program of China (Document No. QWJB-125 to HF) and “Kunlun Talent · Qinghai Scholar” Program of Qinghai Province (2024 to HF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Alveolar echinococcosis (AE) is a severe zoonotic parasitic disease caused by the metacestode larvae of Echinococcus multilocularis (E. m). It is listed by the World Health Organization as a neglected tropical disease of focus [1, 2]. The disease is widely distributed in the Northern Hemisphere, imposing a heavy burden on public health and the economy in endemic areas, including central and western regions of China. AE primarily affects the liver, where the larvae grow invasively by exogenous budding, forming multilocular vesicles. Its pathological features resemble those of malignant tumors, hence the term “worm cancer” [3,4]. AE has a long latent period, a slowly progressive but highly lethal course, with a 10-year mortality rate exceeding 90% in untreated patients [1,5]. The first-line drug, albendazole, only inhibits parasite growth rather than achieving parasiticidal effects, and long-term use carries side effects such as hepatotoxicity, with limited efficacy in advanced patients [4,6]. Therefore, in-depth investigation of the immunopathogenic mechanisms of AE, particularly the interaction between the parasite and the host immune system, is crucial for developing new therapeutic strategies.

The pathogenicity of AE results from the combined effects of direct parasite destruction and the host immune response [7]. During the chronic infection stage, the parasite employs various immune evasion mechanisms to skew the host immune response towards a Th2 type and establishes a microenvironment characterized by immune tolerance, in which regulatory T cells (Tregs) are considered to play a key role [7–10]. Indeed, multiple studies have confirmed significant enrichment of Tregs in the local lesions of AE patients and infected animal models, correlating with disease progression [7, 8, 11]. However, within the complex liver microenvironment, the precise recruitment of Tregs to the parasite lesion site and their subsequent functions remain incompletely understood.

The chemokine system is a key network regulating immune cell migration and positioning. Among these, the chemokine axis composed of CCR8 and its primary ligand CCL1 has been demonstrated in models such as cancer [12,13] and autoimmune disease [14] to guide Tregs to the site of pathology, forming an immunosuppressive microenvironment. In certain parasitic diseases, such as Schistosoma mansoni infection, CCR8 plays a critical role [15]. However, in chronic parasitic diseases like AE that target the liver, whether and how the CCR8/CCL1 axis regulates the local immune microenvironment has not been systematically investigated.

Based on this background, we hypothesize that in chronic E. m infection, activation of the CCR8/CCL1 axis is a key event driving the recruitment of Tregs to liver lesions and establishing an immunosuppressive microenvironment. Therefore, this study aims to address the following key questions: (1) Does E. m infection activate the hepatic CCR8/CCL1 axis? (2) Does CCR8 mediate the recruitment of Tregs and the formation of the immunosuppressive microenvironment? (3) Can targeting CCR8 enhance anti-parasitic immunity and alleviate disease progression? To test this hypothesis, we utilized WT and CCR8 gene knockout mouse models combined with multidisciplinary technical approaches to systematically elucidate the role and mechanism of the CCR8/CCL1 axis in AE liver immunopathology, aiming to provide new targets and a theoretical basis for AE immunotherapy.

Materials and methods

Ethics statement

This study was approved by the Ethics Committee of the Clinical Medical College of Qinghai University (No.P-SL-2023–461), and all procedures were conducted in accordance with the relevant regulations.

Experimental animals and infection model

This study used 6–8 week-old, weight-matched wild-type (WT) mice and CCR8 knockout (CCR8-KO) mice. CCR8-KO mice were purchased from Shanghai Model Organisms Center, Inc. (Shanghai, China), and genotyped by PCR. Female C57BL/6J wild-type mice (6 – 8 weeks old, 18 –22 g) were purchased from Jiangsu Huachuang Sino Pharma Tech Co., Ltd. (Nanjing, China). All experiments were conducted using age-matched female mice. Mice were housed in a specific pathogen-free (SPF) environment with free access to food and water. A liver infection model was established by portal vein injection, with each mouse receiving 4 × 10³ viable E. m protoscoleces (PSC). Control (CON) mice were injected with an equal volume of sterile physiological saline. Experimental groups and sample sizes (n) for each group are specified in the respective figure legends.

