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Pharmacological inhibition of caspase-8 suppresses inflammation-induced lymphangiogenesis and allograft rejection in the cornea  Wenru Su, MD, PhD, Longhui.

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Presentation on theme: "Pharmacological inhibition of caspase-8 suppresses inflammation-induced lymphangiogenesis and allograft rejection in the cornea  Wenru Su, MD, PhD, Longhui."— Presentation transcript:

1 Pharmacological inhibition of caspase-8 suppresses inflammation-induced lymphangiogenesis and allograft rejection in the cornea  Wenru Su, MD, PhD, Longhui Han, MD, PhD, Xiaoqing Chen, MD, PhD, Jianfeng Yu, MD, Xiaokang Cheng, MD, Jingwen Huang, MD, Yichen Xiao, MD, Yunzhe Tian, MD, Nancy Olsen, PhD, Song Guo Zheng, MD, PhD, Dan Liang, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 142, Issue 1, Pages e9 (July 2018) DOI: /j.jaci Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Pharmacological inhibition of caspase-8 suppresses lymphangiogenesis after CT via restraining macrophage recruitment and activation. A, A model of CT was established. B, Diagram of the cornea after CT (lymphatic vessels, red). C, Image of a cornea after CT. D, Merged image of corneal whole-mount of the lymphatic vessels reaching the interface (white arrow) 9 days after CT. E, Topical application of Z-VAD-fmk (a pan-caspase inhibitor) reduced ILA (n = 4). F, The inhibitory effects of caspases inhibition were different in ILA (n = 4). G and H, Caspase-8 blockade decreased CD11b+ macrophages numbers (n = 3). I, Caspase-8 blockade decreased MIP-1 and MCP-1 mRNA expression in grafted corneas (n = 3). J, LPS increased caspase-8 activity in macrophage. K, Caspase-8 blockade inhibited MIP-1 and MCP-1 mRNA expression in macrophage. L, CT increased TLR4 mRNA expression in grafted corneas (n = 3). M and N, In TLR4−/− mice, caspase-8 activity was not increased in grafted corneas (n = 3). O and P, ILA was decreased in TLR4−/− mice after CT (n = 4). Q, LPS increased caspase-8 activity in grafted corneas (n = 3). R, Caspase-8 inhibitor blocked an LPS-mediated increase in ILA (n = 4). Casp-8i, Caspase-8 inhibitor; DAPI, 4′-6-diamidino-2-phenylindole, dihydrochloride; WT, wild-type. The data are presented as the means ± SDs. ∗P < .05, ##P < .01 (compared with normal corneas), **P < .01 (between the indicated groups). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Caspase-8 blockade inhibits NLRP3 inflammasome activation and HMGB1 secretion, reduces mature dendritic cells and effector T cells in DLNs, and prevents allograft rejection. A, CT increased extranuclear HMGB1 expression (n = 3). B and C, HMGB1 inhibition reduced TLR4 expression and caspase-8 activity (n = 3). D and E, ILA was decreased in HMGB1-inhibited mice (n = 4). F and G, Following caspase-8 inhibition, NLRP3, ASC expression, and caspase-1 activity were decreased (n = 3). H, NLRP3 inhibition reduced ILA. I and J, Following caspase-8 inhibition or NLRP3 inhibition, extranuclear HMGB1 expression was decreased in grafted corneas (n = 3). K and L, The inhibition of caspase-8 or NLRP3 in macrophages blocked LPS-induced HMGB1 secretion. M, The lymphatic vessels in corneal isografts and allografts (n = 3). N, Caspase-8 blockade inhibited ILA in corneal isografts (n = 5). O, Topical caspase-8 inhibition reduced the occurrence of CGR (n = 12). P-U, Topical caspase-8 inhibition reduced CD11c+CD80+ DCs and CD11c+CD86+ DCs in the DLNs at day 12 after CT (n = 3). V-Y, Caspase-8 blockade decreased TH17 cells, and increased regulatory T cells (CD4+Foxp3+ cells) in the DLNs (n = 3). Casp-8i, Caspase-8 inhibitor; DC, dendritic cell; DLNs, draining lymph nodes; GA, Glycyrrhizic acid; NLRP3i, NLRP3 inhibitor. The data are presented as the means ± SDs. #P < .05 and ##P < .01 (compared with normal corneas); *P < .05 and **P < .01 (between the indicated groups). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Caspase-8 blockade reduced TNF-α, IL-1β, and VEGF-C production both in vitro and in vivo. A, CD11b+ macrophages, which are rarely in the central cornea, are present in the peripheral cornea (limbus) without CT. B, LPS increased active caspase-8 expression in a murine macrophage cell line (RAW264.7). C, RAW264.7 macrophages treated with LPS were incubated with Z-IETD-fmk at indicated concentration for 24 hours. Then, cell viability was assessed before and after treatment using Trypan blue exclusion. D-F, Caspase-8 blockade inhibited LPS-induced TNF-α, IL-1β, and VEGF-C mRNA expression in macrophages. G-I, Real-time PCR showed increased TNF-α, IL-1β, and VEGF-C mRNA expression in the grafted corneas after CT, but this level was significantly reduced in caspase-8–inhibited corneas (n = 3). J, CT increased TLR4 protein expression in grafted corneas (n = 3). K, TLR4 protein expression is absent in TLR4 knockout mice. Casp-8, Caspase-8; casp-8i, caspase-8 inhibitor; DAPI, 4′-6-diamidino-2-phenylindole, dihydrochloride. The data are presented as the means ± SDs. #P < .05 and ##P < .01 (compared with normal corneas); *P < .05 and **P < .01 (between the indicated groups). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Recombinant HMGB1 increases TLR4 signaling and caspase-8 activity in ILA. A and B, The topical instillation of recombinant HMGB1 protein increased TLR4 mRNA expression and caspase-8 activity 9 days after CT (n = 3). C, Recombinant HMGB1 promoted ILA 9 days after CT, whereas caspase-8 inhibitor blocked a recombinant HMGB1-mediated increase in ILA (n = 4). The data are presented as the means ± SDs. casp-8i, Caspase-8 inhibitor; rHMGB1, recombinant HMGB1. #P < .05 and ##P < .01 (compared with normal corneas); *P < .05 and **P < .01 (between the indicated groups). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 NLRP 3 inflammasome is actively involved in ILA. A-D, CT increased the expression of NLRP3 and ASC and activity of caspase-1; these changes were sustained throughout a 9-day observation period after CT compared with normal corneas (n = 3). E and F, Following caspase-8 inhibition, NLRP3 and ASC expressions were decreased compared with the control group 9 days after CT (n = 3). casp-8i, Caspase-8 inhibitor. The data are presented as the means ± SDs. ##P < .01 (compared with normal corneas) and **P < .01 (between the indicated groups). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 Caspase-8 inhibition restrains NLRP3 inflammasome activation in macrophages. A-C, The inhibition of caspase-8 in macrophages reduced LPS-induced expression of NLRP3 and ASC. D, The inhibition of caspase-8 in macrophages decreased LPS-stimulated caspase-1 activity. casp-8i, Caspase-8 inhibitor. The data are presented as the means ± SDs. ##P < .01 (compared with normal corneas) and **P < .01 (between the indicated groups). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 Pharmacological inhibition of caspase-8 suppresses lymphangiogenesis after CT. A and B, Corneal whole-mount immunostaining of LYVE-1, which shows an increase in the area covered with lymphatic vessels, occurring from day 0 to day 9 after CT in WT mice (n = 3). C, Colorimetric assay revealed significantly increased caspase-8 activity in grafted corneas after CT compared with that in normal corneas (n = 3). D, CT increased active caspase-8 protein expression in grafted corneas (n = 3). E, The efficiency of caspase-8 inhibitor Z-IETD-fmk (30 μM, 60 μM, and 120 μM) on caspase-8 activity. F and G, ILA was significantly decreased by caspase-8 inhibition (60 μM) compared with normal mice (n = 4). Casp, Caspase; casp-8i, caspase-8 inhibition; WT, wild-type. The data are presented as the means ± SDs. #P < .05 and ##P < .01 (compared with normal corneas); *P < .05 and **P < .01 (between the indicated groups). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E6 The role of HMGB1 and TLR4 in corneal allograft rejection. A, Immunofluorescence staining showed that the CT procedure induced the extracellular HMGB1 protein release (white arrow: extracellular HMGB1). B, HMGB1 inhibitor prolonged corneal allograft survival, whereas recombinant HMGB1 promoted CGR. C, The selective knockout of TLR4 also prolonged corneal allograft survival, whereas TLR4 activation by LPS promoted CGR. DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride; GA, glycyrrhizic acid; rHMGB1, recombinant HMGB1. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E7 The proposed pathway and the molecular mechanisms of caspase-8 inhibition in lymphangiogenesis and allograft rejection in the cornea. The CT procedure initially leads to corneal cell damage, resulting in the release of HMGB1. Released HMGB1 bonds with TLR4 to activate caspase-8 signaling in the cornea. The activated caspase-8 signaling mediates macrophage recruitment and activation to induce lymphangiogenesis. Furthermore, caspase-8 regulates NLRP3 inflammasome-mediated HMGB1 secretion in the cornea and macrophage. The secreted HMGB1 may, in turn, interact with TLR4, forming a positive feedback loop to further promote lymphangiogenesis. Finally, new lymphatic vessels transport APCs carrying allograft antigens to activate T cells, thus initiating and promoting allograft rejection. Therefore, the caspase-8/NLRP3 inflammasome pathway functions as an amplification system to promote lymphangiogenesis and allograft rejection, whereas caspase-8 inhibition interrupts this process. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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