Characterization of a Murine Model for Encephalitozoon hellem Infection after Dexamethasone Immunosuppression

Abstract

Background: Encephalitozoon hellem (E. hellem) belongs to a group of opportunistic pathogens called microsporidia. Microsporidia infection symptoms vary and include diarrhea, ocular disorders and systemic inflammations. Traditionally, immunodeficient animals were used to study microsporidia infection. To overcome the difficulties in maintenance and operation using immunodeficient mice, and to better mimic natural occurring microsporidia infection, this study aims to develop a pharmacologically immunosuppressed murine model of E. hellem infection.

Methods: Wild-type C57BL/6 mice were immunosuppressed with dexamethasone (Dex) and then E. hellem spores were inoculated into the mice intraperitoneally. Control groups were the Dex-immunosuppressed but noninoculated mice, and the Dex-immunosuppressed then lipopolysaccharide (LPS)-treated mice. Mice body weights were monitored and all animals were sacrificed at the 15th day after inoculation. Tissue fragments and immune cells were collected and processed.

Results: Histopathological analysis demonstrated that E. hellem inoculation resulted in a disseminated nonlethal infection. Interestingly, E. hellem infection desensitized the innate immunity of the host, as shown by cytokine expressions and dendritic cell maturation. We also found that E. hellem infection greatly altered the composition of host gut microbiota. (4) Conclusions: Dex-immunosuppressed mice provide a useful tool for study microsporidiosis and the interactions between microsporidia and host immunity.

Keywords: Encephalitozoon hellem; dexamethasone; immunity; microsporidia; murine model.

Figure 1

Colonization of E. hellem. (a) Liver and (b) colon samples were collected from E. hellem infected mice, immediately frozen by liquid nitrogen, and then prepared by frozen sectioning. The existences of E. hellem were detected by calcofluor white (CFW) staining, as shown in blue color (also pointed out by white arrow heads) (scale bar = 2 μm). (c) Dendritic cells (DCs) isolated from E. hellem infected mice spleen, the cDNAs were then prepared. A representative image of PCR results using six individual DCs-cDNA samples as template (Lane 1–6), and E. hellem total DNA as positive template control (Lane 7) is shown. All samples showed positive results (as pointed out by arrow), indicating the existence of E. hellem in host DCs. (d) Lane 1 and 2 are representative two DCs-cDNA samples from E. hellem infected mice as template with E. hellem specific primers (arrow shows the positive band at 547 bp); Lane 3 and 4 are representative two DCs-cDNA samples from un-infected mice as template with E. hellem specific primers; Lane 5 is E. hellem total DNA as positive template control (arrow shows the positive band at 547 bp); Lane 6 and 7 are the same two DCs-cDNA samples as in Lane 1 and 2 respectively, and are used as templates with actin specific primers (arrow shows the positive band at 250 bp); Lane 8 and 9 are the same two DCs-cDNA samples from lane 3 and 4 respectively, and are used as templates with actin specific primers (arrow shows the positive band at 250 bp).

Figure 2

Body weights change. Body weights of all three groups of mice (uninfected, E. hellem-infected, and lipopolysaccharide (LPS)-treated) were measured. The first measurements were done on the last day of dexamethasone-injection and then measured again on the day of mice sacrifice, for a total 18 days period of time. The body weight changes over time were calculate and are represented in the figure. LPS-treated mice lost about 0.2 g of body weight over time, while uninfected mice and E. hellem-infected mice gain about 0.2 g of body weight at the end of the experiment. No significant differences among groups were observed. (n = 8 for each group in one experiment, and the experiments have been repeated for three times).

Figure 3

Histopathological analysis. Uninfected control and E. hellem-infected mice organs (colon, spleen and liver) were collected and frozen sectioned respectively. All sections were then subjected to hematoxylin and eosin staining. Here are the representative images of few E. hellem-infected organ samples with histopathological changes. In colon, enlarged microvilli were observed (white arrow heads); In spleen and liver, lymphoepithelioid granulomas were seen (white arrow heads). Images are representatives of all samples from eight control and infected mice respectively. (scale bar = 10 μm).

Figure 4

Cytokines expressions. Plasma samples were prepared from the peripheral blood of uninfected, E. hellem-infected and LPS-treated mice respectively. (a) Cytokine IL-6 expression levels were measured by mouse IL-6 ELISA Kit. Results showed that LPS would significantly upregulate IL-6 expression level, while E. hellem infection did not upregulate IL-6 expression (** = p < 0.01, n = 8 for each group). (b) Il-12 expression levels were quantified by mouse IL-12 ELISA Kit. Results also confirmed that E. hellem infection did not upregulate IL-12 expression, although no statistical differences among groups (n.s = No significance, n = 8 for each group in one experiment, and the experiments have been repeated for three times).

Figure 5

Flow cytometry analysis of DCs maturation. DCs isolated from uninfected control mice, E. hellem-infected mice and LPS-treated mice were subjected to flow cytometry analysis. The amounts of CD40⁺/CD86⁺ double positive DCs were recorded, and the ratios of uninfected to LPS-treated group and E. hellem-infected to LPS-treated group were calculated and presented in figure. As shown, DCs from either E. hellem-infected or uninfected group have significant lower numbers of matured cells. (* = p < 0.05, n = 8 for each group in one experiment, and the experiments have repeated for three times).

Figure 6

Composition of the gut bacteria in E. hellem-infected and uninfected group. The relative abundance of phylum-level (a) and genus-level (b) gut bacteria taxa were analyzed and showed in figure. The results showed E. hellem infection greatly changed the microbiota composition, both on phylum and genus levels.