Sample size justification

The sample size of n = 5 mice per group was determined based on: (1) Established precedent in murine models of chronic hepatic infection and immunology for similar endpoints [16]; (2) Ethical adherence to the 3Rs principle (Reduction) to minimize animal use while ensuring scientific validity; and (3) Post-hoc statistical power analysis of a key mechanistic finding. Specifically, digital image analysis of hepatic CCL1 immunohistochemical staining at 24 weeks post-infection revealed a profound difference between WT and CCR8-KO infected mice (WT: 9.5 ± 1.16% vs KO: 3.95 ± 0.84%). The effect size for this difference is Cohen’s d = 5.51. With this effect size, α = 0.05, and n = 5 per group, the achieved statistical power exceeds 99.99%, confirming that our sample size was more than adequate to detect biologically significant effects.

Sample collection and pathological assessment

Mice were euthanized at 2, 12, and 24 weeks post-infection (wpi), and serum and liver tissues were collected. Liver wet weight was accurately measured, and the liver-to-body weight ratio (liver weight/body weight × 100%) was calculated. A significant increase in liver weight and the liver-to-body weight ratio directly reflects the substantial hepatomegaly caused by parasite infiltration and associated inflammation/fibrosis, serving as an indirect indicator of disease severity and the burden of space-occupying lesions caused by the parasite. Commercial alanine aminotransferase (ALT) and aspartate aminotransferase (AST) detection kits(Nanjing Jiancheng Bioengineering Institute, Cat: ALT C009-2–1; AST C010-2–1) were used according to the manufacturer’s instructions to measure serum ALT and AST activities, assessing the degree of hepatocyte damage.

Ultrasonographic analysis

Livers of anesthetized live mice were scanned using a high-resolution small animal ultrasound imaging system. All ultrasound image analyses were performed independently by at least two researchers blinded to the experimental groups. Given the irregular morphology of AE lesions, we employed a standardized prolate ellipsoid approximation formula widely used in tumor volumetry for comparative analysis. The maximum length (L) and transverse diameter (W) of the lesions were measured, and the lesion volume (V) was estimated as V = 1/2 × L × W² [17,18]. This method provides a reproducible and quantitative metric for the relative comparison of lesion burden between experimental groups.

Quantitative Real-Time PCR (qPCR)

Total RNA was extracted from liver tissue using TRIzol (Invitrogen, Cat 15596026CN) reagent and reverse-transcribed into cDNA. Amplification was performed using SYBR Green premix (Takara, Cat RR820A) on a quantitative PCR instrument. Genes detected included CCR8, CCL1, and FOXP3. GAPDH was used as the reference gene, and the relative mRNA expression levels of target genes were calculated using the 2^{^–ΔΔCt} method. Primer sequences are listed in S1 Table.

Immunohistochemistry and Immunofluorescence

Liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections underwent routine deparaffinization, rehydration, and antigen retrieval. Subsequently, sections were incubated overnight with primary antibodies against CCR8 (Proteintech, 1:100), CCL1 (Abmart, 1:32), and FOXP3 (eBioscience, 1:100), respectively. IHC sections were developed using HRP-conjugated secondary antibodies and DAB chromogen (Abcam), observed under a light microscope, and images were captured. The percentage of positively stained area was analyzed using ImageJ software. For immunofluorescence co-localization experiments, sections were co-incubated with anti-FOXP3 (eBioscience, 1:100) and anti-CCR8 (Proteintech, 1:100) antibodies, followed by corresponding secondary antibodies labeled with different fluorophores. After mounting, sections were observed and photographed under a laser scanning confocal microscope.

Flow cytometry

Mouse liver tissues were mechanically dissociated, and lymphocytes were enriched by Percoll (Biotopped, Cat P1701H) density gradient centrifugation. To detect cytokines, cells were re-stimulated ex vivo for 4 hours with a cell stimulation cocktail (PMA/ionomycin plus Brefeldin A and Monensin, eBioscience, Cat 00-4975-93). After Fc receptor blocking (anti-CD16/32) (Refer to S2 Table for specific antibody clone and catalog numbers), cells were first stained with surface antibodies (against CD45, CD3, CD4, CD8, CD25, CCR8), followed by fixation, permeabilization (Biolegend), and intracellular/nuclear staining with antibodies (against FOXP3, IFN-γ, TNF-α, IL-2, IL-4, IL-17A, IL-10, TGF-β, Granzyme B, Perforin). Gating for CCR8⁺ cells was based on fluorescence-minus-one (FMO) controls. Data were acquired using a flow cytometer and analyzed subsequently with FlowJo_V10.8.1 software. The gating strategy for the CCR8⁺ subsets is detailed in S1 Fig.

