Involvement of corneal lymphangiogenesis in a mouse model of allergic eye disease

Abstract

Purpose: The contribution of lymphangiogenesis (LA) to allergy has received considerable attention and therapeutic inhibition of this process via targeting VEGF has been considered. Likewise, certain inflammatory settings affecting the ocular mucosa can trigger pathogenic LA in the naturally avascular cornea. Chronic inflammation in allergic eye disease (AED) impacts the conjunctiva and cornea, leading to sight threatening conditions. However, whether corneal LA is involved is completely unknown. We addressed this using a validated mouse model of AED.

Methods: Allergic eye disease was induced by ovalbumin (OVA) immunization and chronic OVA exposure. Confocal microscopy of LYVE-1-stained cornea allowed evaluation of corneal LA, and qRT-PCR was used to evaluate expression of VEGF-C, -D, and -R3 in these mice. Administration of VEGF receptor (R) inhibitor was incorporated to inhibit corneal LA in AED. Immune responses were evaluated by in vitro OVA recall responses of T cells, and IgE levels in the serum.

Results: Confocal microscopy of LYVE-1-stained cornea revealed the distinct presence of corneal LA in AED, and corroborated by increased corneal expression of VEGF-C, -D, and -R3. Importantly, prevention of corneal LA in AED via VEGFR inhibition was associated with decreased T helper two responses and IgE production. Furthermore, VEGFR inhibition led a significant reduction in clinical signs of AED.

Conclusions: Collectively, these data reveal that there is a distinct involvement of corneal LA in AED. Furthermore, VEGFR inhibition prevents corneal LA and consequent immune responses in AED.

Figure 1

Involvement of corneal lymphangiogenesis (LA) in the allergic eye disease (AED) model. (A, B) Allergic eye disease was induced via eye drop instillations of OVA, which were administered 2 weeks post OVA sensitization. (A, B) Identification of corneal lymphangiogenesis in AED. Whole-mounted corneas (n = 4–7/group) were stained for LYVE-1 (red) and micrographs captured via fluorescence microscopy. Dashed lines indicate the limbal edge of the excised cornea. (B) Percent of cornea area covered by lymphatic vessels in normal versus AED cornea. Data is presented as the mean ± SEM of data from two independent experiments. (C) Increased corneal VEGF-C, -D, and VEGFR-3 levels detected in AED. Excised corneas (n = 3/group) were analyzed for mRNA expression via qRT-PCR. Data is presented as the mean ± SEM of four experiments. (D) In vitro LEC proliferation is by IL-4, -5 or -13. Cultured LECs were stimulated with the indicated factors and proliferation was measured at 48 hours. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Corneal LA in AED contributes to egress of allergen-laden APCs. (A, B) Cornea APCs (CX3CR1+) capture instilled OVA in the AED setting. Two weeks post OVA sensitization, CX3CR1^eGFP/+ mice were instilled with OVA (Texas-Red) onto the ocular surface and imaged 30 minutes thereafter via multiphoton intravital microscopy (MP-IVM). (B) Colocalization of OVA (red) with eGFP+ APCs (and few eGFP− APCs) was readily detected. (C, D) Corneal LA in AED contributes to egress of allergen-laden APCs. Congenic CD45.2 mice with AED received an injection of OVA-pulsed CD45.1 BMDCs (5 × 10^4 cells) into the corneal stroma. Immediately thereafter, mice were topically administered with isotype control (C) or CCR7 blocking Ab (D). Corneas (n = 6/group) were excised 6 hours post injection and stained for LYVE-1 (red), CD45.1 (green), and DAPI (blue) staining. Asterisks (*) indicate CD45.1+ cells concentrated around lymphatic sprouts. Arrows indicate colocalization (yellow) of CD45.1 cells with lymphatic sprouts.

Figure 3

Vascular endothelial growth factor receptor inhibition prevents corneal LA in AED. (A, B) Vascular endothelial growth factor receptor inhibition reduces corneal lymphangiogenesis. Allergic eye disease was induced and VEGFR inhibitor or vehicle control was administered concurrent with the topical OVA instillation period. Excised corneas (n = 6–11/group) were stained for LYVE-1 (red) and micrographs captured via fluorescence microscopy. Dashed lines in the micrographs show the edge of the excised and radially cut cornea; arrows indicate lymphatic sprouting into the cornea. (B) Percent of cornea area covered by lymphatic vessels in AED mice treated with VEGFR inhibitor was significantly decreased relative to vehicle control treated mice. Data is presented as the mean ± SEM of data from two independent experiments (*P < 0.05).

Figure 4

Vascular endothelial growth factor receptor inhibition inhibits T helper 2 cells in AED. Allergic eye disease was induced and VEGFR inhibitor or vehicle control was administered concurrent with the topical OVA instillation period. Purified T cells from eye draining LN were cocultured with OVA-pulsed BMDCs, and culture supernatants were collected for ELISA analysis of IL-4, -5, and -13 levels. Data (n = 4/group, total n = 12/group) are representative of three independent experiments and presented here as the mean and SEM (*P < 0.05; **P < 0.01; and ***P< 0.001).

Figure 5

Vascular endothelial growth factor receptor inhibition inhibits IgE levels in AED. Allergic eye disease was induced and VEGFR inhibitor or vehicle control was administered concurrent with the topical OVA instillation period. Peripheral blood was drawn, and sera was prepared to measure IgE levels via ELISA. Data (n = 4/group, total n = 12/group) are representative of three independent experiments and presented here as the mean and SEM (*P < 0.05; **P < 0.01; and ***P< 0.001).

Figure 6

Vascular endothelial growth factor receptor inhibition dampens clinical signs of AED. Allergic eye disease was induced and VEGFR inhibitor or vehicle control was administered concurrent with the topical OVA instillation period. Representative clinical images are included. Clinical scoring was performed once per day at 20 minutes post OVA instillation. Mice were scored for tearing/discharge, eyelid edema, chemosis, and hyperemia. Data (n = 4/group, total n = 12/group) are representative of three independent experiments and presented here as the mean and SEM (*P < 0.05).