IL-33-responsive lineage- CD25+ CD44(hi) lymphoid cells mediate innate type 2 immunity and allergic inflammation in the lungs

Abstract

Innate immunity provides the first line of response to invading pathogens and a variety of environmental insults. Recent studies identified novel subsets of innate lymphoid cells that are capable of mediating immune responses in mucosal organs. In this paper, we describe a subset of lymphoid cells that is involved in innate type 2 immunity in the lungs. Airway exposure of naive BALB/c or C57BL/6J mice to IL-33 results in a rapid (<12 h) production of IL-5 and IL-13 and marked airway eosinophilia independently of adaptive immunity. In the lungs of nonsensitized naive mice, IL-33-responsive cells were identified that have a lymphoid morphology, lack lineage markers, highly express CD25, CD44, Thy1.2, ICOS, Sca-1, and IL-7Rα (i.e., Lin(-)CD25(+)CD44(hi) lymphoid cells), and require IL-7Rα for their development. Airway exposure of naive mice to a clinically relevant ubiquitous fungal allergen, Alternaria alternata, increases bronchoalveolar lavage levels of IL-33, followed by IL-5 and IL-13 production and airway eosinophilia without T or B cells. This innate type 2 response to the allergen is nearly abolished in mice deficient in IL-33R (i.e., ST2), and the Lin(-)CD25(+)CD44(hi) lymphoid cells in the lungs are required and sufficient to mediate the response. Thus, a subset of innate immune cells that responds to IL-33 and vigorously produces Th2-type cytokines is present in mouse lungs. These cells may provide a novel mechanism for type 2 immunity in the airways and induction of allergic airway diseases such as asthma.

Figure 1

IL-33 induces airway eosinophilia and Th2 cytokine production in naïve mice lacking adaptive immunity. (A) IL-33 (100 ng/dose) or PBS were administered i.n. to naïve BALB/c and Rag1^−/− mice three times, as indicated by the arrowheads. BAL fluid was collected 48 h after each administration, and the eosinophil number was counted. Data are means ± SEMs of 3-5 mice per group and representative of 2 individual experiments. (B) Lungs were collected 24 h after the third IL-33 administration as described in A, formalin fixed, paraffin embedded, and stained with H&E (top row), PAS (middle row) or anti-MBP (bottom row). (C) IL-33 (100 ng/dose) or PBS were administered i.n. to naïve C57BL and Rag1^−/− mice once. After 12 h, lungs were collected, and the cytokine levels in the lung homogenates were measured. Data shown are means ± SEM of 5-8 mice per group and representative of 3 individual experiments. **, p<0.01 compared to PBS administration in each mouse strain. (D) IL-33 or PBS was administered i.n. to naïve BALB/c mice. Lungs were collected at the indicated time points, and the levels of IL-5 (circles) and IL-13 (squares) in the lung homogenates were measured. Data shown are means ± SEM of 4-8 mice per group and representative of 2 individual experiments. **, p<0.01 compared to PBS administration.

Figure 2

Lung cells cultured with IL-33 produce IL-5 and IL-13 in vitro. (A) Single-cell suspensions were obtained from lungs, spleens, thymi and mediastinal and mesenteric LNs of naive BALB/c mice and cultured for 2 or 4 days with IL-33 (10 ng/ml). The concentrations of IL-5 and IL-13 in the cell-free supernatants were measured. (B) Single cell suspensions of lung cells from naive BALB/c mice were cultured with IL-33 (10 ng/ml) for 48 h and stained for intracellular IL-5 and cell surface markers. Data are representative of 4 different experiments showing similar results.

