To explore the therapeutic effect of anti-interleukin (IL)-12/IL-23 p40 antibody on experimental autoimmune uveitis (EAU) and its mechanism.

Sixty-six SPF female C57BL/6N mice aged 6-8 weeks were selected.EAU model was established in 24 mice through immunization with the interphotoreceptor retinoid-binding protein (IRBP) 651-670.The 24 mice were sacrificed before immunization, and on the 3rd, 12th, and 18th day after immunization, with 6 at each time point.Flow cytometry was used to detect the proportion of IL-17A⁺ interferon-γ (IFN-γ)⁺ CD4⁺ T cells in the spleen, lymph nodes and eyeballs.Another 6 mice were selected to establish EAU model, and fundus images of the mice were taken with a small animal imaging instrument and optical coherence tomography (OCT) 18 days after immunization.The 6 mice were sacrificed after OCT examination and the eyeballs were collected.Hematoxylin-eosin staining was used to observe the retinal inflammation and morphological changes in tissue structure.Flow cytometry was employed to detect the proportion of IL-17A⁺ IFN-γ⁺ CD4⁺ T cells in lymph nodes.The 6 mice were divided into IL-17A⁺ IFN-γ⁺ highly expressed group and IL-17A⁺ IFN-γ⁺ lowly expressed group according to flow cytometry results, and the retinal injury was compared between the two groups.EAU model was established in another 36 mice, which were divided into anti-IL-12/IL-23 p40 group and IgG group by random number table method, with 18 mice in each group.Anti-IL-12/IL-23 p40 or IgG was injected by tail vein at a 3-day inteval according to grouping.On the 12th and 18th day after immunization, 6 mice were selected from each group to collect lymph nodes and eyeballs, and the proportion of T cell subsets was detected by flow cytometry.Eyeballs of 6 mice in each group were extracted on the 24th day after immunization and retinal damage was observed by hematoxylin-eosin staining.The induced differentiation of CD4⁺ T cells in vitro was assayed by flow cytometry.The expressions of IL-17 and IFN-γ were detected by enzyme-linked immunosorbent assay (ELISA) after induced differentiation of IL-17A⁺ IFN-γ⁺ CD4⁺ T cells.The relative expression levels of Th1 transcription factor T-bet and Th17 transcription factor retinoid acid-related orphan nuclear receptor γt (ROR-γt) after induced differentiation of IL-17A⁺ IFN-γ⁺ CD4⁺ T cells were detected by real-time quantitative PCR.The use and care of animals followed the ARVO statement and this study protocol was approved by an Ethics Committee of Experimental Animals of Tianjin Medical University Eye Hospital (No.TJYY2019111019).

There were significant differences in the proportion of IL-17A⁺ IFN-γ⁺ CD4⁺ T cells in lymph nodes, spleen and eyeballs between wild-type mice and EAU mice at the 3rd, 12th and 18th day after immunization (H=9.642, 16.531, 10.385; all at P<0.05). Compared with before immunization, the proportion of IL-17A⁺ IFN-γ⁺ CD4⁺ T cells was significantly increased in lymph nodes of EAU mice on the 12th day following immunization and was significantly increased in spleen and lymph nodes on day 18 after immunization (all at P<0.05). Severe retinal exudation, retinal detachment, severe inflammatory cell infiltration and extensive retinal folds were detected in IL-17A⁺ IFN-γ⁺ highly expressed mice.Mild retinal edema, focal inflammatory cell infiltration and mild retinal folds were found in IL-17A⁺ IFN-γ⁺ lowly expressed mice.The proportion of CD3 and IL-17A⁺ IFN-γ⁺ CD4⁺ T cells in the eyeballs of anti-IL-12/IL-23 p40 group was lower than that in IgG group at the 18th day after immunization, and the differences were statistically significant (t=15.304, 8.080; both at P<0.05). On day 12 after immunization, the percentage of IL-17A⁺ IFN-γ⁺ CD4⁺ T cells in anti-IL-12/IL-23 p40 group was (0.33±0.18)%, which was significantly lower than (4.83±0.45)% in IgG group (t=15.974, P<0.001). Compared with IgG group, the percentage of Th1, Th17, IL-17A⁺ IFN-γ⁺ CD4⁺ T cells and the expression levels of IL-17, IFN-γ, T-bet, ROR-γt in anti-IL-12/IL-23 p40 group were significantly decreased, with statistical significances (all at P<0.05).

Anti-IL-12/IL-23 p40 has a therapeutic effect on EAU by inhibiting IL-17A⁺ IFN-γ⁺ CD4⁺ T cells.

Uveitis; Drug therapy; Interleukin-12 subunit p40; Antibodies, monoclonal; Interphotoreceptor retinoid-binging protein; Mice