Statistical analysis

All data are presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9.5 software. All datasets were first tested for normality using the Shapiro-Wilk test. Comparisons between two groups meeting normal distribution were performed using Student’s t-test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test, which inherently corrects for multiple comparisons. If heterogeneity of variance is present, a non-parametric test should be employed. Paired samples were analyzed using the paired t-test. P < 0.05 was considered statistically significant (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, P > 0.05). The specific P-values for all data are detailed in S1 Data.

Results

E. multilocularis infection induces progressive liver pathological damage in wild-type mice

To assess the direct impact of E. m infection on the liver, we first monitored the hepatic pathological phenotypes in infected mice. Compared to the uninfected control (CON) group, the absolute liver weight of infected wild-type (WT) mice was significantly increased at 12 and 24 weeks post-infection (wpi) (Fig 1A). The liver-to-body weight ratio, calculated to account for body weight effects, was significantly higher than the control group at all time points post-infection (2, 12, 24 wpi) (Fig 1B), indicating substantial hepatomegaly. Consistent with these pathological changes, the levels of serological markers of liver injury – alanine aminotransferase (ALT) and aspartate aminotransferase (AST) – generally showed an increasing trend during the infection course, with statistically significant differences observed at multiple time points (Fig 1C). Together, these data demonstrate that E. m infection successfully induced a model of chronic progressive liver injury in wild-type mice.

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Fig 1. E. multilocularis infection induces progressive liver pathological damage in wild-type mice.

(A) Comparison of absolute liver weight between infected wild-type (WT) mice and uninfected control (CON) group at different time points post-infection (2, 12, 24 weeks). (B) Liver-to-body weight ratio (liver weight/body weight × 100%). (C) Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity. Data are presented as mean ± SD, n = 5 mice/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, P > 0.05. Uninfected control group vs infected WT group.

https://doi.org/10.1371/journal.pntd.0014018.g001

The hepatic CCR8/CCL1 chemokine axis and FOXP3 pathway are specifically activated in the late stage of infection

We next investigated molecular pathways related to immune cell recruitment and functional regulation. Immunohistochemical staining showed that in the late stage of infection (24 wpi), the percentage area positive for the chemokine receptor CCR8, its ligand CCL1, and the Treg key transcription factor FOXP3 was significantly higher in the liver tissues of infected WT mice compared to the uninfected control group (Fig 2A-B). At the gene transcription level, quantitative real-time PCR results confirmed that the relative mRNA expression of CCR8, CCL1, and FOXP3 in the liver was significantly upregulated at 24 wpi; analysis of earlier time points indicated that this upregulation was time-dependent and most prominent in the late stage of infection (Fig 2C). Notably, using immunofluorescence co-localization, we observed FOXP3 and CCR8 co-expressed on infiltrating lymphocytes surrounding the liver lesions, with clear co-localization signals visible under high-magnification fields (Fig 2D). This provides direct morphological evidence for the role of CCR8 in recruiting or stabilizing Tregs at the infection site.

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Fig 2. E. multilocularis infection activates the hepatic CCR8/CCL1 axis and induces FOXP3 expression in the late stage.

(A) Representative immunohistochemical images showing protein expression of CCR8, CCL1, and FOXP3 in liver tissue at 24 weeks post-infection (400x magnification). (B) Quantitative analysis of the positive staining area (percentage). (C) qPCR analysis of the relative mRNA expression levels of CCR8, CCL1, and FOXP3 in the liver at different time points post-infection (normalized to GAPDH). (D) Immunofluorescence double staining shows co-localization of FOXP3 (green) and CCR8 (red) in lymphocytes surrounding liver lesions in the late stage of infection; DAPI (blue) stains nuclei, with an enlarged view. Data are presented as mean ± SD, n = 5 mice/group, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, P > 0.05. Uninfected control group vs infected WT group.

