Lung-resident eosinophils represent a distinct regulatory eosinophil subset

Abstract

Increases in eosinophil numbers are associated with infection and allergic diseases, including asthma, but there is also evidence that eosinophils contribute to homeostatic immune processes. In mice, the normal lung contains resident eosinophils (rEos), but their function has not been characterized. Here, we have reported that steady-state pulmonary rEos are IL-5-independent parenchymal Siglec-FintCD62L+CD101lo cells with a ring-shaped nucleus. During house dust mite-induced airway allergy, rEos features remained unchanged, and rEos were accompanied by recruited inflammatory eosinophils (iEos), which were defined as IL-5-dependent peribronchial Siglec-FhiCD62L-CD101hi cells with a segmented nucleus. Gene expression analyses revealed a more regulatory profile for rEos than for iEos, and correspondingly, mice lacking lung rEos showed an increase in Th2 cell responses to inhaled allergens. Such elevation of Th2 responses was linked to the ability of rEos, but not iEos, to inhibit the maturation, and therefore the pro-Th2 function, of allergen-loaded DCs. Finally, we determined that the parenchymal rEos found in nonasthmatic human lungs (Siglec-8+CD62L+IL-3Rlo cells) were phenotypically distinct from the iEos isolated from the sputa of eosinophilic asthmatic patients (Siglec-8+CD62LloIL-3Rhi cells), suggesting that our findings in mice are relevant to humans. In conclusion, our data define lung rEos as a distinct eosinophil subset with key homeostatic functions.

Figure 1. Characterization of lung rEos in naive C57BL/6 mice.

(A) Dot plot of lung leukocytes according to Siglec-F and CD125 expression. Numbers indicate the percentage of gated cells. Neutros, neutrophils. (B) Photographs of the FACS-sorted cell populations gated in A. (C) Histograms of F4/80 and CCR3 expression in the cell populations shown in B. (D) Representative electron microscopy photographs of rEos (left panel, representative of more than 100 individual cells analyzed) and rEos-associated granules (right panels). Arrows, asterisks, and arrowheads indicate secondary granules with high, intermediate, and low densities, respectively. (E) Phagocytic potential of rEos 6 hours after local i.t. instillation of fluorescent beads. Number indicates the percentage of bead-positive cells. (F) Congo red– and (G) MBP-stained lung sections. Arrows indicate Congo red– and MBP-positive eosinophils, respectively. (H) Kinetics of appearance of lung rEos after birth. (I) Absolute numbers of rEos in the lungs of female and male mice. (H and I) Results represent the mean ± SEM as well as individual values. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA, followed by Tukey’s test for multiple comparisons on log-transformed values (H) or by 2-tailed Student’s t test on square root–transformed values (I). Data in A–G are representative of 1 of more than 8 mice, each giving similar results. Data in H and I were pooled from 2 and 3 independent experiments, respectively (n = 4–6/group and 14/group, respectively). Scale bars: 1 μm (D) and 10 μm (B, E–G). Max, maximum.

Figure 2. Characterization of lung rEos and iEos in HDM-challenged C57BL/6 mice.

(A) Experimental outline. i.n., intranasal. (B) Absolute numbers of lung eosinophil subsets. (C) Dot plot of lung leukocytes according to Siglec-F and CD125 expression. Numbers indicate the percentage of gated cells. (D) Photographs of the 2 lung eosinophil subsets. (E) Histograms of F4/80 and CCR3 expression in the cell populations shown in D. (F) Representative electron microscopy photograph of iEos (>100 individual cells analyzed). Arrows indicate secondary granules. (G) Congo red– and (H) MBP-stained lung sections. Arrows indicate Congo red– and MBP-positive eosinophils, respectively. (I) Quantification of parenchymal and peribronchial eosinophils in Congo red–stained lung sections. Eos, eosinophils. (J) Dot plot of BALF leukocytes according to Siglec-F and CD125 expression. Numbers indicate the percentage of gated cells. (K) Photographs of the gated cell populations in J. (B and I) Results represent the mean ± SEM as well as individual values (n = 5–6/group) and are representative of 1 of more than 4 independent experiments. **P < 0.01 and ***P < 0.001, by (B) a mixed-effects model, followed by Tukey’s test for multiple comparisons on log-transformed values, and (I) a Mann-Whitney U test. (C–H) Data are representative of 1 of more than 8 mice, each of them giving similar results. Scale bars: 10 μm (D, G, and H) and 1 μm (F). Neutros, neutrophils.

Figure 3. Transcriptomic profile of rEos_ss, rEos_i, and iEos.

(A) Summary of differentially expressed genes (P < 0.01; FC >2) in each pairwise comparison showing differentially expressed genes in red and blue in a volcano plot, the total number of differentially expressed genes in the middle of the bidirectional arrows, and arrowheads showing the direction of differential expression for all and highly (FC 4–16) differentially expressed genes. (B) Heatmap showing the relative expression of differentially expressed genes associated with the regulation of inflammatory and immune responses between iEos and rEos.

Figure 4. Identification of discriminating surface markers for mouse lung rEos and iEos.

