Bone marrow type 2 innate lymphoid cells: a local source of interleukin-5 in interleukin-33-driven eosinophilia

Abstract

T helper type 2 (Th2) cells, type 2 innate lymphoid cells (ILC2s) and eosinophil progenitors have previously been described to produce interleukin-5 (IL-5) in the airways upon allergen provocation or by direct administration of IL-33. Eosinophilic airway inflammation is known to be associated with IL-5-dependent eosinophil development in the bone marrow, however, the source of IL-5 remains unclear. T helper cells, ILC2s and CD34⁺ progenitors have been proposed to be involved in this process, therefore, we investigated whether these cells are taking part in eosinophilopoiesis by producing IL-5 locally in the bone marrow in IL-33-driven inflammation. Airway exposure with IL-33 led to eosinophil infiltration in airways and elevated eotaxin-2/CCL24. Importantly, IL-5 production as well as expression of the IL-33 receptor increased in ILC2s in the bone marrow under this treatment. A small but significant induction of IL-5 was also found in CD34⁺ progenitors but not in T helper cells. Similar results were obtained by in vitro stimulation with IL-33 where ILC2s rapidly produced large amounts of IL-5, which coincided with the induction of eosinophil hematopoiesis. IL-33-mediated eosinophil production was indeed dependent on IL-5 as both airway and bone marrow eosinophils decreased in mice treated with anti-IL-5 in combination with IL-33. Interestingly, the responsiveness of ILC2s to IL-33 as well as IL-33-induced eotaxin-2/CCL24 were independent of the levels of IL-5. In summary, we demonstrate for the first time that IL-33 acts directly on bone marrow ILC2s, making them an early source of IL-5 and part of a process that is central in IL-33-driven eosinophilia.

Keywords: IL-33; IL-5; ILC2; bone marrow; eosinophilia.

Figure 1

IL‐33 elicits airway, blood and bone marrow eosinophilia. (a) Experimental model of IL‐33‐induced inflammation. (b) Contrast‐stained cells; arrows indicate eosinophils. (c) Percentage eosinophils in bone marrow, and total eosinophils in blood (d) and bronchoalveolar lavage (BAL) (e) from IL‐33‐ or PBS‐treated mice. Eotaxin‐1/CCL11 (f) and eotaxin‐2/CCL24 (g) protein levels in serum, and eotaxin‐1/CCL11 (h) and eotaxin‐2/CCL24 (i) protein levels in BAL from IL‐33‐ or PBS‐treated mice. (j) Percentage eosinophils in BAL from mice treated with IL‐33 in combination with anti‐eotaxin‐2/CCL24 (αEot‐2) or isotype control. Data are shown as means (±SEM in f–i) (n = 4 to n = 14 per group). *P < 0·05, **P < 0·01, and ***P < 0·001. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

IL‐33 promotes eosinophil maturation in bone marrow and alters T helper (Th) cells but not type 2 innate lymphoid cells (ILC2s). (a) Mature eosinophils (Mat Eos; CD45⁺ SSC^hi CD34⁻ IL5Rα ^lo CCR3^hi), immature eosinophils (Imm Eos; CD45⁺ SSC^int CD34⁻ IL5Rα ^hi CCR3^neg/lo), progenitors (Prog; CD45⁺ SSC^lo CD34⁺), ILC2s (CD45⁺ SSC^lo Lin⁻ CD19⁻ CD11c⁻  NK1.1⁻ FcԑRI⁻ IL2Rα ⁺ IL7Rα ⁺) and T helper cells (Th cell; CD45⁺ SSC^lo CD3⁺ CD4⁺) in bone marrow identified by flow cytometry. Total number of Mat Eos and Imm Eos (b), Prog (c), ILC2s (d), and Th cells (e) in the bone marrow of IL‐33‐treated or PBS‐treated mice. Data are shown as means (n = 5 to n = 7 per group). *P < 0·05, and ***P < 0·001. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Mature eosinophils (Mat Eos), type 2 innate lymphoid cells (ILC2s) and T helper (Th) cells in bone marrow respond to IL‐33 through IL33R/ST2. (a) Representative IL‐33 receptor (ST2) expression on investigated bone marrow populations. Percentage ST2⁺ cells (b) and ST2 receptor density (rMFI) (c) within indicated populations in bone marrow from IL‐33‐treated or PBS‐treated mice. Data are shown as means (n = 5 to n = 7 per group). *P < 0·05, **P < 0·01, and ***P < 0·001. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

IL‐33‐mediated eosinophil hematopoiesis, but not recruitment, is driven by IL‐5. (a) IL‐5 protein in serum from IL‐33‐treated or PBS‐treated mice. (b) Model of IL‐33‐induced inflammation including anti‐IL‐5 (αIL‐5) antibody pre‐treatment. (c) Percentage eosinophils in bone marrow, and total eosinophils in blood (d) and bronchoalveolar lavage (BAL) (e) and, eotaxin‐2/CCL24 protein in serum (f) and BAL (g) from αIL‐5‐ or isotype‐pre‐treated mice challenged with IL‐33 or PBS. Data are shown as means (± SEM in a, f–g) (n = 3 to n = 8 per group). *P < 0·05, and **P < 0·01.

Figure 5

Bone marrow type 2 innate lymphoid cells (ILC2s) and CD34⁺ progenitors, but not T helper (Th) cells, produce interleukin‐5 (IL‐5) in response to IL‐33 in vivo and in vitro. (a) Percentage IL‐5⁺ progenitors, ILC2s and Th cells in vivo in bone marrow of IL‐33‐ or PBS‐treated mice. (b) Percentage IL‐5⁺ progenitors, ILC2s and Th cells of in vitro stimulated bone marrow cells from naive mice. Cells were stimulated with IL‐33 for 3, 6, 12 or 24 hr or left unstimulated as medium control. Fold change of IL5Rα expression (MFI) on eosinophil progenitors (CD34⁺ IL5Rα ⁺) (c) and ST2 expression (MFI) on ILC2s (d) in IL‐33 stimulated wells compared with unstimulated wells. Data are shown as means (± SEM in b–d) (n = 3 to n = 7 per group). *P < 0·05, **P < 0·01, and ***P < 0·001. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Culture supernatants from bone marrow type 2 innate lymphoid cells (ILC2s) stimulate eosinophil differentiation. (a) FACS‐sorted bone marrow ILC2s (Lin⁻ CD45⁺ ICOS⁺ CD25⁺ ST2⁺) stained with Hemacolor rapid stain. (b) IL‐5 protein in cell‐free culture supernatants from in vitro expanded ILC2s stimulated with IL‐33 for 20 hr or left unstimulated as medium control. (c) Percentage eosinophil progenitors (CD34⁺ IL5Rα ⁺) in naive bone marrow cultures supplemented with 5% culture supernatant from stimulated ILC2s or plain medium as control. (d) Fold change of IL5Rα expression (MFI) in stimulated wells (5% ILC2 media) compared with control wells. Data are shown as means (± SEM in b and d) (n = 4 or n = 5 per group). *P < 0·05, and **P < 0·01. [Colour figure can be viewed at wileyonlinelibrary.com]