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CCR8⁺ T lymphocytes expand in the infected liver and exhibit a functionally suppressed phenotype

Based on the above findings, we used flow cytometry to deeply analyze the dynamics of liver T lymphocytes. Analysis revealed that the proportion of CD4⁺ CD25⁺ FOXP3⁺ Tregs in the livers of infected WT mice was significantly increased in the late stage of infection (24 wpi). More importantly, within the Treg population, the subset expressing CCR8 (CD4⁺ CCR8⁺ Tregs) showed significant expansion both in the early (2 wpi) and late (24 wpi) stages of infection (Fig 3A). This expansion was not limited to Tregs. Analysis of the total T cell pool indicated that the proportions of various T cell subsets carrying CCR8, including CD3⁺ CCR8⁺, CD4⁺ CCR8⁺, and CD8⁺ CCR8⁺ T cells, were significantly higher than the control group in the mid and late stages of infection (12 and 24 wpi) (Fig 3B). We first observed that the frequencies of hepatic CCR8 ⁺ T cells in uninfected control mice exhibited dynamic changes. Notably, the CD4 ⁺ CCR8 ⁺ subset peaked at 12 weeks post-infection (33.37 ± 6.98%), a level significantly higher than at 2 weeks (5.60 ± 2.10%) and 24 weeks (3.49 ± 0.86%) (P < 0.01), suggesting regulation by age- or development-associated factors. Superimposed on this physiological fluctuation, chronic infection induced more pronounced and sustained alterations. At both 12 and 24 weeks, the proportions of all CCR8 ⁺ T cell subsets (CD3 ⁺ , CD4 ⁺ , CD8⁺) in infected mice were significantly elevated compared to their time-matched controls (e.g., CD4 ⁺ CCR8⁺ at 12 weeks: 56.10 ± 8.80% vs 33.37 ± 6.98%, P < 0.01). Of critical importance, at 24 weeks, when the frequencies in controls had declined, they remained persistently high in the infected group (e.g., CD4 ⁺ CCR8 ⁺ : 6.40 ± 1.19% vs. 3.49 ± 0.86%, P < 0.01), indicating that the infection not only amplified a physiological wave but also abrogated its normal resolution. Further analysis of the subset composition of the CCR8⁺ T cell population revealed that within the CCR8-positive CD3⁺ T cell population in the liver, the proportion of CD4⁺ T cells was significantly lower than in the CCR8-negative population, suggesting an association between CCR8 expression and T cell subset distribution (Fig 3C). However, this numerically expanded CCR8⁺ T cell population exhibited a markedly suppressed functional state. By assessing their effector function through intracellular cytokine staining, we found that compared to the CCR8-negative population, CCR8-positive CD4⁺ and CD8⁺ T cells generally had a significantly reduced capacity to secrete key cytotoxic mediators (like perforin) and pro-inflammatory cytokines (including IFN-γ, TNF-α, and IL-2) (Fig 3D, Fig 4A). Functional analysis further revealed the immune polarization characteristics of CCR8⁺ T cells: they exhibited a higher tendency to express IL-4 (Th2-type) and immunoregulatory factors (IL-10, TGF-β1), while Th1/Th17-related functions were relatively diminished (Fig 4B).

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Fig 3. CCR8⁺ T cell subsets expand and exhibit a functionally suppressed phenotype in the liver after E. multilocularis infection. (A) Flow cytometric analysis showing the proportions of CD4⁺ CD25⁺ FOXP3⁺ Tregs and their CCR8 + subset in the liver at different time points post-infection. (B) Proportions of CCR8⁺ cells among total CD3⁺, CD4⁺, and CD8⁺ T cells. (C) Comparison of CD4⁺ and CD8⁺ subset distribution between CCR8⁺ and CCR8^- T cells. (D) Comparison of Granzyme B and Perforin expression between CCR8⁺ and CCR8^- CD4⁺ and CD8⁺ T cells. Data are presented as mean ± SD, n = 5 mice/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, P > 0.05. (A)-(B) is compared between the uninfected control group and infected WT groups.

https://doi.org/10.1371/journal.pntd.0014018.g003

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Fig 4. CCR8⁺ T cells exhibit impaired effector function and a regulatory polarization profile. (A) Comparison of perforin, IFN-γ, TNF-α, and IL-2 expression between CCR8⁺ and CCR8^- CD4⁺ and CD8⁺ T cells. (B) Differences in the expression of Th1/Th2/Th17 and related regulatory cytokines (IL-4, IL-17A, IL-10, TGF-β1) between the two cell populations. Data are presented as mean ± SD, n = 5 mice/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, P > 0.05.

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CCR8 deficiency alleviates liver pathological damage by enhancing anti-parasitic immune responses

To functionally confirm the pathogenic role of CCR8, we conducted studies using CCR8 knockout (KO) mice. In vivo ultrasonographic assessment showed that at 12 wpi, the size of parasitic lesions in the livers of infected KO mice was significantly smaller than in the infected WT group (Fig 5A-B). Given that parasitic lesions are the primary cause of hepatomegaly in infected mice, we used liver weight measurements to assess the overall parasite load and pathological damage indirectly. At the macroscopic pathological level, compared to the infected WT group, infected KO mice showed significantly reduced absolute liver weight and standardized liver-to-body weight ratio in the mid and late stages of infection (12 and 24 wpi) (Fig 5C). This result strongly suggests that the absence of CCR8 signaling effectively controlled parasite growth and infiltration, thereby reducing the burden of space-occupying lesions in the liver. Correspondingly, serological markers of liver injury also improved, with infected KO mice showing significantly lower ALT levels than the infected WT group in the late stage of infection (Fig 5D). At the molecular level, both immunohistochemical and qRT-PCR analyses confirmed that the expression levels of CCL1 and FOXP3 in the livers of infected KO mice were significantly lower than in the infected WT group (Fig 5E-G). These phenotypic data clearly demonstrate that the absence of CCR8 signaling effectively curtails the liver pathological damage induced by E. m infection.

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Fig 5. CCR8 deficiency alleviates E. multilocularis-induced liver pathological damage and downregulates immunosuppressive molecule expression.

(A) Representative in vivo liver ultrasound images of infected wild-type (WT) and CCR8 knockout (KO) mice at 12 weeks post-infection. Black arrows indicate parasitic lesions. (B) Quantitative analysis of lesion volume based on ultrasound images (n = 5 mice/group). (C) Absolute liver weight (left) and liver-to-body weight ratio (right, liver weight/body weight × 100%) at 12 and 24 weeks post-infection. The significant reduction in liver weight and liver-to-body weight ratio indirectly reflects the reduced burden of space-occupying lesions in infected CCR8-KO mice due to controlled parasite infiltration and growth. (D) Serum ALT levels at different time points post-infection. (E-F) Representative immunohistochemical images and quantitative analysis showing protein expression of CCL1 and FOXP3 in the livers of infected WT and KO mice at 24 weeks post-infection. (G) qRT-PCR analysis showing the relative mRNA expression levels of CCL1 and FOXP3 in the livers of infected WT and KO mice at 24 weeks post-infection (normalized to GAPDH). All data are presented as mean ± SD. n = 5 mice/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, P > 0.05, infected WT group vs. infected KO group.

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CCR8 deficiency reshapes the liver immune microenvironment and enhances T cell effector functions

We further explored the immunological mechanisms underlying the improved phenotype upon CCR8 knockout. Flow cytometric analysis revealed that CCR8-deficient T cells exhibited a stronger anti-parasitic immune response capacity. At multiple time points post-infection, the proportions of CD4⁺ and CD8⁺ T cells in the livers of infected KO mice secreting IFN-γ and TNF-α were significantly higher than in the infected WT group (Fig 6A). Furthermore, the capacity of CD8⁺ T cells to produce perforin was significantly enhanced in the late stage of infection (Fig 6B). For another key cytokine, IL-2, we found that at 12 wpi, the proportion of CD8⁺ T cells secreting IL-2 was significantly higher in the infected KO group than in the infected WT group, while no significant differences were observed in CD4⁺ T cells or at other time points during infection (Fig 6C). In contrast, the patterns of change for Th2 (IL-4) and Th17 (IL-17A) related cytokines were more complex and mostly non-significant across time points (Fig 7A). The lack of significant modulation of IL-4 and IL-17A in CCR8-KO mice, despite the strong restoration of Th1/cytotoxic functions, suggests that CCR8’s immunosuppressive role in AE is relatively specific to the Th1/cytotoxic axis. This pathway specificity may reflect the particular immune evasion requirements of E. m or indicate that Th2/Th17 responses in this model are regulated by additional, CCR8-independent mechanisms. Notably, in the late stage of infection (24 wpi), Treg-related inhibitory cytokines, such as IL-10 and TGF-β1 produced by CD8⁺ T cells, were significantly downregulated in infected CCR8-KO mice compared to infected WT controls (Fig 7B). Interestingly, at the mid-stage (12 wpi), TGF-β1 production showed a transient increase in KO mice, highlighting the dynamic nature of immune regulation during different phases of infection. These results indicate that CCR8 deficiency, by relieving the suppression of T cell effector functions (e.g., IFN-γ, TNF-α, perforin), partially enhancing their survival/proliferation potential (e.g., IL-2), and partially reversing the immunosuppressive microenvironment, confers upon the host a greater ability to control parasitic infection.

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Fig 6. CCR8 deficiency enhances the effector functions of liver T cells.

(A) Flow cytometric analysis showing the proportions of liver CD4⁺ and CD8⁺ T cells from infected WT and KO mice secreting IFN-γ and TNF-α at different time points post-infection. (B) Flow cytometric analysis showing the proportions of liver CD4⁺ and CD8⁺ T cells from infected WT and KO mice secreting Granzyme B and Perforin at different time points post-infection. (C) Proportions of CD4⁺ and CD8⁺ T cells secreting IL-2 from infected WT and KO mice. Data are presented as mean ± SD, n = 5 mice/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, P > 0.05. Infected WT group vs infected KO group.

https://doi.org/10.1371/journal.pntd.0014018.g006

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Fig 7. Effect of CCR8 deficiency on Th2/Th17 and regulatory cytokine expression.

(A) Flow cytometric analysis showing the proportions of CD4⁺ T cells and CD8⁺ T cells secreting IL-4 and IL-17A. (B) Proportions of CD4⁺ T cells and CD8⁺ T cells secreting IL-10 and TGF-β1. Data are presented as mean ± SD, n = 5 mice/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, P > 0.05. Infected WT group vs infected KO group.

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Discussion

Through systematic experimentation, this study reveals for the first time the crucial role of the CCR8/CCL1 chemokine axis in shaping an immunosuppressive microenvironment in the liver during E. m infection. We found that this axis is specifically activated in the late stage of infection, accompanied by local expansion of FOXP3⁺ Tregs and functional suppression of CCR8⁺ T cells, whereas CCR8 deficiency significantly improves liver pathological indices and indirectly suggests a reduced parasite load by enhancing effector T cell functions. These results not only deepen the understanding of immune evasion mechanisms in AE but also establish CCR8 as a potential target for immunotherapy.

The CCR8/CCL1 axis is a key driver in the formation of the immunosuppressive microenvironment in the AE liver. We observed significant upregulation of CCR8 and CCL1 at both the transcriptional and protein levels in the liver during the late stage of infection. This aligns with observations of CCL1 upregulation in models of liver fibrosis [19,20], hepatocellular carcinoma [21], and certain chronic viral infections [22], suggesting CCL1 may be a common feature of chronic inflammatory microenvironments. Immunofluorescence co-localization showing FOXP3 and CCR8 co-expressed on infiltrating lymphocytes around lesions strongly supports the hypothesis that CCR8 mediates Tregs recruitment to the infection site. Flow cytometry further confirmed significant expansion of CD4⁺CCR8⁺ Tregs in the liver during late infection, highly consistent with the enrichment of CCR8⁺ Tregs observed in cancer [14,23,24]. The delayed activation of the CCR8/CCL1 axis, peaking at 24 wpi, suggests it is a feature of established chronic infection rather than the initial acute response. Early stages likely involve innate immune activation and transient inflammation, with the parasite subsequently inducing this specific chemokine axis to orchestrate a durable immunosuppressive niche, thereby facilitating its long-term survival. These results implicate the CCR8/CCL1 axis in Tregs recruitment during E. m infection, suggesting its unique contribution to immunosuppressive microenvironment formation.

