1. Repeated FcεRI triggering reveals modified mast cell function related to chronic allergic responses in tissue 
Jolien Suurmond, MSc, Kim L.L. Habets, PhD, Zuotian Tatum, MSc, Joris J. Schonkeren, BASc, Peter A.C.'t Hoen, PhD, Tom W.J. Huizinga, MD, PhD, Jeroen F.J. Laros, PhD, René E.M. Toes, PhD, Fina Kurreeman, PhD 
Journal of Allergy and Clinical Immunology 
Volume 138, Issue 3, Pages (September 2016)
DOI: /j.jaci
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Repeated mast cell stimulation through FcεRI. A, Experimental setup of repeated stimulation of mast cells. Human cord blood–derived mast cells were stimulated for 2 weeks with 0.2 μg/mL anti-IgE (Repeated αIgE) or control medium (Naive). Twenty-four hours before each stimulation, mast cells were sensitized with human IgE. At the end of 2 weeks, mast cells were either left untreated (N and R) or stimulated with anti-IgE (N+αIgE and R+αIgE). B, Representative flow cytometric plots for CD63 of naive mast cells (N) or mast cells 3 hours after their first anti-IgE stimulation (R), showing degranulation in response to the first anti-IgE stimulation on day 0. PE, Phycoerythrin. C, Summary of surface CD63 expression as in Fig 1, B, and IL-8 secretion 24 hours after the first anti-IgE stimulation or control medium. MC, Mast cells. D and E, Surface CD63 and CD203c expression in response to 24 hours of anti-IgE stimulation in naive mast cells or mast cells exposed to 2 weeks of stimulation (Repeated αIgE). CD63 staining as in Fig 1, C, is shown, and the CD203c median fluorescence intensity (MFI) ratio is calculated as a ratio of the MFI of CD203c staining divided by the MFI of the isotype control for each individual sample. Data are represented as means ± SEMs from 3 (Fig 1, C) or 6 (Fig 1, D and E) independent experiments. Numbers indicate that no significant differences were found between naive mast cells (N) and cells exposed for 2 weeks to anti-IgE (R) by using the paired t test.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
Dampened genes after repeated anti-IgE. A, Gene expression determined by using RNA sequencing in response to a single 6-hour anti-IgE stimulation (N+αIgE) compared with that seen in control mast cells (N). Log fold change (LogFC) relative to naive (N) mast cells of the differentially expressed genes is shown. B, Genes that were significantly upregulated after a single anti-IgE stimulation were selected as shown in Fig 2, A, and compared with their upregulation after repeated stimulation (R+αIgE). LogFC values were calculated compared with unstimulated mast cells at the end of the 2-week stimulation (N+αIgE/N and R+αIgE/R). C, Genes that were significantly upregulated after a single anti-IgE stimulation were selected as shown in Fig 2, A, where each dot indicates a different gene. Those genes significantly upregulated (top) and tolerized (bottom) after repeated anti-IgE (R+αIgE) compared with a single anti-IgE (N+αIgE) stimulation are indicated. Data are shown as fold change relative to N+αIgE. D, Genes that are significantly downregulated after repeated anti-IgE stimulation compared with a single anti-IgE stimulation, as determined by using differential expression analysis. Each line represents a single gene, and data are shown as relative reads per kilobase per million (RPKM) normalized to single anti-IgE stimulation (N+αIgE). E and H, Gene expression of CSF2 and CCL4 determined by using RNA sequencing. Data are shown as RPKM fold change relative to single anti-IgE stimulation (N+αIgE). F and I, Gene expression of CSF2 and CCL4 determined by using qPCR. Data are shown as relative expression normalized to the housekeeping gene RPL5 as a fold change to single anti-IgE stimulation (N+αIgE). G and J, Protein levels of CSF2 (GM-CSF) and CCL4 (macrophage inflammatory protein 1β [MIP-1β]) in supernatant 24 hours after stimulation with anti-IgE of naive mast cells or mast cells that were exposed to anti-IgE for 2 weeks. K, Expression of genes in the arachidonic acid (AA) metabolism pathway determined by using RNA sequencing. Log fold change (LogFC) relative to naive (N) mast cells of the differentially expressed genes is shown. L and M, Protein levels of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) in the supernatant 1 hour after stimulation with anti-IgE of naive mast cells or mast cells that were exposed to anti-IgE for 2 weeks. Data are represented as means ± SEMs from 3 (Fig 2, B, E, H, and K), 5 (Fig 2, F, G, I, and J), or 6 (Fig 2, L and M) independent experiments. Asterisks indicate significant differences determined by using the paired t test (P < .05; Fig 2, B), a differential expression analysis false discovery rate of less than .05 (Fig 2, C, E, and H), or repeated-measures ANOVA with the Bonferroni post hoc test (P < .05; Fig 2, F, G, I, L, and M).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4. Fig 3
