Liver Fibrosis Is Enhanced by a Higher Egg Burden in Younger Mice Infected with S. mansoni

Abstract

Schistosomiasis affects over 250 million people worldwide, with the highest prevalence at the age of 10-14 years. The influence of the host's age on the severity of liver damage is unclear. We infected male 8, 14, and 20-week-old mice with S. mansoni. Hepatic damage, inflammation, fibrosis, and metabolism were analyzed by RT-qPCR, Western blotting, ELISA, immunohistochemistry, and mechanistic transwell chamber experiments using S. mansoni eggs and human hepatic stellate cells (HSCs) or primary mouse hepatocytes. Major results were validated in human biopsies. We found that hepatosplenomegaly, granuloma size, egg load, inflammation, fibrosis, and glycogen stores all improved with the increasing age of the host. However, serum alanine transaminase (ALT) levels were lowest in young mice infected with S. mansoni. Hepatic carbohydrate exploitation was characterized by a shift towards Warburg-like glycolysis in S. mansoni-infected animals. Notably, S. mansoni eggs stimulated hepatic stellate cells to an alternatively activated phenotype (GFAP⁺/desmin⁺/αSMA^-) that secretes IL-6 and MCP-1. The reduction of fibrosis in older age likely depends on the fine-tuning of regulatory and inflammatory cytokines, alternative HSC activation, and the age-dependent preservation of hepatic energy stores. The current results emphasize the significance of investigations on the clinical relevance of host age-dependent liver damage in patients with schistosomiasis.

Keywords: fibrosis; hepatic stellate cells; host; metabolism; parasite.

Figure 1

Hepatic damage and egg load in S. mansoni-infected mice. (A) Schematic illustration of the animal experiment. Created in BioRender. (B) The expansion of granulomatous tissue decreased in older hosts. Granuloma areas were assessed, as shown in Figure S3. (C) Hepatic egg load and liver-to-body weight ratio (D) were reduced in older S. mansoni-infected mice. (E) The liver-to-body weight ratio correlated well with hepatic egg load in S. mansoni-infected animals. Curve fitting was performed to assess the coefficient of determination and the related probability. (F) Serum ALT levels were raised by S. mansoni infection in all age groups. When animals were infected with S. mansoni at the age of 14 weeks, ALT values increased more prominently compared to the younger animals. There were no changes compared to the older animals. (G) Beyond that, serum ALT was inversely correlated with hepatic egg load across all age groups of S. mansoni-infected mice. Curve fitting analysis was performed to calculate statistical parameters. Color codes indicate the age of infection: blue for 8 weeks, red for 14 weeks, and green for 20 weeks. * indicates p < 0.05, ** indicates p < 0.001. White bars represent uninfected mice, while grey bars represent infected mice. Uninfected controls: n = 6, S. mansoni infected animals: n = 6 (8 w), 10 (14 w), and 8 (20 w). The indicated p-values were calculated by curve fitting analysis or ANOVA and post hoc pairwise comparison of groups using Fisher’s LSD.

Figure 2

S. mansoni-induced splenomegaly in infected mice. (A) Rate of histologically positive findings for eggs in the spleen (grey bars) of infected mice. White bars indicate a negative histological result for eggs in the spleen. Chi-square tests over all groups showed no differences. (B) Infected mice with eggs in the spleen (grey bars) had a higher liver-to-body weight ratio than mice without splenic eggs (white bars). (C,D) Spleen weight (C) and spleen-to-body weight ratio (D) were increased in infected mice (grey bars) and reduced in older infected animals. (E) Lymphatic hyperplasia was assessed histologically by an experienced veterinary pathologist (KK). The number and size of lymphoid follicles, as well as the cellularity of red pulp and occurrence of extramedullary hematopoiesis, were assessed semiquantitatively. Inflammatory lesions were characterized, and parasitic stages were counted. (F) Sirius red staining demonstrated splenic fibrosis within the granuloma around S. mansoni eggs (*). Magnification 200×, bar 100 µm. Uninfected controls: n = 6, S. mansoni infected animals: n = 6 (8w), 10 (14w), and 8 (20w). ** indicates p < 0.001; white bars represent uninfected mice. The indicated p-values were calculated using the Chi²-test or ANOVA and a post hoc pairwise comparison of groups using Fisher’s LSD.

