Fig 1.
H. diminuta-infection is cleared by 11 days post-infection and induces systemic Th2 cytokines in the murine host.
Male BALB/c and C57BL/6 mice were infected with 5 cysticercoids of H. diminuta and assessed at days post-infection (dpi). (A) Murine small intestines were flushed with ice cold PBS to retrieve and enumerate worms. Cytokine ELISAs were performed on supernatants from splenic cells (5x106/mL) from (B, C, D) BALB/c and (E, F, G) C57BL/6 mice stimulated with concanavalin A (2 μg/mL, 48h). Data are mean ± SEM, n = 5-16/group, pooled from 2–3 experiments, * p<0.05 compared to the control (Con) group, analysed by Kruskal Wallis test with Dunn’s test for multiple comparisons.
Fig 2.
H. diminuta-infection induces murine small intestinal tuft cell hyperplasia.
Male BALB/c and C57BL/6 mice were infected with 5 cysticercoids of H. diminuta and assessed at days post-infection (dpi). (A, C) Representative images of mid-jejunal cryosections (10 μm) from control and infected BALB/c and C57BL/6 mice (11 dpi) immunostained for DCLK1 (red) and counterstained with DAPI (blue). “L”–lumen, “V”- villus,” C”- crypt, “LP”- lamina propria and “S”- serosa on the image. DCLK1+ cells were enumerated per villus crypt unit (VCU) and averaged over 20 respective units per mouse. (E, F) Relative mRNA expression (compared to the 18S rRNA housekeeping gene) in mid-jejunal segments from BALB/c and C57BL/6 mice respectively, was assessed by real-time PCR. Data are mean ± SEM values, n = 4-9/group, pooled from 2–3 experiments, * p<0.05 compared to the control (Con) group, analysed by (B, D) Browns Forsythe and Welch’s ANOVA and Dunnett’s test or (E, F) Kruskal Wallis test and Dunn’s test for multiple comparisons; # p<0.05 comparing tuft cell counts at 14 dpi vs at 11 dpi analysed by Welch’s t test (A).
Fig 3.
Tuft cell hyperplasia in response to H. diminuta infection depends on IL-4Rα signalling and adaptive immunity.
Male Il-4rα-/- (A, B) and Rag-1-/- mice (with and without anti-CD90.2 antibody treatment) (C-G) were infected with 5 cysticercoids of H. diminuta and assessed at 11 days post-infection (dpi). For ILC2 depletion, Rag-1-/- mice were treated with 1800 μg anti-CD90.2 antibody as shown in (E). (A, C, F) Small intestines were flushed with ice cold PBS at necropsy to retrieve and enumerate worms (B, D, G) Mid-jejunal and (B) ileal cryosections were immunostained with anti-DCLK1 antibody and counterstained with DAPI. DCLK1+ cells were enumerated per villus crypt unit (VCU). Data are mean ± SEM values, n = 3-9/group, pooled from 2–3 experiments, * p<0.05 compared to control (Con) mice and analysed by (B) Welch’s unpaired t test or (D, G) Browns Forsythe and Welch’s ANOVA test and Dunnet’s post-test for multiple comparisons.
Fig 4.
Tuft cell hyperplasia in response to H. diminuta does not depend on the microbiome.
Male germ free C57BL/6 mice were infected with 10 antibiotic-treated cysticercoids of H. diminuta and assessed at 11 days post-infection (dpi). (A) Murine small intestines were flushed with ice cold PBS at necropsy to retrieve and enumerate worms. (B) Mid-jejunal sections were immunostained with anti-DCLK1 antibody and counterstained with DAPI. DCLK1+ cells were enumerated per villus crypt-unit (VCU) averaged over at least 3 random VCUs/mouse. (C) IL-10 ELISA was performed on supernatants from splenic cells (5x106/mL) stimulated with concanavalin A (2 μg/mL, 48h). Data are mean ± SEM values, n = 5-8/group, pooled from 2 experiments, * p<0.05 compared to control (Con) mice and analysed by Welch’s unpaired t test.
Fig 5.
Tuft cell deficiency delays expulsion of H. diminuta from the murine intestine.
Male (●) and female (◼) Pou2f3-/- (homozygous colony) (A-C), and Pou2f3+/+, +/-, -/- littermate mice (D) were infected with 5 cysticercoids of H. diminuta and assessed at days post-infection (dpi). (A, B) Small intestines were flushed with ice cold PBS to retrieve and enumerate worms. (C) H. diminuta worms from Pou2f3-/- mice were significantly longer at 11 dpi. (D) Unlike their infected wild type littermates that show no detectable worms, Pou2f3-/- mice still harbor H. diminuta worms at 11 dpi. Data in (C) are mean ± SEM, n = 9-21/group, pooled from 2–3 experiments, *p<0.05 analysed by Welch’s t test.
Fig 6.
Tuft cell-deficient mice display local deficiencies in response against H. diminuta.
