1. Volume 46, Issue 5, Pages 863-874.e4 (May 2017)
Mast Cells Are Crucial for Induction of Group 2 Innate Lymphoid Cells and Clearance of Helminth Infections 
Chikako Shimokawa, Takashi Kanaya, Masami Hachisuka, Kenji Ishiwata, Hajime Hisaeda, Yosuke Kurashima, Hiroshi Kiyono, Tomohiro Yoshimoto, Tsuneyasu Kaisho, Hiroshi Ohno 
Immunity 
Volume 46, Issue 5, Pages e4 (May 2017)
DOI: /j.immuni
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2. Immunity 2017 46, 863-874.e4DOI: (10.1016/j.immuni.2017.04.017)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Resistance of Spib−/− Mice to Hp Infection Is Associated with an Increase in Goblet Cells
(A and B) WT and Spib−/− mice were infected with 200 L3 larvae. Eggs in feces (A) and worms in the small intestines (B) were counted at the indicated days after infection. Asterisks denote statistical significance at p < 0.05 using the Mann-Whitney U test.
(C) Hematoxylin- and eosin-stained sections of the duodenum from WT and Spib−/− mice 7 days after Hp infection were counterstained with Alcian blue. The Alcian blue-stained area was quantified using ImageJ, and the number of goblet cells per villus (n = 15) of the duodenum was counted. Data are mean ± SD of five mice. Similar results were obtained from three replicate experiments. Scale bars, 100 μm. Please see also Figure S1.
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4. Figure 2
Increase of IL-13-Producing ILC2 in Lamina Propria of Spib−/− Mice in Response to Hp Infection
(A) mRNAs encoding the indicated genes were quantified in the small intestinal LP lymphoid cells isolated at the indicated days after Hp infection. Values are expression of each gene relative to that of Gapdh.
(B) Flow cytometric profiles showing expression of lineage markers and CD4 by IL-13-producing cells in LP lymphoid cells isolated from the indicated mice 7 days after Hp infection. The data are representative of five mice for each genotype. The absolute number of IL-13+ cells was calculated from the flow cytometric profile for each mouse and is shown in the bar graph.
(C) Gated Lin−CD45+ LP lymphoid cells were analyzed for expression of KLRG-1 and Sca-1. The absolute number of Lin−CD45+Sca-1+KLRG-1+ ILC2 is shown in the bar graphs. Overlaid histogram plots showing production of IL-13 by gated Lin−CD45+Sca-1+KLRG-1+ ILC2. The gray histogram is the isotype control antibody. Data are mean ± SD from three mice. Error bars, ∗p < 0.05, ∗∗p < 0.01, as calculated by Mann-Whitney test or one-way ANOVA. Similar results were obtained from three replicate experiments.
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5. Figure 3
Expanded Granulocytes and Altered Hematopoiesis in Steady State Spib−/− Mice
(A) Percentages of neutrophils (CD11b+Ly-6G+), eosinophils (CD11b+Siglec-F+), basophils (c-Kit+FcεRI−), and mast cells (c-Kit−FcεRI+) among mononuclear cells in the mesenteric lymph nodes of WT and Spib−/− mice are shown based on flow cytometric analyses.
(B) The absolute number of bone marrow cells per femur in WT and Spib−/− mice.
(C) Representative flow cytometric profiles of bone marrow progenitor cells. Lin− cells were separated based on c-Kit and Sca-1 expression. Numbers indicate the percentage of Lin−Sca-1+c-Kit+ cells and Lin−Sca-1−c-Kit+ myeloid progenitors (MPs) (top). MP cells were further divided into CMP, GMP, and MEP based on the expression of CD34 and FcγRII/III (CD16/CD32). Numbers indicate the percentage of each progenitor among the MP (bottom).
(D) The absolute numbers of CMP, GMP, and MEP in a femur. Values are mean ± SD of nine to ten mice. Three replicate experiments were performed. Error bars, SD; N.S., not significant, ∗p < 0.05, ∗∗p < 0.01, as calculated by the Student’s t test. Please see also Figure S3.
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6. Figure 4
Expression of Spi1 and Gata1 mRNA in CMP in the Absence or Presence of Spi-B
(A) The mRNA encoding the indicated transcription factors was quantified in sorted CMP from WT (open bars) and Spib−/− (closed bar) mice in comparison with Gapdh. Values are mean ± SD of the relative amount to WT mice from three mice. Three independent experiments were performed with similar results.
(B) Sorted CMP from WT and Spib−/− mice were transduced with the indicated viruses and recovered for mRNA quantification 3 days later. Values are mean ± SD. Asterisks denote statistical significance at ∗p < 0.05 using the Student’s t test.
