Enhanced innate type 2 immune response in peripheral blood from patients with asthma

Abstract

Background: In mice, group 2 innate lymphoid cells (ILC2s) likely mediate helminth immunity, inflammation, and tissue repair and remodeling. However, the involvement of ILC2s in human diseases, such as asthma, is not well understood.

Objectives: The goals of this study were to investigate whether peripheral blood specimens can be used to monitor innate type 2 immunity in human subjects and to examine whether ILC2s are involved in human asthma.

Methods: PBMCs from subjects with allergic asthma (AA), subjects with allergic rhinitis (AR), or healthy control (HC) subjects were cultured in vitro with IL-25 or IL-33. Flow cytometry and cell sorting were used to identify, isolate, and quantitate ILC2s in PBMCs.

Results: Human PBMCs produced IL-5 and IL-13 when stimulated with IL-33 or IL-25 in the presence of IL-2 without antigens. In addition, IL-7 or thymic stromal lymphopoietin were able to replace IL-2. The cell population with phenotypic ILC2 characteristics, lineage(-)CD127(+)CRTH2(+) cells, responded to IL-33 and produced large quantities of IL-5 and IL-13 but undetectable levels of IL-4. PBMCs from subjects with AA produced significantly larger amounts of IL-5 and IL-13 in response to IL-25 or IL-33 than from subjects with AR or HC. The prevalence of ILC2s in blood was greater in the AA group than in the AR group or the HC group.

Conclusions: Innate type 2 immune responses are increased in asthma but not in AR, suggesting potential differences in the immunopathogenesis of these diseases. Peripheral blood is useful for evaluating innate type 2 immunity in humans.

Keywords: IL-13; IL-25; IL-33; IL-5; ILC2; allergy; asthma; innate immunity.

Figure 1. IL-33 or IL-25 induce antigen-independent type 2 cytokine production by human PBMCs

PBMCs were cultured without antigen for 7 days with increasing concentrations of IL-33 (A) or IL-25 (B) in the presence or absence of 20 U/ml IL-2. IL-5 and IL-13 levels in the supernatants were determined using ELISA. Data are the mean ± SEM of 30 subjects. *p < 0.05; **p < 0.01, compared to IL-2 alone or between groups as indicated by horizontal bars. (C) PBMCs were cultured with medium, IL-33 alone (10 ng/ml), IL-25 alone (0.4 ng/ml), or in combination with IL-2 (20 U/ml), IL-7 (10 ng/ml), or TSLP (10 ng/ml). IL-5 levels in the supernatants were determined. Data are the mean ± SEM of 6 experiments. *p < 0.05 compared to IL-33 alone or IL-25 alone.

Figure 2. Lineage-negative (Lin⁻) cells are necessary for IL-25- and IL-33-induced IL-13 production

PBMCs from subjects with who had allergic asthma and were skin test-positive for house dust mite (HDM) were separated into two fractions, the Lin⁺ with Lin⁻ cell fraction and the Lin⁺ cell alone fraction. The Lin⁺ with Lin⁻ cell fraction contained 82.0% Lin⁺ cells and 18.0% Lin⁻ cells; the Lin⁺ cell alone fraction contained 99.5% Lin⁺ cells and 0.5% Lin⁻ cells. The fractions were cultured with HDM extract (25 μg/ml), IL-25 or IL-33 (10 ng/ml) plus IL-2 (20 U/ml), or combinations of HDM extract and cytokines for 7 days. (A) IL-13 levels in cell-free supernatants of the Lin⁺ with Lin⁻ cell fraction were determined. (B) IL-13 levels in cell-free supernatants of the Lin⁺ cell alone fraction are presented as the ratio of the Lin⁺ cell alone fraction divided by the Lin⁺ with Lin⁻ cell fraction from the same donor. Data are the mean ± SEM of 3 subjects.

Figure 3. Circulating ILC2s in peripheral blood respond to IL-33 ex vivo

(A) Gating strategy to isolate ILC2s and other lineage-negative (Lin⁻) and lineage-positive (Lin⁺) cell populations. Lin⁻ lymphocytic cells were subdivided into three populations based on CD127 and CRTH2 expression. Red boxes (labeled 1–4) indicate the sorted cells. (B) Sorted cell populations from A were stimulated for 5 days with medium alone or IL-33 (10 ng/ml) plus IL-2 (20 U/ml). Levels of IL-5, IL-13, and IFNγ in cell-free supernatants were measured using Milliplex. Data shown are the mean ± SEM of 3 independent experiments. **p < 0.01 compared to medium alone. (C) ILC2s (Box 1 in A) were cultured for 5 days with IL-33 plus IL-2. Cell morphology is shown before and after culture by staining the cytospin preparations with Wright Giemsa. Scale bar = 10 μm.

Figure 4. PBMCs from patients with allergic asthma show enhanced type 2 responses to IL-33 or IL-25

(A) PBMCs from healthy controls (HC), subjects with allergic rhinitis (AR), or subjects with allergic asthma (AA) were cultured for 7 days with medium alone, IL-2 (20 U/ml) alone, IL-2 plus IL-33 (20 U/ml and 10 ng/ml, respectively), or IL-2 plus IL-25 (20 U/ml and 0.4 ng/ml, respectively). IL-5 (left) and IL-13 (right) levels in cell-free supernatants were determined using ELISA. Data shown are box and whisker plots (10^th, 25^th, 50^th, 75^th, and 90^th percentile) of 14 or 15 subjects per group. *p < 0.05; **p < 0.01, between the groups indicated by horizontal bars. (B) ILC2s in PBMCs were identified and enumerated using flow cytometry as described in Figure 2A. Proportion of ILC2s in PBMCs and the total number of PBMCs per ml of blood are shown as the mean ± SEM (n = 6 per group). *p < 0.05, between the groups indicated by the horizontal bar. (C) Total numbers of ILC2s per ml of blood were calculated. Each dot represents one patient, and horizontal bars indicate the mean for each group.