Clonorchis sinensis co-infection potentiates polymorphonuclear-myeloid-derived suppressor cells expansion and metabolic reprogramming in chronic hepatitis B patients

Abstract

BACKGROUND

Clonorchis sinensis (C. sinensis) co-infection is known to accelerate disease progression and induce immune tolerance in patients with hepatitis B virus (HBV), particularly in regions where both infections are endemic. Metabolic reprogramming has emerged as a key driver of immunosuppression. These metabolic shifts are often orchestrated by myeloid-derived suppressor cells (MDSCs), which expand during chronic infections to promote immune tolerance. However, whether C. sinensis co-infection reprograms metabolism to favor MDSC-linked suppression and how this process relates to normal liver enzyme levels remains unclear.

AIM

To investigate whether C. sinensis co-infection induces MDSC- linked metabolic reprogramming in patients with HBV and normal liver enzyme levels.

METHODS

In this cross-sectional study, a total of 117 participants were enrolled and assigned to four groups: Healthy controls (n = 30), C. sinensis mono-infected participants (n = 29), HBV mono-infected participants (n = 28), and co-infected participants (n = 30). Serum cytokine profiles were measured using chemiluminescence immunoassay, amino acid metabolites using liquid chromatography-tandem mass spectrometry, and MDSC subsets using flow cytometry. Spearman correlation and mediation analyses were performed to explore the relationships among inflammation, metabolism, and immunity.

RESULTS

HBV mono-infected and co-infected patients showed no statistically significant differences in liver enzyme and HBV DNA levels. However, compared with the HBV mono-infected group, the co-infected group had higher levels of the inflammatory cytokine: Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), accompanied by markedly lower interferon-γ (IFN-γ) levels. Flow cytometric assay revealed that polymorphonuclear MDSCs (PMN-MDSCs) showed the greatest expansion in the co-infected group, and their proportion correlated strongly with IL-6 and TNF-α levels. Metabolically, the co-infected group had a significantly higher ornithine-to-arginine ratio. Mediation analysis suggested that PMN-MDSCs accounted for approximately 45% of the total effect of IL-6 on IFN-γ suppression.

CONCLUSION

C. sinensis co-infection was associated with immunosuppression through IL-6/TNF-α-linked PMN-MDSC expansion and elevated ornithine-to-arginine ratio, resulting in suppressed IFN-γ production despite normal liver enzyme levels.

Key Words: Clonorchis sinensis; Chronic hepatitis B; Hepatitis B virus-Clonorchis sinensis co-infection; Polymorphonuclear myeloid-derived suppressor cell; Ornithine-to-arginine ratio; Immunosuppression

Core Tip: Compared with hepatitis B virus mono-infected patients with normal liver enzyme levels, Clonorchis sinensis co-infection expands polymorphonuclear-myeloid-derived suppressor cells. This activates the arginine (Arg)-ornithine (Orn) axis, raising the Orn-to-Arg ratio. Consequently, interferon-γ production is suppressed. The Orn-to-Arg ratio outperforms standard liver enzyme levels for immune assessment. Screening for Clonorchis sinensis is warranted even in patients with well-controlled viremia.

INTRODUCTION

Chronic hepatitis B virus (HBV) infection remains a major driver of cirrhosis and liver cancer worldwide[1,2]. Clonorchis sinensis (C. sinensis) is a bile-dwelling trematode classified as a group 1 biological carcinogen by the International Agency for Research on Cancer in 2009[3]. While infections with other trematodes, such as Fasciola hepatica, are more prevalent in Africa and the Middle East, C. sinensis is the dominant liver fluke in East Asia[1]. In southern China, there is a notable epidemiological overlap between chronic HBV and C. sinensis infection[3-5]. Untreated chronic infection can progress to cirrhosis and cholangiocarcinoma. Although the natural history of HBV largely depends on host immunity, co-infection with C. sinensis likely reshapes the immune microenvironment, thereby compromising antiviral efficacy and accelerating disease progression[6-8]. A clinically perplexing phenomenon is frequently observed: Patients co-infected with HBV and C. sinensis often have normal or near-normal liver transaminase levels despite persistent viral replication. This finding suggests that routine liver function tests fail to reveal the true immunopathological status of this high-risk population.

