Alkali injury–induced pathological lymphangiogenesis in the iris facilitates the infiltration of T cells and ocular inflammation
Zheng Liu, Keli Liu, Shunhua Shi, Xun Chen, Xinyu Gu, Weifa Wang, Keli Mao, Rukeye Yibulayi, Wanwen Wu, Lei Zeng, Weibin Zhou, Xiaofeng Lin, Feng Zhang, Bingsheng Lou
Zheng Liu, Keli Liu, Shunhua Shi, Xun Chen, Xinyu Gu, Weifa Wang, Keli Mao, Rukeye Yibulayi, Wanwen Wu, Lei Zeng, Weibin Zhou, Xiaofeng Lin, Feng Zhang, Bingsheng Lou
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Research Article Immunology Ophthalmology

Alkali injury–induced pathological lymphangiogenesis in the iris facilitates the infiltration of T cells and ocular inflammation

Abstract

Inflammatory lymphangiogenesis is intimately linked to immune regulation and tissue homeostasis. However, current evidence has suggested that classic lymphatic vessels are physiologically absent in intraocular structures. Here, we show that neolymphatic vessels were induced in the iris after corneal alkali injury (CAI) in a VEGFR3-dependent manner. Cre-loxP–based lineage tracing revealed that these lymphatic endothelial cells (LECs) originate from existing Prox1+ lymphatic vessels. Notably, the ablation of iridial lymphangiogenesis via conditional deletion of VEGFR3 alleviated the ocular inflammatory response and pathological T cell infiltration. Our findings demonstrate that iridial neolymphatics actively participate in pathological immune responses following injury and suggest intraocular lymphangiogenesis as a valuable therapeutic target for the treatment of ocular inflammation.

Authors

Zheng Liu, Keli Liu, Shunhua Shi, Xun Chen, Xinyu Gu, Weifa Wang, Keli Mao, Rukeye Yibulayi, Wanwen Wu, Lei Zeng, Weibin Zhou, Xiaofeng Lin, Feng Zhang, Bingsheng Lou

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Figure 3

CAI-induced iridial lymphangiogenesis depends on VEGFC/VEGFR3 signaling.

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CAI-induced iridial lymphangiogenesis depends on VEGFC/VEGFR3 signaling....
(A) Schematic showing generation of mice harboring the VEGFR3fl/fl allele and Prox1-CreERT2 transgene and experimental timeline of TAM administration for conditional deletion of the VEGFR3 gene (referred to as VEGFR3iLECko) and CAI in B and C. Mice received CAI or sham treatment at week 6, followed by TAM administered 5 times (80 mg/kg) i.p. between days 13 and 17 and analysis on day 28 after CAI. (B) CD31 and Lyve1 immunostaining images of the iris whole mounts from VEGFR3iLECko and VEGFR3fl/fl mice that received CAI or sham treatment at week 6, followed by TAM administered 5 times (80 mg/kg) i.p. between days 13 and 17 and analysis on day 28 after CAI. Note ablation of lymphatics in the injured VEGFR3iLECko mouse group. Scale bars: 1,000 μm and 100 μm. (C) Quantification of Lyve1+ lymphatic vessel and CD31+ blood vessel coverage in the iris in mice in B. Data are shown as mean ± SEM. n = 7 mice per group. Each dot represents 1 mouse. ***P < 0.001. One-way ANOVA with the Tukey post hoc test. (D) Experimental timeline of VEGFC-156S administration and CAI in E and F. Mice received CAI or sham treatment at week 6, followed by 150 ng administered 3 times VEGFC-156S or PBS intracameral injections between days 16 and 18 and analysis on day 28 after CAI. (E) Lyve1 immunostaining images of the iris whole mounts from WT mice that received CAI treatment at week 6, followed by 150 ng administered 3 times VEGFC-156S or PBS intracameral injections and analysis on day 28 after CAI. Note that intracameral injections with PBS alone could inhibit CAI-induced iridial lymphangiogenesis, compared with no injection controls, and that VEGFC-156S administration could promote CAI-induced iridial lymphangiogenesis. Scale bar: 1,000 μm. (F) Quantification of Lyve1+ lymphatic vessel coverage, branches, and terminal endpoints in the iris in mice in E. Data are shown as mean ± SEM. n = 4 mice per group. Each dot represents 1 mouse. *P < 0.05. Mann-Whitney U test.