IL-33 mediates reactive eosinophilopoiesis in response to airborne allergen exposure

Abstract

Background: Exposure to aeroallergens induces eosinophilic airway inflammation in patients with asthma and allergic airway diseases. The circulating number of eosinophils in peripheral blood is relatively small, leading us to hypothesize that bone marrow needs to be engaged quickly to meet the demands of the tissues.

Methods: To investigate the communication between the lungs and bone marrow, we used acute allergen exposure and airway inflammation models in mice. Gene-deficient mice and cytokine reporter mice as well as in vitro cell culture models were used to dissect the mechanisms.

Results: Naïve BALB/c mice produced increased numbers of eosinophil precursors and mature eosinophils in the bone marrow when their airways were exposed to a common fungal allergen, Alternaria alternata. Expression of IL-5 and IL-33 increased rapidly in the lungs, but not in the bone marrow. Sera from allergen-exposed mice promoted eosinophilopoiesis in bone marrow cells from naïve mice, which was blocked by anti-IL-5 antibody. Mice deficient in the IL-33 receptor ST2 (i.e., Il1rl1(-/-) mice) were unable to increase their serum levels of IL-5 and allergen-induced eosinophilopoiesis in the bone marrow after allergen exposure. Finally, group 2 innate lymphoid cells (ILC2s) in the lungs showed robust expression of IL-5 after Alternaria exposure.

Conclusions: These finding suggests that lung IL-33, through innate activation of ILC2s and their production of IL-5, plays a key role in promoting acute reactive eosinophilopoiesis in the bone marrow when naïve animals are exposed to airborne allergens. Therefore, bone marrow eosinophilopoiesis may be affected by atmospheric environmental conditions.

Keywords: IL-33; IL-5; eosinophilopoiesis; eosinophils.

Figure 1

Alternaria alternata exposure accelerates eosinophilopoiesis in the bone marrow. (A) Naïve BALB/c mice were exposed intranasally (i.n.) to phosphate-buffered saline (PBS) (open circles) or 50 µg Alternaria alternata extract (Alt) (closed circles) on days 0, 3, and 6. After 12 h or 48 h of each exposure, numbers of eosinophils in bronchoalveolar lavage (BAL) fluids were determined by analyzing cytospin preparations. Data (mean ± SEM, n = 3) are representative of two experiments. *P < 0.05 and **P < 0.01, compared to baseline (i.e., day 0). (B) Gating strategy to identify bone marrow eosinophil populations by flow cytometry. Bone marrow cells from naïve mice were cultured with 10 ng/ml IL-5 for 5 days. Siglec-F⁺Gr-1⁺ and Siglec-F⁺Gr-1^lo populations were sorted, and cytospin preparations were stained with Wright–Giemsa stain. (C–F) Naïve BALB.c mice were exposed i.n. to 25 µg Alternaria or PBS, once every day for 6 days. Six hours after the last exposure, bone marrow cells (C) and peripheral blood cells (E) were stained with anti-Siglec-F and anti-Gr-1 antibodies and analyzed as described in Panel B. A summary of results from bone marrow (D) and peripheral blood (F) is shown (mean ± SEM, n = 3 or 4). Data are representative of two independent experiments. *P < 0.05 and **P < 0.01, between the groups indicated by horizontal lines.

Figure 2

Anti-IL-5 Ab inhibits eosinophilopoiesis in response to Alternaria exposure. Naïve BALB/c mice were injected intraperitoneally (i.p.) with 100 µg anti-IL-5 antibody (Ab) or isotype-matched control Ab 7 days and 1 day prior to the first i.n. administration of Alternaria. Mice were then exposed i.n. to 25 µg Alternaria or PBS once every day for 6 days and analyzed as described in Fig. 1. (A) A representative FACS scattergram from each group is shown. (B) A summary of results is shown (mean ± SEM, n = 4). *P < 0.05, between the groups indicated by horizontal lines.

Figure 3

IL-5 is expressed in the lungs, but not in the bone marrow. (A) Wild-type BALB/c mice were exposed once i.n. to either 50 µg Alternaria or PBS. The bone marrow and lungs were collected after 3 h or 24 h, and IL-5 mRNA expression was analyzed by qRT-PCR. Cytokine mRNA expression was normalized to the expression of 18S RNA. Data are shown as mean ± SEM (n = 3). Data are representative of two independent experiments. (B) Wild-type BALB/c mice were exposed i.n. to 50 µg Alternaria or injected i.p. with PBS or 4 µg IL-33. The bone marrow was collected after 6 h, and IL-5 mRNA expression was analyzed by qRT-PCR. Data are shown as mean ± SEM (n = 3). *P < 0.05, compared to mice given either PBS or Alternaria. Data are representative of two independent experiments. (C) Mice were exposed i.n. to 50 µg Alternaria, and sera were collected at the indicated times points. The levels of cytokines were determined by an ELISA. Data are shown as mean ± range (n = 2). *P < 0.05, compared to baseline (i.e., 0 h). (D and E) Mice were exposed i.n. to 50 µg Alternaria or PBS, and peripheral blood was collected at 6 h. Sera were untreated or preincubated with neutralizing anti-mouse IL-5 or control Ab and then added at 10% (v/v) to freshly isolated bone marrow cells from naïve mice. Cells were cultured for 5 days and analyzed as described in Figure 1. (D) A representative FACS scattergram from each group is shown. (E) A summary of results is shown (mean ± SEM, n = 4). *P < 0.05, between the groups indicated by horizontal lines.

Figure 4

Increased serum IL-5 is dependent on the ST2/IL-33 pathway. (A) Naïve BALB/c mice were exposed once i.n. to 50 µg Alternaria, and the lungs were collected at the times indicated. Expression of mRNA for IL-5 and IL-33 was examined by qRT-PCR. Cytokine mRNA expression was normalized to the expression of 18S RNA. Data are shown as mean ± range (n = 2). *P < 0.05, compared to baseline (Base). (B) Naïve BALB/c mice were exposed once i.n. to 50 µg Alternaria, and the bone marrow and lungs were collected at 3 h or 24 h. IL-33 mRNA expression was determined by qRT-PCR. Data are shown as mean ± SEM (n = 3). Data are representative of two independent experiments. *P < 0.05 and **P < 0.01, between the groups indicated by horizontal lines. (C) Naïve wild-type BALB/c mice or Il1rl1^−/− mice (BALB/c background) were exposed once i.n. to 50 µg Alternaria or PBS. IL-5 protein levels in sera and lung tissues were determined after 6 h. Data are shown as mean ± SEM (n = 4). *P < 0.05, between the groups indicated by horizontal lines.

Figure 5

The ST2/IL-33 pathway is necessary for eosinophilopoiesis in response to Alternaria exposure. (A) Naïve wild-type BALB/c mice or Il1rl1^−/− mice (BALB/c background) were exposed i.n. to 25 µg Alternaria or PBS every day for 6 days. Bone marrow was collected 6 h after the last exposure and analyzed by FACS. A representative FACS scattergram from each group is shown. (B) A summary of results is shown (mean ± SEM, n = 4). *P < 0.05 and **P < 0.01, between the groups indicated by horizontal lines.

Figure 6

ILC2s are a major source of serum IL-5 in mice exposed to Alternaria. Naïve IL-5venus cytokine receptor mice (BALB/c background) were exposed once i.n. to PBS or 50 µg Alternaria. (A) Lung single-cell suspensions were stained with a lineage cocktail Ab and analyzed for the IL-5venus signal by FACS. Right panel depicts CD25 and CD44 expression by the lineage-negative and IL-5venus-positive cell population in the Alternaria-exposed mice. (B) Lung single-cell suspensions were stained with a lineage cocktail Ab and Abs for CD25 and CD44, and expression of IL-5venus in the ILC2 population (Lin⁻CD25⁺CD44^hi) was analyzed by FACS. Data are presented as representative of 2–4 mice in each group. (C and D) Wild-type C57BL/6 or Rag1^−/− mice (C57BL/6 background) (C) or wild-type C57BL/6 mice or Il7ra^−/− mice (C57BL/6 background) (D) were exposed i.n. to PBS or 50 µg Alternaria. Sera were collected 6 h later, and IL-5 levels were determined by ELISA. Data are shown as mean ± SEM (n = 4). **P < 0.01, between the groups indicated by horizontal lines.