The Transcription Factor PLZF Is Necessary for the Development and Function of Mouse Basophils

Abstract

Basophils are innate immune cells associated with type 2 immunity, allergic reactions, and host defense against parasite infections. In this study, we show that the transcription factor PLZF, which is known for its essential role in the function and development of several innate lymphocyte subsets, is also important for the myeloid-derived basophil lineage. PLZF-deficient mice had decreased numbers of basophil progenitors in the bone marrow and mature basophils in multiple peripheral tissues. Functionally, PLZF-deficient basophils were less responsive to IgE activation and produced reduced amounts of IL-4. The altered function of basophils resulted in a blunted Th2 T cell response to a protein allergen. Additionally, PLZF-deficient basophils had reduced expression of the IL-18 receptor, which impacted migration to lungs. PLZF, therefore, is a major player in controlling type 2 immune responses mediated not only by innate lymphocytes but also by myeloid-derived cells.

Fig. 1. PLZF expression during myeloid lineage cell development.

Myeloid lineage cells were stained and identified in bone marrow from PLZF-eGFP reporter mice (PEG) as shown in Supplementary Figs. 2–3. PLZF expression, as defined by eGFP expression, was determined by comparison of the identical population obtained from wild type mice (Fig. S1). The progression of development was based on publications, as indicated in the text. Color-coding in the figure represents mean fluorescence intensity (MFI) values for each subset of cells, based on eGFP expression (MFI of eGFP signal divided by the background fluorescence of the identical cell population from a matched control analyzed in the same experiment). Fold change of MFI key is shown in figure.

Fig. 2. PLZF is expressed in basophils.

(A) eGFP expression in basophils from spleen, bone marrow, blood and lung of PEG mice. Basophils were identified as DAPI⁻CD3⁻CD19⁻FcεRI⁺CD49b⁺ cells. (B) Comparison of eGFP expression in basophils, and NKT cells. Wild type basophils are shown as a fluorescence control. (C) Intracellular staining of PLZF in splenic basophils, with CD8 T cells and NKT cells as negative and positive controls, respectively. Quantification of PLZF MFI (fold change over CD8 T cells) is shown (N=3 biological replicates from 2 individual experiments). (D) eGFP expression in basophil progenitors (BaPs) in the bone marrow in PEG mice. BaPs were identified as DAPI⁻CD3⁻CD19⁻CD34⁺FcεRI⁺CD49b⁺cKit⁻ cells. (E) ZsGreen and tdTomato expression in basophils from the bone marrow and spleen in PCre x ZsGreen and PLZF-IRES-GFPCre x tdTomato mice, respectively. Representative FACS plots from 1 of 3 independent experiments are shown.

Fig. 3. Reduced numbers of basophils PLZF deficient mice.

(A) Basophil numbers in the bone marrow, spleen, lung and blood of PLZF-deficient and WT mice. Cells shown are DAPI⁻CD3⁻CD19⁻CD34⁻ population and basophils were identified as CD49b⁺FCεR1⁺. Numbers indicate the percentage of events within the gated area. Percentage and absolute number of basophils are shown in scatter plot graphs. Each symbol indicates a single mouse. N=14 with ~5 independent experiments for each tissue. (B) Basophil progenitor numbers in the bone marrow of PLZF-deficient and WT mice. Cells shown are DAPI⁻CD3⁻CD19⁻CD34⁺ population. Numbers in dot plots show the percentage of events in the gated region. Percentage and absolute number of basophil precursors are shown in scatter plot graphs. Each symbol indicates a single mouse. N=3 with 3 independent experiments. The horizontal lines indicate the mean (±s.e.m.). *P<0.05, **P<0.01, ***P<.001 determined by Student’s t-test.

Fig. 4.. Papain-induced basophil-mediated Th2 responses were decreased in PLZF KO mice.

(A) 50μg of papain was injected into the footpads of PLZF knock out (KO) and wild type (WT) mice. The same amount of heat inactivated (HI) papain was injected into the other footpad as control. 3 days after injection, the draining lymph nodes were removed and stained for basophils. Cells shown were gated are DAPI⁻CD3⁻CD19⁻. Frequency of basophils, identified as CD49b⁺FCεR1⁺ is shown in the dot plot and for multiple experiments in the scatter plot graphs. Each symbol indicates a single mouse. N=8 with 3–5 independent experiments. (B-C) 50ug of papain or HI papain was injected into (B) RAG1 KO (N=3; 3 independent experiments) or (C) CD1d KO (N=5; 3 independent experiments) mice and WT controls. Basophil frequency was determined as in (A). The horizontal lines in the scatter plots indicate the mean (±s.e.m.). *P<0.05, **P<0.01, ***P<.001, n.s. (not significant) was determined by Student’s t-test.

Fig. 5.. Differentiation of Th2 T cells is reduced in the absence of PLZF.

(A) 50μg of papain was injected into the footpads of WT or PLZF KO mice. 4 days after papain injection, the draining lymph nodes were removed. Th2 differentiation, as measured by intracellular staining for GATA3, in CD3⁺ CD4⁺ T cells, with the percent positive cells shown in the dot plots. Scatter plots show the frequency of GATA3⁺ CD4⁺ T cells, with an N = 5 and 4 independent experiments. (B) 50μg of papain was injected into the footpads of WT or CD1d KO mice. The frequency of GATA3⁺ CD4⁺ T cells is shown in the dot plots. FACS is representative of 3 independent experiments. The horizontal lines in the scatter plots indicate the mean (±s.e.m.). *P<0.05, **P<0.01, ***P<.001, n.s (not significant) was determined by Student’s t-test.

Fig. 6.. Function of PLZF deficient basophils.

(A) Peripheral blood from PLZF KO and WT mice was activated with 0.05ng/ml of IgE for 3 hours. Brefeldin A (BFA) was added for the last two hours of activation. The cells were made permeable and stained for intracellular IL-4. CD200R was analyzed to measure activation. Cells shown are CD3⁻CD19⁻FcεRI⁺CD49b⁺. Representative FACS plots from 1 of 4 independent experiments are shown. (B) Peripheral blood from WT or PLZF KO mice was activated with increasing concentrations of IgE for 3 hours. The percentage of basophils expressing IL-4 is shown. (C) Peripheral blood samples from PLZF KO and WT mice were activated with PMA/Ionomycin for 3 hours. BFA was added for the last two hours of activation. The cells were then stained for intracellular IL-4. Basophils were identified as the CD3⁻CD19⁻FcεRI⁺CD49b⁺ population. The scatter plot shows the frequency of basophils that produced IL-4 under these conditions. (D) Fold change of IL-4 Mean Fluorescence Intensity (MFI) of the cytokine producing basophils from (C), normalized to WT. N=10 with 5 independent experiments.

Fig. 7.. PLZF impacts IL-18R expression on basophils and their migration to the lung.

(A) Expression of the indicated proteins was not altered in PLZF deficient spleen basophils. (B) IL-18R expression on basophils from spleen, lung and bone marrow was reduced on PLZF deficient basophils. Representative FACS plots from 1 of 4 independent experiments is shown. The fold change on basophils from each of these tissues is shown in the scatter plot. N=8 with 4 independent experiments. (C) IL-18R expression is shown for spleen T cells (CD3⁺CD8⁺ or CD3⁺CD4⁺) taken from WT mice (grey filled) or from mice ectopically expressing PLZF in all T cells. (D) The frequency of basophils in the lung, spleen and bone marrow from IL-18R KO and WT mice. N=7–12 mice with 3–5 independent experiments.