**4. Discussion**

Increasing amounts of evidence linking the immune system to ocular disease has emerged in the last decade. Studies focusing on glaucoma, AMD, RP, and other RDs have continued to show increased presence of many immune components including monocytes (blood-borne and resident), pro- and anti-inflammatory cytokines, and autoantibodies in many models of these diseases [24–26]. While all of these diseases present an initial insult of genetic and/or environmental origins, these findings overwhelmingly implicate the immune system in the pathogenesis of RDs. For example, the prevalence of single-nucleotide polymorphisms in the genes coding for complement factor H, complement factor I, as well as many other components of the innate immune system bolster the chances of developing AMD in otherwise normal patients [27,28]. In addition, models of glaucoma have shown the presence of macrophages and T-lymphocytes in the eye along with autoantibodies to proteins of the retina [3,4,29,30]. Models of RP have also shown similar features, including monocytic phagocytosis of both diseased and healthy retinal cells as well as numerous cytokines such as interleukin-1, interleuken-6, vascular endothelial growth factor, and others [4,31,32]. While the molecular mechanisms of the primary genetic assault have been teased out by copious amounts of work using cell culture and animal models for many RDs (i.e., loss of outer segmen<sup>t</sup> formation, disrupted visual cycle, etc.) [1,2,33], the full mechanisms of pathogenesis and progression of RDs still need thorough investigation before we fully understand them. In our findings, the *Ter349Glu*/*Ter349Glu* mice exhibited an almost complete loss of photoreceptor function by ERG; however, the *Ter349Glu*/+ mice had heightened a and b waves while also showing a decrease in b wave latency compared to the +/+ animals. This phenomenon might be explained by the expression levels of mutant rhodopsin compared to WT rhodopsin, as Ter349Glu rhodopsin expression levels are less than half of the WT levels [2]. This lower level of expression while still having 50% WT rhodopsin could result in a somewhat thinner outer segmen<sup>t</sup> with less rhodopsin protein in the discs, thus allowing for faster rates of di ffusion of the phototransduction cascade components, decreasing the response latency, while normal WT rhodopsin activates the cascade, enhancing the signal. We also showed that animals with more advanced RD had significant numbers of macrophages, a feature found in many RDs, as well as activation of a known pro-inflammatory pathway (JAK/STAT). Interestingly, the *Ter349Glu*/+ mice showed SOCS3 expression in the inner retinal layers early in life (4 weeks of age), with a dramatic decrease in expression with age, while the +/+ mice had minimal SOCS3 expression, which peaked at 8 weeks of age, and the *Ter349Glu*/*Ter349Glu* mice exhibited a somewhat constant SOCS3 expression in the outer retina and RPE. While the relatively stable SOCS3 expression in the *Ter349Glu*/*Ter349Glu* mice is explainable when compared to the levels of STAT3 phosphorylation which was present throughout the first 3 months of the animals' life, the stark di fference in SOCS 3 expression the *Ter349Glu*/+ mice remains to be elucidated. More experiments examining the activation and deactivation of the proinflammatory JAK/STAT pathway are needed to better understand these expression di fferences. Further work will aim at pinning down the cytokines responsible for pathway activation; experiments inhibiting the pathway activation using antagonists to the JAK/STAT pathway and/or those inhibiting specific cytokine(s) will better underpin this pathway's role in RD. Due to the nature of STAT3 role in numerous developmental pathways [13], a more targeted approach to delivering inhibitory compounds directly to the eye would be necessary. Future experiments will also work to decrease the number of activated macrophages in RD models to attempt to rescue some photoreceptor degeneration due to excessive phagocytosis and/or macrophage-derived cytokines. Work is already underway testing the inhibition of PAD4 in RD models to rescue retinal cells, retinal function, or both [34]. This work will be achieved using PAD4-deficient mice as well as inhibitors of the enzyme. Due to the excessive citrullination observed in *Ter349Glu*/*Ter349Glu* mice, it is not far-fetched to think that lowering or preventing this post-translation modification from occurring could lead to a slower rate of disease progression, allowing for not only longer lasting vision in patients a ffected but also an extended opportunity to correct the genetic insult, thus preventing further retinal degeneration. In all, our work further contributes to the increasing volume of studies indicating a role of the immune system in RDs as well as provides possible targets forthetreatmentofthesedebilitatingblindingdiseases.

**Author Contributions:** Conceptualization, A.K.G. and T.J.H.; methodology, A.K.G. and T.J.H.; investigation, A.K.G. and T.J.H.; resources, A.K.G. and T.J.H.; data curation, A.K.G. and T.J.H.; writing—original draft preparation, T.J.H.; writing—review and editing, A.K.G. and T.J.H.; visualization, A.K.G. and T.J.H.; project administration, A.K.G.; funding acquisition, A.K.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the NIH R01EY019311 and the E. Matilda Ziegler Foundation. Center support for the project was provided by NIH P30EY003039.

**Acknowledgments:** We thank Brian Simms and Meredith Hubbard for technical assistance and W. Clay Smith for the K62-82 rhodopsin antibody.

**Conflicts of Interest:** The authors declare no conflicts of interest.
