Course of <i>Encepahlitozoon cuniculi</i> genotype II infection, including pattern of spore shedding and dissemination of infection to selected organs and tissues.

<p><b>a)</b> SCID mice, <b>b)</b> SCID mice treated with albendazole, <b>c)</b> BALB/c mice and BALB/c mice re-infected in chronic stage of infection, <b>d)</b> BALB/c mice treated with albendazole and BALB/c mice treated with albendazole with following re-infection, <b>e)</b> BALB/c mice immunosuppressed in chronic stage of infection, <b>f)</b> BALB/c mice immunosuppressed after albendazole treatment. <b>Light-gray field</b> – albendazole treatment; <b>dark-gray field</b> – dexamethasone immunosuppression; <b>black line</b> – course of <i>E. cuniculi</i> infection; <b>black dash line</b> - course of <i>E. cuniculi</i> re-infection; <b>cross</b> – <i>E. cuniculi</i> positive organ during primarily infection; <b>ring</b> – <i>E. cuniculi</i> positive organ during re-infection; <b>black square</b> – spores shedding during primarily infection; <b>black circle</b> – spores shedding during re-infection.</p

Design of experiments.

<p><b>^a</b>inoculation with 200 ul sterilized deionised water; <b>^b</b>infection with 10⁷<i>E. cuniculi</i> spores in 0.2 ml of sterilized deionised water; INF – infection; REINF – reinfection (black column); IMSUP – dexamethasone immunosuppression (highlighted in dark grey); TREAT – albendazole treatment (highlighted in light grey); <b>n1–</b> number of used animals; <b>n2</b>– number of dissected animals; <b>NS</b> – not shown; <b>x</b> – not observed due to mouse death; <b>DPI</b> – day post infection;</p

A benign helminth alters the host immune system and the gut microbiota in a rat model system

<div><p>Helminths and bacteria are major players in the mammalian gut ecosystem and each influences the host immune system and health. Declines in helminth prevalence and bacterial diversity appear to play a role in the dramatic rise of immune mediated inflammatory diseases (IMIDs) in western populations. Helminths are potent modulators of immune system and their reintroduction is a promising therapeutic avenue for IMIDs. However, the introduction of helminths represents a disturbance for the host and it is important to understand the impact of helminth reintroduction on the host, including the immune system and gut microbiome. We tested the impact of a benign tapeworm, <i>Hymenolepis diminuta</i>, in a rat model system. We find that <i>H</i>. <i>diminuta</i> infection results in increased interleukin 10 gene expression in the beginning of the prepatent period, consistent with induction of a type 2 immune response. We also find induction of humoral immunity during the patent period, shown here by increased IgA in feces. Further, we see an immuno-modulatory effect in the small intestine and spleen in patent period, as measured by reductions in tissue immune cells. We observed shifts in microbiota community composition during the patent period (beta-diversity) in response to <i>H</i>. <i>diminuta</i> infection. However, these compositional changes appear to be minor; they occur within families and genera common to both treatment groups. There was no change in alpha diversity. <i>Hymenolepis diminuta</i> is a promising model for helminth therapy because it establishes long-term, stable colonization in rats and modulates the immune system without causing bacterial dysbiosis. These results suggest that the goal of engineering a therapeutic helminth that can safely manipulate the mammalian immune system without disrupting the rest of the gut ecosystem is in reach.</p></div

Hematology line graph panel figure for leukocytes.

<p>Abundance of leukocytes in blood samples of rats infected with <i>Hymenolepis diminuta</i> (dark grey, n = 4) and healthy, uninfected rats (light grey, n = 4). The samples were taken before infection, during the pre-patent period (after infection before establishment), and at two time points during the patent period (after the establishment of the infection). The error bars represent the 95% confidence interval of the mean cell counts for each group of rats at each time point. The asterisks represent a significant difference in the mean cell counts at α = 0.05 level for t-tests implemented at each time point. (*): 0.01 ≤ p < 0.05, (**): 0.001< p < 0.01.</p

Experiment timeline.

<p>The entire experiment included five periods during which fecal, blood and tissue samples were collected: (i) acclimatization–rats were co-housed for a month prior to the start of experiment to acclimate laboratory conditions; (ii) before infection–the fecal samples for microbial analyses and for ELISA analyses were collected; (iii) infection–we infected rats in three consecutive days to prevent negativity of some animals from experimental group; (iv) prepatent period–period from infection to adults maturation, i.e. releasing of eggs in feces, (v) patent period–adults were present in the intestine; during prepatent and patent period were collected fecal, blood and tissue samples.</p

ELISA line graph for fluctuation of IgA antibodies from feces.

<p>Mean absorbance values for rats infected with <i>Hymenolepis diminuta</i> (dark grey, n = 4) and control rats (light grey, n = 4). Other notes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182205#pone.0182205.g003" target="_blank">Fig 3</a>.</p

List of anti-rat antibodies used in flow cytometry analyses.

<p>List of anti-rat antibodies used in flow cytometry analyses.</p

Flow cytometry boxplot panel figure—general leukocyte populations.

<p>Abundance of leukocyte populations in flow cytometric samples from rats infected with <i>Hymenolepis diminuta</i> (dark grey, n = 4) and control rats (light grey, n = 4). The error bars represent the 95% confidence intervals for the mean cell counts at each sample location for each group. The asterisks represent a significant difference in the mean cell counts at α = 0.05 level for t-tests implemented at each time point. (*): 0.01 ≤ p < 0.05, (**): 0.001< p < 0.01. [the numbers of cells are in the chart are given in 10⁴ in duodenum & ileum, colon, in case of spleen in 10⁶].</p

Composition of the gut microbiota shifted in response to <i>Hymenolepis diminuta</i> infection.

<p>Principle coordinate plots of the Bray-Curtis dissimilarity metric. A) All samples (N = 219) colored by treatment group and shaped according to time period. The seven samples are intestinal mucosa samples (triangles), and the rest are from feces. B-D) Fecal microbiota over the time course of infection. Gray = control and black = <i>H</i>. <i>diminuta</i> treatment. Each rat is represented by a different symbol/color combination. P-values for PERMANOVA with rat nested in treatment group are shown, and full results are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182205#pone.0182205.s008" target="_blank">S1B Table</a>) Before <i>H</i>. <i>diminuta</i> infection: Day 1 to Day 30 (n = 80), C) prepatent period of <i>H</i>. <i>diminuta</i> infection: Day 38 to Day 50 (n = 32). D) patent period of <i>H</i>. <i>diminuta</i> infection: Day 51 to Day 82 (n = 80).</p

Relative expression of interleukin 10 during <i>Hymenolepis diminuta</i> infection.

<p>IL-10 gene expression in rat blood significantly increased during the early phase of prepatent period. In late prepatent period and patent period, expression of IL-10 dropped to basal levels. Expression is shown in relation to the UBC as the housekeeping gene. Error bars represent the standard errors from four independent biological replicates. (***): P<0.0001.</p