Giardia-induced Type 2 mucosal immunity attenuates intestinal inflammation caused by co-infection or colitis in mice

Abstract

Diarrhoeal diseases are the second leading cause of death in children worldwide. Epidemiological studies show that co-infection with the protozoan parasite Giardia intestinalis decreases diarrhoeal severity. Here we show a high incidence of asymptomatic Giardia infection in school-aged children from Nigeria. In a mouse model, Giardia induced a Type 2 mucosal immune response, characterized by antigen-specific Th2 cells, IL-25, Type 2 cytokines, and goblet cell hyperplasia. Single-cell RNA sequencing and multiparameter flow cytometry revealed expansion of IL-10-producing Th2 cells, which promoted parasite persistence and protected against Toxoplasma gondii-induced ileitis and dextran sulfate sodium-induced colitis. This protective effect was STAT6 dependent, as IL-4R blockade or STAT6 deficiency impaired IL-10⁺ Th2 responses, resulting in Th1/Th17-driven tissue damage, inflammation and clearance of Giardia infection. Our findings demonstrate that Giardia reshapes mucosal immunity toward a Type 2 response, facilitating parasitism and conferring mutualistic protection from inflammatory pathologies, highlighting a key role for protists in mucosal defence regulation.

Figure 1.. Giardia-infected mice induce a Th2 immune response in the lamina propria at the small and large intestines.

(A) Experimental schematic. Female C57BL/6 mice were perorally infected with 1×10⁶ Giardia GS/M strain trophozoites. (B) Scatter plot graphs indicating the absolute number of Th1 (Foxp3⁻Tbet⁺IFN-γ⁺), Th2 (Foxp3⁻GATA3⁺IL-13⁺), and Th17 (Foxp3⁻RORγt⁺IL-17⁺) cells in the small intestine lamina propria of Giardia-infected mice (7 d.p.i.). Gated on Live CD45⁺TCRβ⁺CD4⁺. (C) IFN-γ, IL-4, IL-13, and IL-17 levels in the small intestine tissue homogenate of Giardia-infected mice (7 d.p.i.) measured by Luminex. (D) IFN-γ, IL-4, IL-13, and IL-17 levels produced by small intestine isolated lamina propria cells after 24 hours of in vitro stimulation with Giardia soluble antigens (10 μg/mL). (E) Representative image of the jejunum from naïve or Giardia-infected mice stained with Periodic Acid Schiff (PAS) for mucus production by Goblet cells (7 d.p.i.). Scale bars represent 100 μm. (F) IL-25 levels in the small intestine tissue homogenate of Giardia-infected mice (7 d.p.i.) measured by ELISA. (G) Scatter plot graphs indicating the frequency and the absolute number of Th2 cells (Foxp3⁻GATA3⁺) in the colonic lamina propria of Giardia-infected mice (7 d.p.i.) (gated on Live CD45⁺TCRβ⁺CD4⁺), and IL-4 levels in the colon (7 d.p.i.). (H) Giardia burden in the small intestine (duodenum) and colon of C57BL/6 infected mice (7 d.p.i.). Data are represented as mean ± SEM and significance was calculated with non-parametric Mann-Whitney test. *p≤0.05, **p≤0.01, ***p≤0.001. Data are representative of four (B-C) or two (D-H) independent experiments.

Figure 2.. Giardia induces secretion of IL-10 and expansion of IL-10-producing Th2 and GATA3⁺ Treg cells in the small intestine.

IL-10 levels in the (A) small intestine and (B) colon of Giardia-infected mice (7 d.p.i.) measure by Luminex. (C) IL-10 levels produced by small intestine isolated lamina propria cells after 24 hours of in vitro stimulation with Giardia soluble antigens (10 μg/mL). (D) Scatter plot graphs indicating the frequency and the absolute number of IL-10-producing Treg cells (Foxp3⁺GATA3⁻IL-10⁺) in the small intestine lamina propria of Giardia-infected mice (7 d.p.i.) (gated on Live CD45⁺TCRβ⁺CD4⁺). (E) Experimental schematic. Female C57BL/6 IL-10 GFP reporter mice were perorally infected with 1×10⁶ Giardia GS/M strain trophozoites. (F) Frequency and absolute number of IL-10-producing cells in the small intestine lamina propria of Giardia-infected mice (7 d.p.i.). Gated on: CD4⁺ T cells (Live CD45⁺IL-10⁺TCRβ⁺CD19⁻CD4⁺), CD8⁺ T cells (Live CD45⁺IL-10⁺TCRβ⁺CD19⁻CD8⁺), Macrophages (Live CD45⁺IL-10⁺TCRβ⁻CD19⁻CD11b⁺CD64⁺), Dendritic Cells (DCs) (Live CD45⁺IL-10⁺TCRβ⁻CD19⁻CD11c⁺MHCII⁺), B cells (Live CD45⁺IL-10⁺TCRβ⁻CD19⁺), and Innate Lymphoid Cells (ILCs) (Live CD45⁺IL-10⁺TCRβ⁻CD19⁻CD90.2⁺). (G) Scatter plot graphs indicating the frequency and the absolute number of IL-10-producing Th2 (Foxp3⁻GATA3⁺), GATA3⁺ Treg (Foxp3⁺GATA3⁺), and non-Th2/GATA3⁺ Treg (Foxp3⁻GATA3⁻) cells in the small intestine lamina propria of Giardia-infected mice (7 d.p.i.) (gated on Live CD45⁺TCRβ⁺CD4⁺). Data are represented as mean ± SEM and significance was calculated with non-parametric Mann-Whitney test. *p≤0.05, **p≤0.01, ***p≤0.001. Data are representative of two independent experiments.

Figure 3:. Single-cell transcriptional profiling of Giardia-induced IL-10-producing CD4⁺ T cells.

(A) Experimental schematic. Small intestine lamina propria cells were isolated from Giardia-infected IL-10 GFP reporter mice (7 d.p.i.), and IL-10-producing CD4⁺ T cells were sorted for further analysis by single-cell RNA-seq. (B) Representative dot plots of sorted TCRβ⁺CD4⁺IL-10⁺ from naïve and Giardia-infected mice (7 d.p.i.). (C) UMAP plots showing the identity of IL-10 producing CD4⁺ T cell subpopulations by RunUMAP and clustering analysis, and PoW plots indicating the frequency of T helper and T regulatory cell subpopulations in the naïve or Giardia-infected TCRβ⁺CD4⁺IL-10⁺ sorted population. (D) Heatmap showing the expression of the most significantly 20 enriched transcripts in each subpopulation cluster and pathway enrichment analysis using the top 25 markers in Th2 cells. (E) and (F) Violin plots indicating IL-4 and IL-13 transcripts expression in CD4⁺ T cell subpopulations. Data are representative of one experiment using 8 naïve and 12 Giardia-infected mice.

Figure 4:. Type 2 signaling blockage impairs Giardia-induced IL-10 and shifts the mucosal immune response to a Type 1/17 phenotype that promotes parasite clearance.

(A) Experimental schematic. Female WT or STAT6^−/− mice (C57BL/6 background) were perorally infected with 1×10⁶ Giardia GS/M strain trophozoites. (B) Scatter plot graphs indicating the absolute number of Th2 (Foxp3⁻GATA3⁺IL-13⁺), IL-10⁺ Th2 (FoxP3⁻GATA3⁺IL-10⁺), and IL-10⁺ GATA3⁺ Treg (FoxP3⁺GATA3⁺IL-10⁺) cells in the small intestine lamina propria of Giardia-infected mice (7 d.p.i.) (gated on Live CD45⁺TCRβ⁺CD4⁺). (C) Scatter plot graphs indicating the absolute number of Th17 (FoxP3⁻RORγt⁺IL-17A⁺), Th1 (Foxp3⁻GATA3⁻Tbet⁺IFN-γ⁺), and IL-10⁺ Treg (FoxP3⁺GATA3⁻IL-10⁺) cells in the small intestine lamina propria of Giardia-infected mice (7 d.p.i.) (gated on Live CD45⁺TCRβ⁺CD4⁺). (D) IL-4, IL-10, and IL-17A levels in the small intestine of WT or STAT6^−/− Giardia-infected mice (7 d.p.i.). (E) Representative image of the jejunum from WT or STAT6^−/− Giardia-infected mice stained with Alcian Blue/Periodic Acid Schiff (AB/PAS) for mucus production by goblet cells (7 d.p.i.). Scale bars represent 100 μm. (F) IL-25 levels in the small intestine tissue homogenate of WT or STAT6^−/− Giardia-infected mice (7 d.p.i.) measured by ELISA. (G) Giardia burden in the small intestine (duodenum) of WT or STAT6^−/− Giardia-infected mice (7 d.p.i.). Data are represented as mean ± SEM and significance was calculated with one-way ANOVA test followed by Sidak’s multiple comparisons test. *p≤0.05, **p≤0.01, ***p≤0.001. Data are representative of two independent experiments.

Figure 5:. Previous infection with Giardia induces protection against inflammatory bowel-like diseases.

(A) Experimental schematic. Female C57BL/6 mice were perorally infected with 1×10⁶ Giardia GS/M strain trophozoites and three days later mice were perorally co-infected with 10 cysts of Toxoplasma gondii Type II 76K GFP-Luc strain. (B) Body weight loss of mice single infected with Toxoplasma or co-infected with Giardia and Toxoplasma were monitored daily. (C) Representative image of hematoxylin-and-eosin staining (H&E) of the proximal small intestine (swiss roll) from naïve, Giardia-, Toxoplasma- or Giardia+Toxoplasma-infected mice (8 days-post Toxoplasma infection, 8 d.p.T.i.). Scale bars represent 160 μm. (D) IFN-γ and IL-10 levels in the small intestine of singly or co-infected mice (8 d.p.T.i.) measured by Luminex. (E) Scatter plot graphs indicating the frequency of Th1 (FoxP3⁻Tbet⁺), total Treg (FoxP3⁺), GATA3⁺ Treg (GATA3⁺FoxP3⁺), Th2 (GATA3⁻Foxp3⁺), and IL-10⁺ Th2 (FoxP3⁻GATA3⁺IL-10⁺) cells in the small intestine lamina propria of singly or co-infected mice (8 d.p.T.i.). Gated on Live CD45⁺TCRβ⁺CD4⁺. (F) Experimental schematic. Female C57BL/6 mice were perorally infected with 1×10⁶ Giardia GS/M strain trophozoites and three days later mice were administered 2% DSS drinking water or normal drinking water for 7 consecutive days. From day 7 to day 8 mice were placed back on normal drinking water, and euthanasia was performed on day 8 (8 days-post treatment, 8 d.p.t.). (G) Body weight loss of DSS-treated mice infected or not with Giardia was monitored daily. (H) Representative image of H&E staining of the small and large intestine sections from DSS-treated mice infected or not with Giardia. Scale bars represent 250 μm. (I) IL-10 and IL-6 levels in the distal small and proximal large intestines of DSS-treated mice infected with or without Giardia (8 d.p.t.) measured by Luminex. (J) Scatter plot graphs indicating the frequency of Th2 (FoxP3⁻GATA3⁺IL-13⁺), IL-10⁺ Th2 (Foxp3⁻GATA3⁺IL-10⁺), and Th17 (RORγt⁺IL-17A⁺) cells in the colonic lamina propria of DSS-treated mice infected or not with Giardia (8 d.p.t.). Gated on Live CD45⁺TCRβ⁺CD4⁺. Data are represented as mean ± SEM and significance was calculated with one-way ANOVA test followed by Sidak’s multiple comparisons test. *p≤0.05, **p≤0.01, ***p≤0.001. Data are representative of three (A-E) and two (G-J) independent experiments.

Figure 6:. Type 2 signaling blockage reduces Giardia-mediated protection against inflammatory bowel-like diseases.

