Lineage(-)Sca1+c-Kit(-)CD25+ cells are IL-33-responsive type 2 innate cells in the mouse bone marrow

Abstract

IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cultures of bone marrow cells from Rag1 KO and Kit(W-sh/W-sh) mice also responded similarly; hence, eliminating the possible contributions of T, B, and mast cells. Rather, intracellular staining revealed that the IL-5- and IL-13-positive cells display a marker profile consistent with the Lineage(-)Sca-1(+)c-Kit(-)CD25(+) (LSK(-)CD25(+)) cells, a bone marrow cell population of previously unknown function. Freshly isolated LSK(-)CD25(+) cells uniformly express ST2, the IL-33 receptor. In addition, culture of sorted LSK(-)CD25(+) cells showed that they indeed produce IL-5 and IL-13 when cultured with IL-33 plus IL-2 and IL-33 plus IL-7. Furthermore, i.p. injections of IL-33 or IL-25 into mice induced LSK(-)CD25(+) cells to expand, in both size and frequency, and to upregulate ST2 and α(4)β(7) integrin, a mucosal homing marker. Thus, we identify the enigmatic bone marrow LSK(-)CD25(+) cells as IL-33 responsive, both in vitro and in vivo, with attributes similar to other type 2 innate cells described in peripheral tissues.

FIGURE 1

Cytokine production from mouse bone marrow cells in response to IL-33 and IL-7. A, Bone marrow cells were harvested from C57BL/6 mice and cultured in media only or media supplemented with 10 ng/ml IL-33, 10 ng/ml IL-7, or 10 ng/ml IL-33 + 10 ng/ml IL-7. Culture supernatants were sampled daily, and IL-4, IL-5, IL-13, and IFN-γ levels were measured by ELISA. Error bars represent the SEM calculated from triplicate wells. B, Bone marrow cells from C57BL/6, Rag-1^{−/ −}, and IL-7Rα ^{−/ −} mice were cultured as in A, and day 5 supernatants were analyzed by ELISA. Error bars represent the SEM calculated from 9 wells in three independent experiments. C, Bone marrow cells from C57BL/6 and Kit^W-sh/W-sh mice were cultured as in A, and day 5 supernatants were analyzed by ELISA. Error bars represent the SEM calculated from 6 wells in two independent experiments.

FIGURE 2

IL-5– and IL-13–producing cells in an in vitro culture of mouse bone marrow cells in media containing IL-33 + IL-7 are positive for progenitor and hematopoietic markers. Bone marrow cells from C57BL/6 mice were harvested as in Fig. 1 and were cultured in media supplemented with 10 ng/ml IL-33 + 10 ng/ml IL-7. BD GolgiPlug was added to culture media 12 h before intracellular cytokine staining and subsequent analysis by flow cytometry. Cells were stained for intracellular IL-5, or with corresponding isotype control Ab, and for surface expression of lineage markers (B220, CD3ε, CD4, CD8α, NK1.1, CD11b, CD11c, Gr-1, TER-119), Sca-1, and CD25 (A); or c-Kit, FcεRIα, SiglecF, CD45.2, or CD44 (B); or intracellular IL-13 (C). Gates in B and C were set using data from stains with corresponding isotype control Abs. All data shown are representative of at least two independent experiments.

FIGURE 3

ST2 expression on lineage low populations in the bone marrow. Bone marrow cells from 8-wk-old C57BL/6 mice were analyzed by flow cytometry. A, Gating strategy. Lineage low cells (low to no expression of B220, CD3ε, CD4, CD8α, DX5, CD11b, CD11c, Gr-1, and TER-119) were subdivided into four populations, based on their expression of Sca-1 and c-Kit. B, Expression of ST2 on the various lineage low subpopulations. Open histograms show anti-ST2 staining, and gray shaded histograms show isotype control Ab staining. Plots representative of stains from three different mice. C, ST2 and other cell surface markers expression on CD25⁻ and CD25⁺LSK⁻ cells. Quadrants were drawn on the basis of isotype control Ab stains. All data shown are representative of stains from two to three mice.

FIGURE 4

LSK⁻CD25⁺ cells in mutant and knockout mice. A, Bone marrow cells were harvested from 8- to 11-wk-old WT C57BL/6, Rag-1 KO, IL-7Rα KO, IL-2Rγ KO, and Kit^W-sh/W-sh or from 8- to 9-wk-old WT C57BL/6 and Foxn1^nu/nu mice (B). Cells were stained as in Fig. 3 for LSK⁻CD25⁺ cells and for their expression of ST2 and IL-7Rα. Plots representative of two to three mice per mouse type. C, The frequency and absolute cell number of LSK⁻CD25⁺ cells in the bone marrow from two femurs and two tibias of each mouse. Error bars represent the SEM calculated from two to three mice per mouse type.

FIGURE 5

Sorted LSK⁻CD25⁺ cells cultured in IL-33 + IL-2 and IL-33 + IL-7 produce IL-5 and IL-13 and lose their lymphoid morphology. Bone marrow cells from C57BL/6 mouse were lineage depleted using the Lineage Cell Depletion Kit (mouse) from Miltenyi Biotec. Lineage-depleted cells were stained and sorted for LSK⁻CD25⁻ and LSK⁻CD25⁺ cells. A, Purity of sorted cells. B, Sorted cells were cultured in media only or media supplemented with 10 ng/ml IL-33, 10 ng/ml IL-2, 10 ng/ml IL-7, 10 ng/ml IL-33 plus 10 ng/ml IL-2, or 10 ng/ml IL-33 plus 10 ng/ml IL-7. Culture supernatants were harvested on day 5 of culture, and IL-4, IL-5, IL-13, and IFNγ productions were assayed by ELISA. Error bars represent the SEM calculated from 3 wells per condition. C, Wright–Giemsa stained LSK⁻CD25⁺ cells preculture and after 5 d culture in media supplemented with IL-33 plus IL-2 or IL-33 plus IL-7. Photos were taken with ×1000 magnification. Scale bars, 10 μm. All data shown are representative of three independent experiments.

FIGURE 6

LSK⁻CD25⁺ cells are responsive to IL-25 and IL-33 stimulation in vivo. PBS, 400 ng IL-25, or 400 ng IL-33 in PBS was injected i.p. into 8-wk-old WT C57BL/6 mice daily for 4 consecutive days. On day 5, nucleated bone marrow cells were harvested and stained for flow cytometry as in Fig. 3. A, Mean frequency and absolute cell number of LSK⁻CD25⁻ and LSK⁻CD25⁺ cells per mouse in each treatment group. Error bars represent the SEM from four mice per treatment group (*p < 0.05, **p < 0.01 compared with PBS treatment by unpaired t test with Welch correction). B, ST2, IL-7Rα, and α₄β₇ integrin expression on LSK⁻CD25⁻ and LSK⁻CD25⁺ cells across treatments. C, Mean fluorescence intensity (MFI) of CD25, ST2, IL-7Rα, and α₄β₇ integrin expression on LSK⁻CD25⁺ cells across treatments. Error bars represent the SEM calculated from four mice per treatment group (*p < 0.05, **p < 0.01, ***p < 0.001 compared with PBS treatment by unpaired t test with Welch correction). D, Forward scatter and side scatter of LSK⁻CD25⁺ cells across treatments. All data shown in B and D are representative of data from four mice per treatment group in two independent experiments.

FIGURE 7

ST2 high cells in the bone marrow of mice treated with IL-25 or IL-33 are LSK⁻CD25⁺ cells. Total live bone marrow cells from Fig. 6 were analyzed for ST2 expression. Subgating on the ST2 high population shows that they are mostly LSK⁻ CD25⁺ cells. Plots are representative of data from four mice per treatment group in two independent experiments.