Identification of an interleukin (IL)-25-dependent cell population that provides IL-4, IL-5, and IL-13 at the onset of helminth expulsion

Abstract

Type 2 immunity, which involves coordinated regulation of innate and adaptive immune responses, can protect against helminth parasite infection, but may lead to allergy and asthma after inappropriate activation. We demonstrate that il25(-/-) mice display inefficient Nippostrongylus brasiliensis expulsion and delayed cytokine production by T helper 2 cells. We further establish a key role for interleukin (IL)-25 in regulating a novel population of IL-4-, IL-5-, IL-13-producing non-B/non-T (NBNT), c-kit+, FcepsilonR1- cells during helminth infection. A deficit in this population in il25(-/-) mice correlates with inefficient N. brasiliensis expulsion. In contrast, administration of recombinant IL-25 in vivo induces the appearance of NBNT, c-kit+, FcepsilonR1- cells and leads to rapid worm expulsion that is T and B cell independent, but type 2 cytokine dependent. We demonstrate that these IL-25-regulated cells appear rapidly in the draining lymph nodes, implicating them as a source of type 2 cytokines during initiation of worm expulsion.

Figure 1.

il25^−/− mice fail to expel N. brasiliensis efficiently and have delayed type 2 cytokine production. (A) Infected mice were killed at the times indicated to obtain intestinal worm counts. (B) Fecal egg counts were determined at the times indicated. Epg, eggs per gram of feces. (C) Cytokine production after infection. MLN cells from wild-type and il25 ^−/− animals after infection with N. brasiliensis were stimulated with N. brasiliensis antigen. Cytokines were analyzed by ELISA. (D) Total serum IgE expression from wild-type and il25 ^−/− animals after infection. (E) Mouse mast cell protease-1 (MMCP-1) activity was measured from sera. (F) Determination of goblet cell number and enumeration of PAS positive goblet cells per villus crypt unit. (G) Blood basophils were enumerated after FACS analysis of NBNT, c-kit⁻, FcɛR1⁺, SSC^low cells. (H) Blood eosinophils were enumerated after FACS analysis of NBNT, CCR3⁺, SSC^high. White bars, wild type; black bars, il25 ^−/−. Cohorts of five to six infected mice were used per group. Data are presented as means plus standard error and are representative of two to three experiments. ND, not detected.

Figure 2.

Administration of rIL-25 induces rapid worm expulsion that is independent of T and B cells, but dependent on type 2 cytokine production. Cohorts of mice (n = 3–5 per group) were infected with N. brasiliensis in the presence or absence of rIL-25 and killed on days 5 or 10 to obtain intestinal worm counts. Data are presented as means plus standard error and are representative of two to three experiments. ND, not detected. (A) Wild-type and il25 ^−/− mice. (B) Wild-type and rag1 ^−/− mice. (C) Wild-type and il4 ^−/− il5 ^−/− il9 ^−/− il13 ^−/− mice.

Figure 3.

IL-25 induces NBNT, c-kit⁺, FcɛR1⁻ cell population in the MLN. (A) NBNT, c-kit⁺, FcɛR1⁻ cells are deficient in the MLN of il25 ^−/− animals 5 d after infection with N. brasiliensis (Nb). Cohorts of wild-type and il25 ^−/−-infected mice (n = 5–6) were killed and the NBNT, c-kit⁺, FcɛR1⁻ cell population assessed by flow cytometry. (B) BALB/c mice (n = 3–5) were administered with rIL-25 and flow cytometry was used to analyze NBNT, c-kit⁺, FcɛR1⁻ cells in the MLN and spleen 4 d later. (C) Cohorts of wild-type and rag1 ^−/− mice (n = 3–5) were administered with rIL-25 and flow cytometry used to analyze NBNT, c-kit⁺, FcɛR1⁻ cells in the MLN 4 d later. (D) Cohorts of wild-type and il4 ^−/− il5 ^−/− il9 ^−/− il13 ^−/− mice (n = 3–5) were administered with rIL-25 and flow cytometry used to analyze NBNT, c-kit⁺, FcɛR1⁻ cells in the MLN 4 d later. Data are representative of two to three experiments.

