IL-33 facilitates rapid expulsion of the parasitic nematode Strongyloides ratti from the intestine via ILC2- and IL-9-driven mast cell activation

Abstract

Parasitic helminths are sensed by the immune system via tissue-derived alarmins that promote the initiation of the appropriate type 2 immune responses. Here we establish the nuclear alarmin cytokine IL-33 as a non-redundant trigger of specifically IL-9-driven and mast cell-mediated immunity to the intestinal parasite Strongyloides ratti. Blockade of endogenous IL-33 using a helminth-derived IL-33 inhibitor elevated intestinal parasite burdens in the context of reduced mast cell activation while stabilization of endogenous IL-33 or application of recombinant IL-33 reciprocally reduced intestinal parasite burdens and increased mast cell activation. Using gene-deficient mice, we show that application of IL-33 triggered rapid mast cell-mediated expulsion of parasites directly in the intestine, independent of the adaptive immune system, basophils, eosinophils or Gr-1+ cells but dependent on functional IL-9 receptor and innate lymphoid cells (ILC). Thereby we connect the described axis of IL-33-mediated ILC2 expansion to the rapid initiation of IL-9-mediated and mast cell-driven intestinal anti-helminth immunity.

Fig 1. IL-33 reduces intestinal S. ratti burden.

(A) Experimental procedure: BALB/c mice were treated i.n. with PBS (open circles), 1 μg rec. IL-33 (closed circles), 5 μg rec. HpARI (closed squares) or 5 μg rec. CCP1/2 (closed triangles) in 20 μl PBS 3 h before and 24 h post s.c. infection with 2000 S. ratti L3. (B and C) Shown are combined results from 3 independent experiments (n = 4–5 per experiment and group), each symbol represents an individual mouse, bars show the mean and asterisk indicate statistically significant differences of the means of untreated (PBS) or treated mice (B: one-way ANOVA, C: students t-test).

Fig 2. IL-33 reduces intestinal S. ratti burden independent of the tissue migration phase.

(A) Experimental procedure: BALB/c mice were treated i.p. (B,C,F, and G) or i.n. (D and E) with PBS (open circles) or 1 μg rec. IL-33 (closed circles). Treatment was performed either 3 h before and 24 h after S. ratti infection (black circles, B,C,D, and F) or after the tissue migration phase i.e. 4 days and 5 days after S. ratti infection (blue circles, E and G). (B) Mice were sacrificed at the indicated time points and L3, L4 and adults in the intestine were counted and (C) S. ratti-derived DNA in the feces was quantified by qPCR at the indicated time points. Shown are the combined results of 2 independent experiments (n = 4 per experiment, group, and time point), symbols show the mean and error bars indicate SEM (D-G). Adults in the intestine were counted at day 6 p.i. Shown are combined results from 2–3 independent experiments (n = 3–5 each), each symbol represents an individual mouse, bars show the mean and asterisks indicate statistically significant difference of the means (B and C: two-way ANOVA, D-G: students t-test).

Fig 3. IL-33 treatment induces rapid mast cell activation that mediates accelerated intestinal parasite expulsion.

(A) BALB/c mice were either left uninfected (white and blue bars) or infected with 2000 S. ratti L3 s.c. (black and red bars). Mice received PBS (white bars, black bars) or 1 μg rec. IL-33 (blue bars, red bars) either 3 h before and 24 h post S. ratti infection or mock infection. mMCPT-1 in the serum was quantified by ELISA at the indicated time points. Graph shows combined results of 2–3 independent experiments (n = 3–5 per experiment and time point, n = 2 for PBS control), bars indicate the mean and error bar show SEM. Asterisks indicate statistically significant differences of the mean of S. ratti + PBS to PBS in black; IL-33 to PBS in red: S. ratti + IL-33 to S. ratti + PBS in blue and S. ratti + IL-33 to IL-33 in green (one-way ANOVA performed for the 4 groups at each time point separately). (B and C) BALB/c mice received PBS (open circles) or (B) 5 μg rec. HpARI (closed squares) or (C) 5 μg rec. CCP1/2 (closed triangles) i.p. 3 h before and 24 h post s.c. infection with 2000 S. ratti L3. mMCPT-1 in the serum was quantified by ELISA at the indicated time points. (D) SCID mice or (E) Cpa3^Cre mice (squares) and wildtype littermates (circles) were treated with 1 μg rec. IL-33 (closed symbols) or with PBS (open symbols) 3 h before and 24 h post S. ratti infection. Mice were sacrificed day 6 p.i. to count adults in the intestine. Graphs show the combined results of 2–4 independent experiments (n ≥ 3–5 per experiment and group), each symbol represents an individual mouse, bars show the mean and asterisks indicate statistically significant differences of the mean, numbers indicate p value (B,C, and E: students t-test D: Mann-Whitney test).

Fig 4. IL-33 mediates accelerated intestinal expulsion of S. ratti parasites independent of basophilic, eosinophilic and neutrophilic granulocytes but dependent of IL-9.

(A) BALB/c Mcpt8^Cre mice (squares) and non-transgenic littermates (circles) or (B) ΔdblGATA mice (squares) and co-housed BALB/c mice (circles) or (C) BALB/c mice treated with anti Gr-1 mAb (depletion protocol and control is shown in S3 Fig) (squares) or isotype control, and (D) BALB/c IL-9 receptor-deficient mice (squares) and co-housed BALB/c mice (circles) were treated i.p. with 1 μg of IL-33 (closed symbols) or with PBS (open symbols) 3 h before and 24 h post S. ratti infection. mMCPT-1 concentration in the sera was quantified day 2 p.i. and parasitic adults in the intestine counted day 6 p.i. Graphs show combined results from 2–4 independent experiments (n = 3–8 per experiment and group). Each symbol represents an individual mouse, bars show the mean, asterisks indicate statistically significant differences of the mean, numbers indicate p value (Mann-Whitney and students t-test).

Fig 5. ILC promote IL-33-mediated S. ratti expulsion from the intestine.

(A-D) BALB/c RAG^-/- mice (circles) and BALB/c RAG^-/- γc^-/- mice (squares) were treated i.p. with 1 μg of IL-33 (closed symbols) or with PBS (open symbols) 3 h before and 24 h post S. ratti infection. (A) Representative dot blots showing frequency of ILC2 in spleens of BALB/c RAG^-/- mice or BALB/c RAG^-/- γc^-/- mice with or without IL-33 treatment. Cells were measured using an LSRII Cytometer (BD, Germany) and analyzed by FlowJo software. (B) Frequencies of lung, spleen and PEC cells day 6 p.i. Graphs show combined results from 1 (lung), 2 (spleen) or 3 (PEC) independent experiments. (C) Parasitic adults in the intestine were counted 6 days p.i. and (D) mMCPT-1 concentration in the sera was quantified at indicated time points p.i. Graphs show combined results from 3 independent experiments (n = 4 per experiment and group) or 1 experiment (day 6 mMCPT-1). Each symbol represents an individual mouse, bars show the mean, asterisks indicate statistically significant differences of the mean, numbers indicate p value (Mann-Whitney test (B and D) and students t-test (C).

Fig 6. Role of IL-33 during S. ratti infection.

The cartoon illustrates the proposed functions of IL-33 during S. ratti infection. (i) Migrating S. ratti larvae induce IL-33 that (ii) activates ILC2 to produce IL-9 that further activates ILC2, (iii) IL-9 directly and/or indirectly activates mucosal mast cells (iv) independently of T and B cells, eosinophils, basophils or neutrophils, (v) to promote ejection of S. ratti from the intestine.