Previous studies have demonstrated that this apparent dissociation between liver enzyme levels and viral activity may be associated with C. sinensis excretory-secretory products, which act synergistically with HBV-related antigens to promote myeloid-derived suppressor cell (MDSC) accumulation and shift the cytokine balance toward a T-helper 2 (Th2)-biased phenotype[9,10]. However, the in vivo mechanisms remain unclear. First, which MDSC subset is preferentially expanded in co-infected patients? Second, given that MDSCs suppress T cells using metabolic enzymes[11], primarily those involved in the kynurenine (Kyn)-tryptophan (Trp) axis (indoleamine 2,3-dioxygenase-mediated) and the arginine (Arg)-ornithine (Orn) axis (arginase-mediated), does C. sinensis co-infection trigger a pathogen-specific metabolic bias that preferentially activates one of these axes? This metabolic selectivity may be key to understanding how immune tolerance is established without concurrent hepatocellular injury.

Given these unresolved issues, the present study aimed to explore the immunometabolic regulatory mechanisms underlying HBV and C. sinensis co-infection in vivo. Specifically, this study sought to identify the dominant MDSC subset and characterize the key metabolic pathway mediating immunosuppression in co-infected patients, thereby providing novel mechanistic insights and a theoretical basis for targeted clinical intervention and management of HBV-C. sinensis co-infection.

MATERIALS AND METHODS

Participants and study design

This cross-sectional study included 117 participants consecutively recruited from the Third Affiliated Hospital of Sun Yat-Sen University from January 1, 2025 to December 31, 2025). The sample size was estimated based on prior studies investigating MDSC frequencies in parasitic and viral co-infections[11]. Considering a medium effect size (f = 0.35), a two-sided significance level (α) of 0.05, and a desired power (1-β) of 80% for comparisons among four groups, at least 28 participants per group were required. Participants were stratified into four groups: Healthy controls (HC, n = 30), C. sinensis mono-infected participants (C. sinensis group, n = 29), HBV mono-infected participants (HBV group, n = 28), and participants co-infected with C. sinensis and HBV (co-infected group, n = 30). Each condition was diagnosed on clinical presentation and laboratory findings. All participants were selected according to the following inclusion and exclusion criteria.

Inclusion criteria: HC group: Seronegative for HBV and C. sinensis, with normal liver function test results. C. sinensis group: C. sinensis eggs detected by stool microscopy and HBV ruled out. HBV group: Positive for HBV surface-antigen, with HBV DNA ≥ 10 IU/mL, receiving ongoing antiviral therapy, and negative for C. sinensis. Co-infected group: C. sinensis eggs detected in stool and confirmed HBV infection, with ongoing antiviral therapy.

Exclusion criteria: This study excluded individuals who were younger than 18 or older than 70 years, were pregnant, or had any of the following: Liver fibrosis, cirrhosis, or liver cancer; co-infection with other parasites or hepatitis virus genotypes; metabolic liver disease, drug-induced liver injury, acute infection, malignancy, human immunodeficiency virus, thyroid dysfunction, or autoimmune disease. The study protocol was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval No.[2020]02-045-01) and the study was conducted in accordance with the Declaration of Helsinki.

Liver function tests

Peripheral blood was collected from each participant and centrifuged at 3000 × g for 10 minutes at room temperature to obtain serum. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TB) were measured using a Hitachi 7600 automated biochemical analyzer (Hitachi, Tokyo, Japan). ALT and AST were determined using an enzymatic rate method, and TB was determined using an oxidation method, according to the manufacturer’s protocols. All samples were analyzed in duplicate, and quality control samples were included in each batch to monitor accuracy.

Cytokine assays

Peripheral blood was collected from each participant and centrifuged it at 3000 × g for 10 minutes at 4 °C to obtain serum. Six cytokines - interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-10, IL-1β and IL-4 - were measured on a Hotgen automated chemiluminescence immunoassay analyzer (Beijing Hotgen Biotech Co., Ltd., Beijing, China) using commercially available reagent kits from the same manufacturer and according to the manufacturer’s protocols. All samples were run in duplicate to ensure consistency.