A core finding of this study is the revelation of the “paradox” of the CCR8⁺ T cell population – numerical expansion coupled with functional suppression. We found that CCR8⁺ CD4⁺ and CCR8⁺ CD8⁺ T cells were significantly inferior to the CCR8⁻ population in secreting key effector molecules like IFN-γ, TNF-α, and perforin, displaying an “exhausted” or “anergic” phenotype. It is noteworthy to consider whether this CCR8-associated functional suppression is distinct from the classical T cell exhaustion state mediated by multiple inhibitory receptors (e.g., PD-1 [25], LAG-3 [26], TIM-3 [27]). CCR8 might mark a unique T cell subset (or ‘pre-exhausted’ precursor) that is more prone or susceptible to being induced into a functionally suppressed state under the combined effects of chronic antigen stimulation and a specific chemokine microenvironment. This finding is significant for understanding the heterogeneity of immune cell dysfunction in chronic infections. Potential mechanisms underlying this phenomenon include: (1) CCR8 signaling itself might directly transmit inhibitory signals, or synergize with immune checkpoints like PD-1 [14,28,29]; (2) CCR8⁺ T cells might inherently represent a specific subset predisposed to functional exhaustion (“exhaustion precursors”), whose recruitment to the high-antigen-load lesion site accelerates functional impairment [23,30–32]; (3) The CCL1/CCR8 axis, by attracting Tregs, indirectly suppresses the function of neighboring effector T cells through a “bystander suppression” mechanism [33,34]. However, a key limitation of this study is that we have not been able to distinguish whether CCR8 signaling is the direct cause of T cell functional suppression or merely a marker of functionally exhausted T cells. Future research requires in vitro T cell activation experiments to investigate the direct effects of CCR8 agonists/antagonists on T cell effector function, and the use of techniques like single-cell RNA sequencing to further resolve the transcriptomic characteristics and cellular heterogeneity of CCR8⁺ T cells.

CCR8 gene deletion significantly enhances anti-parasitic immunity and alleviates liver injury, highlighting its potential as a therapeutic target. KO mice exhibited smaller ultrasound lesion sizes, lower liver weights, and reduced liver-to-body weight ratios in the mid and late stages of infection. These improvements in macroscopic pathological indicators collectively demonstrate that CCR8 deficiency effectively suppressed parasite growth and infiltration, thereby reducing the host’s disease burden. This result echoes the efficacy shown by CCR8-targeting antibodies in cancer immunotherapy [24,35,36]. Mechanistically, CCR8 deficiency led to a significant enhancement in the ability of effector T cells to secrete IFN-γ, TNF-α, and perforin, while some inhibitory factors (e.g., IL-10, TGF-β1) were downregulated, suggesting a shift in immune balance towards a Th1-type response. IFN-γ is a key factor in controlling various intracellular parasites [37–40], and our results further affirm its central role in controlling E. m infection. Although our study yields encouraging results in a mouse model, whether the CCR8/CCL1 axis plays a similar role in human AE patients requires further validation. Future investigations measuring serum CCL1 levels and the infiltration of CCR8⁺ T cells and Tregs in lesion tissues from AE patients could validate the clinical relevance of our findings and provide a basis for advancing CCR8-targeted therapies towards clinical application.

Our study nominates CCR8 as a promising immunotherapeutic target for AE. To translate this finding, future work should correlate serum CCL1 levels and CCR8 ⁺ T cell/Treg infiltration in patient liver biopsies with disease progression or treatment response, validating the axis’s clinical relevance and enabling patient stratification for future trials. However, specific safety considerations arise when modulating CCR8 in a chronic infectious setting, as opposed to oncology. Long-term, rather than transient, inhibition may be required. Its effects on systemic immune homeostasis, hepatic tissue repair mechanisms, and the control of latent or concurrent infections must be thoroughly evaluated in preclinical models to de-risk clinical translation.

