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

Nature Microbiology volume 10, pages 1886–1901 (2025)Cite this article

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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.

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Fig. 1: Giardia induces a Type 2 immune response in the small and large intestines of mice.
Fig. 2: Giardia induces secretion of IL-10 and expansion of IL-10-producing Th2 and GATA3+ Treg cells in the small intestine.
Fig. 3: Single-cell transcriptional profiling of Giardia-induced IL-10-producing CD4+ T cells.
Fig. 4: Type 2 signalling blockage impairs Giardia-induced IL-10 and shifts the mucosal immune response to a Type 1/17 phenotype that promotes parasite clearance.
Fig. 5: Previous infection with Giardia induces protection against bystander intestinal inflammation.
Fig. 6: Type 2 signalling blockage reduces Giardia-mediated protection against bystander intestinal inflammation.

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Data availability

The data supporting the findings of this study are available in the Article, Extended Data and Source Data. The raw single-cell RNA-seq data generated in this study have been deposited in the NCBI SRA database under BioProject ID PRJNA1082359, available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1082359. The processed Seurat objects and the associated R code are available at https://doi.org/10.5281/zenodo.15390320 (ref. 72). Source data are provided with this paper.

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Acknowledgements

We thank B. Joshua, D. Samuel, M. Ali, N. Victor, A. Onekutu and E. Effanga who coordinated the samples collection in Nigeria, as well as administered the questionnaire used to establish whether infection was associated with symptomatic disease. We also thank C. de Oliveira Silva Souza and P. Loke from the Type 2 Immunity Section, LPD, NIAID, for providing us the STAT6−/− mice, and S. Ganesan from the Biological Imaging Section, RTB, NIAID, for assistance with histology image scanning. This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health, and NIH extramural grant AI109591 to S.M.S.

Author information

Author notes

  1. Marc Y. Fink

    Present address: Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA

Authors and Affiliations

  1. Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Aline Sardinha-Silva, Pedro H. Gazzinelli-Guimaraes, Oluwaremilekun G. Ajakaye, Tiago R. Ferreira, Eliza V. C. Alves-Ferreira, Erick T. Tjhin, Beth Gregg & Michael E. Grigg

  2. Department of Microbiology, Immunology and Tropical Medicine, School of Medicine and Health Sciences, George Washington University, Washington, DC, USA

    Pedro H. Gazzinelli-Guimaraes & Fabricio M. S. Oliveira

  3. Department of Animal and Environmental Biology, Adekunle Ajasin University, Akungba Akoko, Nigeria

    Oluwaremilekun G. Ajakaye

  4. Department of Biology, Georgetown University, Washington, DC, USA

    Marc Y. Fink & Steven M. Singer

  5. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Camila H. Coelho

  6. Center for Vaccine Research and Pandemic Preparedness, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Camila H. Coelho

  7. Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Camila H. Coelho

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  1. Aline Sardinha-Silva
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Contributions

A.S.-S., C.H.C., S.M.S. and M.E.G. conceptualized the project. A.S.-S., P.H.G.-G., C.H.C. and T.R.F. designed the methodology. A.S.-S., P.H.G.G., O.G.A., F.M.S.O., T.R.F., E.V.C.A.-F., E.T.T., B.G. and M.Y.F. conducted investigation. A.S.-S. and P.H.G.G. wrote the original manuscript draft. A.S.-S., P.H.G.-G., S.M.S. and M.E.G. reviewed and edited the manuscript. S.M.S. and M.E.G. acquired funding and resources. M.E.G. supervised the project.

Corresponding authors

Correspondence to Aline Sardinha-Silva or Michael E. Grigg.

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Nature Microbiology thanks Peter Cook and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Nigeria map showing sampled states.

1. Benue (latitude: 7° 19’ 60.00” N and longitude: 8° 44’ 59.99” E); 2. Cross River (latitude: 4° 34’ 59.99” N and longitude: 8° 24’ 59.99” E); 3. Enugu (latitude: 6° 27’ 9.60” N and longitude: 7° 30’ 37.20” E); 4. Jigawa (latitudes 11.00°N to 13.00°N and longitudes 8.00°E to 10.15°E); 5. Kano (latitude: 12° 00’ 0.43” N and longitude: 8° 31’ 0.19” E); 6. Plateau (latitudes 8°24’ N to 10°30’ N and longitudes 8°32’ E to 10°38’).

Source data

Extended Data Fig. 2 Cytokine production kinetics across the three segments from the small intestine in Giardia-infected mice.

