Crosstalk between ILC2s and Th2 cells varies among mouse models

Abstract

Type 2 T helper (Th2) cells and group 2 innate lymphoid cells (ILC2s) provide protection against helminth infection and are involved in allergic responses. However, their relative importance and crosstalk during type 2 immune responses are still controversial. By generating and utilizing mouse strains that are deficient in either ILC2s or Th2 cells, we report that interleukin (IL)-33-mediated ILC2 activation promotes the Th2 cell response to papain; however, the Th2 cell response to ovalbumin (OVA)/alum immunization is thymic stromal lymphopoietin (TSLP) dependent but independent of ILC2s. During helminth infection, ILC2s and Th2 cells collaborate at different phases of the immune responses. Th2 cells, mainly through IL-4 production, induce the expression of IL-25, IL-33, and TSLP, among which IL-25 and IL-33 redundantly promote ILC2 expansion. Thus, while Th2 cell differentiation can occur independently of ILC2s, activation of ILC2s may promote Th2 responses, and Th2 cells can expand ILC2s by inducing type 2 alarmins.

Keywords: CP: Immunology; IL-25; IL-33; ILC2-deficient mice; ILC2s; Nippostrongylus brasiliensis; TSLP; Th2 cell-deficient mice; Th2 cells; airway inflammation; group 2 innate lymphoid cells; thymic stromal lymphopoietin; type 2 alarmins; type 2 immune responses; type 2 T helper cells.

Figure 1.. ILC2-dependent and -independent Th2 cell differentiation

(A) Experimental procedure for acute and chronic papain models. (B) Flow cytometric histogram overlay showing the expression of KLRG1 on lung ILC2s (CD45⁺Lin⁻IL-7Rα⁺GATA3⁺T1/ST2⁺) and Th2 cells (CD45⁺CD4⁺Foxp3⁻GATA3⁺T1/ST2⁺) from WT mice chronically challenged with papain. (C) ILC2s in the pulmonary tissue were compared between Gata3^fl/fl and Klrg1^CreGata3^fl/fl mice at the steady state by flow cytometry. (D) Gata3^fl/fl (WT) and Klrg1^CreGata3^fl/fl (ILC2-deficient) mice were acutely challenged with papain. The total numbers of eosinophils infiltrated into BALF and lung were counted and plotted (mean ± SEM; n = 9–14; ****p < 0.0001, Student’s t test). (E) Mice were chronically challenged with papain followed by flow cytometric analysis of eosinophilic infiltration in BALF. The total numbers of eosinophils infiltrated into BALF and lung were counted and plotted (mean ± SEM; n = 7–14; ****p < 0.0001, Student’s t test). (F) Mice were chronically challenged with papain. The total numbers of Th2 cells in lung were counted and plotted (mean ± SEM; n = 4–6; ****p < 0.0001, Student’s t test). (G) Shown are the microscopic images of periodic acid-Schiff-stained lung tissue sections. (H) Eosinophilic scores and total lung histopathology scores were plotted (mean ± SEM; n = 5–10; *p < 0.05, Student’s t test). (I) Experimental procedure for OVA-induced pulmonary allergy model. (J) The total cell number of eosinophils and Th2 cells in mice treated in (I) were measured by flow cytometry, and the values were plotted (mean ± SEM; n = 4–6; ns, not significant, Student’s t test). (K) The ILC2-deficient mice were injected with ILC2s followed by chronic papain challenges. Th2 cells in the lung were analyzed by flow cytometry, and the total Th2 cell numbers were counted and plotted (mean ± SEM; n = 3–4; *p < 0.05, **p < 0.01, Student’s t test). (L) Experimental procedure for the generation of antigen-specific Th2 cells by papain:2W1S immunization. (M) 2W1S-specific 2W1S:I-A^b tetramer⁺ CD4⁺ T cells were assessed by flow cytometry. (N) The total numbers of 2W1S:I-A^b tetramer⁺ CD4⁺ T cells from (M) were counted and plotted (mean ± SEM; n = 3–5; ns, not significant, **p < 0.01, Student’s t test). (O) Flow cytometric analysis of GATA3 expression by 2W1S:I-A^b tetramer⁺ CD4⁺ T cells. Results are representative of two (M–O) and three (B–H, J, and K) independent experiments. See also Figures S1–S3.

Figure 2.. IL-33 is required for ILC2-mediated enhancement of Th2 cell response

(A) WT mice were challenged i.n. with PBS or papain and then sacrificed on day 3 (acute) or 16 (chronic). IL-25, IL-33, and TSLP were measured by ELISA (mean ± SEM; n = 8–14; ns, not significant, **p < 0.01, ****p < 0.0001, Student’s t test). (B) WT mice sensitized with OVA/alum or alum alone on days 0 and 7, followed by four doses of OVA i.n. challenges on days 11–14. These mice were sacrificed on days 11, 13, and 15. IL-25, IL-33, and TSLP were measured by ELISA (mean ± SEM; n = 8–11; ns, not significant, *p < 0.05, **p < 0.01, ****p < 0.0001, Student’s t test). (C and D) WT mice and mice deficient for CIKS, IL-33, and TSLPR are acutely challenged with PBS or papain, and mice were sacrificed on day 3. (C) Eosinophils in BALF and lung were analyzed by flow cytometry. The total numbers were counted and plotted (mean ± SEM; n = 6; ****p < 0.0001, ns, not significant, Student’s t test). (D) Lung cells were stimulated with PMA/ionomycin, and then IL-13-producing ILC2s were analyzed by flow cytometry. Percentages of IL-13⁺ ILC2s were calculated and plotted (mean ± SEM; n = 5–6; ****p < 0.0001, ns, not significant, Student’s t test). (E–G) Mice were chronically challenged with papain or PBS and sacrificed on day 16. (E) Eosinophils in BALF and lung were analyzed by flow cytometry. The total numbers were counted and plotted (mean ± SEM; n = 5; ****p < 0.0001, ns, not significant, Student’s t test). (F) Th2 cells in the lung were analyzed by flow cytometry. The total numbers were counted and plotted (mean ± SEM; n = 5; *p < 0.05, ns, not significant, Student’s t test). (G) ILC2s in the lung were analyzed by flow cytometry. The total numbers were counted and plotted (mean ± SEM; n = 5; ****p < 0.0001, ns, not significant, Student’s t test). (H) Shown are the microscopic images of lung tissue sections stained with periodic acid-Schiff. (I) Eosinophilic scores and total lung histopathology scores were plotted (mean ± SEM; n = 4–10; *p < 0.05, **p < 0.01, Student’s t test). Results are representative of at least two independent experiments. See also Figures S2 and S4.

Figure 3.. TSLP is important for ILC2-independent Th2 cell response

(A–I) OVA/alum-induced airway inflammation model was used as described in Figure 1I. The total cell numbers of eosinophil and Th2 cells were measured by flow cytometry, and the values were plotted (mean ± SEM; n = 4–12; **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t test). (J) A modified experimental procedure for OVA-induced pulmonary allergy model for testing the effect of TSLP administration. (K and L) The total cell numbers of Th2 cells in mice treated in (J) were measured by flow cytometry, and the values were plotted (mean ± SEM; n = 5–6; *p < 0.05, **p < 0.01, Student’s t test). Results are representative of two (G–I and K–L) to three (A–F) independent experiments. See also Figure S4.

Figure 4.. ILC2 expansion during chronic papain challenges requires Th2 cells

(A–D) WT and Rag1^−/− mice were intranasally challenged with PBS or papain and then sacrificed on days 3 (acute) and 16 (chronic). (A–C) Eosinophils in the BALF and lung and ILC2s in the lung were analyzed by flow cytometry. The total numbers of eosinophils in BALF (A) and lung (B) were counted and plotted (mean ± SEM; n = 7–12; ***p < 0.001, ****p < 0.0001, Student’s t test). The total numbers of ILC2s (C) were counted and plotted (mean ± SEM; n = 9–15; *p < 0.05, ****p < 0.0001, Student’s t test). (D) Lung cells were stimulated with PMA/ionomycin, and then IL-5/IL-13-producing ILC2s were analyzed by flow cytometry. The percentage of ILC2s expressing IL-5/IL-13 were counted and plotted (mean ± SEM; n = 7–13; ns, not significant, Student’s t test). (E–H) Gata3^fl/fl mice and hCD2^CreGata3^fl/fl mice were challenged i.n. with PBS or papain and then sacrificed on days 3 (acute) and 16 (chronic). (E and F) Eosinophils in BALF (E) and lung (F) were analyzed by flow cytometry. The total numbers were counted and plotted (mean ± SEM; n = 9–20; ns, not significant, ****p < 0.0001, Student’s t test). (G) ILC2s in the lung were analyzed by flow cytometry. The total numbers were counted and plotted (mean ± SEM; n = 7–16; *p < 0.05, ****p < 0.0001, Student’s t test). (H) Lung cells were stimulated with PMA/ionomycin, and then IL-5/IL-13-producing ILC2s were analyzed by flow cytometry. The percentage of ILC2s expressing IL-5/IL-13 were counted and plotted (mean ± SEM; n = 5–14; ns, not significant, Student’s t test). (I) Shown are the microscopic images of periodic acid-Schiff-stained lung tissue sections. (J) Eosinophilic scores and total lung histopathology scores were plotted (mean ± SEM; n = 4–10; ns, not significant, ***p < 0.001, Student’s t test). (K) OVA-induced airway inflammation was performed with WT mice. ILC2s in the lung were analyzed by flow cytometry. (L) The total numbers of ILC2s in (K) were counted and plotted (mean ± SEM; n = 6–7, **p < 0.01, Student’s t test). (M) Experimental procedure for pulmonary inflammation induced by in-vitro-differentiated OT-II-Th2 cell adoptive cell transfer. (N) Eosinophils, ILC2s, and IL-5/IL-13-expressing ILC2s were analyzed by flow cytometry. The total numbers were then counted and plotted (mean ± SEM; n = 6; ***p < 0.001, ****p < 0.0001, Student’s t test). Results are representative of three independent experiments. See also Figure S5.

