Figures and data in Concerted IL-25R and IL-4Rα signaling drive innate type 2 effector immunity for optimal helminth expulsion

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6 figures, 1 table and 1 additional file

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Figure 1

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IL-25R signaling is required for expulsion of H.

*polygyrus* from BALB/c mice. *H. polygyrus*-infected BALB/c or *Il17rb*-deficient (IL25R^–/–) BALB/c mice were analyzed at day 14 post-infection for fecal egg counts (A) and intestinal adult worm burden (B) or day 28 post-infection for fecal egg counts (C) and intestinal adult worm burden (D). MLN cells underwent intracellular cytokine staining (ICCS) to compare the number of Lin^-ICOS⁺innate lymphoid cells (ILCs) (E) and IL-13⁺ ILC2s (F) by flow cytometry in the different naïve and day 14 infected genotypes. Peritoneal lavage cells (PL) were stained with Siglec-F, CD11b and Ly6G/C to compare the number of Siglec-F^-CD11b⁺Ly6G/C⁺ monocytes (G) in the different naïve and day 14 infected genotypes. Percentages of eosinophils (H) and RELMα⁺ alternatively-activated macrophages (Ι) were also determined over a 4 week infection timecourse. ELISA of supernatants from MLN cells incubated with media or 1 μg HES for 72 hr was performed to compare IL-4 (J), IL-5 (K) and IL-13 (L) production in the different naïve and day 14 infected genotypes. ICCS of MLN allowed a comparison of the proportion of IL-4⁺, IL-5⁺ and IL-13⁺ CD4⁺ T cells by flow cytometry in the different naïve and day 14 infected genotypes (**M–O**). Results shown are one representative of three experiments with n ≥ 4 mice/group (**A–D**), pooled data from two experiments with n ≥ 4 mice/group (**C–F, M–O**) or one representative of two experiments with n ≥ 4 mice/group (**G–L**). Data were analysed by unpaired t test or one way ANOVA, where *=p≤0.05,**=p≤0.01,***=p≤0.001 and ns = not significant. Error bars represent Standard Error of the Mean.

https://doi.org/10.7554/eLife.38269.002

Figure 2

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IL-25R signaling is required within the hematopoietic compartment for efficient expulsion.

Bone marrow chimeras generated from BALB/c (WT) or *Il17rb*-deficient (KO) donor and BALB/c (WT) or *Il17rb*-deficient (KO) recipient mice were infected with *H. polygyrus* and intestinal adult worm burden (A) and fecal egg burden (B) performed at day 28 post-infection. Results shown are pooled data from two experiments performed with n ≥ 3 mice/group, and data from all individual mice are presented. Data were analysed by unpaired t test, where *=p≤0.05,**=p≤0.01,***=p≤0.001 and ns = not significant. Error bars represent Standard Error of the Mean.

https://doi.org/10.7554/eLife.38269.003

Figure 3

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IL-25R signaling synergises with IL-4Rα within the innate immune compartment to facilitate efficient worm expulsion.

*H. polygyrus*-infected *Rag1^-/-* mice (RAG^-/-) were given 0.4 µg recombinant IL-25 i.p. days 14–17 (late) post-infection with or without a complex of 5 μg rIL-4 and 25 μg anti-IL-4 (IL-4C) on days 13, 16 and 19 post-infection, according the schedule shown in (A). Mice were analyzed at 28 days post-infection for fecal egg burden (B) and intestinal adult worm burden (C). *H. polygyrus*-infected *Rag1^-/-* mice were given IL-25 and IL-4C according to the same schedule, as well as 200 µg of anti-CD90.2/Th1.2 antibody or rat IgG2b control (days 12, 15, 18 and 21). The peritoneal lavage was analyzed at 28 days post-infection for CD45⁺lin^- (CD3, CD5, CD8α, CD11c, CD19, DX5, F4/80, GR-1, TCRβ, CD11b), ICOS and ST2 staining by flow cytometry as shown (D) and the number of CD45⁺lin^– (E), CD45⁺lin^–ST2^– (F) and CD45⁺lin^–ST2⁺ (G) ILCs was determined. Mice were analysed at 28 days post-infection for fecal egg burden (H) and intestinal adult worm burden (I). Results shown are one representative of two experiments with n = 4 mice/group (**D–G**), or pooled data from two experiments with n ≥ 3 mice/group (**B,C,H,I**). Data were analysed by unpaired t test, where *=p≤0.05,**=p≤0.01,***=p≤0.001 and ns = not significant. Error bars represent Standard Error of the Mean.

https://doi.org/10.7554/eLife.38269.004

Figure 4

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IL-25 induces protective immune responses in the late stage of infection.

*H. polygyrus*-infected C57BL/6 mice were given 0.4 μg recombinant IL-25 i.p. at day 1–4 (early) or day 14–17 (late) post-infection according to the schedule shown in (A). Before administration of IL-25, intestinal egg burden was analysed in two groups at day 14 post-infection (B). Mice were then analysed at day 28 post-infection for fecal egg counts (C) and intestinal adult worm burden (D) following administration of IL-25 early or late. *H. polygyrus*-infected C57BL/6 mice were given 0.4 μg recombinant IL-25 i.p. at day 14–17 (late) post-infection. Day 18 post-infection, MLN cells underwent ICCS to compare the proportion of IL-4⁺CD4⁺ (E) and IL-13⁺CD4⁺ (F) T cells, as well as the number of ILCs (G) and Lin-IL-13 + ILC2 s (H) by flow cytometry. Results shown are data pooled from three experiments with n ≥ 3 mice/group (**B–D**), or are representative of two experiments with n = 4 mice/group (**E,F**) or pooled from two experiments with n = 4 mice/group (**G, H**). Data were analysed by unpaired t test, where *=p≤0.05,**=p≤0.01,***=p≤0.001 and ns = not significant. Error bars represent Standard Error of the Mean.

https://doi.org/10.7554/eLife.38269.005

Figure 5

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IL-25 drives alternative activation and IL-13 expression of macrophages.