(A) Experimental schematic. Female WT or STAT6^−/− mice (C57BL/6 background) were perorally infected with 1×10⁶ Giardia GS/M strain trophozoites and three days later mice were perorally co-infected with 10 cysts of Toxoplasma gondii. Euthanasia was performed 8 days post-Toxoplasma infection (8 d.p.T.i.). (B) Body weight loss of WT or STAT6^−/− mice co-infected with Giardia and Toxoplasma were monitored daily. (C) Representative image of H&E staining of the proximal small intestine from WT or STAT6^−/− mice co-infected with Giardia and Toxoplasma (8 d.p.T.i.). (D) IFN-γ, IL-6, IL-13, and IL-10 levels in the proximal small intestine of WT or STAT6^−/− co-infected mice (8 d.p.T.i.) measured by Luminex. (E) Scatter plot graphs indicating the frequency of Th1 (Foxp3⁻Tbet⁺), Treg (Foxp3⁺GATA3⁻), GATA3⁺ Treg (GATA3⁺FoxP3⁺), Th2 (GATA3⁻Foxp3⁺), and IL-10⁺ Th2 (FoxP3⁻GATA3⁺IL-10⁺) cells in the small intestine lamina propria of WT or STAT6^−/− co-infected mice (8 d.p.T.i.). Gated on Live CD45⁺TCRβ⁺CD4⁺. (F) Experimental schematic. WT or STAT6^−/− mice were perorally infected with 1×10⁶ Giardia GS/M strain trophozoites and three days later mice were administered 2% DSS drinking water or normal drinking water for 7 consecutive days. From day 7 to day 8 mice were placed back on normal drinking water, and euthanasia was performed on day 8 (8 d.p.t.). (G) Body weight loss of WT or STAT6^−/− Giardia-infected and DSS-treated mice was monitored daily. (H) Representative image of H&E straining of the colon from Giardia-infected and DSS-treated mice. Scale bars represent 200 μm (8 d.p.t.). (I) IL-17, IL-10, IL-13, and IL-25 levels in the distal small and proximal large intestines of WT or STAT6^−/− Giardia-infected mice and treated with DSS (8 d.p.t.) measured by Luminex. (J) Scatter plot graphs indicating the frequency of Th2 (FoxP3⁻GATA3⁺IL-13⁺), IL-10⁺ Th2 (Foxp3⁻GATA3⁺IL-10⁺), Th17 (RORγt⁺IL-17A⁺), and Th1 (Tbet⁺IFN-γ⁺) cells in the colonic lamina propria of WT or STAT6^−/− Giardia-infected mice and treated with DSS (8 d.p.t.). Gated on Live CD45⁺TCRβ⁺CD4⁺. (K) Experimental schematic. Female C57BL/6 mice were perorally infected with 1×10⁶ Giardia GS/M strain trophozoites (day −3) or treated with recombinant IL-10 (250 ng/mouse) and/or recombinant IL-4 and IL-13 (5ug/mouse each) every two days (days 0, 2, 4, and 6), and on day 0 all groups were perorally infected with 10 cysts of Toxoplasma gondii. Euthanasia was performed 8 days post-Toxoplasma infection (8 d.p.T.i.). (L) Representative image of H&E staining of the proximal small intestine from Toxoplasma-infected mice that were co-infected with Giardia or treated with rIL-10 and/or rIL-4/rIL-13 (8 d.p.T.i.). (M) Body weight loss of mice was monitored daily. (N) Scatter plot graph indicating the frequency of IFN-γ⁺ CD4⁺ T cells in the small intestine lamina propria of Toxoplasma-infected mice that were co-infected with Giardia or treated with rIL-10 and/or rIL-4/rIL-13 (8 d.p.T.i.). Gated on Live CD45⁺TCRβ⁺CD4⁺. Data are represented as mean ± SEM and significance was calculated with non-parametric Mann-Whitney test (D-E, I-J) and one-way ANOVA test followed by Sidak’s multiple comparisons test (B, M, N). *p≤0.05, **p≤0.01, ***p≤0.001. Data are representative of two independent experiments.