Flow cytometry

A 10-color flow cytometric assay was used to quantify MDSCs and lymphocyte subsets. The following fluorochrome-conjugated antibodies were used: CD45-KRO (clone J33), CD19-APC (J3-119), CD56-PECY7 (N901), HLA-DR-FITC (Immu-357), CD33-PC5.5 (D3HL60.251), CD3-APC-A750 (UCHT1), CD16-APC-A700 (3G8), and CD14-PE (RMO52) (all from Beckman Coulter, Brea, CA, United States); CD11b-BV605 (D12) from BD Biosciences (San Jose, CA, United States); and CD15-PB (HI98) from 4A Biotech (Beijing, China).

Red blood cells were lysed using BD FACS™ Lysing Solution (BD Biosciences, San Jose, CA, United States). Total MDSCs were defined as CD45⁺CD3^-CD19^-CD56^-CD16^-HLA^-DR^-CD33⁺CD11b⁺ cells[12]. Within this gate, CD14⁺ cells were classified as monocytic MDSCs (M-MDSCs) and CD15⁺ cells as polymorphonuclear MDSCs (PMN-MDSCs). T lymphocytes were defined as CD3⁺, B lymphocytes as CD3^- CD19⁺, natural killer (NK) lymphocytes as CD3^- CD56⁺, NK T lymphocytes as CD3⁺ CD56⁺. After staining, samples were fixed with 2% paraformaldehyde and acquired on a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, United States) on the same day. Data were analyzed using Kaluza Analysis 2.2 (Beckman Coulter, Brea, CA, United States).

Liquid chromatography-tandem mass spectrometry analysis

Serum amino acid metabolites were quantified using liquid chromatography-tandem mass spectrometry. The LC system consisted of an ExionLC AD (SCIEX, Framingham, MA, United States) equipped with a SHIMADZU Shim-pack VP-ODS column (2.0 mm × 150 mm, 5 μm; Shimadzu, Kyoto, Japan). The mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The flow rate was 0.3 mL/minute, the column temperature 40 °C, and the injection volume 2 μL. Mass spectrometry was performed on a SCIEX AB4500MD triple quadrupole mass spectrometer (SCIEX, Framingham, MA, United States) using positive electrospray ionization, with data acquired in multiple reaction monitoring mode. Analyst software (version 1.6.3; SCIEX, Framingham, MA, United States) was used for data acquisition and processing. Amino acid standards and reagents were purchased from Sigma-Aldrich (St. Louis, MO, United States). For quality control, blank serum was spiked with known concentrations of amino acid standards, and these quality control samples were analyzed alongside the study samples to monitor assay performance.

Statistical analysis

SPSS (version 26.0; IBM Corp., Armonk, NY, United States) and GraphPad Prism (version 9.0; GraphPad Software, La Jolla, CA, United States) were used for statistical analysis. All continuous variables were entered into the analyses as raw, untransformed measurements. Normally distributed continuous variables are reported as the mean ± SD; whereas nonnormally distributed data as the median (minimum-maximum). Group comparisons were performed using one-way analysis of variance with Tukey’s post hoc test for parametric data and the Kruskal-Wallis H test with Dunn’s correction for nonparametric data. Categorical variables were compared using the χ² test or Fisher’s exact test, as appropriate. Pearson correlation was applied to normally distributed variables, and Spearman correlation was applied to nonparametric variables. Missing data were minimal (< 2% for all analytes) and handled by complete-case analysis without imputation. No formal subgroup analyses, interaction tests, or sensitivity analyses were conducted beyond the primary comparisons among the four predefined diagnostic groups. Groups were balanced for age and sex at the design stage to minimize confounding. Post-hoc power analysis indicated > 80% power for the primary comparisons. A two-sided P < 0.05 was considered statistically significant.

RESULTS

Comparable baseline characteristics among groups

A total of 117 participants were enrolled and assigned to four groups: HC (n = 30), C. sinensis (n = 29), HBV (n = 28), and co-infected (n = 30). Table 1 summarizes the demographic characteristics. Age and gender distributions were comparable among the groups (all P > 0.05).

Table 1 Baseline characteristics, median (interquartile range).

	HC group (n = 30)	Clonorchis sinensis group (n = 29)	HBV group (n = 28)	Co-infected group (n = 30)
Age, years (mean ± SD)	47.77 ± 9.49	48.59 ± 14.26	46.82 ± 9.83	47.87 ± 10.16
Gender (male/female)	30/2	30/3	28/3	30/2
HBV DNA log10 copies/mL	Negative	Negative	2.00 (1.08-7.20)	2.00 (1.43-8.85)c
Clonorchis sinensis eggs (n/g)	Negative	200 (100-1400)	Negative	200 (100-1100)
ALT (U/L)	19.00 (8.00-44.00)	28.00 (8.00-95.00)	51.00 (7.00-225.00)a	37.50 (5.00-986.00)a,b
AST (U/L)	20.00 (14.00-27.00)	23.00 (11.00-60.00)	50.50 (16.00-138.00)a	36.50 (11.00-485.00)a,b
TB (μmol/L)	5.90 (3.20-24.20)	6.90 (4.00-262.60)	12.80 (6.00-635.50)a	14.80 (3.00-115.40)a,b

^aP < 0.05 vs healthy control group.

^bP < 0.05 vs Clonorchis sinensis group.

^cP < 0.05 vs hepatitis B virus group.

Age and sex distributions were comparable across the four groups (all P > 0.05). HC: Healthy controls; HBV: Hepatitis B virus mono-infected participants; Co-infected: Co-infected participants; Clonorchis sinensis: Clonorchis sinensis mono-infected participants; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TB: Total bilirubin.

Serum ALT, AST, and TB levels were elevated in both the HBV and co-infected groups relative to the HC group (Kruskal-Wallis H test with Dunn’s post-hoc test, all P < 0.05). The co-infected group also exhibited higher levels of these markers than the C. sinensis group (all P < 0.05), whereas no significant difference was observed between the HBV and co-infected groups (all P > 0.05).

Regarding infection status, participants in the C. sinensis and co-infected groups had C. sinensis eggs detected in stool. Participants in the HBV and co-infected groups were positive for HBV surface antigen and exhibited comparable low-level HBV DNA levels (mean 2 Log₁₀ copies/mL, P > 0.05), whereas those in the HC and C. sinensis groups were negative.

Elevated pro-inflammatory cytokines and suppressed IFN-γ in co-infected patients

Serum cytokine levels were measured to assess the immune profile (Figure 1). Compared with HCs, all three infected groups exhibited significantly elevated levels of the proinflammatory cytokines IL-6 and TNF-α (all P < 0.05). The co-infected group had the highest levels - IL-6 and TNF-α were significantly higher than those in the HBV group (both P < 0.05).

Figure 1 Serum cytokine levels in the four groups. Scatter plots show serum concentrations. A: Interleukin-6; B Tumor necrosis factor-α; C: Interferon-γ; D: Interleukin-10; E: Interleukin-1β; F: Interleukin-4. HC: Healthy controls; C. sinensis: Clonorchis sinensis mono-infected participants; HBV: Hepatitis B virus mono-infected participants; Co-infected: Co-infected participants; IL: Interleukin; TNF-α: Tumor necrosis factor-α; IFN-γ: Interferon-γ.

In contrast, IFN-γ levels were lower in the HBV and co-infected groups than in the HC group (P < 0.05), and this suppression was more pronounced in the co-infected group than in the HBV group (P < 0.05). IL-10 levels were significantly higher in all three infected groups than in the HC group (P < 0.05), whereas IL-1β and IL-4 levels did not differ significantly among the four groups (P > 0.05).

To better visualize the overall cytokine pattern, the levels of the six cytokines were normalized to those in the HC group (set to 1) and plotted on a radar chart (Figure 2). The co-infected group occupied a larger area on the radar chart, mainly because of higher pro-inflammatory cytokine levels, particularly IL-6 and TNF-α, than those in the other two infected groups. This pattern indicates a distinct inflammatory response associated with co-infection.

Figure 2 Radar chart of cytokine levels among the four groups. Relative levels of interleukin-6, tumor necrosis factor-α, interferon-γ, interleukin-10, interleukin-1β, and interleukin-4 are shown after normalization to the healthy controls group (set to 1). Each axis represents one cytokine; the area covered by each group reflects its overall cytokine profile. HC: Healthy controls; C. sinensis: Clonorchis sinensis mono-infected participants; HBV: Hepatitis B virus mono-infected participants; Co-infected: Co-infected participants; IL: Interleukin; TNF-α: Tumor necrosis factor-α; IFN-γ: Interferon-γ.

