Research ArticleMUCOSAL IMMUNOLOGY

BATF acts as an essential regulator of IL-25–responsive migratory ILC2 cell fate and function

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Science Immunology  10 Jan 2020:
Vol. 5, Issue 43, eaay3994
DOI: 10.1126/sciimmunol.aay3994
  • Fig. 1 Batf is expressed by lung ILC2 cells and is predicted to regulate distinct gene sets in KLRG1pos populations.

    WT C57BL/6 mice were injected intraperitoneally with IL-33 daily for 4 days, and pulmonary KLRG1neg (non-ILC2s) and KLRG1pos (ILC2s) were sorted on day 5 and used for RNA-seq. (A) Diagonal scatter plot comparing differentially expressed genes (Padj < 0.05) among KLRG1neg and KLRG1pos cells. (B) Heat map of ILC2-related genes and S1P receptor genes up-regulated in KLRG1pos ILCs. *P ≤ 0.05. (C) Transcription factors predicted by PASTAA analysis to be involved in regulating genes that were enriched among KLRG1pos, compared with KLRG1neg populations. (D) Normalized enrichment scores (NES) of the predicted transcription factors (TF) as assessed with gene set enrichment analysis of KLRG1pos ILC2s compared with the KLRG1neg population. Binding motifs as reported by TRANSFAC are also included in the table, where S = C or G, W = A or T, R = A or G, Y = C or T, M = A or C, and N = any base. RNA-seq analysis was compiled from three separate experiments with n = 3 to 6 mice per group.

  • Fig. 2 BATF is required by IL-25–responsive KLRG1high pulmonary ILC2s.

    (A and B) Intestinal helminth burden after PBS, IL-25, or IL-33 administration to (A) Rag−/−, WT, or Batf−/− mice or (B) Rag−/−Batf−/− mice 5 days after infection with N. brasiliensis. Data combined from six (A) or two (B) independent experiments. (C to E) WT or Batf−/− mice were infected with N. brasiliensis, and pulmonary LinCD90+ ILC2s were assessed 5 days later by flow cytometry. (C) Flow cytometry plots of different CD90+ ILC subsets based on KLRG1 expression. (D) Number and frequency of KLRG1high ILC2s combined from seven independent experiments. (E) Representative histograms and compiled gMFI of various surface markers and transcription factors in the indicated ILC populations from infected WT mice. n = 6 to 18 mice pooled from two to seven separate experiments. Means ± SEM, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data were analyzed by two-tailed unpaired t test (D) and one-way ANOVA with Tukey post test (A, B, and E).

  • Fig. 3 KLRG1mid, but not KLRG1high, pulmonary ILC2s express high levels of Arg1.

    (A to D) Arg1YFP and Batf−/−Arg1YFP mice were infected with N. brasiliensis, and pulmonary ILC2s were assessed 5 days later by flow cytometry. (A) Contour plots and combined data of the percentage of KLRG1high iILC2s. (B) Histograms displaying the percentage of YFP+ cells in the gated populations depicted in the contour plot. (C) Compiled data showing the percentage of cells that are YFP+ in (B). (D) Compiled data of the gMFI of YFP of each population in (B). The dashed line represents the gMFI of the reporter negative mouse. Data in (A) to (D) are from a single experiment with n = 2 to 4 mice per group, representative of four individual experiments. (E) Arg1YFP mice were given consecutive daily HDM (10 μg) by oropharyngeal aspiration for 4 days and rested on day 5, and pulmonary ILC2s were assessed by flow cytometry on day 6. Representative contour plots display the frequency of KLRG1high iILC2s in the lung, and the bar graph shows combined data from n = 6 to 7 mice per group compiled from two separate experiments. **P ≤ 0.01, ***P ≤ 0.001, as determined by one-way ANOVA and Tukey post hoc analysis (A, C, and D) or two-tailed unpaired t test (E).

  • Fig. 4 Cell-intrinsic requirement of BATF for pulmonary KLRG1high ILC2s.

    (A) Schematic depicting the generation and infection of bone marrow chimeric mice. Cells were analyzed 5 days after N. brasiliensis infection. (B) Contour plots showing congenic markers delineating LinCD90+KLRG1high lung cells that arose from CD45.1 Batf−/− or CD45.2 WT donors or CD45.1/CD45.2 residual host cells. (C) Combined results from two separate experiments; n = 7. *P ≤ 0.05, **P ≤ 0.01, as determined by one-way ANOVA and Tukey post hoc analysis.

  • Fig. 5 KLRG1high iILC2s are responsible for type 2 cytokine production in the lung 5 days after N. brasiliensis infection.

    (A to C) RNA-seq was performed on KLRG1pos and KLRG1neg LinCD90+ lung cells from mice treated with IL-33 as described in Fig. 1. (A and B) Genes that are significantly different and increased at least twofold in KLRG1pos relative to KLRG1neg cells were used in (A) Gene Ontology molecular function analysis and (B) KEGG pathway analysis. Results are shown as −log10 P values to denote pathways that are significantly associated with genes up-regulated in KLRG1high ILC2s. Activity groups or pathways that relate to cytokine signaling are highlighted in red. (C) Quantification of type 2 cytokine expression in KLRG1pos and KLRG1neg populations. (D to G) IL44get and Batf−/−IL44get and (H to K) IL13yetcre13 and Batf−/−IL13yetcre13 mice were infected with N. brasiliensis, and pulmonary LinCD90+ ILC2s were assessed 5 days later by flow cytometry. (D and H) Contour plot showing IL-4 (GFP) (D) or IL-13 (YFP) (H) cytokine reporter expression of KLRG1mid (blue) and KLRG1high (red) ILC2s. Gates were set according to a reporter negative control. (E and I) Bar graphs comparing the percent of KLRG1mid and KLRG1high ILC2s that express either IL-4 (GFP) with n = 8 mice combined from three separate experiments (E) or IL-13 (YFP) with n = 9 to 10 mice combined from four experiments (I). (F and J) Representative contour plots of IL-4 (GFP) (F) or IL-13 (YFP) (J) and KLRG1 expression on LinCD90+ lung ILC2s. (G and K) Bar graphs comparing the percent of KLRG1+ ILCs expressing IL-4 (GFP) with n = 6 to 8 mice combined from three separate experiments (G) or IL-13 (YFP) with n = 6 to 7 mice combined from three separate experiments (K). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, as determined by two-tailed unpaired t test.

  • Fig. 6 Appearance of KLRG1high pulmonary iILC2s in the lung is blocked by FTY720.

    (A to C) RNA-seq was performed on KLRG1pos and KLRG1neg LinCD90+ lung cells from mice treated with IL-33 as described in Fig. 1. (A) Gene Ontology molecular function and (B) KEGG pathway analyses highlighting gene groups and pathways that are significantly associated with genes that increased at least twofold in KLRG1pos relative to KLRG1neg populations, as performed in Fig. 5. Gene groups and pathways associated with cell trafficking are highlighted in red. (C) Heatmap of chemokine receptor expression in indicated populations from three separate RNA-seq experiments. Significant differential expression is denoted by an asterisk. (D to G) IL13yetcre13 mice were infected with N. brasiliensis on day 0 and given saline control or FTY720 on days 0, 2, and 4 after infection. Pulmonary LinCD90+ ILC2s were assessed on day 5 by flow cytometry. (D) Representative contour plots of the frequency of KLRG1mid and KLRG1high lung ILC2s. (E) Combined frequency and number of KLRG1high cells; n = 3 from a single experiment representative of three independent experiments. (F) Contour plots of YFP (IL-13 mRNA) expression within the KLRG1+ subset of LinCD90+ ILC2s. (G) Combined frequency and number of YFP+ LinCD90+KLRG1high cells; n = 3 from a single experiment representative of three independent experiments. *P ≤ 0.05, **P ≤ 0.01, as determined by two-tailed unpaired t test.

  • Fig. 7 BATF is required for ILC2 function in the small intestine after helminth infection.

    (A and B) WT or Batf−/− mice were infected with N. brasiliensis or remained uninfected. LinCD90+KLRG1+ ILC2s in the SILP were assessed 5 days after infection by flow cytometry. (A) Contour plots of SILP ILC2s in indicated mice before and after infection. (B) Compiled data comparing the frequency of LinCD90+KLRG1+ cells in the SILP between infected WT (blue) and Batf−/− (red) mice; n = 7 to 9 mice, combined from three separate experiments. (C and D) IL13yetcre13 and Batf−/−IL13yetcre13 were infected with N. brasiliensis or remained uninfected. LinCD90+KLRG1+ ILC2s in the SILP were assessed 5 days later by flow cytometry. (C) Contour plots of YFP (IL-13) expression versus KLRG1 expression. (D) Compiled data comparing the frequency of LinCD90+KLRG1+ cells that express YFP (IL-13) in the SILP between infected WT (blue) and Batf−/− (red) mice; n = 5 mice, combined from two separate experiments. (E) Representative histograms of various surface markers and transcription factors in SILP ILC2s from infected WT mice. (F and G) WT and Batf−/− mice were infected with N. brasiliensis or remained uninfected, and LinCD90+KLRG1+ ILC2s in the SILP were assessed 5 days later by flow cytometry. (F) Representative contour plots showing the percentage of LinCD90+KLRG1+ ILC2s expressing BATF. Gates were set according to Batf−/− mice. (G) Bar graph showing the percentage of LinCD90+KLRG1+ ILC2s expressing BATF; n = 6 to 8 mice, combined from three separate experiments. (H and I) Arg1YFP and Batf−/−Arg1YFP mice were infected with N. brasiliensis, and SILP ILC2s were assessed 5 days later by flow cytometry. (H) Representative contour plots showing the percentage of LinCD90+KLRG1+ ILC2s that express YFP (Arg1 mRNA). Gates were set according to reporter negative mice. (I) Bar graph showing the percentage of LinCD90+KLRG1+ ILC2s that express YFP (Arg1) between infected WT (blue) and Batf−/− (red) mice; n = 6 mice, combined from two separate experiments. **P ≤ 0.01, ***P ≤ 0.001, as determined by two-tailed unpaired t test.

  • Fig. 8 BATF-deficient mice have reduced tuft cells and mucin-producing epithelial cells after helminth infection.

    WT and Batf−/− mice were infected with N. brasiliensis, and immunohistochemistry was performed on the small intestine 8 days later. (A) Representative image of DAPI (blue) and DCLK (red) staining. (B) Bar graphs depicting the number of DCLK+ cells per millimeter of villus; n = 28 to 77 individual villi counted from four individual mice per group, combined from two separate experiments. (C) Representative image of DAPI (blue) and MUC2 (red) staining. (D) Bar graphs depicting the number of MUC2+ foci per millimeter of villus; n = 22 to 23 individual villi from two mice per group from one experiment. Scale bar, 100 μm. ****P ≤ 0.0001, as determined by two-tailed unpaired t test.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/43/eaay3994/DC1

    Fig. S1. Gating strategy for sorting cells for RNA-seq.

    Fig. S2. Gating strategy for murine pulmonary and SILP ILCs.

    Fig. S3. Quantification of KLRG1neg and KLRG1mid pulmonary ILC2s.

    Fig. S4. Induction of pulmonary ILC2s by alarmins.

    Fig. S5. Bone marrow chimeric mice reveal that KLRG1high ILC2s require BATF and expand even in the absence of infection.

    Fig. S6. Quantification of type 2 cytokine protein reporter production by lung ILC2s.

    Fig. S7. FTY720 treatment does not affect nILC2s in the lung or any ILC2 population in the SILP.

    Fig. S8. Graphical summary.

    Table S1. Raw data spreadsheet.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Gating strategy for sorting cells for RNA-seq.
    • Fig. S2. Gating strategy for murine pulmonary and SILP ILCs.
    • Fig. S3. Quantification of KLRG1neg and KLRG1mid pulmonary ILC2s.
    • Fig. S4. Induction of pulmonary ILC2s by alarmins.
    • Fig. S5. Bone marrow chimeric mice reveal that KLRG1high ILC2s require BATF and expand even in the absence of infection.
    • Fig. S6. Quantification of type 2 cytokine protein reporter production by lung ILC2s.
    • Fig. S7. FTY720 treatment does not affect nILC2s in the lung or any ILC2 population in the SILP.
    • Fig. S8. Graphical summary.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data spreadsheet.

    Files in this Data Supplement:

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