*H. polygyrus*-infected C57BL/6 mice were given 0.4 μg recombinant IL-25 i.p. at day 14–17 (late) post-infection. At day 18 post-infection, PL cells were taken and stimulated with 10 μg Brefeldin A to determine the expression intensity (geomean) of IL-13 (A) or were unstimulated to determine the expression intensity (geomean) of RELM-α (**B, C**) within Siglec-F^–CD11b⁺F4/80⁺ monocytes by flow cytometry, as shown by the example flow cytometry plots in B and compared to fluorescence minus one (FMO) samples stained from the Hp:IL-25 group. Total Siglec-F⁺CD11b⁺ eosinophil (D) numbers were calculated from populations shown in the example flow cytometry plots (E), numbers of Siglec-F^–CD11b⁺ (F) monocytes. Bone marrow-derived macrophages from C57BL/6 mice were generated in vitro and stimulated with 10 ng/ml IL-4, 200 ng/ml IL-25 or a combination of both for 16 hr before analysis of the percentage of RELM-α expression (**G, H**) and the mean fluorescence intensity (geometric mean) of Arginase-1 expression (**I, J**) within CD11b⁺F4/80⁺ cells by flow cytometry. Results shown are one representative of 2 experiments with n = 4 mice/group (**A–F, I, J**) or pooled from two experiments with n = 4 replicates/group (**G, H**). Data were analysed by unpaired t test, where *=p≤0.05,**=p≤0.01,***=p≤0.001 and ns = not significant. Error bars represent Standard Error of the Mean.

https://doi.org/10.7554/eLife.38269.006

Figure 6

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IL-4Rα expression is required for activation of the immune system by IL-25.

*H. polygyru*s-infected *Il4ra*^-/- mice (IL-4R^-/-) were given 0.4 μg recombinant IL-25 i.p. days 14–17 (late) post-infection (A). Mice were analyzed at day 28 post-infection for intestinal adult worm burden (B). *H. polygyrus*-infected BALB/c and *Il4ra*^-/- mice (IL-4R^-/-) were given 0.4 μg recombinant IL-25 i.p. days 14–17 (late) post-infection. Day 18 post-infection, PL cells were taken to determine the percentage of eosinophils (C) and percentage of RELM-α expression within Siglec-F^-CD11b⁺F4/80⁺ macrophages by flow cytometry (D). Results shown are one representative of 2 experiments with n ≥ 4 mice/group (**A–D**). Data were analysed by unpaired t test, where *=p≤0.05,**=p≤0.01,***=p≤0.001 and ns = not significant. Error bars represent Standard Error of the Mean.

https://doi.org/10.7554/eLife.38269.007

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers
Mus musculus, BALB/c	Il17rb^-/- (IL-25R^-/-)	Neill et al. (2010) Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 464:1367–1370
Mus musculus, C57BL/6	Rag1^-/-	Mombaerts,P., Iacomini,J., Johnson, R.S., Herrup, K., Tonegawa,S., and Papaioannou, V. E. 1992. Cell. Vol 68: 869–877. PMID: 1547488
Mus musculus, C57BL/6	Il4ra-/-	Noben-Trauth N, Shultz LD, Brombacher F, Urban JF, Jr., Gu H, Paul WE. 1997. An interleukin 4 (IL-4)-independent pathway for CD4 + T cell IL-4 production is revealed in IL-4 receptor-deficient mice. Proc Natl Acad Sci USA 94:10838–10843.
Parasite	Heligmosomoides polygyrus bakeri	Johnston et al., 2015. Cultivation of Heligmosomoides polygyrus: an immunomodulatory nematode parasite and its secreted products. Journal of Visualized Experiments 98:e52412.
Antibody	Anti-IL-4	BioXCell	Clone 11B11
Antibody	Anti-IL-13	BioLegend	JES10-5A2
Antibody	Anti-CD90.2	BioXCell	30H12
Antibody	Anti-SiglecF	BD Pharmingen	E50-2440
Antibody	Anti-CD11b	BioLegend	M1/70
Antibody	Anti-F4/80	BioLegend	BM8
Antibody	Anti-Ly6G	BioLegend	1A8
Antibody	Anti-Ly6C	BioLegend	AL-21
Antibody	Anti-RELMα	R and D Systems	22603
Antibody	Anti-Arginase-1 (Polyclonal)	R and D Systems	IC5868P
Antibody	Anti-Lineage	BioLegend	17A2,RB6-8C5,RA3-6B2,Ter-119,M1/70
Peptide, recombinant protein	IL-4	Peprotech	214–14
Peptide, recombinant protein	IL-25	BioLegend	587302
Commercial assay or kit	Foxp3 Staining Kit	eBioScience	88–8118
Other	Control IgG2b immunoglobulin	BioXcell	LTF-2
Other	Anti-CD90.2 Microbeads	Miltenyi	130-049-101