Chitin activates parallel immune modules that direct distinct inflammatory responses via innate lymphoid type 2 and γδ T cells

Abstract

Chitin, a polysaccharide constituent of many allergens and parasites, initiates innate type 2 lung inflammation through incompletely defined pathways. We show that inhaled chitin induced expression of three epithelial cytokines, interleukin-25 (IL-25), IL-33, and thymic stromal lymphopoietin (TSLP), which nonredundantly activated resident innate lymphoid type 2 cells (ILC2s) to express IL-5 and IL-13 necessary for accumulation of eosinophils and alternatively activated macrophages (AAMs). In the absence of all three epithelial cytokines, ILC2s normally populated the lung but failed to increase IL-5 and IL-13. Although eosinophils and AAMs were attenuated, neutrophil influx remained normal without these epithelial cytokines. Genetic ablation of ILC2s, however, enhanced IL-1β, TNFα, and IL-23 expression, increased activation of IL-17A-producing γδ T cells, and prolonged neutrophil influx. Thus, chitin elicited patterns of innate cytokines that targeted distinct populations of resident lymphoid cells, revealing divergent but interacting pathways underlying the tissue accumulation of specific types of inflammatory myeloid cells.

FIGURE 1. Chitin induces focal clustering of type 2 innate immune cells

(A) Intranasal chitin dosing regimen. (B) Total lung eosinophils and (C) Arg1⁺ macrophages from Yarg reporter mice (Arg1^Yarg) after intranasal administration of equivalent numbers of indicated bead types. PS=polystyrene; PMMA=polymethylmethacrylate. Total live cell subset numbers were calculated from cell counts and flow cytometric percentages as described in Figure S1. (D) Lung histology from Il4^4get reporter and Il4^4getGata1^{ΔdblGATA/ΔdblGATA} mice at indicated time points after intranasal chitin administration. Red=rhodamine-conjugated chitin-binding probe; Green=4get⁺ cells; Blue=DAPI⁺ cell nuclei. (E) Localization of Arg1⁺ cells (green) within chitin inflammatory clusters (red) in lung sections from Yarg reporter mice at indicated time points. Far right panel, yellow=PS bead (90 μm). Data are representative of at least two independent experiments, and results from similar treatment groups were pooled in (B) and (C) to represent mean±SEM, n=4/group; **p<0.0001 (unpaired t-test), compared to PBS-treated control. Scale bar=30 μm. See also Figure S1 and Movies S1–S5.

FIGURE 2. Induction of IL-13 from ILC2 in response to chitin contributes to AAM and eosinophil accumulation

(A) Total eosinophil numbers and (B) Arg1⁺ macrophages in lungs of indicated mice (on a Yarg reporter background) treated with chitin as described in Figure 1. (C) Expression of IL-13 (huCD4) on lung ILC2 from wild-type (WT) and Il13^Smart reporter (S13) mice 24 hours after intranasal chitin challenge. Number indicates percentage of ILC2 (Lin-CD25⁺Thy1⁺) positive for huCD4 marker. (D) IL-13 protein levels in culture supernatant from sorted ILC2 after in vivo PBS or chitin treatment. ND=none detected. (E) Total ILC2 numbers in the lungs of indicated mice and (F) cell surface expression of indicated markers among ILC2 48 hours after PBS or chitin treatment. MFI values are indicated above histograms for isotype (left), PBS- (middle), or chitin-treated populations (right). Flow cytometry results shown in (C) and (F) are representative of 3 independent experiments, and (A), (B), (D), and (E) represent mean±SEM, n=4–6/group; *p<0.001; **p<0.0001 (unpaired t-test), compared to PBS-treated control. See also Figures S2 and S3.

FIGURE 3. Chitin administration increases IL-5 expression among lung ILC2 in vivo and mediates eosinophil, but not AAM, accumulation

(A) Expression of IL-5 (tdTomato) among lung ILC2 from wild-type (WT) and heterozygous Il5^Red5/+ reporter (R/+) mice before and 24 hours after intranasal chitin challenge. Number indicates percentage of ILC2 positive for Red5 reporter, and mean fluorescence intensity (MFI) values, where indicated, represent entire ILC2 population. (B) IL-5 protein levels in culture supernatant from sorted ILC2 after in vivo PBS or chitin treatment. (C) Flow cytometric analysis of IL-5 (tdTomato) MFI among ILC2 populations from wild-type or SPAM transgenic mice on a heterozygous Il5^Red5/+ (R/+) background 24 hours after in vivo PBS or chitin treatment. (D) Lung micrograph from chitin-challenged Il5^Red5/Red5 mouse co-stained with VCAM-1 and DAPI. Green=VCAM-1; Blue=DAPI⁺ cell nuclei; Red=IL-5 (Red5)⁺ ILC2. (E) Serum IL-5 levels after in vivo PBS or chitin treatment in WT or homozygous Red5 (Il5^Red5/Red5) mice. Total numbers of ILC2 (F), eosinophils (G), and Arg1⁺ macrophages (H) in lungs of indicated mice (on a Yarg reporter background) after chitin treatment as described in Figure 1. Results shown in (A) and (D) are representative of 3 independent experiments, and (B, C), and (E–H) represent mean±SEM, n=4–6/group; *p<0.001; **p<0.0001 (unpaired t-test), compared to PBS-treated (B, C, E) or WT control (F–H). ND=none detected. Scale bar=30 μm. See also Figures S4, S5 and Movies S6–S9.

