Supplementary Figure 1. H-PGDS deficiency does not affect GI tract functions and anaphylactic reaction. (a) Representative pictures of H&E-stained
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1 Supplementary Figure 1. H-PGDS deficiency does not affect GI tract functions and anaphylactic reaction. (a) Representative pictures of H&E-stained jejunum sections ( 200 magnification; scale bar, 100 µm). (b) Length of villi or crypt and ratio of villi/crypt in jejunum (n = 4). (c) Intestinal permeability was measured by plasma FITC-dextran concentrations in naïve and tenth OVA-challenged mice (n = 5-7). * P < 0.05, ** P < Tukey s test was performed for comparisons. (d) mrna expression of several cytokines in the colon of naïve mice. The data are presented as the ratio to WT (n = 4). (e) Serotonin content in the colon of naïve mice (n = 4). (f) Maximum temperature drop after 5 min antigen (DNP: DNP-BSA 500 ng) injection in IgE-α-DNP sensitized mice (n = 5 each). Data are presented as mean ± SEM. 1
2 Supplementary Figure 2. High dosage of OVA induces comparable diarrhea incidence between Balb/c WT and H-PGDS -/-. (a) Diarrhea occurrence of 1, 10 and 50 mg OVA challenged WT mice (n = 6, 8, 9, respectively). (b) Diarrhea occurrence of 10 mg OVA challenged WT and H-PGDS -/- mice (n = 9 each). Data are represented as the percentage over the number of OVA challenges. 2
3 Supplementary Figure 3. H-PGDS deficiency increases mast cell infiltration into colonic lamina propria in response to OVA regardless mice strain. (a) Mean number of mast cells per high power field (HPF) in each tissue of naïve and 1 day before OVA challenge (n = 4-5). (b) The percentage of CD4 + or CD8 + T cells, B220 + B cells, CD11c + cells and c-kit + /FcεRI + mast cells in the colonic lamina propria after the tenth OVA-challenged mice (n = 4). (c) Mean number of mast cells per high power field (HPF) in each tissue of Balb/c mice at tenth 1 mg OVA challenge (n = 4-5). Data are presented as mean ± SEM. * P < Tukey s test was performed for comparisons. 29 3
4 Supplementary Figure 4. H-PGDS deficiency does not increase the expression of Th2 cytokines. mrna expression of IL-4 (a) and IL-13 (c) in the colon following the tenth saline or OVA challenge (n = 4). IL-4 (b), IL-13 (d) content in the colon following the tenth OVA challenge (n = 4). There was no statistical significance in mrna expression of IL-4 between tenth OVA challenged WT and H-PGDS -/- mice (P = ). Data are presented as mean ± SEM. * P < Tukey s test was performed for comparisons. 38 4
5 Supplementary Figure 5. Kit W-sh/W-sh mice exhibits scratching behavior in response to OVA. Scratch score was monitored in Kit W-sh/W-sh mice (n = 7). Data are presented as mean ± SEM. 43 5
6 Supplementary Figure 6. Single transfer of BMMCs is insufficient to cause food allergic manifestations. (a) Experimental outline. Kit W-sh/W-sh mice were reconstituted with BMMC by single injection. Feces score (b) and systemic score (c) were monitored in Kit W-sh/W-sh mice reconstituted with BMMCs derived from WT or H-PGDS -/- by single injection (n = 5 each). (d) Representative pictures of CAE-stained colon sections following the tenth OVA challenge in mice ( 200 magnification; scale bar, 50 µm). Almost all CAE-positive mast cells were localized in the submucosal region and muscle (square, 400 magnification). (e) Mean number of mast cells per HPF in the colon of mice following the tenth OVA challenge (n = 5). There was no difference in the number between both lines of mice. Data are presented as mean ± SEM. 6
7 Supplementary Figure 7. H-PGDS deficiency does not affect the content of mast cell growth factors in colon. (a) The mrna expression of SCF, TNF-α, SDF-1α, IL-9 and MMP-9 in the tenth OVA-challenged colon (n = 4-5). Data are presented as the ratio to WT and as mean ± SEM. ** P < Tukey s test was performed for comparisons. (b) Image of gelatin zymogram of pro-mmp-9 (105 kda) and active (act)-mmp-9 (95 and 88 kda) in the tenth OVA-challenged colon of 3 independent mice. Standard (Std) indicates mouse MMP-9 recombinant protein. 64 7
