Regulatory role of the intestinal microbiota in the immune response against Giardia

Abstract

Giardia duodenalis is one of the most commonly found intestinal parasites in mammalian hosts. Infections can generally be cleared by mounting an adequate protective immune response that is orchestrated through IL-17A. This study was aimed to investigate if and how the intestinal microbiome affects the protective Th17 response against Giardia by analysing and comparing the immune response following a G. muris and G. duodenalis infection in antibiotic treated and untreated mice. Depletion of the intestinal flora by antibiotic treatment had a severe effect on the infection dynamics of both Giardia species. Not only duration of infection was affected, but also the parasite burden increased significantly. Markers associated with a protective immune response, such as IL-17A and mannose binding lectin 2 were still significantly upregulated following infection in the antibiotic-treated mice, despite the lack of protection. On the other hand, the antibiotic treatment significantly decreased the level of IgA in the intestinal lumen by affecting its transporter and by reducing the number of IgA⁺ B-cells at the Peyer's patches. Furthermore, the depletion of the gut microbiota by antibiotics also significantly lowered the intestinal motility. The combination of these factors likely results in a decreased clearance of the parasite from the intestinal tract.

Figure 1

Dynamics of Giardia infections in mice following antibiotic treatment. (A) G. duodenalis cysts present in the feces of antibiotic-treated and untreated mice were monitored daily until day 21 p.i. Mean numbers of cysts per gram faeces obtained from 5 mice at every time point are depicted, with SEM as error bars. (B) G. duodenalis trophozoite numbers in the small intestine of antibiotic-treated C57Bl/6 mice at indicated time points. No trophozoites were detected in the untreated mice. (C) G. muris cysts present in the faeces of antibiotic-treated and untreated mice were monitored from day 4 p.i. until day 21 p.i. Mean numbers of cysts per gram faeces obtained from 5 mice at every time point are depicted, with SEM as error bars. (D) Area under the curve analysis of cyst counts between antibiotic treated and untreated C57Bl/6 mice infected with G. muris (E) G. muris trophozoite numbers in the small intestine of antibiotic-treated and untreated C57Bl/6 mice at indicated time points. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 2

Effect of antibiotic treatment on the bacterial load in mice determined by qPCR on DNA extract of fecal (A–D) pellets and intestinal content (E). Results are shown for Total Bacteria, Bacteroidetes, Firmicutes and Beta proteobacteria as the mean of 5 mice with SEM errors bars. (**p ≤ 0.01).

Figure 3

Effect of antibiotic treatment on the transcription of anti-microbial peptides and MBL2 in non-infected control mice without (black bar) and with (white bar) after 5 days of antibiotic cocktail treatment (A) α-defensins (defa-tot), (B) MMP-7, (C) defb1, (D) MBL2 and (E) ANG4. Relative mRNA expression levels were measured by qPCR. Mean transcription level from 5 mice are shown with SEM as error bars. (*p ≤ 0.05, **p ≤ 0.01).

Figure 4

Kinetics of the intestinal IL-17A and MBL2 response following a G. duodenalis (A, B) and G. muris (C, D) infection in C75Bl/6 mice without (black bar) and with (white bar) antibiotic cocktail treatment. Relative mRNA expression levels were measured by qPCR at indicated time points. Mean transcription level from 5 mice are shown with SEM as error bars. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 5

Effect of antibiotic treatment and G. muris infection on the intestinal production and secretion of IgA. (A) Relative percentage of IgA producing B-cells within the CD45⁺ cell population isolated from the Peyer’s patch tissue. (B) Relative percentage of total B-cells within the CD45⁺ cell population isolated from the Peyer’s patch tissue. (C) Relative mRNA expression levels of PIgR measured by qPCR at indicated time points. (D) Total intestinal IgA levels. Results are shown as the mean from 5 mice with SEM as error bars. (*p ≤ 0.05, **p ≤ 0.01).

Figure 6

Effect of antibiotic treatment and G. muris infection on the intestinal motility. (A) Picture of murine small intestine, 20 min after oral administration of 10% charcoal solution. (B) Kinetics of intestinal transit following a G. muris infection in C75 BL/6 mice without (black bar) and with (white bar) antibiotic treatment. Results are shown as the mean from 5 mice with SEM as error bars. (*p ≤ 0.05, **p ≤ 0.01).