Figure 3

Lin⁻CD25⁺CD44^hi lymphoid cells in the lungs produce a large quantity of IL-5 and IL-13 in response to IL-33 in vitro. (A) Four populations of lung cells, including Lin⁺ cells, Lin⁻CD25⁻CD44⁻ cells, Lin⁻ CD25⁻CD44⁺ cells and Lin⁻CD25⁺CD44^hi cells were isolated from naïve BALB/c mice by FACS sorting; the upper panels show the gating strategy. Sorted and unsorted lung cells were cultured with medium alone or with 10 ng/ml IL-33 for 96 h, and the levels of cytokines in the supernatants were measured by ELISA. Data shown are means ± SEMs of 4-9 independent experiments. *, p<0.05 compared to cells cultured with medium alone. (B) FACS-sorted Lin⁻CD25⁺CD44^hi lung cells were cultured with medium alone or 10 ng/ml IL-33 for 96 h and examined under electron microscopy. Original magnifications; 25,000x (medium alone, top) and 12,000x (IL-33, bottom). (C) Expression of various cell surface markers on the Lin⁻CD25⁺CD44^hi lung cell population was examined by flow cytometry. Solid line depicts staining with antibody to the indicated marker; filled grey areas are isotype control staining. Data are representative of three experiments showing similar results.

Figure 4

The roles of IL-2 and IL-7 in proliferation of and cytokine production by Lin⁻ CD25⁺CD44^hi lung cells. (A) Isolated Lin⁻CD25⁺CD44^hi cells (1×10⁴ cells/well) were cultured with medium alone or with IL-2 (50 ng/ml), IL-7 (10 ng/ml), or IL-33 (10 ng/ml) or their combinations for up to 10 days. Expansion of Lin⁻CD25⁺CD44^hi cells was determined by counting the cell number at each time point. Data from one of three experiments showing similar results are presented. (B) Isolated Lin⁻CD25⁺CD44^hi cells were cultured with medium alone or with IL-2 (50 ng/ml), IL-7, IL-25, IL-33 (10 ng/ml each) or their combinations for 96 h. The levels of IL-5 and IL-13 in the supernatants were determined by ELISA. Data are representative of three experiments showing similar findings. (C) Single-cells suspensions of lung cells from naive C57BL, Rag1^−/− and Il7r^−/− mice were stained for lineage markers, CD44, and CD25 or CD127 (IL-7Rα), and Lin⁻ lung cells were analyzed as described in Figure 3A. (D) Single-cell suspensions of lung cells from BALB/c and ST2^−/− mice were stained with lineage markers, CD44, CD25 and ST2. Top panels: Lin⁻ lymphocytic cells were analyzed as described in Figure 3A. Bottom panel: expression of ST2 on Lin⁻CD25⁺CD44^hi cells is depicted in BALB/c (black line) and ST2^−/− (red line) mice.

Figure 5

Airway exposure of mice to Alternaria extract induces rapid Th2-type cytokine responses through IL-33-dependent innate immune mechanism(s). (A) Naïve BALB/c mice were exposed i.n. three times to Alternaria extract, as described in Materials and Methods. Lungs were collected 24 h after the third exposure, formalin fixed, paraffin embedded and stained with H&E. (B) Naïve BALB/c or Rag1^−/− mice were exposed i.n. three times to Alternaria extract or PBS on days 0, 3, and 6. BALs were collected 24 h after the first exposure and 48 h after the second and third exposure, and the eosinophil numbers were determined. Data shown are means ± SEMs of 3-5 mice per group and representative of 3 individual experiments. (C) Naïve BALB/c mice were exposed i.n. once to Alternaria extract or PBS. BAL fluid was collected 1 or 12 h later, and the levels of IL-25, IL-33, and TSLP in the supernatants were determined. Data shown are means ± SEMs of 3-6 mice per group and representative of 3 individual experiments. *, p<0.05; **, p<0.01. (D) Naïve C57BL or Rag1^−/− mice were exposed i.n. once to Alternaria extract or PBS. Lungs were collected 12 h later and the levels of IL-5 and IL-13 in lung homogenates were examined. Data shown are means ± SEMs of 4-8 mice per group and representative of 2 individual experiments. *, p<0.05; **, p<0.01. (E) Naïve BALB/c or ST2^−/− mice were exposed i.n. once to Alternaria extract or PBS. Lungs were collected 12 h later, and the levels of cytokines in lung homogenates were determined. Data are shown as means ± SEMs of 8-16 mice per group, a pool of 2 individual experiments. **, p<0.01. (F) Naïve BALB/c or ST2^−/− mice were exposed i.n. three times to Alternaria extract or PBS as described in B. BAL fluid was collected 48 h after the second exposure (i.e. Day 5) or 24 h after the third exposure (i.e. Day 7), and the numbers of eosinophils were determined. Data are shown as means ± SEMs of 8-16 mice per group from a pool of 2 individual experiments. **, p<0.01.