8 Supplementary Figure 8. H-PGDS deficiency does not affect mast cell maturation and function. (a) The proliferation rate of BMMCs (n = 4). (b) The percentage of c-kit + /FcεRI + cells in 2-, 4-, and 6-week-old BMMCs (n = 4). (b) The degranulation rate of sensitized BMMCs stimulated by DNP-HSA (10 ng ml -1 ) or ionomycin (0.5 µm). (d) The amount of cystenyl leukotrienes (cys-lts) released from sensitized BMMCs stimulated by DNP-HSA (10 ng ml -1 ) (n = 4). Data are presented as mean ± SEM. ** P < Tukey s test was performed for comparisons. 74 8
9 Supplementary Figure 9. CXCR4 antagonism attenuates systemic score in H-PGDS -/- mice. AMD3100 (300 µg/mouse) was administered to mice intraperitoneally before each OVA challenges. Systemic scores were monitored (n = 5 10). Data are presented as mean ± SEM. * P < Mann Whitney U test was performed for comparisons. 81 9
10 82 Supplementary Table 83 Supplementary Table 1. Sequences of the primers used for real-time RT-PCR Target Forward primer Reverse primer IL-1β TGACGTTCCCATTAGACAGC TGGGGAAGGCATTAGAAACA IL-4 ATGCCTGGATTCATCGATAAG GCTCAGTACTACGAGTAATCC IL-9 ATCTGAAGGATGATCCACCGTC TCTGTGTGGCATTGGTCAGC IL-13 CTGAGCAACATCACACAAGACC TTGCAATTGGAGATGTTGGTCAG TNF-α AGCCTGTAGCCCACGTCGTAG GTAGACAAGGTACAACCCATCG CCL-2 AATGCTAACGCCACCGAGAG CCTTGTTCTGCTCCTCATAGTCC SCF CCTTAGGAATGACAGCAGTAGCA GCCAATTACAAGCGAAATGAGAG SDF-1α CTGCATCAGTGACGGTAAACC CAGCCGTGCAACAATCTGAAG 5-LO ACCAAACCCCTGGAGAGAGTA GCGATACCAAACACCTCAGAC LTA 4 H ATTTGTGGACGGTTGTTTGG CATAGGGGATGGAGGAATAGG MMP-9 CATTTCGACGACGACGAGT AGTGGTGCAGGCAGAGTAGG 18S rrna GACTCAACACGGGAAACCTCAC CACCCACGGAATCGAGAAAG 5-LO, 5-lipoxygenase; LTA 4 H, Leukotriene A 4 hydrolase; MMP, matrix metalloproteinase. 10
11 84 Supplementary Methods 85 FITC-dextran permeability assay 86 Intestinal permeability was assessed by luminal enteral administration of FITC-dextran kda (TdBCons). All mice were subjected to gavage with FITC-dextran (20 mg/mouse) and 88 whole blood was obtained by cardiac puncture after 4 h. The plasma was collected and the 89 absorption was measured at 488 nm using a fluorometer. Dilutions of FITC-dextran in PBS were 90 used as a standard curve Degranulation assay 93 Six-week-old BMMCs ( ) were sensitized with DNP-IgE (100 ng ml -1 ) for 24 h 94 and then stimulated with DNP-HSA (3-10 ng ml -1 ) or ionomycin (0.5 µm) for 30 min at 37 C. A 95 p-nitrophenyl N-acetyl-β-D-glucosamide solution (3.5 mg ml -1, 100 µl) was added to the 96 supernatant (50 µl) of the BMMCs. After 90 min incubation at 37 C, glycine solutions (400 mm, µl) were added and the absorbance was measured at 405 nm Flow cytometry 100 Inflamed colon tissues were digested by collagenase IA (Wako) and dispase (Roche) for min at 37 C in RPMI containing 10% FBS. The collected cells were washed with PBS and 11
12 102 stained with the following antibodies (Biolegend) in PBS containing 5% FBS and 0.5% NaN for 30 min at 4 C: Alexa Fluor 488 anti-mouse CD45 (30-F11), PE anti-mouse CD4 (GK1.5), 104 Alex Fluor 647 anti-mouse CD8 (53-6.7), PE anti-mouse B220 (RA3-6B2), Alex Fluor anti-mouse CD11c (N418), PE anti-mouse CD117 (2B8), and Alex Fluor647 anti-mouse FcεRI 106 (MAR-1). Each antigen-positive cell among live leukocytes (CD45-positive and 7-AAD 107 negative) was analyzed on a BD Accuri C6 (BD Bioscience). 108 For analysis of BMMC maturation, the cells ( ) were stained with FITC 109 anti-cd117 and PE anti-fcεri antibodies as described above. Cells double-positive for both 110 antigens were defined as mature BMMCs Passive systemic anaphylaxis 113 Mouse monoclonal IgE anti-dinitrophenyl (DNP) antibody (3 µg in 100 µl saline) was 114 administrated intravenously by the tail vein. After 24h, DNP-BSA (500 ng in saline) was injected 115 into the tail vein and body temperature was measured using a rectal thermometer (Physitemp 116 Instruments Inc, BAT-12) 5 min after antigen challenge. 12
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