Crosstalk Between TGFβ2 and TLR4 in the Trabecular Meshwork

Abstract

Purpose : The trabecular meshwork (TM) regulates aqueous humor outflow and intraocular pressure (IOP). The effects of TGFβ signaling pathways on the TM extracellular matrix (EÇM) have been extensively studied. Recently, we identified TGFβ2 and toll-like receptor 4 (TLR4) signaling crosstalk regulates changes in the TM ECM and mutation in Tlr4 rescues TGFβ2 -induced ocular hypertension in mice. Here, we investigated the role of an endogenous TLR4 ligand, FN-EDA, and a downstream signaling molecule of TLR4, NFκB, in TGFβ2 -induced ocular hypertension in mice.

Methods : B6.FN-EDA^-/- (n=18), B6.FN-EDA^+/+/TLR4^-/- (n=7), B6.FN-EDA^-/-/ TLR4^-/- (n=15), and C57BL/6J (n=11) mice were intravitreally injected with 2.0 μL Ad5.TGFβ2 (2.5x10⁷ pfu) in one eye and the contralateral uninjected eye was used as a negative control. Likewise, we tested mice lacking the p50 subunit of NFκB (B6.Cg-NFκB1^tm1Bal/J) (n=7) and C57BL/6J (n=10) mice. IOP was measured using a TonoLab rebound tonometer on isoflurane-anesthetized mice for 42 days post-injection. Significance determined by one-way ANOVA at each time point.

Results : Ad5.TGFβ2 significantly induced ocular hypertension in C57BL/6J mice in both experiments. In the first experimental cohort, Ad5.TGFβ2 significantly induced ocular hypertension in C57BL/6J mice starting at 7-days post injection and remained significant until 42 days post-injection (p<0.0001). IOP peaked at 35 d (Ad5.TGFβ2 injected eye, 21.7 ± 0.5 mmHg; uninjected eye, 13.1 ± 0.2 mmHg; p<0.0001). Mutation in TLR4 and FN-EDA blocked Ad5.TGFβ2 induced ocular hypertension with no significant IOP elevation at any time point. In the second experimental cohort, Ad5.TGFβ2 significantly induced ocular hypertension in C57BL/6J mice starting at 14-days post injection and remained significant until 42 days post-injection (p<0.0001). IOP peaked at 21 d (Ad5.TGFβ2 injected eye, 20.6 ± 0.6 mmHg; uninjected eye, 13.6 ± 0.2 mmHg; p<0.0001). Mutation in NFκB blocked Ad5.TGFβ2 induced ocular hypertension with no significant IOP elevation at any time point.

Conclusions : These findings demonstrate that TLR4, the endogenous TLR4 ligand FN-EDA, and NFκB are necessary for TGFβ2 induced ocular hypertension in mice. These data provide potential new targets to lower IOP and to further explore the molecular mechanisms involved in the development of glaucomatous TM damage.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.