Selective expansion of PMN-MDSCs in co-infected patients

Whole blood samples were analyzed by flow cytometry to detect MDSCs. The gating strategy (Figure 3) began with the exclusion of doublets and dead cells using forward and side scatter (forward scatter and side scatter-area). Total MDSCs were defined as CD45⁺CD3^-CD19^-CD56^-CD16^-HLA-DR^-CD33⁺CD11b⁺ cells. Within this gate, CD14⁺ cells were classified as M-MDSCs and CD15⁺ cells as PMN-MDSCs.

Figure 3 Gating strategy for myeloid-derived suppressor cell and lymphocyte subsets. Total myeloid-derived suppressor cells (MDSCs) were defined as CD45⁺CD3^-CD19^-CD56^-CD16^-HLA-DR^-CD33⁺CD11b⁺ cells. Within this gate, CD14⁺ cells are monocytic MDSCs and CD15⁺ cells are polymorphonuclear MDSCs. Lymphocyte subsets: CD3⁺ (T cells), CD3^-CD19⁺ (B cells), CD3^-CD56⁺ (natural killer cells), and CD3⁺CD56⁺ (natural killer T cells). MDSC: Myeloid-derived suppressor cells; FSC-H: Forward scatter-height; SSC-A: Side scatter-area; WBC: White blood cell; APC: Allophycocyanin; HLA: Human leukocyte antigen; FITC: Fluorescein isothiocyanate; PMN-MDSC: Polymorphonuclear myeloid-derived suppressor cells; M-MDSC: Monocytic myeloid-derived suppressor cells; PE: Phycoerythrin.

Table 2 summarizes the proportions of MDSC subsets across groups. PMN-MDSC proportions were significantly higher in all three infected groups than in the HC group (P < 0.05). The co-infected group demonstrated the most pronounced PMN-MDSC expansion, with a median proportion of 0.38%, which was significantly higher than that in the HBV group (median: 0.20%, P < 0.05). For M-MDSCs, only the co-infected group showed a significant increase compared with the HC group (P < 0.05). The co-infected group also had a higher M-MDSC proportion than the C. sinensis group (P < 0.05) but did not differ significantly from the HBV group.

Table 2 Myeloid-derived suppressor cell and lymphocyte subsets by group, median (interquartile range).

	HC group (n = 30)	Clonorchis sinensis group (n = 29)	HBV group (n = 28)	Co-infected group (n = 30)
PMN-MDSCs (%)	0.09 (0.01-0.27)	0.33 (0.00-0.76)a	0.20 (0.04-0.61)a	0.38 (0.10-1.12)a,c
M-MDSCs (%)	0.10 (0.01-0.29)	0.07 (0.01-0.85)	0.14 (0.01-0.55)	0.17 (0.05-2.50)a,b
T lymphocytes (%)	67.83 (38.26-81.54)	68.56 (43.92-87.07)	65.65 (52.87-81.95)	70.74 (49.41-89.78)
B lymphocytes (%)	10.45 (4.38-22.92)	12.95 (2.90-35.81)	13.33 (2.16-28.10)	15.12 (4.25-24.34)a
NK lymphocytes (%)	16.07 (5.76-42.67)	12.73 (4.08-35.29)	15.48 (5.95-39.80)	11.20 (3.31-34.52)a
NK T lymphocytes (%)	4.55 (0.49-21.37)	4.28 (0.47-13.22)	3.38 (1.54-11.44)	2.79 (0.47-25.92)

^aP < 0.05 vs healthy control group.

^bP < 0.05 vs Clonorchis sinensis group.

^cP < 0.05 vs hepatitis B virus group.

HC: Healthy controls; HBV: Hepatitis B virus mono-infected participants; Co-infected: Co-infected participants; Clonorchis sinensis: Clonorchis sinensis mono-infected participants; PMN-MDSCs: Polymorphonuclear myeloid-derived suppressor cells; M-MDSCs: Monocytic myeloid-derived suppressor cells; NK: Natural killer.

Lymphocyte subset analysis showed no differences in the overall proportion of T-cells among the four groups. However, the co-infected group had a higher proportion of B cells and a lower proportion of NK cells than the HC group (both P < 0.05). The C. sinensis and HBV groups showed no significant changes in B- or NK- cells proportions relative to the HC group. Correlation analysis showed that the proportion of PMN-MDSCs was positively correlated with IL-6 concentration (r = 0.459, P < 0.05), and TNF-α concentration (r = 0.376, P < 0.05) (Figure 4).