(A and B) Representative flow cytometric histograms of CD62L and CD101 expression on (A) rEos_ss from naive C57BL/6 mice and (B) rEos_i and iEos from HDM-treated allergic C57BL/6 mice. (C) Quantification of CD62L and CD101 expression levels on the surface of rEos_ss, rEos_i, and iEos, expressed as the fold change (FC) increase in mean fluorescence intensity (MFI) as compared with the control MFI. Data represent the mean ± SEM as well as individual values and are pooled from 3 to 5 independent experiments (n = 7–13/group). (D) Histograms of CD62L and CD101 expression on rEos_i (blue) and iEos (red) and total lung eosinophils (Siglec-F^int/hiCD125^int, black) from HDM-treated allergic C57BL/6 mice. (C) *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA, followed by Tukey’s test for multiple comparisons. FMO, fluorescence minus 1.

Figure 5. Identification of rEos-like and iEos-like eosinophils in the blood of naive and HDM-treated C57BL/6 mice.

White blood cells from (A) naive and (B) HDM-treated allergic C57BL/6 mice were analyzed by flow cytometry (see Figure 2A for experimental outline). Living singlet CD45.2⁺SSC^hi cells were analyzed according to Siglec-F and CD125 expression, and representative flow cytometric histograms of CD62L and CD101 expression in the gated populations are shown. Data shown are representative of 1 of more than 8 mice analyzed from 3 independent experiments, each of them giving similar results. FMO, fluorescence minus 1; FSC, forward scatter; SSC, side scatter.

Figure 6. Sensitivity and responsiveness of eosinophil subsets to in vivo α–IL-5 and in vitro rIL-5 treatments.

(A) Experimental outline for data shown in B–D. (B–D) Effects of in vivo α–IL-5 (TRFK5) treatment on the numbers of (B and D) lung and (C) blood eosinophil subsets in naive and HDM-treated allergic C57BL/6 mice. (E) Phosphorylation of ERK1/2 was assessed by phospho-flow cytometry on freshly isolated eosinophils and AMs stimulated in vitro with rIL-5 or vehicle. Representative flow cytometric histograms and quantification of p-ERK levels. (F) Survival (as assessed by incorporation of 7-AAD) of FACS-sorted lung eosinophil subsets stimulated ex vivo, with or without rIL-5, for the indicated times. More than 90% of the cells were negative for 7-AAD before stimulation. (B–F) Data represent the mean ± SEM as well as individual values and are pooled from 2 to 3 independent experiments (n = 5–11/group). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test (B and C, left panels, and D) or a mixed-effects model, followed by Tukey’s test for multiple comparisons (B and C, right panels) on log-transformed values and (E and F) by Welch’s t test (for comparison of vehicle vs. IL-5–treated groups) or 1-way ANOVA, followed by Tukey’s test for multiple comparisons (for comparison of IL-5–treated subsets). cpm, counts per minute.

Figure 7. Assessment of rEos immunosuppressive functions using eosinophil-deficient ΔdblGATA mice and DC coculture experiments.

(A) Experimental outline for B and C. Briefly, the indicated groups of mice were sensitized i.n. with 5 μg HDM or injected with saline, and the MLN cell response to HDM was assessed 5 day later. Some WT mice also received an i.p. injection of α–IL-5 or isotype Abs 1 hour before HDM instillation. (B) Proliferation of MLN cells restimulated for 3 days with or without HDM. (C) Cytokine concentrations in culture supernatants of MLN cell cultures restimulated with HDM. (D) Expression of the indicated maturation markers on the surface of OVA_LPS-pulsed BMDCs cultured alone or cocultured with rEos or iEos for 12 hours. (E) Experimental outline for F–H. (F) Total and eosinophil cell counts in the BALF. (G) Proliferation of LN cells restimulated for 3 days with or without OVA. (H) Cytokine concentrations in culture supernatants of LN cell cultures restimulated with OVA. Data shown represent the mean ± SEM and were (B and C) pooled from 3 independent experiments (n = 6 biological replicates/group) and (D–H) are representative of 1 of 4 different batches of BMDCs cocultured with primary rEos, AMs, or IMs and 1 of 3 different batches of BMDCs cocultured with primary rEos or iEos isolated from independent cohorts of mice. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA (B, F, and G) or 1-way ANOVA (C, D, and H), followed by Tukey’s test for multiple comparisons. Iso, isotype Abs; Sal, saline.

Figure 8. Localization, morphology, and phenotype of lung rEos and iEos in humans.

(A) Representative Congo red– and MBP-stained lung sections of healthy human donors and asthmatic patients. Arrows indicate Congo red– and MBP-positive eosinophils, respectively. (B) Quantification of parenchymal and peribronchial eosinophils in Congo red–stained lung sections. Eos, eosinophils. (C–E) Parenchymal eosinophils from normal lung tissue (rEos) and eosinophils from the sputa of eosinophilic asthmatic patients (iEos) were analyzed morphologically and phenotypically. (C) Dot plots of living singlet CD45⁺SSC^hi cells according to surface Siglec-8 and CD125 expression. Insets depict a representative photograph of FACS-sorted rEos and iEos. (D) Representative flow cytometric histograms of Siglec-8, CD62L, CD101, and IL-3R expression on rEos and iEos. (E) Quantification of Siglec-8, CD62L, CD101, and IL-3R expression levels on the surface of rEos and iEos, expressed as the FC increase in MFI as compared with the control MFI. Number are the P values for comparisons that were not significant (P > 0.05). (B and E) Data shown represent the mean ± SEM as well as individual values. Each dot represents a single individual analyzed (n = 5–8/group) (see also Supplemental Methods and Supplemental Tables 1–3). *P <0.05 and **P < 0.01, by nonparametric Mann-Whitney U test. Scale bars: 10 μm and 2 μm (insets in A).