Type 2 immunity is controlled by IL-4/IL-13 expression in hematopoietic non-eosinophil cells of the innate immune system

Abstract

Nippostrongylus brasiliensis infection and ovalbumin-induced allergic lung pathology are highly interleukin (IL)-4/IL-13 dependent, but the contributions of IL-4/IL-13 from adaptive (T helper [Th]2 cells) and innate (eosinophil, basophils, and mast cells) immune cells remain unknown. Although required for immunoglobulin (Ig)E induction, IL-4/IL-13 from Th2 cells was not required for worm expulsion, tissue inflammation, or airway hyperreactivity. In contrast, innate hematopoietic cell-derived IL-4/IL-13 was dispensable for Th2 cell differentiation in lymph nodes but required for effector cell recruitment and tissue responses. Eosinophils were not required for primary immune responses. Thus, components of type 2 immunity mediated by IL-4/IL-13 are partitioned between T cell-dependent IgE and an innate non-eosinophil tissue component, suggesting new strategies for interventions in allergic immunity.

Figure 1.

Worm expulsion requires expression of IL-4/IL-13 from cells of the innate immune system. (A) 10⁷ MACS-purified naive CD4 T cells from IL-4/IL-13– or 4get/STAT6-deficient mice were adoptively transferred into RAG-deficient mice. Transferred mice and indicated control mice were infected with N. brasiliensis 4 d after T cell transfer as described in Materials and methods. Adult worms were counted 9 d after infection in the small intestine. Three to five mice per group were analyzed. (B) 10⁷ MACS-purified naive CD4 T cells from BALB/c or IL-4/IL-13–deficient mice were adoptively transferred into TCR-Cα– or IL-4/IL-13/TCR-Cα–deficient mice. Reconstituted mice were infected with N. brasiliensis 4 d after transfer and analyzed for worm expulsion and serum IgE levels on day 9 after infection. 3–9 mice per group were analyzed. ND, not detectable. Both experiments were repeated once with similar results.

Figure 2.

Accumulation of Th2 cells in the lung requires IL-4/IL-13 production from cells of the innate immune system. (A) 10⁷ MACS-purified naive CD4 T cells from 4get mice were transferred into TCR-Cα– (Cα^−/−) or IL-4/IL-13/TCR-Cα–deficient (IL-4/IL-13/Cα^−/−) mice, and the groups were infected with N. brasiliensis 4 d later. The frequency of Th2 cells in the lung or paratracheal lymph nodes was analyzed by flow cytometry on day 9 after infection. Expression of GFP indicates the expression of IL-4 in these reporter mice. One representative experiment of four is shown. (B) 10⁶ naive KJ1-26⁺ CD4 T cells from DO11.10/4get/TCR-Cα–deficient mice were transferred into BALB/c or IL-4/IL-13–deficient mice, which were inoculated with N. brasiliensis and OVA 1 d after transfer, challenged intranasally with OVA on day 6, and analyzed by flow cytometry on day 9. Bars indicate the frequency of IL-4/GFP-expressing cells among antigen-specific KJ1-26⁺ cells and the frequency of KJ1-26⁺ cells among total CD4 T cells in the lung or lymph nodes of WT (WT) recipients (closed bars) or IL-4/IL-13–deficient recipients (open bars). The experiment shows the combined results from two independent experiments with a total of five mice per group. Error bars indicate the standard deviation. The p-value was determined by Student's t test. (C) Total numbers of clonotypic KJ1-26⁺ cells in the lungs of reconstituted BALB/c mice (closed bars) or IL-4/IL-13–deficient mice (open bars). (D) Activation of antigen-specific T cells as assessed by expression of CD44 and CD25 in the lung and lymph node.

Figure 3.

Bone marrow–derived cells and CD4 T cells regulate type 2 immunity in vivo. (A) Bone marrow cells from 4get mice were transferred into lethally irradiated WT, IL-4–deficient, or IL-4/IL-13–deficient recipient mice. 8 wk after reconstitution, three mice per group were infected with N. brasiliensis and analyzed on day 9 after infection by four-color flow cytometry for the presence of Th2 cells, eosinophils, and basophils in the lung after staining for CD4, IgE, and CCR3. Error bars indicate the standard deviation. (B) Single cell suspensions of the lung of 4get, 4get/MHC class II^−/−, or 4get/RAG^−/− mice were stained for CD4, IgE, and CCR3 and analyzed 9 d after N. brasiliensis infection by flow cytometry. (C) The blood, spleen, and bone marrow of naive 4get mice were analyzed by flow cytometry using the markers indicated above.

Figure 4.

Eosinophils and basophils are the major innate IL-4–expressing cells recruited to the lung during OVA-induced lung inflammation. (A) 4get mice were primed with OVA or saline and challenged for three consecutive days with OVA as described in Materials and methods. Single cell suspensions of the lung were stained and analyzed as described in Fig. 3. (B) Total numbers of eosinophils, basophils, and Th2 cells in the lung of five OVA-challenged mice or three saline-treated control mice were calculated based on the frequency of each population among total cells in the lung. (C) 4get/RAG^−/− mice were reconstituted with 10⁷ CD4 T cells from BALB/c or IL-4/IL-13–deficient mice. A group of five mice was sensitized and challenged with OVA (closed bars), whereas another group of three mice was treated with saline (open bars). The total number of eosinophils and basophils in the lung was determined 1 d after the last challenge. p-values were determined by Student's t test and are indicated in the figure. (D) Pulmonary resistance was determined in both groups of mice in anesthetized mice by injecting increasing doses of acetylcholine 1 d after the last challenge of 4get/RAG^−/− mice reconstituted with BALB/c (circles) or IL-4/IL-13–deficient (triangles) T cells. Error bars indicate the standard deviation. P < 0.001 for WT saline versus OVA, and P = 0.027 for IL-4/IL-13–deficient saline versus OVA. The experiment was performed twice with similar results.

Figure 5.

Attenuated induction of type 2 immunity in IL-5tg mice. (A) The lungs of normal (4get) or IL-5tg 4get (4get/IL-5) mice were analyzed by flow cytometry before (naive) or 9 d after (infected) N. brasiliensis infection by staining with anti-CD4 to identify Th2 cells (CD4⁺IL-4/GFP⁺). (B) Frequency of Th2 cells in paratracheal lymph nodes and lungs of 4get (open bar) and 4get/IL-5tg (closed bar) mice on day 9 after infection. (C) Analysis of serum IgE levels before (naive) or 9 d after (infected) infection of 4get or 4get/IL-5tg mice. Error bars indicate the standard deviation. (B and C) Combined results from three independent experiments with a total of six mice per group. p-values were determined with Student's t test.

Figure 6.

Eosinophils are not required for Th2 or basophil recruitment to the lung, increased serum IgE levels, or worm expulsion after N. brasiliensis infection. (A) WT 4get or eosinophil-deficient 4get/ΔdblGATA mice were infected with N. brasiliensis and analyzed 9 d later for Th2 cell polarization in paratracheal lymph nodes, the frequency of eosinophils and basophils in the blood, and infiltration of CD4 T cells, basophils, and eosinophils into the lung parenchyma by flow cytometry. The dot plots from the lung and lymph node are gated on total live cells, and the lung and blood samples are gated on CD4⁻GFP⁺ cells. (B) Frequency and total cell number of eosinophils, basophils, and Th2 cells in the lungs of BALB/c (WT) and ΔdblGATA mice on day 9 after infection. (C) Total serum IgE from BALB/c (WT) and ΔdblGATA mice (three mice per group) determined by ELISA on day 9 after infection. Error bars indicate the standard deviation.

Figure 7.

Eosinophils contribute to a secondary immune response against N. brasiliensis larvae. 4get or 4get/ΔdblGATA mice were infected with N. brasiliensis, rechallenged 5 wk later, and analyzed for worm expulsion on day 7 after secondary infection. One set of mice from each group was depleted of CD4 cells 3 d before the second infection. The graph shows the combined results from two independent experiments. The p-value was determined by Student's t test.