Limitations and Future Directions. First, we used systemic CCR8-KO mice, which prevents distinguishing the relative contributions of CCR8 on T cells versus non-T cells (e.g., myeloid cells, liver sinusoidal endothelial cells). Future use of T cell-specific conditional knockout mice will allow more precise dissection of the cell-specific functions of CCR8. Second, we have not identified which parasite component triggers CCL1 expression. Additionally, a key limitation of this study is the reliance on indirect measures (liver weight, ultrasound) to infer parasite burden. To directly quantify the parasitic load and further substantiate our findings, future studies will employ methods such as measuring the dry weight of carefully dissected parasitic vesicles or quantifying parasite-specific genomic DNA in liver tissues. Furthermore, the systemic knockout of CCR8 precludes attribution of the observed phenotypes specifically to T cells, as CCR8 is also expressed on myeloid cells and other populations. To precisely dissect the cell-specific role of CCR8, future investigations will utilize T cell–conditional CCR8 knockout mice (e.g., CD4-Cre; CCR8^flow/flow) or adoptive transfer experiments with WT and CCR8-KO T cells into infected hosts. Then, the function of CCR8 signaling in liver-resident cells like hepatic stellate cells and Kupffer cells also warrants further investigation. Finally, a related outstanding question is whether CCR8 signaling directly causes T cell dysfunction or merely marks a pre-exhausted subset. To establish direct causality, in vitro experiments are needed where sorted T cells are stimulated in the presence of CCR8 agonists (e.g., CCL1) or antagonists, followed by assessment of effector cytokine production and proliferation. The CCR8-dependent immunosuppressive mechanism revealed in this study not only deepens the understanding of AE pathogenesis but may also provide new perspectives for understanding immune evasion strategies in other chronic parasitic infections (e.g., cystic echinococcosis, leishmaniasis).

In summary, we propose the following working model: In wild-type mice, E. m infection induces hepatic CCL1 production, which recruits FOXP3⁺ Tregs and functionally suppresses CCR8⁺ T cells via CCR8, collectively constructing an immunosuppressive niche that promotes parasite survival and disease progression. In CCR8-KO mice, this pathway is blocked, T cell effector functions are restored, and the immune balance shifts towards a Th1-type response, thereby effectively controlling the infection. This model provides a new perspective for understanding the chronicity mechanisms of AE and lays a theoretical foundation for CCR8-targeted immunotherapeutic strategies. Developing humanized CCR8 antagonists, or combining them with existing drugs, holds promise for offering new treatment options for patients with advanced AE.

Supporting information

S1 Data. The Excel spreadsheet contains the P-values for Figs 1–7, with each figure’s data stored in separate worksheets.

https://doi.org/10.1371/journal.pntd.0014018.s001

(XLSX)

S1 Table. Mouse primer sequences for qRT-PCR.

https://doi.org/10.1371/journal.pntd.0014018.s002

(DOCX)

S2 Table. Antibodies for flow cytometry.

https://doi.org/10.1371/journal.pntd.0014018.s003

(DOCX)

S1 Fig. Representative flow cytometry gating strategy for identification of CCR8 ⁺ T cell subsets.

https://doi.org/10.1371/journal.pntd.0014018.s004

(TIF)

Acknowledgments

During my time conducting scientific research, I would like to express my gratitude to all the faculty members and classmates in the laboratory for their support. I want to thank my advisor for his invaluable guidance and patient mentorship.

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Figures

Abstract

Author summary

Introduction

Materials and methods

Ethics statement

Experimental animals and infection model

Sample size justification

Sample collection and pathological assessment

Ultrasonographic analysis

Quantitative Real-Time PCR (qPCR)

Immunohistochemistry and Immunofluorescence

Flow cytometry

Statistical analysis

Results

E. multilocularis infection induces progressive liver pathological damage in wild-type mice

The hepatic CCR8/CCL1 chemokine axis and FOXP3 pathway are specifically activated in the late stage of infection

CCR8+ T lymphocytes expand in the infected liver and exhibit a functionally suppressed phenotype

CCR8 deficiency alleviates liver pathological damage by enhancing anti-parasitic immune responses

CCR8 deficiency reshapes the liver immune microenvironment and enhances T cell effector functions

Discussion

Supporting information

S1 Data. The Excel spreadsheet contains the P-values for Figs 1–7, with each figure’s data stored in separate worksheets.

S1 Table. Mouse primer sequences for qRT-PCR.

S2 Table. Antibodies for flow cytometry.

S1 Fig. Representative flow cytometry gating strategy for identification of CCR8 ⁺ T cell subsets.

Acknowledgments

References

CCR8⁺ T lymphocytes expand in the infected liver and exhibit a functionally suppressed phenotype