Systemically dispersed innate IL-13-expressing cells in type 2 immunity

Abstract

Type 2 immunity is a stereotyped host response to allergens and parasitic helminths that is sustained in large part by the cytokines IL-4 and IL-13. Recent advances have called attention to the contributions by innate cells in initiating adaptive immunity, including a novel lineage-negative population of cells that secretes IL-13 and IL-5 in response to the epithelial cytokines IL-25 and IL-33. Here, we use IL-4 and IL-13 reporter mice to track lineage-negative innate cells that arise during type 2 immunity or in response to IL-25 and IL-33 in vivo. Unexpectedly, lineage-negative IL-25 (and IL-33) responsive cells are widely distributed in tissues of the mouse and are particularly prevalent in mesenteric lymph nodes, spleen, and liver. These cells expand robustly in response to exogenous IL-25 or IL-33 and after infection with the helminth Nippostrongylus brasiliensis, and they are the major innate IL-13-expressing cells under these conditions. Activation of these cells using IL-25 is sufficient for worm clearance, even in the absence of adaptive immunity. Widely dispersed innate type 2 helper cells, which we designate Ih2 cells, play an integral role in type 2 immune responses.

Fig. 1.

Lineage-negative cells are systemically distributed at rest and possess a unique surface phenotype. (A) Gating scheme for lineage-negative cells in 4get mice. Example shown is resting liver. Box denotes lineage-negative gate. Numbers denote percentage of boxed cells of total. (B) Staining with a lineage-marker antibody mixture (CD3ε, Ly-6G/Ly-6C, CD11b, CD45R, and TER-119). Eosinophils are used as a positive control, whereas basophils are used as a negative control. (C) Surface-marker antibody staining (black lines) compared with isotype control (gray) on lineage-negative cells from resting liver. (D) Numbers of lineage-negative cells in organs and tissues at rest. Experiment was independently repeated three times, and data were combined for graph (n = 3–14 mice per group). (E) Gating of lineage-negative cells in 4get mice compared with 4get × common-γ^−/− mouse liver. Lineage-negative cells collect in side-scatter (SSC)-lo, CD49b-negative gate. Numbers are percentages of total non-CD4 GFP-positive cells.

Fig. 2.

Lineage-negative cells increase in number after IL-25 administration and during infection. (A) Numbers of lineage-negative cells and eosinophils in designated organs after four daily doses of 500 ng IL-25 or IL-33. (B) Numbers of lineage-negative cells after infection with N. brasiliensis. Experiments were repeated three times, and data were compiled for graphs (n = 3–14 mice per group). Bars are means with SEM.

Fig. 3.

Lineage-negative cells are a distinct Th2-associated cell type. (A) Relative gene expression in Th2 cells (two samples), lineage-negative cells (three samples), and basophils (three samples) isolated from N. brasiliensis-infected mice. Heat map shows log2 (sample intensity/mean intensity for all eight samples) for all microarray probes with differential expression (false discovery rate (FDR) < 0.05) in any pairwise comparison between cell types. (B) Grouping of microarray probes based on their expression in lineage-negative cells, Th2 cells, and basophils. Signal intensity cutoff of 2⁸ is ~2³-fold higher than the median signal obtained with randomized negative-control probes. *, Itga4, integrin alpha 4; Jag1, Jagged 1; IL9R, IL-9 receptor; IL1R1, IL-1 receptor type 1; Ptger3, prostaglandin E receptor 3; CCR3, chemokine receptor 3; Btk, Bruton agammaglobulinemia tyrosine kinase; Gata3, GATA binding protein 3; Ikzf3, IKAROS family zince finger 3 (Aiolos); IL2ra, IL-2 receptor alpha chain; Rgs18, regulator of G protein signaling 18; Tph1, tryptophan hydrolase 1; Chdh, choline dehydrogenase; Stat-6, signal transducer and activator of transcription 6 (C). Diagram showing the numbers of probes in each of the seven groups listed in B.

Fig. 4.

Lineage-negative cells are major innate IL-13–expressing cells. (A) Gating of YFP+ lineage-negative cells from YetCre13 Rosa-YFP amplifier mice. Example shown is of liver after administration of IL-25. Gates and percentages are as in Fig. 1. (B) Numbers of YFP+ lineage-negative cells after IL-25 administration. Experiment was repeated two times, and results were compiled (n = 2–3 mice per group). Bars are means with SEM. (C) Time course showing YFP+ lineage-negative cells from designated organs during infection with N. brasiliensis. Experiment was repeated two times, and results were compiled (n = 3–5 mice per group). Bars are means with SEM. (D) Gating of non-CD4+ YFP+ cells showing percentages of mast cells (c-kit^hiIgE⁺) and lineage-negative (c-kit^loIgE⁻) cells in multiple organs after IL-25 administration (Upper) or after infection with N. brasiliensis (Lower).

Fig. 5.

Lineage-negative cells contribute to eosinophilia and mediate IL-25–dependent worm clearance. (A) Gating of GFP+CD4- cells in IL-25–treated 4get YetCre13-Rosa-DTA deleter mice or 4get YetCre13 littermates. Percentages shown are eosinophils (SSC^hiCD49b^lo), basophils (SSC^loCD49b⁺), and lineage-negative cells (SSC^loCD49b⁻). (B) Numbers of lineage-negative cells and eosinophils in multiple organs in 4get YetCre13-DTA deleter mice or 4get YetCre13 controls after administration of IL-25. Experiment was repeated two times, and results were compiled (n = 3–5 mice per group). Bars denote means with SEM. (C) Rag2^−/−, common-γ^−/− × Rag2^−/−, or common-γ^−/− × Rag2^−/− mice adoptively transferred with 500,000 lineage-negative cells 2 d previously were infected with N. brasiliensis. Five mice from each group received IL-25 on days 0–4. Worm counts from the small intestine and numbers of eosinophils (SSC^hiCD11b⁺Siglec-F⁺) were quantified on day 5. Experiment was repeated two times, and a representative experiment is shown.