Inverse Correlation of Th2-Specific Cytokines with Hepatic Egg Burden in S. mansoni-Infected Hamsters

Abstract

Schistosomiasis, a parasitic disease caused by Schistosoma spp., affects more than 250 million people worldwide. S. mansoni in particular affects the gastrointestinal tract and, through its eggs, induces a Th2 immune response leading to granuloma formation. The relationship between egg load and immune response is poorly understood. We investigated whether the quantity of parasitic eggs influences the immune response in S. mansoni-infected hamsters. The hepatic and intestinal egg load was assessed, and cytokine expression as well as the expression of three major egg-derived proteins were analyzed in monosex- and bisex-infected animals by qRT-PCR. Statistical correlations between egg load or egg-derived factors Ipse/alpha-1, kappa-5, and omega-1, and the immune response were analyzed in liver and colon tissue. Surprisingly, no correlation of the Th1 cytokines with the hepatic egg load was observed, while the Th2 cytokines Il4, Il5, and Il13 showed an inverse correlation in the liver but not in the colon. A longer embryogenesis of the parasitic eggs in the liver could explain this correlation. This conclusion is supported by the lack of any correlation with immune response in the colon, as the intestinal passage of the eggs is limited to a few days.

Keywords: S. mansoni; cytokines; immune response; schistosomiasis.

Figure 1

S. mansoni infection caused individual hepatic and colon egg burdens with different patterns of Ipse/α1 expression. (A) Experimental setting. Created in BioRender. Roderfeld, M. (2023) BioRender.com/o55m552. (B) Uniform distribution of eggs within a liver lobe. The liver lobes of bs-infected hamsters were stained with HE in total cross-section. Overview of a liver lobe in cross-section with parasitic eggs (red circles). Enlargement with eggs (→) and granulomas delineated by (---). (C,D) Regression analysis demonstrated that the correlation of egg load and Ipse/α1 fitted best with an exponential curve in the liver and a linear dependence in the colon. The expression of hepatic and colon Ipse/α1 was analyzed by qRT-PCR in the bisex group and evaluated using the 2^−∆∆CT method (liver n = 22, colon n = 14). Three technical replicates were performed for each tissue. Relative quantification was calculated from qRT-PCR data using the 2^−∆∆CT method [24] and normalized to the monosex-infected group or, in the case of S. mansoni genes, to an artificially introduced control of cDNA derived from mRNA isolated from S. mansoni eggs.

Figure 2

(A–D) S. mansoni infection-induced Th1 cytokine response was independent of hepatic egg burden. (A) Increased Ifnγ expression in livers of ms- (n = 10) and bs-S. mansoni-infected hamsters (n = 22). (B) No correlation between Ifnγ expression and hepatic egg load in bs-infected hamsters was observed. (C) Tnfα expression increased in livers of ms- (n = 10) and bs-S. mansoni-infected hamsters (n = 22). (D) No correlation was found between Tnfα expression and hepatic egg load in bs-infected hamsters. After performing qRT-PCR, the analysis of the data was performed using the 2^−∆∆CT method. (E) Ifnγ expression was induced in the colon of bisex-infected hamsters. (F) Ifnγ expression in the colon exponentially correlated to the egg load in the colon. (G) Tnfα expression was induced in the colon of bisex-infected hamsters. (H) Tnfα expression showed no correlation to egg burden in the colon. The ni group (n = 6) served as control. Three technical replicates were performed, and their mean values were determined. The equal distribution of expression values between the groups was tested using the Kruskal–Wallis test (p < 0.001) (A,C,E,G). In the regression analyses, egg load served as the independent variable, and fold increase in Ifnγ mRNA as the dependent variable.

Figure 3

Inverse egg load-dependence of the hepatic Th2 cytokines IL-4, IL-5, and IL-13. (A,C,E) Increased expression (transcript) levels of Il4 (A), Il5 (C), and Il13 (E) in the liver of infected hamsters. (B,D,F) Negative correlation between Il4 (B), Il5 (D), and Il13 mRNA (F) and the respective egg load. Relative quantification was calculated from qRT-PCR data using the 2^−∆∆CT method and normalized to the ms- (A–D) or ni-groups (E,F). Mean values were calculated from three technical replicates each; biological replicates covered bs-infected (n = 22), ms-infected (n = 10), and ni (n = 6). The equal distribution of expression values between the groups was tested using the Kruskal–Wallis test ((A) p < 0.001; (C) p < 0.001). The linear correlation was determined by linear regression analyses.

Figure 4

S. mansoni infection-induced Il4 expression was independent of intestinal egg burden. (A,C,D) An increased expression of Il4 (A), but not of Il5 (C) and Il13 (D), was observed in the bowel of infected hamsters. (B) No correlation was found between Il4 and the respective intestinal egg load. The relative quantification was calculated from qRT-PCR data using the 2^−∆∆CT method and normalized to the ms-infected group (A,C,D). Mean values were calculated from three technical replicates each; biological replicates covered bs-infected (n = 14), ms-infected (n = 7), and ni (n = 7) animals. The equal distribution of expression values between the groups was analyzed using the Kruskal–Wallis test ((A) p < 0.001; (C) p < 0.001).

Figure 5

Inverse correlation between hepatic Il4 (A–C) or Il13 mRNA (D–F) and the gene expression of the different soluble egg-dependent factors Ipse/α1, Kappa5 and Omega1.

Figure 6

Expression of Il10 in liver and colon was independent of the egg burden. (A) An increased expression of Il10 in livers of ms- and bs-infected hamsters was observed. (B) We found no correlation between Il10 and the hepatic egg load. (C) In the colon of bs-infected hamsters, we found an increased expression of Il10. (D) Also in the colon, we found no correlation between Il10 and egg load. (A–D) mRNA quantification was performed by qRT-PCR and the 2^−∆∆CT method (normalization to non-infected group). Liver samples were obtained from bs-infected (n = 22), ms-infected (n = 10), and ni (n = 6) animals. Colon samples were obtained from bs-infected (n = 14), ms-infected (n = 7), and ni (n = 7) animals. Mean values were calculated from three replicates. The equal distribution of expression values between the groups was analyzed using the Kruskal–Wallis test ((A) p < 0.001). In the regression analysis, egg load served as the independent variable, and Il10 as the dependent variable (B).