Contribution of parasite and host genotype to immunopathology of schistosome infections

Abstract

Background: The role of pathogen genotype in determining disease severity and immunopathology has been studied intensively in microbial pathogens including bacteria, fungi, protozoa, and viruses, but is poorly understood in parasitic helminths. The medically important blood fluke Schistosoma mansoni is an excellent model system to study the impact of helminth genetic variation on immunopathology. Our laboratory has demonstrated that laboratory schistosome populations differ in sporocyst growth and cercarial production in the intermediate snail host and worm establishment and fecundity in the vertebrate host. Here, we (i) investigate the hypothesis that schistosome genotype plays a significant role in immunopathology and related parasite life history traits in the vertebrate mouse host and (ii) quantify the relative impact of parasite and host genetics on infection outcomes.

Methods: We infected BALB/c and C57BL/6 mice with four different laboratory schistosome populations from Africa and the Americas. We quantified disease progression in the vertebrate host by measuring body weight and complete blood count (CBC) with differential over an infection period of 12 weeks. On sacrifice, we assessed parasitological (egg and worm counts, fecundity), immunopathological (organ measurements and histopathology), and immunological (CBC with differential and cytokine profiles) characteristics to determine the impact of parasite and host genetics.

Results: We found significant variation between parasite populations in worm numbers, fecundity, liver and intestine egg counts, liver and spleen weight, and fibrotic area, but not in granuloma size. Variation in organ weight was explained by egg burden and by intrinsic parasite factors independent of egg burden. We found significant variation between infected mouse lines in cytokines (IFN-γ, TNF-α), eosinophil, lymphocyte, and monocyte counts.

Conclusions: This study showed that both parasite and host genotype impact the outcome of infection. While host genotype explains most of the variation in immunological traits, parasite genotype explains most of the variation in parasitological traits, and both host and parasite genotype impact immunopathology outcomes.

Keywords: BALB/c mouse; C57BL/6 mouse; Schistosoma mansoni; host-parasite interaction; immunopathogenesis.

Figure 1:. Experimental timeline.

We investigated the influence of parasite and host genotype on disease progression during schistosome infection in BALB/c and C57BL/6 mice. Four laboratory schistosome populations from Africa (SmEG) and the Americas (SmOR, SmLE, and SmBRE) were used to infect the mice. Over a 12-week infection period, we quantified disease progression in the vertebrate host by monitoring body weight and complete blood count (CBC). Upon sacrifice, we measured multiple parasitological, immunopathological, and immunological traits (see box, top right and main text).

Figure 2:. Parasitological outcome is driven by parasite rather than host genotype.

Box and whisker plots showing cercarial penetration rate (A), worm burden (B), liver and intestine egg counts (C) and fecundity (D) for infections with SmBRE, SmEG, SmLE and SmOR in BALB/c (left) and C57BL/6 mice (right). Parasite populations marked with different letters are significantly different in comparisons using Kruskal-Wallis (K-W) followed by Dunn’s or ANOVA followed by Tukey HSD post hoc test. Use of ANOVA or Kruskal-Wallis is shown at the top of each graph. For (C), stats are shown for liver eggs only.

Figure 3:. Impact of parasite and host on mouse immunopathology.

(A) Longitudinal plots of weight gain in mice infected with the 4 parasite populations (solid lines) and uninfected controls (dotted lines). Means shown with standard error. (B) Liver weight or (C) spleen weight from euthanized BALB/c (left) and C57BL/6 mice (right) infected with the 4 parasite populations or uninfected controls. Photos (right) show representative organ samples from mice infected with 3 of the 4 parasite populations and the control group for reference. (D) Fibrotic area and (E) granuloma area measured from liver sections. For all box plots, statistical comparisons are between infected groups only. Parasite populations marked with different letters are significantly different in comparisons using Kruskal-Wallis (K-W) followed by Dunn’s or ANOVA followed by Tukey HSD post hoc test. Differences between BALB/c (left) and C57BL/6 mice (right) in the pathology traits were calculated with Wilcoxon rank-sum or Student’s t-test and are shown by: # P < 0.05, ## P < 0.01, ### P < 0.001.

Figure 4:. White blood cells and reticulocytes are influenced by host background.

Longitudinal data of (A) eosinophil, (B) basophil, (C) lymphocyte and (D) monocyte levels in BALB/c (top) and C57BL/6 mice (bottom) infected with the 4 infected parasite populations (solid lines) or uninfected controls (dotted lines). For all plots, statistical comparisons are between infected groups only. Mice with missing data points were excluded from the analysis. Groups not connected by the same letter and separated by host strain are significantly different (Friedman’s test followed by Conover’s post hoc test). # P < 0.05, ## P < 0.01, ### P < 0.001: values are significantly different between infected (parasite lines combined) host strains using Wilcoxon rank-sum test.

Figure 5:. Cytokine production in response to schistosome infection.

Box and whisker plots showing cytokine production of (A) IFN-γ, (B) TNF-α, (C) IL-4, (D) IL-5, and (E) IL-6 in BALB/c (left) and C57BL/6 mice (right) infected with the 4 parasite populations or uninfected controls. For all plots, statistical comparisons are between infected groups only using Kruskal-Wallis (K-W) followed by Dunn’s post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001: values are significantly different between host strains as calculated by Wilcoxon rank-sum test.

Figure 6:. Effect Size by Parasite and Host Genotype on Disease Parameters.

Heatmap showing effect size measures (Eta squared) as calculated by ANOVA. Parasite includes the 4 parasite populations, host includes both mouse strains, and interactions applies to the interaction between the two. Traits are organized in rows and assigned to the parasitology, immunopathology, or immunology category. * P < 0.05, ** P < 0.01, *** P < 0.001: values are significant per ANOVA result.