Male (●) and female (◼) littermate Pou2f3+/+, +/-, -/- mice were infected with 5 cysticercoids of H. diminuta and assessed at days post-infection (dpi). (A) Goblet cells (PAS+) were enumerated in mid-jejunal sections and are represented as number of cells/villus crypt unit (VCU) averaged over at least 3 full VCUs/mouse. (B) Small intestinal transit was measured as % of the small intestine covered by Evan’s blue dye 15 min after oral gavage. (C) Eosinophils were enumerated in mid-jejunal sections and presented as cells per HPF. (D) Mast cell protease-1 concentrations were measured by ELISA in mid-jejunal homogenates. (E) Small intestinal epithelial cell isolations from naïve mice and H. diminuta-infected mice (11 dpi) were used for mRNA analysis. Gene expression data were normalized within each sample to the housekeeping gene 18S rRNA and then to uninfected wild type mice. Data are mean ± SEM, n = 2-9/group, pooled from 2–3 experiments, (A-D) * represents p<0.05 compared to uninfected mice and (E) * and # represent p<0.05 analysed by two-way ANOVA and Tukey’s post-test, where * compared to uninfected mice of the respective genotype and # compared to infected wild type (WT) control.
Fig 7.
Tuft cell-deficient mice display systemic Th2 immune response following infection with H. diminuta.
Male (●) and female (◼) Pou2f3+/+, +/-, -/- mice were infected with 5 cysticercoids of H. diminuta (H.d) and assessed at days post-infection (dpi). In (E, F), mice were treated with praziquantel (PZQ; 1 mg/mouse by oral gavage) at 8 dpi prior to necropsy on 11 dpi. (A, B, C, E) Cytokine ELISAs for IL-4, IL-10, and IL-13 were performed on supernatants from splenic cells (5x106) stimulated with concanavalin A (2 μg/mL, 48h) or (D, F) a PBS-soluble crude extract of adult H. diminuta (HdAg, 200 μg/mL, 96h). Data are mean ± SEM, n = 3-6/group, pooled from 1–3 experiments, * p<0.05 compared to control (Con) uninfected mice, or as indicated on the graph, analysed by (A, B, C) Two-Way ANOVA and Dunnet’s/ Tukey’s post-test or (D) multiple Welch’s t tests.
Fig 8.
Immunophenotyping reveals subtle changes in infected wild type mice compared to infected Pou2f3-/- mice.
Male (●) and female (◼)Pou2f3+/+, +/-, -/- mice were infected with 5 cysticercoids of H. diminuta and assessed at days post-infection (dpi). In (C, D), mice were treated with praziquantel (PZQ; 1 mg/mouse by oral gavage) at 8 dpi prior to necropsy. GATA3+ CD4+ lymphocytes and Foxp3+ CD4+ lymphocyte populations were analysed by flow cytometry using single cell suspensions isolated from the (A-D) mesenteric lymph nodes and (E-H) Peyer’s patches (without ConA stimulation) and presented as (A, C, E, G) a percentage of total live CD4+ lymphocytes and (B, D, F, H) as cell numbers. Data are mean ± SEM, n = 3-10/group, pooled from 1–3 experiments, * p<0.05 compared to genotype matched uninfected (Con) mice analysed by two-way ANOVA followed by Dunnett’s post-test and # p<0.05 analysed by multiple t tests with Hom-Sidak correction comparing between genotypes.
Fig 9.
H. diminuta-infection 10 days prior to H. bakeri provides protection against the nematode.
(A) Male C57BL/6 mice were infected with 5 cysticercoids of H. diminuta (H.d.), then 200 L3 H. bakeri (H.b.) 10 days later and necropsied at 24 days post-infection (dpi) with H. diminuta. Single parasite-infected and naïve mice served as controls. (B-E) In this experimental setting, prior infection with H. diminuta resulted in reduced H. bakeri egg output, fewer worms, and more granulomas. (F) DCLK1+ tuft cells were enumerated per high power field (HPF) of view (40x objective) in swiss rolls made from the first 10 cm of small intestine. Data are mean ± SEM; n = 10/group, pooled from 2 experiments, *p <0.05 compared to H. bakeri only mice or control (con) uninfected mice (B, D, E) by Welch’s unpaired t-test or (F) Browns Forsythe and Welch’s ANOVA test and Dunnet’s post- test for multiple comparisons.
Fig 10.
H. diminuta-induced protection against secondary infection with H. bakeri is tuft cell independent.
Male C57BL/6 or Pou2f3-/- mice were infected with 5 cysticercoids of H. diminuta (H.d.), then 200 larvae of H. bakeri (H.b.) 10 days later and necropsied at 24 days post-infection (dpi) of H. diminuta. Single parasite infected and naïve mice served as controls. Co-infected C57BL/6 and Pou2f3-/- mice show (A-C) reduced egg burden and increased numbers of granulomas. (D) Co-infected C57BL/6 but not Pou2f3-/- mice show reduced adult luminal worms. Data are mean ± SEM values, n = 5-6/group, * and # represent p <0.05 analysed by Browns Forsythe and Welch’s ANOVA test and Dunnett’s post- test for multiple comparisons, where * compared to H. bakeri only mice of respective strain and # compared to wild-type H. bakeri only mice.