Type 2 innate lymphoid cells control eosinophil homeostasis

Abstract

Eosinophils are specialized myeloid cells associated with allergy and helminth infections. Blood eosinophils demonstrate circadian cycling, as described over 80 years ago, and are abundant in the healthy gastrointestinal tract. Although a cytokine, interleukin (IL)-5, and chemokines such as eotaxins mediate eosinophil development and survival, and tissue recruitment, respectively, the processes underlying the basal regulation of these signals remain unknown. Here we show that serum IL-5 levels are maintained by long-lived type 2 innate lymphoid cells (ILC2) resident in peripheral tissues. ILC2 cells secrete IL-5 constitutively and are induced to co-express IL-13 during type 2 inflammation, resulting in localized eotaxin production and eosinophil accumulation. In the small intestine where eosinophils and eotaxin are constitutive, ILC2 cells co-express IL-5 and IL-13; this co-expression is enhanced after caloric intake. The circadian synchronizer vasoactive intestinal peptide also stimulates ILC2 cells through the VPAC2 receptor to release IL-5, linking eosinophil levels with metabolic cycling. Tissue ILC2 cells regulate basal eosinophilopoiesis and tissue eosinophil accumulation through constitutive and stimulated cytokine expression, and this dissociated regulation can be tuned by nutrient intake and central circadian rhythms.

Figure 1. Innate cells produce IL-5 in tissues at rest

a, Schematic of targeting construct. b-c, Flow cytometry of tissues, previously gated on CD45+CD90.2+ cells in wild-type and R5/+ (b) or CD90.2+ cells in R5/R5 (c) naïve mice. d, Serum IL-5. Data representative of two independent experiments with two mice per group (b-d) or pooled from three independent experiments for 7 (wild-type), 4 (Red5), or 8 (others) mice per group (c). LN, lymph nodes; ND, none detected; NS not significant; *, p < 0.05.

Figure 2. ILC2 expand after birth and persist in collagen-rich structures

a, Percent of lung Lin-CD90.2+ cells R5+T1/ST2 on day 1, day 8, or week 8. b, Percent BrdU+ of R5+ ILC2 and total CD4+ cells in lung after four weeks BrdU. c, Representative multiphoton images of tdTomato fluorescence (red) in naïve R5/R5 actin-CFP mice; CFP and autofluorescence in blue and green, respectively. A=airway. V=vasculature. Collagen second harmonic appears blue. Scale bars 100 µm. Data pooled from three independent experiments for 5 (Day 1), 6 (Day 8), or 4 (Adult) mice per group (a); or pooled from two independent experiments for 5 (week 0), 6 (week 1), or 3 (others) mice per group (b), represented as mean +/− SEM. Images represent 8 regions taken from two mice. Lin, Lineage markers (B220, CD5, CD11b, CD11c, Ly6G, FcεRI, and NK1.1); ***, p < 0.01 by Student’s t test.

Figure 3. IL-5 and IL-13 co-expression in lung ILC2

a, Lung IL-5 and IL-13 reporter expression before and after infection. b, Flow cytometry of CD90.2+ lung cells and percent with ILC2 surface markers (CD90.2 and either KRLG1, T1/ST2 or CD25) at rest. c, ILC2 (left lung) and CCL11 concentration (right lung) after IL-2, IL-33, and IL-13 treatment. Data representative of three independent experiments with 4 (naïve R5+S13+), 5 (infected R5+S13+), or 2 (others) mice per group (a), pooled from three independent experiments for 6 (R5/R5 bone marrow and spleen) or 9 (all others) mice per group (b), or pooled from two independent experiments for 8 (R5/R5 + IL-2/IL-33), 5 (R5/R5 Deleter + IL-2/IL-33/IL-13), or 3 (others) mice per group (c). Represented as mean +/− SEM. huCD4, human CD4; BM, bone marrow; NS, not significant; *, p < 0.05; ***, p < 0.001 by Student’s t test.

Figure 4. ILC2 respond to circadian and metabolic cues

a-b, Serum IL-5 and blood eosinophils at 10:00, 22:00 or at 10:00 after fasting. c-d, Flow cytometry of small intestine ILC2 (Lin-CD127+ICOS+) and percent of R5-hi ILC2 expressing S13 at 8:00 in mice on nighttime (black) or daytime (white) feeding (c) or in fasted mice given food (black) or water (white) by oral gavage (d). e, Supernatant IL-5 from intestinal Lin-CD45+KLRG1+ ILC2 cultured in IL-7 alone or with indicated reagents. f, Expression of Vpac1 and Vpac2 in sorted cells, relative to Rps17. Data pooled from independent experiments for 19 (AM), 6 (PM), or 5 (fasted) mice per group (a); 7 (AM), 4 (PM), or 8 (fasted) mice per group (b); 8 mice per group (c); 6 mice per group (d); or pooled averages of duplicate cultures from 6 (IL-7 alone, + VIP, + VPAC2 agonist) or 3 (all others) cell sorts from independent mice (e), or representative of two experiments of independent cell sorts (f). Represented as mean +/− SEM. Lin, Lineage markers (B220, CD11b, CD11c, Ly6G, FcεRI, and NK1.1); Rps17, 40S ribosomal protein S17; NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student’s t test.