Figure 3

S. mansoni-induced hepatic cytokine expression was highest in younger animals. (A) Inflammatory infiltration led to the formation of granulomas (indicated by dashed line) around S. mansoni eggs (*) and the accumulation of leukocytes within the liver tissue (indicated by arrowhead). A representative liver slice stained with H&E is shown, magnification 200×, scale bar 100 µm. (B) Hepatic mRNA levels of the leukocyte marker Cd45. (C–F) Hepatic mRNA levels of the cytokines Tnfα, Ifnγ, Il4, and Il10 increased with infection but decreased with the age of the host at the time of infection. Uninfected controls: n = 6, S. mansoni infected animals: n = 6 (8w), 10 (14w), and 7 (20w, one sample from this group was excluded from qPCR due to mRNA degradation) and 3 technical replicates each. ** indicates p < 0.001, white bars represent uninfected mice, and grey bars are infected mice. The indicated p-values were calculated by ANOVA and post hoc pairwise comparison of groups using Fisher’s LSD.

Figure 4

S. mansoni–induced hepatic fibrosis is highest among the younger infected. (A) S. mansoni infection-induced hepatic hydroxyproline content, which is a quantitative measure of fibrillar collagen accumulation and, therefore, hepatic fibrosis. Due to the material-consuming analysis and its good reproducibility, hydroxyproline quantification was performed once only. (B) The number of eggs/mg liver tissue correlated well with hepatic hydroxyproline level in S. mansoni-infected animals as determined by curve fitting analysis. (C) Hepatic mRNA levels of type I collagen, Col I, were induced by S. mansoni infection. (D) Representative liver slices stained with Sirius red/fast green visualize granulomatous fibrosis. Magnification 200×, scale bar 100 µm. (E,F) The hepatic protein content of αSMA and desmin were analyzed by Western blotting and subsequent densitometric assessment of signal intensities. Representative blots are shown. Uninfected controls: n = 5–6, S. mansoni infected animals: n = 6 (8w), 8–10 (14w), and 7 (20w) and 3 technical replicates each. ** indicates p < 0.001, white bars represent uninfected mice, and grey bars represent infected mice. The indicated p-values were calculated by curve fitting analysis or ANOVA and post hoc pairwise comparison of groups using Fisher’s LSD.

Figure 5

S. mansoni eggs activated HSCs towards a GFAP⁺/desmin⁺/αSMA⁻ phenotype. (A) Schematic abstract of the transwell co-culture experiments with S. mansoni eggs and the human hepatic stellate cell line LX-2 for subsequent analysis of proliferation and transdifferentiation markers, as well as secreted factors. Created in BioRender. Representative Western blots for HSC markers are depicted below. Quantification of HSC markers is presented in box and whisker plots: (B–D) S. mansoni eggs decrease αSMA expression (B) in LX-2 cells and induced GFAP (C) and desmin (D) expression in a dose-dependent manner. (E,F) Protein levels of human IL-6 and MCP-1/CCL-2 in the supernatant of S. mansoni egg-stimulated LX-2 cells. White bars represent no eggs, while grey bars represent increasing numbers of eggs. Collagen I coated 24 well plates, 1.4 × 10⁵ cells per well cultured overnight before 48 h co-culture in DMEM, Transwell: Costar #3421. n = 6–7 and 3 technical replicates. The indicated p-values were calculated by ANOVA and post hoc pairwise comparison of groups using Fisher’s LSD.

Figure 6

Host age-dependent dysregulation of the hepatic carbohydrate metabolism by S. mansoni eggs. (A) Total liver glycogen increased with age but was reduced by infection with S. mansoni. (B) Hepatic glycogen was inversely dependent on the number of S. mansoni eggs. (C–E) Hepatic mRNA levels of hexokinase 2, Hk2 (C), pyruvate kinase M2, Pkm2 (D), and glucose-6-phosphate dehydrogenase, G6pdh (E) were induced by S. mansoni infection. (F) Notably, G6pdh expression was dependent on Il4 expression. Curve fitting analysis was performed. Uninfected controls: n = 6, S. mansoni infected animals: n = 6 (8w), 10 (14w), and 7 (20w) and 3 technical replicates each. * indicates p < 0.05, ** indicates p < 0.001, white bars represent uninfected mice, while grey bars represent infected mice. The indicated p-values were calculated using curve fitting analysis or ANOVA and post hoc pairwise comparison of groups using Fisher’s LSD.