Figure 6

Lung Lin⁻CD25⁺CD44^hi cells are activated when mice are exposed i.n. to IL-33 or Alternaria extract in vivo. (A) Naïve BALB/c mice were exposed i.n. once to PBS, IL-33 or Alternaria extract, and lungs were collected 12 h later. The cell surface expression of CD25 and CD44 on lung Lin⁻ cells was analyzed, as described in Figure 2A. One of two experiments with similar results is shown. (B) Left panel: Representative histograms of CD25 intensity in Lin⁻CD25⁺CD44^hi cells from mice exposed to PBS (grey filled histogram), IL-33 (green line) or Alternaria (blue line) are shown. Right panel: Mean fluorescence intensity (MFI) of CD25 staining in Lin⁻CD25⁺CD44^hi cells was determined from flow cytometry, and data from 3 mice per group are summarized (means ± SEMs). *, p<0.05 compared to PBS administration. (C) Naïve BALB/c or ST2^−/− mice were exposed i.n. once to PBS or Alternaria extract, and lungs were collected 12 h later. The cell surface expression of CD25 and CD44 on lung Lin⁻ cells was analyzed, as described in Figure 2C. One of two experiments showing similar results is shown. (D) Left panel: Representative histograms of CD25 intensity in Lin⁻CD25⁺CD44^hi cells from mice exposed to PBS (BALB/c, grey filled histogram; ST2^−/−, dashed black line) or Alternaria (BALB/c, blue line; ST2^−/−, green line) are shown. Right panel: MFI of CD25 staining in Lin⁻ CD25⁺CD44^hi cells was determined by flow cytometry, and data from 3 mice per group are summarized (means ± SEMs). *, p<0.05 compared to PBS administration.

Figure 7

Adoptive transfer of Lin⁻CD25⁺CD44^hi cells from wild type mice reconstitutes the Alternaria-induced immune responses in Il7r^−/− mice. (A) Lin⁻ cells were isolated from the lungs of naïve C57BL mice as described in Materials and Methods, and they (1×10⁶ cells/mouse) were adoptively transferred intravenously to naive Il7r^−/− mice. Control naïve C57BL and Il7r^−/− mice received PBS. Twenty-four h later, mice were exposed i.n. once to PBS or Alternaria extract. Lungs were collected 24 h later, and the expression of CD25 and CD44 on lung Lin⁻ cells was analyzed, as described in Figure 2C. (B) MFI of CD25 staining in Lin⁻CD25⁺CD44^hi cells was determined by flow cytometry. (C) Lin⁻ICOS⁺ cells were isolated from the lungs of naïve C57BL mice as described in Materials and Methods, and they (1×10⁵ cells/mouse) were adoptively transferred intravenously to naive Il7r^−/− mice. Control naïve C57BL and Il7r^−/− mice received PBS. Beginning 24 h later, mice were exposed i.n. three times to PBS or Alternaria extract on days 0, 3, and 6. BAL fluid was collected 24 h after the last exposure, and the cell number and differentials were determined. Data shown are means ± SEMs from 2-4 mice per group. *, p<0.05 compared to the same strain of mice treated with PBS. #, p<0.05 compared to Il7r^−/− mice without Lin⁻ICOS⁺ cell transfer but exposed to Alternaria extract. (D) The levels of IL-5 and IL-13 in the BAL fluid supernatants of the mice as described in C were determined. *, p<0.05 compared to the same strain of mice treated with PBS. (E) Representative histology (upper panels: H&E staining, lower panels: PAS staining) of the mice as described in C is presented.