MicroRNA-4443 regulates mast cell activation by T cell–derived microvesicles Irit Shefler, PhD, Pazit Salamon, PhD, Francesca Levi-Schaffer, PharmD, Adam.

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MicroRNA-4443 regulates mast cell activation by T cell–derived microvesicles Irit Shefler, PhD, Pazit Salamon, PhD, Francesca Levi-Schaffer, PharmD, Adam Mor, MD, PhD, Alon Y. Hershko, MD, PhD, Yoseph A. Mekori, MD Journal of Allergy and Clinical Immunology Volume 141, Issue 6, Pages 2132-2141.e4 (June 2018) DOI: 10.1016/j.jaci.2017.06.045 Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 mvT*s downregulate PTPRJ expression in MCs. A-D, LAD2 cells (Fig 1, A-C) or CBMCs (Fig 1, D) were incubated with mvTs or mvT*s (Fig 1, A and C) or activated by FcεRI cross-linking (Fig 1, B). PTPRJ mRNA levels were analyzed by using qRT-PCR and normalized to the levels of GUSB. Data are relative to untreated cells. E and F, PTPRJ protein levels were assessed by using Western blot analysis (Fig 1, E) and quantitated by using densitometric analysis (Fig 1, F). Results are shown from 3 independent experiments (means ± SEMs). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Expression of miR-4443 in MCs stimulated by mvT*s. A, NanoString technology of RNA isolated from LAD2 cells activated by mvTs or mvT*s for 24 hours. B-D, LAD2 cells (Fig 2, B and C) or CBMCs (Fig 2, D) were incubated as indicated, and miR-4443 levels were measured by using qRT-PCR and normalized to RNU44 levels. Data are relative to untreated cells. E, Resting or phorbol 12-myristate 13-acetate (PMA)–activated Jurkat T cells or their microvesicles were analyzed for their miR-4443 content by using qRT-PCR. F, LAD2 cells were incubated with mvT*s in the presence of 50 μg/mL α-amanitin, and the selected miRs were evaluated by using qRT-PCR. Results are shown from 3 independent experiments (means ± SEMs). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 miR-4443 regulates PTPRJ gene expression. Luciferase assay of LAD2 cells cotransfected with PTPRJ–3′-UTR reporter plasmid (LightSwitch) or empty vector and mimic miR-4443 or mimic. NC, Negative control. Results are representative of 3 different experiments. Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 miR-4443 mimic or inhibitor regulates PTPRJ expression. A-C, LAD2 cells were transfected with 30 nmol/L miR-4443 mimic, its negative control (NC; Fig 4, A and B), or 30 nmol/L miR-4443 inhibitor or NC inhibitor (Fig 4, C) for 24 hours. Then the transfected cells were activated with mvTs or mvT*s for 24 hours (Fig 4, B and C). PTPRJ mRNA levels were measured by using qRT-PCR and normalized to levels of GUSB. Data are compared with mimic/inhibitor-NC–transfected cells. D, The PTPRJ protein level was measured by using Western blot analysis and was normalized to α-tubulin. This is a representative experiment of 3 different experiments. E, Densitometry presented as a percentage of mimic NC response. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 miR-4443 regulates ERK phosphorylation in mvT*-activated MCs. LAD2 cells were transfected with 30 nmol/L miR-4443 mimic, mimic negative control (NC), miR-4443 inhibitor, or NC inhibitor for 24 hours. Thereafter, cells were stimulated with mvT*s for 24 hours. A, Levels of phosphorylated ERK (pERK) and α-tubulin were analyzed by using Western blotting. B, Densitometry from 1 representative experiment of relative bands of phospho-ERK presented as a percentage of mimic or inhibitor negative control (NC) response. C, LAD2 cells were transfected with 5 μg of PTPRJ expression plasmid or with the empty vector phosphorylated enhanced green fluorescent protein as a control for 48 hours, followed by activation with mvT*s for 24 hours. Levels of phosphorylated ERK, PTPRJ, and α-tubulin were analyzed by means of Western blotting. This is a representative experiment of 2 different experiments. D, Densitometry presented as a percentage of LAD2 cells with mvT*s. Results are shown from 2 independent experiments (means ± SEMs). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 miR-4443 regulates IL-8 release in mvT*-activated MCs. LAD2 cells were transfected with 30 nmol/L miR-4443 mimic, mimic negative control (NC) (A), miR-4443 inhibitor, or NC inhibitor (B) for 24 hours. Thereafter, cells were stimulated with mvT*s for 24 hours. IL-8 release was assayed by means of ELISA. Results are shown from 3 independent experiments (means ± SEMs). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Proposed model of the effect of miR-4443 on mvT*-activated MCs. Activated T cells release microvesicles carrying miRNAs. These microvesicles are internalized into MCs, resulting in increased miR-4443 levels. High levels of miR-4443 downregulate PTPRJ gene expression, leading to increased ERK phosphorylation and augmented cytokine release. Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 miR expression in MCs treated by mvT*s. RNA isolated from LAD2 cells activated by mvTs or mvT*s was analyzed for miR expression by using NanoString technology (A) or by using qRT-PCR and normalized to RNU44 levels (B). Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Time course of miR-4443 and PTPRJ expression in MCs treated by mvT*s. LAD2 cells were activated with 50 μg/mL mvT*s at different time points, and expression of miR-4443 and PTPRJ was measured by using qRT-PCR and normalized to the levels of RNU44 or GUSB, respectively. Data are relative to untreated cells. Results are shown from 3 independent experiments (means ± SEMs). Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Expression of miR-4443 in MCs treated with anti-CD3/CD28–activated human peripheral blood T lymphocytes (mvTCD3/CD28). LAD2 cells were activated by microvesicles derived from resting cells or mvTCD3/CD28. miR-4443 levels were measured by using RT-PCR and normalized to RNU44 levels. Data are relative untreated cells. Results are shown from 3 independent experiments (means ± SEMs). **P < .01. Journal of Allergy and Clinical Immunology 2018 141, 2132-2141.e4DOI: (10.1016/j.jaci.2017.06.045) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions