Ocular Microbiology and Immunology Group
Back to OMIG Main Page

Previous | 2024 Agenda and Abstracts | Next

2024 OMIG Abstract

IL-6 Amplifies Alloimmunity by Inducing Dysfunctional IFN-γ Secreting Treg through the VEGFA-VEGFR1 Axis

Seokjoo Lee, Akitomo Narimatsu, Hamid Alemi, Parisa Dashti, Mark Krauthammer, Shilpy Bhullar, Yihe Chen, Sunil K. Chauhan, Thomas H. Dohlman, Reza Dana

Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear,
Harvard Medical School, Boston, MA

Purpose: To investigate the mechanistic role of IL-6 in inducing dysfunctional IFN-γ-secreting regulatory T cells (Treg) leading to corneal allograft rejection.

Methods: Treg isolated from BALB/c spleens via magnetic sorting were treated with IL-6 and VEGFA to evaluate their effects on FoxP3, IL-10, and IFN-γ expression with or without VEGFR1 blockade over a 24-hour in vitro period. Following treatment, Real-Time RT-PCR, immunoblotting, and flow cytometry were used to analyze these expressions. To assess corneal cell death, whole corneal tissues cultured with fresh Treg or Treg previously exposed to IL-6 and VEGFA with or without VEGFR1 blockade were either mounted on slides and stained with annexin V to identify apoptotic cells. In corneal transplantation, fresh Treg or Treg previously exposed to IL-6 and VEGFA with or without VEGFR1 blockade (1x105 cells in 50 µL of sterile PBS) were injected subconjunctivally, and graft survival was monitored for up to 8 weeks by measuring corneal opacity using slit lamp biomicroscopy.

Results: IL-6 treatment significantly decreased FoxP3 expression (p=0.0015) and increased VEGFR1 levels (p=0.0041) in Treg. Cotreatment with IL-6 and VEGFA further reduced FoxP3 (p=0.0005) and the anti-inflammatory cytokine IL-10 (p<0.0001), while increasing the pro-inflammatory cytokine IFN-γ (p<0.0001). Blocking VEGFR1 during exposure to IL-6 and VEGFA significantly mitigated the reduction in FoxP3 (p=0.0004) and IL-10 (p=0.0105), and also decreased IFN-γ secretion (p=0.0005) from Treg. In whole mount corneal staining, corneas exposed to fresh Treg cells showed no dead cells, while those exposed to dysfunctional Treg (previously exposed to IL-6 and VEGFA) exhibited a higher percentage of apoptotic cells, as indicated by increased annexin V staining. However, corneas treated with Treg previously exposed to IL-6, VEGFA, and VEGFR1 blockade had fewer apoptotic cells. In corneal transplantation, injection with dysfunctional Treg decreased graft survival rates, with all grafts rejected by 3 weeks. Conversely, injection with Treg previously exposed to IL-6, VEGFA, and VEGFR1 blockade showed higher survival rates compared to groups injected with dysfunctional Treg. After follow-up, corneal grafts were harvested and stained with annexin V. The rejected corneas exhibited a higher percentage of apoptotic cells compared to the accepted ones.

Conclusions: IL-6 impairs Treg by increasing VEGFR1 expression, leading to enhanced IFN-γ secretion through the VEGFA-VEGFR1 axis, which damages corneal cells and causes graft rejection.


Disclosure:

S