Characterization of an inducible, conditional HIF-1α knockout mouse strain and induction of experimental chronic autoimmune uveitis in the strain

Abstract

Purpose : Long-lived, uveitogenic memory CD4⁺ T cells express high levels of hypoxia-inducible factor-1α (HIF-1α). Herein, we aim to develop a tamoxifen-inducible, CD4-conditional HIF-1α knockout mouse strain to investigate the roles of HIF-1α in governing memory CD4⁺ T cells in experimental chronic autoimmune uveitis (CAU).

Methods : The CD4-CreER^T2 mice (JAX) were crossed with Hif1a^fl/fl mice (JAX), and the resultant F1 B6.CD4CreER^T2-Hif1a^fl/- strain was then backcrossed to Hif1a^fl/fl strain. The offspring were genotyped and examined using fundoscopy, optical coherence tomography (OCT), and electroretinogram. Mice were intraperitoneally injected with tamoxifen, and clinically followed for 4 weeks. Thereafter, lymph nodes (LN), spleen (SP) and peripheral blood (BL) were collected for flow cytometry. In addition, CAU was induced by immunization with interphotoreceptor retinoid-binding protein peptides emulsified in complete Freund’s adjuvant, with Bordetella pertussis toxin injection, and evaluated at 12 weeks.

Results : The desired B6.CD4CreER^T2-Hif1a^fl/fl strain along with control strains, B6.CD4-Hif1a^fl/fl and B6.CD4-Hif1a^fl/- were generated. All strains exhibited normal ocular structures. Subsequent tamoxifen treatment did not induce retinal infiltrates or dysfunction in any strain. Additionally, it had no significant impact on the CD11b⁺ myeloid (2.1±0.2% vs 2.8±0.2% in LN, 2.4±0.3% vs 2.7±0.5% in SP, 3.5±0.2% vs 3.6±1.2% in BL) or CD3⁺ lymphoid pool (40±4% vs 40±2% in LN, 20±4% vs 24±3% in SP, 10±2% vs 8±3% in BL) in either the desired or control strains. Furthermore, it did not affect CD4 (47±3% vs 46±1% in LN, 49±4% vs 48±1% in SP, 45±5% vs 40±3% in BL) or CD8 (47±3% vs 48±3% in LN, 39±2% vs 44±1% in SP, 46±5% vs 49±1% in BL) subsets in any strain. At 12 weeks post-immunization, both the desired strain and littermate controls exhibited comparable retinal infiltration and structural damage as documented by fundoscopy (3.3±0.5 vs 2.8±0.5 on a scale of 0-4) and OCT (3.2±0.4 vs 3.0±0.3 on a scale of 0-4).

Conclusions : The B6.CD4CreER^T2-Hif1a^fl/fl strain shows normal ocular development and robust CAU induction, and tamoxifen treatment does not significantly alter pools of major immune subsets, rendering this strain a novel and validated tool to study the function of HIF-1α specifically expressed by memory CD4⁺ T cells in chronic uveitis.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.