Figure 5.. Type 2 alarmins induced by Th2 cells play a redundant role in ILC2 expansion

(A) PCA of RNA-seq data from sorted lung ILC2s after chronic papain challenges on day 16 (highlighted by pink) or with PBS as controls (highlighted by blue). (B) Volcano plot showing few differentially expressed genes after papain challenges. (C) Heatmap showing the expression of selected genes that are associated with type 2 immune responses. Heatmap color indicates the Z score. (D) Type 2 alarmins in the BLAF of papain challenged mice on days 3 (acute) and 16 (chronic) were measured by ELSIA and plotted (mean ± SEM; n = 6–14; ****p < 0.0001, ns, not significant, Student’s t test). (E) Type 2 alarmins in the BLAF of WT mice that received OT-II Th2 cells and were challenged with OVA for 5 days were measured by ELISA (mean ± SEM; n = 12–15; ****p < 0.0001, Student’s t test). (F) The total cell numbers of ILC2s from mice that received OT-II Th2 cells and were challenged with OVA for 5 days were counted and plotted (mean ± SEM; n = 5–6; ns, not significant, Student’s t test). (G) The total cell numbers of ILC2s from mice that received OT-II Th2 cells and were challenged with OVA with or without indicated antibody treatment for 5 days were counted and plotted (mean ± SEM; n = 6; ****p < 0.0001, Student’s t test). (H) Flow cytometric analysis of IL-5 and IL-13 expression by pulmonary ILC2s from WT naive mice treated with various type 2 alarmins in vitro. Results for RNA-seq experiment (A–C) are representative of one single experiment with biological duplicates (PBS) or triplicates (chronic papain). Results are representative of two (F and G) and three (D, E, and H) experiments.

Figure 6.. IL-4 is necessary and sufficient to induce ILC2 expansion and activation via regulating type 2 alarmins

(A) A modified experimental procedure for pulmonary inflammation induced by adoptive transfer of in-vitro-differentiated OT-II-Th2 cells when IL-4 was neutralized. (B) The type 2 alarmin levels in the BALF were measured by ELISA, and the values were plotted (mean ± SEM; n = 10; ****p < 0.0001, Student’s t test). (C) The cell numbers of eosinophils and ILC2s measured by flow cytometry and plotted (mean ± SEM; n = 10; ***p < 0.001, ****p < 0.0001, Student’s t test). (D) Mouse model to study IL-4 and IL-13 effect on ILC2 response. (E) The cell numbers of eosinophils and ILC2s for WT (top panel) and Rag1^−/− (bottom panel) mice were measured by flow cytometry, and the values were plotted (mean ± SEM; n = 5; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t test). (F) The type 2 alarmin levels in the BALF were measured by ELISA. The values for WT (top panel) and Rag1^−/− (bottom panel) mice were plotted (mean ± SEM; n = 5; ns, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t test). (G) The cell numbers of eosinophils and ILC2s in mice challenged i.n. with IL-4 were measured by flow cytometry, and the values were plotted (mean ± SEM; n = 8; ****p < 0.0001, Student’s t test). (H) The cell numbers of eosinophils and ILC2s in mice challenged i.n. with IL-4 were measured by flow cytometry, and the values were plotted (mean ± SEM; n = 6–7; ns, not significant, Student’s t test). (I) The cell numbers of eosinophils and ILC2s in mice challenged i.n. with IL-4 were measured by flow cytometry, and the values were plotted (mean ± SEM; n = 4–5; ****p < 0.0001, Student’s t test). Results are representative of two independent experiments. See also Figure S6.

Figure 7.. Temporally specific functions of ILC2s and Th2 cells and their crosstalk during N. brasiliensis infection

(A) Mice model of N. brasiliensis infection, egg enumeration, and worm counts. (B–H) Mice were infected with L3 stage larvae. The fecal egg count was monitored throughout the study. Some mice were sacrificed on the indicated days to assess cellular phenotype in different organs. The number of adult worms was enumerated in the small intestine. (B) The excreted eggs were enumerated and plotted as eggs per gram of fecal matter (EPG) (mean ± SEM; n = 4–10). (C) Worm burden in the small intestine (worm count/SI) on different days post-infection (mean; n = 6, **p < 0.01, ****p < 0.0001, Student’s t test). (D) The excreted eggs were enumerated up to 30 days. (E) The excreted eggs were enumerated up to 200 days. (F) The total number of eosinophils in the BALF and lung was measured by flow cytometry and plotted (mean; n = 3–6). (G) The total number of Th2 cells in different organs was measured by flow cytometry and plotted (mean; n = 3–6). (H) The total number of ILC2s in different organs was measured by flow cytometry and plotted (mean; n = 3–6). Results are representative of two (D and E) and three (B, C, and F–H) independent experiments. See also Figure S7.