(C) Flow cytometric analyses for PU.1. Histograms indicate expression of PU.1 in gated Venus+-transduced CMP. Numbers indicate percentage of PU.1+ cells among the gated cells. Three independent experiments were performed with similar results.
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7. Figure 5
The Role of Mast Cells in Resistance to Infection with Hp in Spib−/− Mice
(A) The number of mast cells among LP lymphoid cells of the small intestine was calculated based on flow cytometric analyses for expression of c-Kit and FcεRI as described in STAR Methods.
(B) The duodenal tissues from Hp-infected WT and Spib−/− mice were stained with toluidine blue to visualize mast cells. Arrows indicate LP mast cells. Scale bars, 100 μm.
(C–E) Spib−/− mice were treated with the anti-c-Kit antibody ACK, followed by infection with Hp. The number of LP mast cells in WT and Spib−/− mice with or without ACK-treatment was counted as in (A) at day 5 after Hp infection (C). Egg production in feces of ACK-treated and isotype control IgG2b-treated mice was evaluated as in Figure 1A (D). The number of ILC2s in WT and Spib−/− mice with or without ACK-treatment was counted as in (A) at day 7 after Hp infection (E). Data are mean ± SD from three mice. Asterisks denote statistical significance at ∗p < 0.05 using Student’s t test or two-way ANOVA. Similar results were obtained from three replicate experiments.
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8. Figure 6
Mast Cell Activation by Extracellular ATP Released from Apoptotic Intestinal Epithelium during Hp Infection
(A) mRNA encoding IL-33 in sorted LP mast cells from WT and Spib−/− mice at the indicated days 7 after Hp infection was quantified using real-time RT-PCR. Asterisks denote statistical significance at ∗p < 0.05 using one-way ANOVA.
(B) Representative flow cytometric profiles of IL-33 expression by gated c-Kit+FcεRI+ LP mast cells. Purple and red lines indicate mast cells from WT and Spib−/− mice, respectively, at the indicated days after Hp infection. Gray indicates background staining with the isotype control antibody.
(C) WT, IL33−/−, C57BL/6-KitW-sh/W-sh (Wsh), Wsh reconstituted with WT (WT→Wsh) or IL-33-deficient (IL33−/−→Wsh) BMMCs were infected with Hp. The mice were then monitored by counting egg production in feces at 9 (left) and 14 (right) days after infection as in Figure 3A. Data are mean ± SD from three mice. Error bars, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, calculated by one-way ANOVA followed by Tukey’s multiple comparisons test. Similar results were obtained from three replicate experiments.
(D and E) Measurement of Il33 and Il13 transcript expressions and ILC2 numbers in the adoptive transfer experiment shown in (C). mRNA encoding IL-33 and IL-13 in the small intestinal tissues (D) and ILC2 in LP (E) isolated from the indicated mice or recipients 7 days after infection with Hp were quantified. Data are mean ± SD from three mice. Error bars, ∗p < 0.05 calculated by one-way ANOVA followed by Tukey’s multiple comparisons test.
(F) Sorted mast cells from uninfected WT and Spib−/− mice were cultured with ATP in the absence or presence of suramin. Cultured mast cells and culture supernatants were analyzed for surface expression of the CD63 degranulation marker by flow cytometry (left) and for production of IL-33 using ELISA (right), respectively. Asterisks denote statistical significance at ∗p < 0.05 using two-way ANOVA.
(G) Small intestinal tissues isolated from WT and Spib−/− mice at the indicated hours after Hp infection were cultured for 1 hr and the ATP concentration in the culture supernatants was measured.
(H) The same tissues were immediately stained with DAPI (blue) and assayed by TUNEL (red). Scale bars, 100 μm. Data are mean ± SD from three mice. Similar results were obtained from three replicate experiments.
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9. Figure 7
Involvement of the P2X7 ATP Receptor for Mast Cell Activation in Resistance to Hp Infection in Spib−/− Mice
WT and Spib−/− mice treated with a specific P2X7 inhibitor brilliant blue G (BBG) were infected with Hp, and infection was monitored by counting egg production in feces (A) as in Figure 3A. The number of LP mast cells (B), the expression of IL-33 in gated mast cells (C), the number of ILC2 (D), and ILC2 IL-13 production (E) were analyzed 7 days after infection. Data are mean ± SD from three mice. Error bars, ∗p < 0.05, ∗∗p < 0.01, as calculated by Mann-Whitney test or one-way ANOVA. Similar results were obtained from three replicate experiments.
Immunity  , e4DOI: ( /j.immuni )
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