Levels of IFN-γ (a), IL-4 (b), IL-13 (c), IL-17A (d), and IL-10 (e) in the three different sections of the small intestine tissue homogenate (normalized by 100 mg of tissue) from naïve (n = 3) or Giardia-infected. (n-5) mice at 5-, 7-, 9-, 12-, and 21-days post-infection (measured by Luminex). Data are represented as mean ± SEM for each time point 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.

Source data

Extended Data Fig. 3 Gating strategy for immunophenotypic analysis by Flow Cytometry.

(a) The gating strategy 1 was used for the overall immunophenotypic analysis of the experiments reported in Figs. 1b, 1g, 2d, 2g, 4b, 4c, 4i, 5f, 5k, 6f, 6l, and Extended Data Fig. 6b. Briefly, singlets (a) were gated followed by subsequent lamina propria cell gating by FSC-A vs SSC-A (b). (c) Live hematopoietic cells were gated as CD45/Alexa Fluor700+ and Live/Dead/UV496−. (d) B cells and myeloid cells were then excluded by gating as CD19/PE-Cy5− and CD11b/BV605−. (e) CD4+ T cells were further gated as TCRβ/BUV737+ and CD4/BV510+. Subsets of CD4+ T cells were further subdivided into either the single or co-expression of FoxP3/BV421 in combination with single or co-expression of Tbet/PE-Cy7 (f), GATA3/APC (h) or RORγt/PE-CF594 (k). Finally, IFN-γ/BV650 (g), IL-10/BV711 (i), IL-13/PE (j), and IL-17A/PerCP-Cy5.5 (l) were analyzed into the populations of Tbet+ Th1 cells, GATA3+ Th2 cells, and RORγt+ Th17 cells, respectively. (b) The gating strategy 2 was design specifically for the identification of the major sources of IL-10 in the lamina propria of either naïve- and Giardia-infected IL-10 GFP reporter mice (Fig. 2f). For this, live CD45/Alex Fluor 700+ lamina propria cells (a, b) were gated for the IL-10 GFP expression (d), using a WT mouse as a control for a negative gate (c). (e) IL-10 producing hematopoietic cells were further characterized as either TCRβ/BUV737+ T cells, or CD11b/BV605+ cells or TCRβ−CD11b− cells. (f) T cells were subdivided as CD4/BV510+ cells or CD8β/APC+ cells. (g) CD64/BV421+ macrophages were identified within the CD11b+ cells; and (h) CD64/BV421−, CD11c+/PE-CF596 + , MHCII+/BV711+ cells were classified as Dendritic Cells (DCs). (i) Non-T and non-myeloid cells were further gated as CD19/PE-Cy5+ cells or TCRγδ/PerCP.Cy5.5 T cells. (j) Finally, IL-10 producing CD45+TCRβ−CD11b−CD19−TCRγδ−CD90/BV785+ cells were classified as innate lymphoid cells (ILCs).

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Extended Data Fig. 4 Molecular characterization of IL-10 producing CD4+ T cells in the small intestine lamina propria.

(a) UMAP plot showing the clustering analysis of the merged dataset of all sorted IL-10 producing CD4+ T subsets in the small intestine lamina propria from both naïve and Giardia-infected IL-10-GFP reporter mice. The plot distinctly identifies five key clusters, corresponding to Th1, Th2, Th17, Treg, and Th2 Treg cells subset. (b) Dot plot graph highlighting the top10 highly expressed genes within each cluster of IL-10 producing CD4+ T cells in the small intestine lamina propria from naïve and Giardia-infected mice. (c) UMAP plot of Giardia-induced IL-10-producing CD4+ T cells five clusters mapped against to public available scRNA-seq data set for CD4+ T cells isolated from lamina propria of mice infected with different pathogens (Kiner et al. 2021). (d) dot plot graph highlighting the top20 highly expressed genes in the Th2 cells induced by Heligmosomoides polygyrus (Kiner et al., 2021) within each cluster of IL-10 producing CD4+ T cells in the small intestine lamina propria from naïve and Giardia-infected mice.

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Extended Data Fig. 5 Giardia induces expansion of ILC2s in the small intestine in a STAT6-dependent manner.