Figure 4 Polymorphonuclear myeloid-derived suppressor cells expansion and its correlations with pro-inflammatory cytokines in the four groups. A: Frequency of circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs); B: Correlation between PMN-MDSC frequency and serum interleukin-6 concentration; C: Correlation between PMN-MDSC frequency and serum tumor necrosis factor-α concentration. Spearman’s correlation coefficient (r) and P values are indicated. PMN-MDSC: Polymorphonuclear myeloid-derived suppressor cells; HC: Healthy controls; C. sinensis: Clonorchis sinensis mono-infected participants; HBV: Hepatitis B virus mono-infected participants; Co-infected: Co-infected participants; IL: Interleukin; TNF-α: Tumor necrosis factor-α.

Selective activation of the Arg-Orn axis over the Kyn-Trp axis in co-infected patients

Serum amino acid profiles were analyzed to identify metabolic changes (Figure 5). Orn levels were significantly elevated in the co-infected group than in both the HC and HBV groups (P < 0.05). Conversely, Arg levels were significantly reduced in the co-infected group than in the HBV group (P < 0.05), culminating in a markedly higher Orn-to-Arg ratio (Orn/Arg ratio) (3.46 vs 0.92; P < 0.05).

Figure 5 Comparison of serum metabolites in the arginine-ornithine and kynurenine-tryptophan pathways among the four groups. A: Ornithine; B: Arginine; C: Ornithine-to-arginine ratio; D: Kynurenine; E: Tryptophan; F: Kynurenine-to-tryptophan ratio. Horizontal bars indicate medians. The co-infected group exhibited a markedly elevated ornithine/arginine ratio compared with the hepatitis B virus group (3.46 vs 0.92, P < 0.001; Kruskal-Wallis H test with Dunn’s correction). Orn: Ornithine; Arg: Arginine; Kyn: Kynurenine; Trp: Tryptophan; HC: Healthy controls; C. sinensis: Clonorchis sinensis mono-infected participants; HBV: Hepatitis B virus mono-infected participants; Co-infected: Co-infected participants.

Trp catabolism was evaluated by measuring serum levels of Trp and Kyn and calculating the Kyn-to-Trp ratio (Kyn/Trp ratio). Both Kyn and Trp levels were higher in the co-infected group than in the HBV group (P < 0.05), but the Kyn/Trp ratio did not differ significantly among the four groups (P > 0.05).

Linking inflammation to Arg-Orn axis by PMN-MDSCs

A Spearman correlation heatmap was constructed to assess the relationships among inflammation, metabolism, and immunosuppression (Figure 6). The PMN-MDSC proportion was positively correlated with IL-6 (r = 0.46, P < 0.05) and TNF-α (r = 0.38, P < 0.05). The PMN-MDSC proportion was also associated with amino acid metabolism: It was positively correlated with Orn and the Orn/Arg ratio and negatively correlated with Arg. Notably, Orn was negatively correlated with IFN-γ (r = -0.30, P < 0.05), prompting further investigation of whether PMN-MDSCs mediate the relationship among these variables.

Figure 6 Spearman correlation heatmap of immune and metabolic indicators. The heatmap illustrates the correlations among polymorphonuclear myeloidderived suppressor cells, cytokines (interleukin-6, tumor necrosis factor-α, interferon-γ), and amino acid metabolites (ornithine, arginine, ornithine/arginine ratio). Red indicates positive correlation; blue indicates negative correlation. P < 0.05 (two-sided; Spearman correlation). IL: Interleukin; TNF-α: Tumor necrosis factor-α; PMN-MDSC: Polymorphonuclear myeloid-derived suppressor cells; Orn: Ornithine; Arg: Arginine; IFN-γ: Interferon-γ.

To clarify the direction of these relationships, we performed a mediation analysis (Figure 7). The results revealed that PMN-MDSCs accounted for approximately 45% of the total effect of IL-6 on IFN-γ suppression. IL-6 was strongly associated with an increased PMN-MDSC proportion (coefficient = 0.008, P < 0.001), and PMN-MDSC proportion was inversely associated with IFN-γ (coefficient = -0.507, P = 0.005).

