Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity

Abstract

Innate immunity provides the first line of defence against invading pathogens and provides important cues for the development of adaptive immunity. Type-2 immunity-responsible for protective immune responses to helminth parasites and the underlying cause of the pathogenesis of allergic asthma-consists of responses dominated by the cardinal type-2 cytokines interleukin (IL)4, IL5 and IL13 (ref. 5). T cells are an important source of these cytokines in adaptive immune responses, but the innate cell sources remain to be comprehensively determined. Here, through the use of novel Il13-eGFP reporter mice, we present the identification and functional characterization of a new innate type-2 immune effector leukocyte that we have named the nuocyte. Nuocytes expand in vivo in response to the type-2-inducing cytokines IL25 and IL33, and represent the predominant early source of IL13 during helminth infection with Nippostrongylus brasiliensis. In the combined absence of IL25 and IL33 signalling, nuocytes fail to expand, resulting in a severe defect in worm expulsion that is rescued by the adoptive transfer of in vitro cultured wild-type, but not IL13-deficient, nuocytes. Thus, nuocytes represent a critically important innate effector cell in type-2 immunity.

Figure 1. IL-25 and IL-33 induce IL-13-producing nuocytes

a, Detection of Il13eGFP⁺ NBNT cells in mLN of IL-25 or IL-33-treated mice. b, Cell surface marker expression of Il13eGFP⁺ NBNT cells in mLN following IL-25 administration. c, Immunofluorescence detection of Il13eGFP⁺ cells in small intestine of IL-25 and IL-33 treated mice. a – c, Data representative of 5 experiments with >3 mice per group. d, Nuocyte number. Data representative of two independent experiments with >4 mice per group. e, Cluster analysis for freshly isolated nuocytes (ex vivo) or day 9 in vitro expanded nuocytes (single data sets are shown for clarity).

Figure 2. IL-25 and IL-33 play partially redundant roles for nuocyte induction and worm expulsion

a, Quantification of Il13eGFP⁺ cells five days p.i. with N. brasiliensis. Data are representative of two independent experiments with >5 mice per group. b, Intestinal worm burden of N. brasiliensis-infected mice. c, Quantification of Il13eGFP⁺ cells in N. brasiliensis-infected mice at day 5 p.i. Data are representative of two independent experiments with >5 mice per group. d, Quantification of nuocytes in N. brasiliensis-infected mice. * = p < 0.05, ** = p < 0.01. Data are representative of two independent experiments with >5 mice per group.

Figure 3. Adoptive transfer of cultured nuocytes into il17br^−/- mice restores an IL-25-responsive phenotype

a, Morphology of Giemsa-stained nuocytes. b, Quantification of nuocyte growth in vitro. CC, cytokine cocktail. c, Flow cytometric analysis of interferon (IFN)-γ, IL-4, IL-5 and IL-13 intracellular staining of nuocytes after 7 days culture with IL-7 and IL-33. a – c, Data are representative of three independent experiments. d, Quantification of eosinophil infiltration of the peritoneal cavity following nuocyte (nuo) transfer. e, Transverse histological jejunum sections stained with Periodic acid-Schiff (PAS) for goblet cells. Data are representative of two independent experiments with >5 mice per group.

Figure 4. Adoptive transfer of wildtype nuocytes, but not IL-13-deficient nuocytes, restores rapid worm expulsion in N.brasiliensis infected il17br^−/− mice

a, Intestinal worm burdens. b, Quantification of nuocyte numbers in tissues. Data are representative of three independent experiments with >6 mice per group. c - d, Intestinal worm burdens. Data are from single experiments with 6 - 7 mice per group. e, Intestinal worm burdens. f, N. brasiliensis antigen-specific IL-13 production. g, Intestinal worm burden. h, Quantification of nuocyte numbers. e – h, Data are representative of two independent experiments with >6 mice per group. * = p < 0.05, ** = p < 0.01, *** = p < 0.005.