Persistent upregulation of genes after repeated anti-IgE stimulation. A, Overlap of genes upregulated after repeated anti-IgE stimulation without an additional stimulation (R > N) or with an additional stimulation (R+αIgE > N+αIgE), as determined by using differential expression analysis. B, Heat map showing relative expression of all genes in Fig 3, A. Each row represents a single gene. Relative expression in 3 independent experiments is shown. C, E, and G, Gene expression of TREM2, OLFM4, and CCL18, as obtained by using RNA sequencing. Data are shown as reads per kilobase per million (RPKM) fold change relative to naive mast cells (N). D, F, and H, Gene expression of TREM2, OLFM4, and CCL18, as determined by using qPCR. Data are shown as relative expression normalized to the housekeeping gene RPL5 as a fold change to naive mast cells (N). I, Protein levels of CCL18 in the supernatant 24 hours after stimulation with anti-IgE of naive mast cells or mast cells that were exposed to anti-IgE for 2 weeks. J, Protein levels of CCL18 in the supernatant 6 hours after stimulation with anti-IgE of naive mast cells (left) or 2 weeks after stimulation with repeated anti-IgE or control medium (right). Data are represented as means ± SEMs from 3 (Fig 3, C, E, G, and J), 5 (Fig 3, D, F, and H), or 4 (Fig 3, I) independent experiments. Asterisks indicate significant differences determined by using a differential expression analysis false discovery rate of less than 0.05 (Fig 3, C, E, and G), repeated-measures ANOVA with the Bonferroni post hoc test (P < .05; Fig 3, D, F, H, and I), or the paired t test (P < .05; Fig 3, J). N, Naive mast cells; N+αIgE, single 6-hour stimulation of naive mast cells with anti-IgE; R, repeated anti-IgE stimulation for 2 weeks; R+αIgE, 6-hour stimulation with anti-IgE after repeated anti-IgE stimulation.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5. Fig 4
Overlap in genes and enriched pathways between repeated and single stimulation of mast cells. A and B, Interaction network of GO terms enriched in genes upregulated after repeated anti-IgE (Fig 4, A) or single anti-IgE (Fig 4, B) stimulation. Node size reflects the significance/number of genes, and line thickness indicates the connectivity between 2 GO terms. Pathways that are unique for either of the gene sets are highlighted in red. C and D, Protein expression of HLA-DR determined by using flow cytometry. Data are shown as Δ median fluorescence intensity (MFI) of HLA-DR compared with the isotype control. Data are represented as means ± SEMs from 5 (Fig 4, C) independent experiments. Asterisks indicate significant differences determined by using repeated-measures ANOVA with the Bonferroni post hoc test (P < .05; Fig 4, C).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6. Fig 5
Autocrine signaling and transcription factors. A-F, Mast cells were stimulated for 2 weeks with 0.2 μg/mL anti-IgE (Repeated αIgE) or control medium (Naive) through direct stimulation (left) or using the supernatant from activated mast cells collected 24 hours after activation (right). Gene expression of CCL4, CSF2, EMR3, TMEM45B, TREM2, and CCL18 determined by using qPCR. Data are shown as relative expression normalized to the housekeeping gene RPL5 as a fold change to cells stimulated for 6 hours with anti-IgE (CCL4, CSF2, EMR3, and TMEM45B) or naive mast cells (TREM2 and CCL18). G and H, RNA expression analysis of the transcription factors BATF (Fig 5, G) and RXRA (Fig 5, H) and their target genes as identified by using Cscan enrichment analysis. Expression of these genes in mast cells exposed to repeated anti-IgE stimulation is shown as fold change relative to naive mast cells. Data are represented as means ± SEMs from 4 independent experiments (Fig 5, A-F) or from 3 independent experiments (Fig 5, G and H). Asterisks indicate significant differences determined by using repeated-measures ANOVA with the Bonferroni post hoc test (P < .05).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7. Fig 6
Association of upregulated genes with chronic allergy. A and C, Heat map showing relative expression of genes upregulated in patients with atopic allergy or those with psoriasis in mast cells exposed to repeated anti-IgE stimulation. B and D, Overlap of genes upregulated in patients with atopic allergy or those with psoriasis with random genes (top), genes most highly expressed by mast cells (second row), genes upregulated in mast cells after a single anti-IgE stimulus (third row), or genes upregulated in mast cells after repeated anti-IgE (bottom). Overlap was calculated as a percentage of the mast cell or random gene list. Asterisks indicate significant differences determined by using the Fisher exact test (P < .05; Fig 6, B and D).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions