Additional file 1 of Neuronal FcεRIα directly mediates ocular itch via IgE-immune complex in a mouse model of allergic conjunctivitis

Additional file 1: Figure S1. Expression of FcεRIα in MrgprA3+ pruriceptors. A, B Expression of FcεRIα in trigeminal ganglion (A) and spleen (B) of FcεRIα−/− mice. Scale bar: 50 μm. C Immunostaining by isotype IgG in the TG of WT mice. Scale bar: 50 μm. D Detection of FcεRIα by immunostaining in the TG of Mrgpra3GFP-cre mice. Scale bar: 100 μm. E Proportion of FcεRIα+ neurons among Mrgpra3+ pruriceptors. F–H Detection of FcεRIα by immunostaining in the conjunctiva of Mrgpra3GFP-Cre; ROSA26tdTomato mice. Scale bar: 20 μm. Figure S2. IgE-IC directly activates MrgprA3+ pruriceptors in vitro. A Identification of MrgprA3+ trigeminal neuron by fluorescent view. B–D Representative fluorescent view and Fura-2 ratiometric imaging of dissociated MrgprA3+ neuron (red arrow) and MrgprA3− neuron (white arrow). Scale bar = 50 μm. Figure S3. IgE-IC does not cause immune cell infiltration in mouse TG. A Mice were ocular instilled with IgE-IC (1, 10, 50 μg/ml; 5 μl), monomeric IgE (50 μg/ml; 5 μl), OVA (100 mg/ml, 5 μl) or vehicle (PBS; 5 μl), and eye-towards wiping bouts were counted over 1–12 h. The baseline was identified as recorded without any instillation. n = 8–10 mice per group; 2-way ANOVA for repeated measures followed by Bonferroni’s post hoc test. B–D Representative images of trigeminal ganglions which were taken 1 h after ocular instillation with either PBS, monomeric IgE, or IgE-IC and stained for Ly6C/G, IBA1, and CD3. Scale bar: 100 μm. E Quantification showed no significant differences in fluorescence intensity of markers among treatment groups. n = 4 per group; one-way ANOVA followed by Bonferroni’s post hoc test comparisons. F Representative images of trigeminal ganglions which were taken 1 h after ocular instillation with either PBS, monomeric IgE, or IgE-IC and stained for FITC-avidin. Scale bar: 100 μm. G Quantitative analysis of mast cells number in the TG after different treatments. n = 4 per group; one-way ANOVA followed by Bonferroni’s post hoc test comparisons. H Proportions of activated mast cells (with degranulation) in TG after different treatments. n = 4 per group; one-way ANOVA followed by Bonferroni’s post hoc test comparisons. Figure S4. ACJ does not cause immune cell infiltration in mouse TG. A Representative images of mouse TG sections stained for Ly6C/G (green) and PGP9.5 (red) 12 h after ACJ induction or control-treated mice. B Quantification showed no significant difference in fluorescence intensity of Ly6C/G between ACJ and control-treated mice. n = 4 mice per group; n.s. no significance, Student’s t-test. C Representative images of mouse TG sections stained for IBA1 (green) and PGP9.5 (red) 12 h after ACJ induction or control-treated mice. D Quantification showed no significant difference in fluorescence intensity of IBA1 between ACJ and control-treated mice. n = 4 mice per group; n.s. no significance, Student’s t-test. E Representative images of mouse TG sections stained for CD3 (green) and PGP9.5 (red) 12 h after ACJ induction or control-treated mice. F Quantification showed no significant difference in fluorescence intensity of CD3 between ACJ and control-treated mice. n = 4 mice per group; n.s. no significance, Student’s t-test. G Representative images of mouse TG sections stained for FITC-avidin (green) and PGP9.5 (red) 12 h after ACJ induction or control-treated mice. H Quantitative analysis of mast cells number in TG from ACJ mice or control-treated mice. n = 4 per group. n.s. no significance, Student’s t-test. I Proportions of activated mast cells (with degranulation) in TG from ACJ mice or control-treated mice. n = 4 per group. n.s. no significance, Student’s t-test. Figure S5. Contribution of neuronal FcεRIα in conjunctival inflammation at 12th hour following ACJ. Representative conjunctiva sections from mice infected by AAV9-Pirt-NC-EGFP or AAV9-Pirt-shFcεRIα-EGFP after OVA challenge and stained for Ly6C/G (A), IBA1 (C), and CD3 (E). Scale bar, 100 µm. The embedded boxes showed the enlarged views. B Quantification for fluorescence intensity of Ly6C/G showed no significant differences as comparing Control- or ACJ-treated mice between two viruses’ groups (Left). Quantitative analysis of Ly6C/G+ cell numbers showed no significant differences as comparing Control- or ACJ-treated mice between two viruses’ groups (Right). n = 4 per group; Student’s t-test. D Quantification for fluorescence intensity of IBA1 showed no significant differences as comparing Control- or ACJ-treated mice between two viruses’ groups (Left). Quantitative analysis of IBA1+ cell numbers showed no significant differences as comparing Control- or ACJ-treated mice between two viruses’ groups (Right). n = 4 per group; Student’s t-test. F Quantification for fluorescence intensity of CD3 showed no significant differences as comparing Control- or ACJ-treated mice between two viruses’ groups (Left). Quantitative analysis of CD3+ cell numbers showed no significant differences as comparing Control- or ACJ-treated mice between two viruses’ groups (Right). n = 4 per group; Student’s t-test. n.s. no significance, AAV9-Pirt-NC-EGFP vs. AAV9-Pirt-shFcεRIα-EGFP. Figure S6. IgE enhances Ca2+ response of MrgprA3+ trigeminal neurons to IgE-IC. A The percentage of IgE-IC (0.1 μg/ml) responsive MrgprA3+ neurons was significantly increased as cultured with IgE (5 μg/ml). *P < 0.05, IgE vs. Control, χ2 test. B Magnitude of IgE-IC-evoked Ca2+ responses was significantly increased for MrgprA3+ neurons cultured with IgE (5 μg/ml) in MrgPra3GFP-Cre mice. *P < 0.05, IgE vs. Control, Student’s t-test. Figure S7. Incremental sensitization gradually upregulates trigeminal FcεRIα in small-diameter neurons. A Schematic diagram of experimental design for incremental sensitization model. B Determination of anti-OVA IgE in the serum of each group. *P < 0.05, vs. Control, one-way ANOVA followed by Bonferroni’s post hoc test comparisons. C Typical images for the immunofluorescence staining of FcεRIα (green) and PGP9.5 (red) in the Control, Sensitized 1w, Sensitized 2w and Sensitized 3w mice. Scale bar: 25 μm. D Percentage of FcεRIα-immunopositive TG neurons from mice receiving sensitization of different times (n > 100 neurons total for each group). *P < 0.05, vs. Control; #P < 0.05, vs. sensitized 1w, χ2 test.