FIGURE 4. Genetic deletion of ILC2 ablates both eosinophil and AAM accumulation in lung tissue in response to chitin administration

(A) Total ILC2, (B) eosinophil and (C) Arg1⁺ macrophages in lung tissue of indicated mice (on a Yarg reporter background) after treatment with chitin as described in Figure 1. WT=wild-type. YYDD=IL-13 deleter mice (Il13^{YetCre/YetCre}Gt(Rosa)26^DTA/DTA). RRDD=IL-5 deleter mice (Il5^Red5/Red5Gt(Rosa)26^DTA/DTA). Data are presented as mean±SEM, n=4–6/group; **p<0.0001 (unpaired t-test), as compared to WT control.

FIGURE 5. ILC2 mediate suppression of inflammatory cytokine and innate-like T cell responses after chitin stimulation

(A) IL-1β and IL-17A protein levels in whole lung lysates from wild-type (WT) mice 6 hours after intranasal treatment with PBS or chitin. (B) Expression of IFN-γ (Great; YFP) and IL17A (Smart17; hNGFR) reporters among CD3ε⁺CD4⁺ T cells and CD3ε⁺GL3⁺ γδ T cells from lungs of Ifng^Great/GreatIl17a^Smart/Smart dual reporter mice 18 hours after intranasal treatment with PBS, chitin, or IL-1β and IL-23 (10 ng each cytokine). (C) Cytokine levels in whole lung lysates from WT or IL-5 deleter (RRDD; Il5^Red5/Red5Gt(Rosa)26^DTA/DTA) mice at indicated time points after single intranasal challenge with chitin. (D) Expression of CD69 and IL17A (hNGFR) reporter among CD3ε⁺GL3⁺ γδ T cells in the lungs of Il17a^Smart/Smart reporter mice 48 hours after single intranasal challenge with PBS (top) or chitin (bottom). (E) Total numbers of CD3ε⁺GL3⁺ γδ T cells (left) and percentage expressing CD69 (right), as well as total neutrophil numbers (F) in the lungs of indicated mice before (0) and 4 days after a single intranasal chitin dose. Numbers in each quadrant from flow cytometry plots in (B) and (D) indicate percentage of total T cell subset; data are representative of 3 independent experiments. (A), (C), (E), and (F) represent mean±SEM, n=3–6/group; *p<0.05; **p<0.01 (unpaired t-test), compared to PBS-treated (A) or wild-type control at corresponding time point (C, E, F).

FIGURE 6. Expression of type 2 epithelial cytokines and receptor components in response to chitin stimulation

(A) IL-33 and TSLP protein levels in whole lung lysate and (B) IL-25 levels in bronchoalveolar lavage (BAL) fluid from wild-type mice at indicated times after intranasal chitin. (C) Cell surface expression of indicated markers on ILC2 from wild-type (WT) mice in comparison with cells from Il1rl1^−/− mice (left panel), Crlf2^−/− mice (middle), or cells stained with isotype control antibody (Iso; right). (D) Total ILC2 numbers in the lungs of indicated mice. Flow cytometry results shown in (C) are representative of 3 independent experiments, and (A, B, D) represent mean±SEM, n=3–5/group; *p<0.001; **p<0.0001 (unpaired t-test), compared to untreated control.

FIGURE 7. Epithelial cytokines control chitin-induced eosinophil and AAM accumulation via regulation of IL-5 and IL-13 production from ILC2

(A) Total ILC2, (B) eosinophil and (C) Arg1⁺ macrophage numbers in the lungs of indicated mice (on a Yarg reporter background) treated with PBS or chitin as described in Figure 1. WT=wild-type. (D) Total neutrophils (CD11b⁺Ly6G⁺ Siglec F⁻; also see Figure S1) in left lung lobe tissue and (E) IL-1β protein levels in lung tissue lysate 24 hours after intranasal treatment with PBS or chitin. (F) IL-5 and IL-13 protein levels in supernatant from sorted lung ILC2 from WT or Crlf2^−/−Il25^−/−Il1rl1^−/− triple-deficient mice after in vivo chitin treatment, cultured in the presence of IL-7 or (G) ionomycin/PMA. NS=not statistically significant. ND=none detected. Data are presented as mean±SEM, n=3–5/group; *p<0.001; **p<0.0001 (unpaired t-test), compared to chitin-treated WT control.