IgE antibodies, FcεRIα, and IgE-mediated local anaphylaxis can limit snake venom toxicity  Philipp Starkl, PhD, Thomas Marichal, DVM, PhD, Nicolas Gaudenzio, PhD, Laurent Lionel Reber, PhD, Riccardo Sibilano, PhD, Mindy Tsai, DMSc, Stephen Joseph Galli, MD  Journal of Allergy and Clinical Immunology  Volume 137, Issue 1, Pages 246-257.e11 (January 2016) DOI: 10.1016/j.jaci.2015.08.005 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 RVV can induce local MC degranulation, recruitment of innate inflammatory cells, and hypothermia. A and B, Toluidine blue–stained (Fig 1, A) and hematoxylin and eosin–stained (Fig 1, B) back skin sections. Fig 1, A, shows the extent of MC degranulation (mean + SD). C and D, Flow cytometric plots (Fig 1, C) and quantification (Fig 1, D; mean + SD from 3 mice representative of 2 experiments) of CD45+ skin cells. E and F, Temperature (Fig 1, E) and survival (Fig 1, F) after RVV injection. G and H, Temperature (right panel magnifies the area in the dashed box; Fig 1, G) and survival (Fig 1, H) of RVV-treated mice pretreated with antihistamine (ie, triprolidine), PAF receptor antagonist (ie, CV-6209), or both. P values were determined as follows: χ2 test (Fig 1, A), Student t test (Fig 1, D, E, and G), and Mantel-Cox test (Fig 1, H). Symbols in Fig 1, E, show comparison of the group in that color with vehicle-treated mice for that time point. Fig 1, E-H, show data pooled from 2 to 3 experiments. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 MCs can contribute to innate resistance and behavioral responses to RVV. Experimental outline (A), body temperature (B and E), survival (C and F), and scratching attempts (D and G) of MC-deficient Cpa3-Cre+; Mcl-1fl/fl (Fig 2, B-D) and KitW-sh/W-sh (Fig 2, E-G) mice and corresponding control mice after RVV injection. In Fig 2, D and G, data are shown as individual values and mean ± SEM. P values were determined as follows: Student t test (Fig 2, B, D, E, and G) and Mantel-Cox test (Fig 2, C and F). Data were pooled from 2 to 4 experiments (n = 5-21 per group). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 IgE can contribute to acquired resistance to RVV. A, Outline of experiments with IgE-deficient (Igh-7−/−) and control (Igh-7+/+) C57BL/6 mice (Fig 3, B-E). B and C, Serum RVV-specific IgG1 (Fig 3, B) and total IgE (Fig 3, C). D and E, Body temperature (Fig 3, D) and survival (Fig 3, E). F, Outline of serum transfer experiments in C57BL/6 mice (Fig 3, G-J). G and H, Serum RVV-specific IgG1 (Fig 3, G) and RVV-specific IgE (Fig 3 H). I and J, Body temperature (Fig 3, I) and survival (Fig 3, J). Data were pooled from 3 to 4 experiments (n = 9-25 per group). In Fig 3, B, C, G and H, data are shown as individual values and mean ± SEM. P values were determined as follows: Mann-Whitney test (Fig 3, B, C, G, and H), Student t test (Fig 3, D and I), and Mantel-Cox test (Fig 3, E and J). *P < .05, **P < .01, and ***P < .001. n.d., Not detectable. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 FcεRIα and FcεRIα-bearing cells can contribute to acquired resistance to RVV. A, Outline of experiments with Fcer1a−/− and control (Fcer1a+/+) C57BL/6 mice (Fig 4, B-E). B and C, Serum RVV-specific IgG1 (Fig 4, B) and total IgE (Fig 4, C) levels. D and E, Body temperature (Fig 4, D) and survival (Fig 4, E). F, Outline of serum transfer experiments involving MC-deficient C57BL/6 mice (Fig 4, G and H). G and H, Body temperature (Fig 4, G) and survival (Fig 4, H). Data were pooled from 3 experiments (n = 9-17 per group). In Fig 4, B and C, data are shown as individual values and mean ± SEM. P values were determined as follows: Mann-Whitney test (Fig 4, B and C), Student t test (Fig 4, D and G), and Mantel-Cox test (Fig 4, E and H). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 IgE-dependent passive cutaneous anaphylaxis to an irrelevant antigen can increase resistance to a potentially lethal challenge with RVV. A, Experimental outline. B and C, Body temperature (Fig 5, B) and survival (Fig 5, C) of C57BL/6 mice treated with 3 subcutaneous injections of saline alone or containing 50 ng of anti-DNP IgE, IgG1, or IgG2b antibody and challenged 18 hours later with 2 subcutaneous injections, each containing 37.5 μg of RVV and 0.5 μg of DNP-HSA. Data were pooled from 2 to 5 independent experiments (n = 10-25 per group). P values were determined as follows: Student t test (Fig 5, B) and Mantel-Cox test (Fig 5, C). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Influence of genetic background and immunization regimen on acquired resistance to RVV. A, Experimental outline. B-G, Serum RVV-specific IgG1 (Fig 6, B and C), total IgE (Fig 6, D and E), and RVV-specific IgE (Fig 6, F and G) levels. H and I, Body temperature. J and K, Survival. Data were pooled from 3 to 4 experiments (n = 11-16 per group). In Fig 6, B-G, data are shown as individual values and mean ± SEM. P values were determined as follows: Mann-Whitney test (Fig 6, B-G), Student t test (Fig 6, H and I), or Mantel-Cox test (Fig 6, J and K). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Influence of genetic background and immunization regimen on acquired resistance to BV. A, Experimental outline. B-G, Serum BV-specific IgG1 (Fig 7, B and C) and total IgE (Fig 7, D and E) on day 35 from the same mice shown in Fig 7, H-K and bvPLA2-specific IgE (Fig 7, F and G) from separate groups of mice on day 42. H and I, Body temperature. J and K, Survival. Data were pooled from 3 experiments (n = 9-11 per group). In Fig 7, B-G, data are shown as values from the individual mice in the 3 different experiments performed and mean ± SEM (B-E) or as values for the pooled sera obtained from separate groups of mice in each of the 3 different experiments performed and mean ± SEM. P values were determined as follows: Mann-Whitney test (Fig 7, B-G), Student t test (Fig 7, H and I), and Mantel-Cox test (Fig 7, J and K). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Subcutaneous RVV induces systemic neutrophilia. A, Flow cytometric plots of blood neutrophils (CD45+Ly6GhighF4/80−). PI, Propidium iodide. B, Percentages (means + SDs, n = 3) of blood neutrophils at indicated times after injection. Numbers indicate percentages of gated cells in total subpopulation. Data in Fig E1, A, are from 1 mouse per panel and representative of 3 mice per group. P values were determined as follows: Student t test. **P < .01. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Effects of triprolidine or CV-6209 pretreatment on responses to RVV in BALB/c mice. A, Experimental outline. i.p., Intraperitoneal; i.v., intravenous. B, Temperature (right panel magnifies area in dashed box). C, Survival. P values were determined as follows: Student t test (Fig E2, B) and Mantel-Cox test (Fig E2, C). Data were pooled from 2 experiments. Symbols in Fig E2, B, indicate comparison of the group in that color with vehicle-treated mice for that time point. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Assessment of roles of basophils, MCs, and neutrophils in type 2 humoral responses to RVV. A, Experimental outline (Fig E3, B-E). B-E, Serum RVV-specific IgG1 (Fig E3, B and D) and total IgE (Fig E3, C and E) levels: in Fig E3, B and C, basophil-deficient Mcpt8-Cre+/−; DTAfl/− and control basophil-sufficient Mcpt8-Cre+/−; DTA−/− mice were used, and in Fig E3, D and E, MC- and basophil-deficient Cpa3-Cre+; Mcl-1fl/fl and corresponding control mice were used. F, Experimental outline (Fig E3, G and H). i.p., Intraperitoneal. G and H, Serum RVV-specific IgG1 (Fig E3, G) and total IgE (Fig E3, H) levels in neutrophil-depleted anti–GR-1–treated (see the Methods section for extent of neutrophil depletion), isotype antibody control–treated, and PBS-treated C57BL/6 mice. Data are pooled from 2 to 4 experiments (n = 6-18 per group). In Fig E3, B-E, G and H, data are shown as individual values and mean ± SEM. P values were determined as follows: Mann-Whitney test. *P < .05, **P < .01, and ***P < .001. ns, Not significant (P > .05). Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 IgE contributes to acquired resistance to RVV-induced toxicity in BALB/c mice. A, Outline of experiments with IgE-deficient (Igh-7−/−) and Igh-7+/+ mice. B and C, Serum RVV-specific IgG1 (Fig E4, B) and total IgE (Fig E4, C) levels. D and E, Body temperature (Fig E4, D) and survival (Fig E4, E). Data are pooled from 3 experiments (n = 16-19 per group). In Fig E4, B and C, data are shown as individual values and mean ± SEM. P values were determined as follows: Mann-Whitney test (Fig E4, B-C), Student t test (Fig E4, D), and Mantel-Cox test (Fig E4, E). *P < .05, **P < .01, and ***P < .001. n.d., Not detectable. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Passive cutaneous anaphylaxis to an irrelevant antigen can increase resistance to a potentially lethal challenge with RVV. A-D, C57BL/6 and BALB/c mice received 3 subcutaneous injections of 50 μL of saline alone or containing 50 ng of anti-DNP IgE antibody on the shaved back skin. Eighteen hours later, mice were injected intravenously with 200 μL of PBS containing 1% Evan blue dye and challenged 30 minutes later by means of 2 subcutaneous injections of 50 μL of PBS containing 0.5 μg of DNP-HSA administered to the site of prior injection of anti-DNP IgE or saline. Fig E5, A and C, Body temperature after DNP-HSA challenge. Fig E5, B and D, Extravasation of intravascular fluid into the skin at the site of DNP-HSA challenge 1 hour after challenge, as assessed based on levels of Evan blue dye. E and F, Body temperature (Fig E5, E) and survival (Fig E5, F) of C57BL/6 mice after 2 subcutaneous injections of 37.5 μg of RVV mixed with 0.5 μg of DNP-HSA or vehicle. G, Experimental outline for Fig E5, H and I. H and I, Body temperature (Fig E5, H) and survival (Fig E5, I) of BALB/c mice treated with 3 subcutaneous injections of 50 μL of saline alone or containing 50 ng of anti-DNP IgE antibody and challenged 18 hours later with 2 subcutaneous injections, each of 50 μL of PBS containing 37.5 μg of RVV and 0.5 μg of DNP-HSA. Data are pooled from 2 to 3 experiments (n = 6-15 per group). In Fig E5, B and D, data are shown as individual values and mean ± SEM. P values were determined as follows: Student t test (Fig E5, B, D, and H) and Mantel-Cox test (Fig E5, I). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Eliciting passive cutaneous anaphylaxis–mediated resistance against RVV depends on using an antigen-specific IgE. A, Experimental outline. B and C, Body temperature (Fig E6, B) and survival (Fig E6, C) of IgE-deficient (Igh-7−/−) C57BL/6 mice treated with 3 subcutaneous injections of saline alone or containing 50 ng of anti-DNP IgE alone, 50 ng of anti-DNP IgE mixed with 500 ng of anti-OVA IgE, or 500 ng of anti-OVA IgE antibody alone and challenged 18 hours later with 2 subcutaneous injections, each containing 37.5 μg of RVV and 0.5 μg of DNP-HSA. Data are pooled from 3 independent experiments (n = 7 per group). P values were determined as follows: Student t test (Fig E6, B) and Mantel-Cox test (Fig E6, C). *P < .05. Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 BV induces similar degranulation responses in BMCMCs derived from C57BL/6 or BALB/c mice after their sensitization with BV-immune serum derived from either C57BL/6 or BALB/c mice. A and B, BMCMCs were sensitized overnight with pooled immune serum collected on day 42 from PBS-PBS–, BV-PBS–, or BV-BV–immunized C57BL/6 or BALB/c mice (these were the same sera used for measurements of phospholipase A2–specific IgE in Fig 7, F-G). Percentage of β-hexosaminidase released was measured 1 hour after exposure to BV. Fig E7, B, shows selected data from Fig E7, A, to allow side-by-side comparison of results from C57BL/6 and BALB/c BMCMCs; none of the responses to any of the concentrations of BV tested were significantly different in the BMCMCs derived from the 2 mouse strains. Data (mean ± SEM, n = 3) were pooled from 3 independent experiments by using the same batch of BMCMCs tested on different days but each of the 3 experiments with a different batch of pooled serum from each of the 3 independent immunizations. P values were determined as follows: Student t test. *P < .05, **P < .01, and ***P < .001. ns, Not significant (P > .05). Journal of Allergy and Clinical Immunology 2016 137, 246-257.e11DOI: (10.1016/j.jaci.2015.08.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions