We found that parasite and host genotype impact different categories of disease parameters during infection and post sacrifice. Out of four parasitological traits, parasite population strongly influenced the number of worms, liver egg burden and fecundity, whereas host influenced liver egg burden only. In contrast, host genotype impacted seven of 13 traits related to the immunological profile, with no discernible impact from the parasite population. In the immunopathology category (six traits), parasite genotype influenced three traits (liver weight, spleen weight, and fibrosis), while host genotype influenced five traits (spleen weight, intestine length, weight gain, fibrotic and granuloma area). Hence, the severity of immunopathology observed is dependent on both parasite and host.
Parasite population effects on immunopathology
Egg burden is measured in epidemiological surveys because it is an important predictor of immunopathology in schistosomiasis [44–46]. Parasite genotype contributes to immunopathology in two ways in our experiments. First, we see significant differences between parasite populations in worm burden and fecundity. These jointly contribute to large differences in egg burden between parasite populations and impact liver and spleen weight and fibrosis. However, we also observe intrinsic differences between parasite lines, that impact immunopathology independent of egg burden. Our GLM analysis (Table 2) demonstrated that while egg burden contributed to variation in liver and spleen weight, parasite genotype also significantly impacted these traits. This is clearly shown by examining correlations between egg burden and organ weight (Additional file 5: Fig. S5).
This study confirms and extends our previous findings from studies of two Brazilian populations, SmBRE and SmLE, that show striking differences in cercarial shedding and sporocyst growth in the intermediate snail host [16, 17, 47]. These two parasite populations also showed the most extreme phenotype differences during infection in the vertebrate host. Despite comparable penetration success, we counted fewer worms and eggs and calculated lower fecundity in mice infected with SmBRE. Additionally, parameters such as liver weight, spleen weight, and fibrotic area were also lower in SmBRE-infected mice compared to the other schistosome populations characterized in the present study. We have previously demonstrated that fibrotic area and granuloma size were reduced in mice infected with parasite progeny from SmBRE and SmLE genetic crosses selected for low or high cercarial shedding from the aquatic snail [17]. While the experimental design in this study was rather different, we saw comparable differences in fibrotic area (lower in SmBRE than in SmLE), although granuloma size was unaffected. These observations underscore the profound impact of parasite factors on multiple phenotypes including immunopathology.
Host genetic influences on susceptibility and immune profiles during infection
We did not detect differences in susceptibility and parasite fecundity between the mouse host strains. This supports findings from Alves et al. [41] who used SmLE and found no effect of mouse genetic background on worm and egg burden (Table 1). However, this is inconsistent with reports by Incani et al. [21], who documented higher worm but lower egg burden in BALB/c compared to C57BL/6 mice using Venezuelan and Brazilian S. mansoni populations, and Bin Dajem et al. [48] who found increased worm burden in C57BL/6 hosts with an Egyptian parasite. These differences could be explained by (i) the number of cercariae used for the infection (Alves: 30, Incani: 60, Bin Dajem: 100), (ii) the method of infection (tail immersion vs abdominal skin), (iii) statistical methods (log transformation vs non-parametric tests), (iv) the duration of the study, or (v) even genetic variation in the mouse colonies maintained at the different institutions [49].
IFN-γ, TNF-α, IL-6, monocyte and eosinophil levels were strongly influenced by host genotype as indicated by GLM and ANOVA analyses. This aligned with our expectations since C57BL/6 and BALB/c mice are known to induce different immune responses to many pathogens and are typically described as IFN-γ high/IL-4 low and IFN-γ low/IL-4 high responders respectively [22]. We observed comparable levels of IL-4 in BALB/c and C57BL/6 mice, but significantly increased IFN-γ (a known antifibrogenic agent) levels in infected BALB/c mice [50]. We hypothesize that this outcome represents hyperproduction in BALB/c mice, inducing protective immunity in response to schistosome infection [51, 52]. This could prevent BALB/c mice from mounting a potent type 2 immune response, impact granuloma formation, and explain the mortality we observed in BALB/c and not in C57BL/6 mice [53–55].
We observed larger granulomas, which are predominantly driven by a type 2 immune response, in C57BL/6 mice [40, 43, 56]. In contrast, Alves et al. [41] measured larger granulomas in BALB/c mice. In addition to high IFN-γ levels, we also saw significantly more eosinophils at week 10 post infection in the blood of BALB/c mice compared to C57BL/6 mice. This could imply that eosinophils in C57BL/6 mice were recruited to the liver tissue, because granulomas surrounding S. mansoni eggs are mainly composed of eosinophils [56, 57]. However, that eosinophil depletion did not change granuloma size in schistosome infected mice contradicts this idea [58]. Future studies could follow up on this observation using blood and whole spleen flow cytometry to distinguish the different immune cell populations in both S. mansoni infected mouse hosts.
Both GLM and ANOVA revealed a significant impact of host-parasite interactions on IL-5 secretion. IL-5 influences various cell types including B cells and eosinophils in a pleiotropic manner [59, 60]. This suggests that the four schistosome populations may differ in antigenicity, resulting in varying levels of induced inflammation affected by mouse host genetics. The fact that IL-5 is involved in fibrosis regulation, coupled with our analyses revealing a nearly significant impact of parasite population on fibrosis, supports this notion [61].
Limitations of this study
The influence of parasite genetics on immunopathological parameters measured in this study is likely to be conservative. Laboratory schistosome populations maintain surprisingly high levels of genetic and phenotypic variation [62, Jutzeler et al., in prep]. By infecting mice with genetically diverse schistosome larvae from four parasite populations, we captured an average phenotype for each population, so extreme phenotype traits will be masked. We expect that use of genetically homogeneous parasite populations would result in much larger effects of parasite genotype. Future studies could focus on phenotype measures in mice infected with inbred schistosome lines generated by serial inbreeding over several generations. Such inbred schistosome lines are not currently available, but could provide a valuable resource for investigating the role of parasite genetics in determining outcome of infection.
Similarly, we probably overestimated the role of host genetics by conducting these experiments using two inbred mouse strains known to show divergent immune responses to infection. Future work on host influences on schistosome immunopathology could use inbred mouse lines from the collaborative cross to determine the host genes involved [63].