Figure 7 Mediation analysis of the association between interleukin-6 and interferon-γ through polymorphonuclear myeloid-derived suppressor cells. The path model demonstrates that interleukin-6 (IL-6) promotes polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) expansion (a = 0.008, P < 0.001), and PMN-MDSCs in turn suppress interferon-γ (IFN-γ) (b = -0.507, P = 0.005). After adjusting for PMN-MDSCs, the direct effect of IL-6 on IFN-γ was attenuated and no longer significant (c’ = -0.005, P = 0.186), whereas the indirect effect via PMN-MDSCs remained significant. The total effect of IL-6 on IFN-γ was -0.009 (P = 0.017), with PMN-MDSCs mediating approximately 45% of this effect. IL-6: Interleukin-6; IFN-γ: Interferon-γ; PMN-MDSC: Polymorphonuclear myeloidderived suppressor cell.

DISCUSSION

In this cross-sectional study of patients coinfected with HBV and C. sinensis, a distinct immunological pattern emerged. Despite markedly elevated IL-6 and TNF-α levels, the co-infected group showed no increase in liver enzyme levels (ALT, AST or TB) compared with the HBV mono-infected group. This finding suggests that systemic inflammation does not directly translate into hepatocellular injury in these patients. Instead, the coinfected group exhibited a marked expansion of PMN-MDSCs that correlated strongly with IL-6 and TNF-α levels, together with a selective metabolic shift toward Arg catabolism (an increased Orn/Arg ratio) without activation of the indoleamine 2,3-dioxygenase-mediated Trp catabolism. Statistical modeling was consistent with a mediating role for PMN-MDSCs in the association between IL-6 and suppressed IFN-γ production, accounting for approximately 45% of the total effect. However, due to the cross-sectional nature of this study, causal inferences cannot be definitively established. Rather than representing a definitive causal pathway, these findings delineate a robust statistical association pointing to an IL-6/TNF-α-associated, MDSC-linked, arginase-dependent immunosuppressive signature that appears disconnected from overt hepatocellular injury.

A key observation requiring further interpretation is the selective metabolic reprogramming that favors the Arg-Orn axis over the Kyn-Trp axis. Although indoleamine 2,3-dioxygenase-mediated Trp catabolism is a well-documented mechanism of MDSC-mediated suppression in cancer and viral infections[12,13], the stable Kyn/Trp ratio among groups in this study suggests that C. sinensis co-infection uniquely reshapes myeloid metabolism. This selectivity may be driven by C. sinensis. Previous study has demonstrated that C. sinensis excretory-secretory products synergize with hepatitis B e antigen (HBeAg) to promote MDSC accumulation and shift the immune response toward a Th2-biased phenotype[14]. Mechanistically, a secretory protein of C. sinensis contain pathogen-associated molecular patterns that activate Toll-like receptors on myeloid progenitors[15,16], triggering IL-6 and TNF-α production through MyD88- and TRIF-dependent pathways[17,18]. In the present study, co-infected patients had markedly higher IL-6 and TNF-α levels than patients with HBV mono-infection. Upon binding to its receptor on common myeloid progenitors, IL-6 promotes differentiation toward the granulocytic lineage (PMN-MDSCs)[19-21]. Although the IL-6-STAT3 axis has been extensively characterized in neoplastic settings, the selective expansion of PMN-MDSC observed here provides clinical evidence of its relevance in human helminth-virus co-infection. Furthermore, IFN-γ levels in the co-infected group were significantly lower than those in the HBV mono-infected group. Previous studies have shown that PMN-MDSCs express high levels of arginase-1, which converts Arg to Orn and urea. Arg depletion subsequently impairs T-cell function and reduces IFN-γ production[22-25]. Notably, a recent study of the hepatocellular carcinoma immune microenvironment in patients with dual HBV and C. sinensis infection also reported significantly elevated arginase-1 expression in tumor-infiltrating myeloid cells[26]. Although this observation was made in advanced-stage neoplastic tissue rather than peripheral blood, it is consistent with our findings and suggests that arginase-mediated suppression is a conserved feature of C. sinensis co-infection across disease stages, from chronic inflammation to malignant transformation. Together, these complementary lines of evidence span the disease continuum, illustrating how C. sinensis may exploit the arginase pathway to establish immune tolerance early and potentially promote immune escape later.