(a) Gating strategy for immunophenotypic analysis of ILCs by flow cytometry. (b-d) Scatter plot graphs indicating the frequency of ILC1 (CD90.2+IFN-γ+), ILC2 (CD90.2+IL-13+), and ILC3 (CD90.2+IL-17A+) cells in the small intestine lamina propria of naïve (n = 4) Giardia-infected (n = 4) mice (7 d.p.i.). Gated on Live CD45+CD19−CD11b−TCRβ−CD4−. Data are represented as mean ± SEM for each time point 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.

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Extended Data Fig. 6 The effect of Giardia chronicity in the co-infection with T. gondii.

(a) Bioluminescent detection in photons/sec/cm2 shows Toxoplasma burden in vivo (7 d.p.T.i.) in mice co-infected (n = 18) or not (n = 18) with Giardia GS/M strain three days before. (b) 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 naïve (n = 4), Giardia- (three days before, d-3) (n = 4), Toxoplasma- (n = 4), or Giardia (d-3)+Toxoplasma-infected (n = 4) mice 4 days post-Toxoplasma infection (4 d.p.T.i.). Gated on Live CD45+TCRβ+CD4+. (c) IL-10 levels in the proximal small intestine of naïve (n = 4) and Giardia-chronically infected (n = 5 per group) mice (4 weeks post-infection, 4 wks). (d) Representative image of H&E staining of the proximal small intestine from naïve, Toxoplasma-, Giardia (d-3)+Toxoplasma-, or Giardia (4 wks)+Toxoplasma-infected mice (8 days-post Toxoplasma infection). Scale bars represent 100 μm. Data are represented as mean ± SEM for each time point 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 (a-b) and one (c-d) experiment.

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Extended Data Fig. 7 IL-4R blockage reduces Giardia-mediated protection against bystander intestinal inflammation.

(a) Experimental schematic. Female WT 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, followed by intraperitoneal injection of anti-IL-4R (50 μg) or anti-IL10R (100 μg) for five consecutive days. Euthanasia was performed 8 days post-Toxoplasma infection (8 d.p.T.i.). Created with BioRender. (b) Body weight loss of WT mice co-infected with Giardia and Toxoplasma followed treatment with isotype control (n = 5), anti-IL-4R (n = 5) or anti-IL-10R (n = 5) were monitored daily. (c) Representative image of H&E staining of the jejunum from WT mice co-infected with Giardia and Toxoplasma treated with isotype control (n = 5), anti-IL-4R (n = 5) or anti-IL-10R (n = 5) and scatter plot graph showing the histopathology score (8 d.p.T.i.). Scale bars represent 500 μm (top) and 300 μm (bottom). (d) Experimental schematic. Female WT mice (C57BL/6 background) were perorally infected with 1×106 Giardia GS/M strain trophozoites and three days later mice were administered 2% DSS drinking water for 7 consecutive days, followed by intraperitoneal injection of anti-IL-4R (50 μg) or anti-IL10R (100 μg) for five consecutive days. Euthanasia was performed on day 8 (8 d.p.t.). Created with BioRender. (e) Body weight loss of WT Giardia-infected and DSS-treated mice followed isotype control (n = 4), anti-IL-4R (n = 5) or anti-IL-10R (n = 5) injection. (f) Representative image of H&E staining of the colon from WT mice Giardia-infected and DSS-treated mice followed isotype control (n = 4), anti-IL-4R (n = 5) or anti-IL-10R (n = 5) injection and scatter plot graph showing the histopathology score (8 d.p.t.). Scale bars represent 500 μm (top) and 300 μm (bottom). Figure symbols in c and f: [*] inflammatory infiltrate in the mucosa; [$] inflammatory infiltrate in the submucosa; [#] inflammatory infiltrate in the muscle layer; [arrow] mucosal ulceration; [short arrow] mucosal desquamation. Data are represented as mean ± SEM and significance was calculated with one-way ANOVA test followed by Sidak’s multiple comparisons test (c,f). *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Data are representative of one independent experiment (n = 5).

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Supplementary Tables 1–3

Supplementary Table 1. Giardia intestinalis prevalence in children ≤10 years old in Nigeria. Table 2. Asymptomatic vs Symptomatic infected children ≤10 years old in Nigeria. Table 3. Scoring criteria for semi-quantitative histopathological analysis.

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Sardinha-Silva, A., Gazzinelli-Guimaraes, P.H., Ajakaye, O.G. et al. Giardia-induced Type 2 mucosal immunity attenuates intestinal inflammation caused by co-infection or colitis in mice. Nat Microbiol 10, 1886–1901 (2025). https://doi.org/10.1038/s41564-025-02051-2

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