Figure 4.

NBNT, c-kit⁺, FcɛR1⁻ cells induced by IL-25 in vivo produce IL-4, IL-5, and IL-13. (A) After administration of IL-25, NBNT, c-kit⁺, FcɛR1⁻ cells were purified using flow cytometry and the expression of il4, il5, and il13 determined using Taqman real-time PCR. Mast, PMA/ionomycin-activated, bone marrow–derived mast cells. NBNT, c-kit⁺, FcɛR1⁻ sorted cells. Th1, splenocytes stimulated under Th1 cell differentiation conditions for 48 h. (B) After N. brasiliensis infection NBNT, c-kit⁺ cells were purified using flow cytometry and the expression of il4, il5, and il13 was determined using Taqman real-time PCR. Mast, PMA/ionomycin-activated, bone marrow–derived mast cells. NBNT, NBNT, c-kit⁺ sorted cells. Th1, splenocytes stimulated under Th1 cell differentiation conditions for 48 h. (C) 4get mice were administered with rIL-25 and the frequency of GFP⁺ NBNT, c-kit⁺, FcɛR1⁻ cells was analyzed by flow cytometry. (D) 4get mice were infected with N. brasiliensis (Nb) and the frequency of GFP⁺ NBNT, c-kit⁺ cells was analyzed by flow cytometry at the time points indicated. (E) 4get mice were infected with N. brasiliensis and the frequency of GFP⁺ CD4⁺ cells was analyzed by flow cytometry at the time points indicated. (F) Numbers of GFP⁺, NBNT, c-kit⁺, FcɛR1⁻ cells in MLN after infection. (G) Numbers of GFP⁺, CD4⁺ cells in MLN after infection are shown.

Figure 5.

Analysis of purified NBNT, c-kit⁺, FcɛR1⁻ cells from IL-25–treated wild-type mice. (A) NBNT, c-kit⁺, FcɛR1⁻ cells were isolated by cell sorting and cytospin preparations of sorted cells stained with Giemsa or Toludine. (B) Flow cytometry of cell surface marker expression. (C) Analysis of gene expression. neg, no template control; mast, bone marrow–derived mast cells; c-kit⁺, NBNT, c-kit⁺, FcɛR1⁻ cells; Th2, splenocytes stimulated under Th2 cell differentiation conditions for 48 h; pos, image clone used as positive control; CCR, chemokine receptor; MCP, mast cell protease; EPO, eosinophil peroxidase. EPO positive control was IMAGE clone 30044595. CCR1 positive control was IMAGE clone 3992755.

Figure 6.

IL-25 induces type-2 cytokine production from NBNT, c-kit⁺, FcɛR1⁻ MLN cells in vitro. (A) NBNT, c-kit⁺, FcɛR1⁻ cells from the MLN of naive mice were purified by flow cytometry and treated in vitro with rIL-25 for 4 d and cytokine expression was analyzed by ELISA. (B) NBNT, c-kit⁺, FcɛR1⁻ cells from the spleens of naive mice were purified by flow cytometry and treated in vitro with rIL-25 for 4 d and cytokine expression was analyzed by ELISA. (C) NBNT cells were purified from bone marrow and treated in vitro with rIL-25 for 4 d and cytokine expression analyzed by ELISA. ND, not detected. (D and E) NBNT, c-kit⁺, FcɛR1⁻ cells were purified from rIL-25–treated il13 ^−/− mice and cultured in triplicate with highly purified wild-type CD3⁺ cells, on anti-CD3–coated plates, in the presence or absence of IL-25. D and E are two independent experiments. Differences in IL-13 production between untreated and IL-25–treated cells were analyzed using Student's t test.