Distinguishing between “clinical stability” and “high-risk latent state” is critical for clinical decision-making in this setting. In classic chronic HBV mono-infection, persistently normal ALT/AST can reflect either immune tolerance (HBeAg-positive, high HBV DNA, minimal inflammation - a relatively stable phase) or immune control/inactive carriage (HBeAg-negative, low HBV DNA, favorable long-term outcome)[27]. In the canonical model of immune clearance, hepatocellular injury, reflected by elevated ALT/AST, serves as a hallmark of cytotoxic T-lymphocyte-mediated viral control[28]. In the present study, however, PMN-MDSCs effectively masked this signature: Arg depletion impaired T-cell receptor signaling and IFN-γ production, thereby limiting immune-mediated hepatocellular necrosis[22]. Compounding this effect, dendritic cell maturation in co-infected patients is skewed toward a Th2-biased phenotype characterized by high IL-10 and low IL-12 level, further blunting Th1-type antiviral immunity[14]. Notably, our mediation analysis was statistically consistent with this masking effect, indicating that PMN-MDSCs accounted for approximately 45% of the total effect linking IL-6 to suppressed IFN-γ production. This immune tolerance preserves hepatic architecture at the expense of antiviral surveillance. Consequently, normal liver enzyme levels in co-infected patients should not be interpreted as evidence of immune health or clinical stability. Rather, they appear to signify a high-risk state of parasite-driven immune evasion and ongoing HBV replication. These patients could be misclassified as having “inactive carrier” status under current guidelines[29], thereby delaying antiparasitic treatment and allowing viral replication to continue unchecked. In summary, co-infection challenges the reliability of normal ALT and AST as markers of immune control. Accordingly, these findings support routine parasitological screening and vigilant monitoring to avoid inadvertent delays in initiation of antiparasitic or antiviral therapy.

It is worth noting that all patients in the HBV mono-infected and co-infected groups were receiving ongoing antiviral therapy and that both groups had comparable low-level HBV DNA levels (mean, 2 Log₁₀ copies/mL). Although nucleos(t)ide analogues effectively suppress HBV reverse transcription and slow fibrosis progression, they rarely achieve hepatitis B surface antigen loss or restore HBV-specific T-cell function[30]. Indeed, prolonged analogue therapy has been shown to reduce hepatitis B surface antigen load without restoring the phenotypic and functional characteristics of MDSCs and Tregs[11,31], indicating that the immunosuppressive cascade initiated during chronic infection can persist despite potent viral suppression. In the present study, antiviral therapy effectively controlled viral replication in both HBV mono-infected and co-infected patients but failed to eliminate the distinct immunological phenotype observed in the co-infected group. Crucially, the persistence of PMN-MDSC expansion and the selective shift toward Arg metabolism in the co-infected group, despite equivalent virological suppression, indicate that C. sinensis imposes an additional layer of immune modulation beyond that driven by residual viral replication. The clinical implication is concerning: In endemic regions, clinicians should remain alert to the possibility of C. sinensis co-infection even in patients with well-controlled viremia. Whether parasite eradication can reverse arginase-mediated suppression and restore antiviral immunity remains an open and clinically relevant question.

In summary, this study proposes a model in which C. sinensis co-infection in chronic hepatitis B patients is associated with a PMN-MDSC-dominant, arginase-dependent immunosuppressive network. From a clinical perspective, these results imply that, in endemic regions, screening for C. sinensis should be considered even in chronic hepatitis B patients with normal liver enzyme levels and well-controlled viremia. The Orn/Arg ratio, as a metabolic readout of MDSC activity, offers a practical addition to routine laboratory assessment for detecting immune dysfunction in this population.

CONCLUSION

HBV and C. sinensis co-infection is associated with immunosuppression through IL-6/TNF-α-linked PMN-MDSC expansion and elevated Orn/Arg ratio - without elevating standard liver enzyme levels (ALT/AST). Clinically, normal liver function test results do not rule out significant immune dysfunction in co-infected patients from endemic regions. The Orn/Arg ratio, which reflects arginase activity and MDSC-linked immunosuppression, appears to be more informative than ALT/AST for assessing immune status in this population. Future studies should explore whether parasite eradication or arginase blockade can restore antiviral immunity.

ACKNOWLEDGEMENTS

The authors thank all participants for their contribution to this study.