Culture medium from TNF-α–stimulated mesenchymal stem cells attenuates allergic conjunctivitis through multiple antiallergic mechanisms Wenru Su, MD,

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Culture medium from TNF-α–stimulated mesenchymal stem cells attenuates allergic conjunctivitis through multiple antiallergic mechanisms Wenru Su, MD, PhD, Qian Wan, MD, Jingwen Huang, MD, Longhui Han, MD, Xiaoqing Chen, MD, Guihua Chen, MD, PhD, Nancy Olsen, MD, Song Guo Zheng, MD, PhD, Dan Liang, MD, PhD Journal of Allergy and Clinical Immunology Volume 136, Issue 2, Pages 423-432.e8 (August 2015) DOI: 10.1016/j.jaci.2014.12.1926 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 MSC-CMT treatment attenuates EAC. A, Experimental protocol. B and C, Inflammatory scores of EAC (Fig 1, B) and scratch times (Fig 1, C) were evaluated at the indicated time points after challenge in different experimental groups. D, Representative images of ocular symptoms in the indicated experimental group 30 minutes after the last challenge. Red arrow, Lid swelling; white arrow, conjunctival edema. n = 6 mice for each group. *P < .05 and **P < .01 between the MSC-CMT and EAC groups. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Treatment with MSC-CMT attenuates inflammation and alters the cytokine profile in mice with EAC. A, Representative images of hematoxylin and eosin staining of conjunctival samples from mice in the indicated experimental groups. Scale bars represent 200 μm (upper panels) and 600 μm (lower panels). B and C, Quantification of cellular components (Fig 2, B) and eosinophils (Fig 2, C) in the conjunctivas of mice in the indicated experimental groups. D-G, TNF-α, IL-4, IL-1β, and TGF-β mRNA expression in conjunctival tissue was determined by using real-time PCR. H-K, Phosphorylated NF-κB p65, phosphorylated p38 MAPK, phosphorylated STAT6, and phosphorylated STAT3 levels in conjunctival tissue was determined by means of Western blotting. *P < .05 and **P < .01 between the MSC-CMT and EAC groups. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 MSC-CMT inhibits IgE release by B cells through a COX2-dependent mechanism. A, OLF was collected after the last challenge, and IgE levels were determined by using ELISA. BALB/c splenic B cells were cultured with different media for 24 hours. B and C, After stimulation with LPS/IL-4 for 5 days, IgE (Fig 3, B) and IgG1 (Fig 3, C) levels in supernatants were determined by using ELISA. D, Cell viability was assayed by using trypan blue exclusion. E, MSC-CMT was added with specific neutralizing antibodies for TGF-β1 or IL-10 (10 μg/mL, isotype used as controls), or MSC-CMT was collected after pretreating BMMSCs with a specific IDO inhibitor (1-MT, 500 μmol/L), a specific NO inhibitor (L-NAME, 1 mmol/L), or a specific COX2 inhibitor (NS-398, 1 μmol/L). F and G, Exogenous TNF-α–induced dose-dependent increases in COX2 expression and PGE2 production in BMMSCs. H, MSC-CMT was collected after pretreating BMMSCs with COX2 siRNA (dCMT) or control siRNA (cCMT). **P < .01. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Attenuation of EAC by MSC-CMT is associated with its inhibitory effects on MCs. A-C, MCs were identified by using toluidine blue staining, and activated MCs were identified based on their irregular shape (red arrows) in conjunctivas of normal mice, mice with EAC, and MSC-CMT–treated mice with EAC, as indicated. Scale bars represent 200 μm (upper panels) and 600 μm (lower panels). D, Histamine levels in OLF were determined by using EIA (n = 6). E and H, MCs were cultured with MSC-CMT or MSC-CM for 24 hours and then stimulated with PMACI for an additional 12 hours. TNF-α levels in supernatants were determined by means of ELISA. F and G, IκB-α and NF-kB p65 expression levels in BMMSCs were determined by means of Western blotting. Fig 4, H, MSC-CMT was collected after pretreating BMMSCs with COX2 siRNA (dCMT) or control siRNA (cCMT). **P < .01. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Antihistamine effects of MSC-CMT. A, Conjunctival vascular permeability was evaluated by using the Evans blue assay (n = 6). B, Representative images of eyes after Evans blue injection. C and F, LVECs were cultured with MSC-CMT for 24 hours. After stimulation with histamine for 30 minutes, the vascular permeability assay was performed. D and E, Total VE-cadherin (Fig 5, D) and phosphorylated VE-cadherin (Fig 5, E) expression was determined by using Western blotting. Fig 5, F, MSC-CMT was collected after pretreating BMMSCs with COX2 siRNA (dCMT) or control siRNA (cCMT). **P < .01. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 COX2 signaling in BMMSCs is essential for MSC-CMT–mediated inhibition of EAC. EAC was induced as above, and MSC-CMTs collected after pretreating BMMSCs with COX2 siRNA (dCMT) or control siRNA (cCMT) were administered to mice with EAC. A and B, Inflammatory scores (Fig 6, A) and scratch times (Fig 6, B) were evaluated at the indicated time points after challenge (n = 6). C-G, IgE production (Fig 5, C), MC enrichment and activation (Fig 5, D and E), histamine production (Fig 5, F), and TNF-α expression (Fig 5, G) in EAC were measured as above at the indicated time points after challenge. Topical 16,16-dimethyl PGE2 (dmPGE2; 20-80 ng/mL) was administered on days 10 to 14 in mice with EAC. H and I, Inflammatory scores (Fig 5, H) and scratch times (Fig 5, I) were evaluated at indicated time points after challenge (n = 6). **P < .01 between the MSC-CMT and dMSC-CMT groups. ##P < .01 between the indicated groups. ΔΔP < .01 between the MSC-CMT and PGE2 80 ng/mL groups. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 TNF-α, IL-10, IL-4, IL-13 and IFN-γ levels in MSC-CMT. A, After TNF-α depletion with antibody, the TNF-α level was detected by using ELISA in MSC-CMT. B, IL-10, IL-4, IL-13, and IFN-γ levels were measured by using ELISA in MSC-CMT. **P < .01. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Treatment with MSC-CMT reduces TNF-α, IL-4, and IL-5 production and caspase-1 activation in mice with EAC. A-C, TNF-α (Fig E2, A), IL-4 (Fig E2, B), and IL-5 (Fig E2, C) protein expression in conjunctival tissue was determined by means of ELISA. D, Caspase-1 expression in conjunctival tissue was determined by means of Western blotting. **P < .01. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 MSC-CMT inhibits IgG1 release by B cells through a COX2-dependent mechanism. B cells were cultured with different media for 24 hours. After stimulation with LPS/IL-4 for 5 days, IgG1 levels in supernatants were determined by using ELISA. A, MSC-CMT was added with specific neutralizing antibodies for TGF-β1 or IL-10 (10 μg/mL, isotype used as controls), or MSC-CMT was collected after pretreating BMMSCs with a specific IDO inhibitor (1-MT, 500 μmol/L), a specific NO inhibitor (L-NAME, 1 mmol/L), or a specific COX2 inhibitor (NS-398, 1 μmol/L). B, MSC-CMT was collected after pretreating BMMSCs with COX2 siRNA (dCMT) or control siRNA (cCMT). **P < .01. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 MSC-CMT inhibits IL-4 release by MCs through a COX2-dependent mechanism. A and C, MCs were cultured with MSC-CMT or MSC-CM for 24 hours and then stimulated with PMACI for an additional 12 hours. IL-4 levels in supernatants were determined by using ELISA. B, Cell viability was assayed by using trypan blue exclusion. Fig E4, C, MSC-CMT was collected after pretreating BMMSCs with COX2 siRNA (dCMT) or control siRNA (cCMT). **P < .01. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 COX2 signaling in BMMSCs is essential for MSC-CMT–mediated inhibition of inflammatory cell infiltration and IL-4 production. MSC-CMTs collected after pretreating BMMSCs with COX2 siRNA (dCMT) or control siRNA (cCMT) were administered to mice with EAC. Inflammatory cell infiltration (A) and IL-4 expression (B) in EAC were measured as above at the indicated time points after challenge. **P < .01. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 MSC-CMT reduces inflammatory cytokine release by lung epithelial cells. LA-4, a murine lung epithelial cell line, was cultured with MSC-CMT or MSC-CM for 24 hours and then stimulated with LPS for an additional 12 hours. TNF-α (A) and IL-1β (B) levels in the supernatants were determined by using ELISA. **P < .01. Error bars = means ± SEMs. Journal of Allergy and Clinical Immunology 2015 136, 423-432.e8DOI: (10.1016/j.jaci.2014.12.1926) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions