To our knowledge, this is the second study to evaluate the effect of intravitreal injection of anti-IL-6 on EAU in mice. We conducted this study based on the preliminary study of Tode et al. [29] with slightly different characteristics: our study involved a larger number of mice, the injections were administered three times instead of twice and the control antibody used was an isotype rat antibody. Moreover, in this study setup, functional in addition to morphological parameters were used.

The progression of EAU with the administration of anti-IL-6 was studied in 17 female mice of the B10.RIII strain over an 18-day period. The B10.RIII strain is known to be the most susceptible strain for EAU development and is suitable for exploring new therapeutic approaches for non-infectious posterior uveitis in humans [33, 34]. The course of EAU during this study period corresponds with findings in the literature stating that EAU typically develops in the first week, reaches an inflammation peak towards the end of the second week, and then shows improvement after three weeks [30]. More than half of all mice showed severe to very severe uveitis.

In contrast to the pilot project, the injections in this study were performed at a later time point and were conducted three times in total. The first injection was administered on day 10, with subsequent injections at three-day intervals until day 16. This decision was made in consideration of the fact that EAU usually develops in the first week with apparent clinical symptoms after day 7. The data gathered here demonstrate that antibody administration at a later stage in the acute phase of inflammation also has a significantly positive impact on the clinical and functional course of EAU.

Currently, there is a lack of information regarding the stability and duration of the biological activity of the anti-IL-6 antibody in the mouse eye. Unlike Vascular Endothelial Growth Factor (VEGF) -inhibitors used intravitreally for treating exudative age-related macular degeneration, which have a short half-life in the eye of just a few days [35], no similar data exists for the anti-IL-6 antibody. Therefore, the injection intervals (three-day interval) and dosage (3.6 µg) in this study were chosen based on the pilot study done by Tode et al. (2017).

As a control antibody a murine IgG1 isotype antibody from the rat was used. Isotype antibodies are structurally similar to therapeutic antibodies, the main difference being that there are no binding sites for the target molecule (IL-6) in the Fab region. This feature gives isotype antibodies the advantage of serving as negative controls while attenuating potential immunological responses originating from non-specific binding sites such as Fc receptors. The outcomes from the control eyes were similar to the control eyes of the pilot study where PBS was injected. This indicated that the isotype antibody did not affect the natural course of EAU in terms of clinical evaluations. As a result, the isotype antibody proves to be a suitable negative control for intravitreal applications, distinguishing itself from other control agents like PBS or Saline by preventing immunologically mediated reactions caused by non-specific antibody binding.

Non-infectious uveitis is often treated with systemic drugs [36]. Among those, Tocilizumab, a recombinant humanized anti-IL-6 receptor antibody, has been shown to be effective [28, 37]. However, intravitreal injections would offer the advantage of achieving a higher drug concentration in the eye while reducing the incidence of systemic side effects [38].

The EAU serves as a model for autoimmune diseases induced systemically. T-cells become activated and primed in the periphery, subsequently migrating to the eye and initiating an autoimmune inflammatory cascade within the normally immune-privileged eye structures [15]. As a result, many EAU treatment approaches involve the systemic administration of anti-inflammatory or immune-modulating agents [39]. The local action of intravitreally applied anti-IL-6 is anticipated, focusing on intraocular inflammatory processes. Nevertheless, experiences with intraocular anti-VEGF treatment indicate that intravitreally administered antibodies may minimally breach the blood-retina barrier, appearing in peripheral blood [31, 40]. Consequently, a marginal systemic effect of intravitreally injected anti-IL-6 cannot be completely dismissed. However, if the primary effect were systemic, a decrease in uveitis activity would be expected in both the treated and control eyes. In the current study, 71% of control eyes exhibited grade 3 or 4 uveitis, whereas only 41% of treated eyes in the same individuals had uveitis scores of 3 and 4. Thus, we propose that the systemic impact of intravitreally administered anti-IL-6 has no significant influence on uveitis in the fellow eye. Even if there were systemic effects, the intraocular injections, initiated 10 days after EAU induction, would not affect the initial stages of EAU development based on the temporal sequence of events.

Besides clinical uveitis evaluation via funduscopy and fluorescein angiography, we additionally chose to measure visual acuity with the OptoDrum system on each examination day. This decision was based on the recognition that clinical observations of the eyes only cannot provide a comprehensive picture of anti-IL-6 therapy on EAU.

It proved to be a simple, rapid, and objective tool to assess functional limitations in awake and untrained mice [41]. As a result, the assessment of visual acuity not only confirmed the clinical efficacy of the anti-IL-6 antibody but also demonstrated its functional benefits.

The results generated in this study confirm the results of the mentioned pilot study and indicate that triple intravitreal administration of an anti-IL-6 antibody during the acute phase of EAU is both functionally and clinically superior to isotype antibody administration.

Measurements of visual acuity at day 0 in both groups ranged from 0.4 cycles per degree (cpd) to 0.6 cpd, which is comparable to values documented in previous studies for visual acuity in healthy mice [42]. Following the initial antibody treatment on day 10, both groups experienced a reduction in visual acuity, reaching its peak of inflammation on day 13. Nevertheless, the findings demonstrate that the administration of the anti-IL-6 antibody resulted in a less significant decline in median visual acuity within the treatment group compared to the control group, leading to a lower level of visual loss. Moreover, visual acuity showed a more notable improvement over time in the treatment group. Consequently, intravitreal anti-IL-6 treatment appears to be a promising approach for effectively targeting and managing inflammation, thereby enhancing the quality of life.

The EAU score data showed a statistically significant superiority for anti-IL-6 therapy in terms of reduction of inflammatory activity. On days 13, 16, and 18 the number of eyes with lower EAU-score was significantly higher in the treatment group. At the same time, the additional measurement of visual acuity demonstrated that the visual impairment of the treatment group was significantly lower than in the control group. The combination of visual acuity measurement and EAU score determination was shown to be beneficial in determining the degree of inflammation and eliciting functional impact. Angiography results supported this observation and showed a lower severity of vascular involvement in the treatment group.

The results of this study show that the intravitreal anit-IL-6 therapy is a promising therapeutic approach for the treatment of non-inflammatory uveitis.

A limitation of our study is some ambiguity concerning the timing of injections and the dosing. Future studies should aim to prevent or ideally eliminate disease activity. The long-term goal should be the complete recovery of the disease. Thus, optimal dosage and intervals of treatment should be examined.

The dosage used in the pilot project and in this study has already shown a significant effect. It may be of interest to increase the dosage as this would potentially increase the effectiveness of the antibody. An alternative approach could be in the form of a boost therapy on day 10 to mitigate the peak EAU on day 13 and thus have a positive impact on the course of the disease.

Optimization of the treatment regimen will also require adjustment of the intervals between the injections. As only one preclinical study has investigated intravitreal administration of an anti-IL-6 antibody, the total number of injections required for a sustained therapeutic effect remains unclear.

The isotype antibody used in this study proved to be a reliable negative control. One way to assess the efficacy of the anti-IL-6 antibody compared with an already established therapy could be to introduce a positive control. Corticosteroids could serve as an appropriate choice in this regard.

Future investigations should also assess anti-IL-6 and IL-6 blood levels in treated mice to confirm the absence of any systemic effects.