Stimulating the Melanocortin System in Uveitis and Diabetes Preserves the Structure and Anti-Inflammatory Activity of the Retina

The endogenous neuropeptide α-Melanocyte Stimulating Hormone (α-MSH) is a potent suppressor of inflammation and has an essential role in maintaining the normal anti-inflammatory microenvironment of the retina. While the therapeutic use of α-MSH peptide in uveitis and diabetic retinopathy models has been demonstrated, its short half-life and instability limit its use as a therapeutic drug. A comparable analog, PL-8331, which has a stronger affinity to melanocortin receptors, longer half-life, and, so far, is functionally identical to α-MSH, has the potential to deliver melanocortin-based therapy. We examined the effects of PL-8331 on two mouse models of retinal disease, Experimental Autoimmune Uveoretinitis (EAU) and Diabetic Retinopathy (DR). PL-8331 therapy applied to mice with EAU suppressed EAU and preserved retinal structures. In diabetic mice, PL-8331 enhanced the survival of retinal cells and suppressed VEGF production in the retina. In addition, retinal pigment epithelial cells (RPE) from PL-8331-treated diabetic mice retained normal anti-inflammatory activity. The results demonstrated that the pan-melanocortin receptor agonist PL-8331 is a potent therapeutic drug to suppress inflammation, prevent retinal degeneration, and preserve the normal anti-inflammatory activity of RPE.

In both uveitis and diabetic retinopathy, there is a progressive loss of cells in the retina. The inflammation of uveitis and the ischemia/reperfusion caused by capillary occlusions and hyperglycemia in diabetes strain cell survival and initiates programmed cell death [23,24]. There is a great need for therapy that can suppress inflammation and cell death in the retina. Melanocortin-based therapy is a potentially beneficial therapy.

Effects of PL-8331 Treatment on EAU
The mice were immunized to induce EAU, and the retinas were clinically scored by fundus microscopy for 10 weeks after immunization. On week 4, as the eyes entered the chronic phase of EAU, the mice received two consecutive intraperitoneal injections of PL-8331 at the molar equivalent of a therapeutic injection of α-MSH peptide [26] or vehicle (PBS). The PL-8331-treated EAU mice had significantly suppressed EAU clinical scores 6 weeks after treatment compared to vehicle-injected EAU mice ( Figure 1). The eyes were collected to see if PL-8331 treatments affected retinal structures. The eyes were collected on week 10, along with the eyes of age-matched naive mice. They were sectioned and stained for histological analysis ( Figure 2). The retinas of vehicle-injected EAU mice ( Figure 2B) had increased infiltration of immune cells in the vitreous and visibly shortened photoreceptor lengths with disruption of the outer nuclear layer (ONL). In addition, the retinas showed inflammatory cells in the subretinal space near the disrupted ONL. In contrast, the retinas from the PL-8331-treated EAU mice ( Figure 2C) retained almost normal retinal structures compared to retinas from naive mice ( Figure 2A). Therefore, treating EAU with the α-MSH-analog PL-8331 suppresses the inflammation of uveitis and can preserve retinal structures. melanocortin receptors except MC2r and is more like α-MSH in regulating in including uveitis [22,26]. In addition, PL-8331 has 33-, 8-, 3-, and 21-fold great activity than α-MSH on MC1r, MC3r, MC4r, and MC5r, respectively, and w vivo stability [22]. Therefore, we assayed for the effects of augmenting the m signal with PL-8331 on the preservation of retinal cells and structure in two m of retinal degeneration, experimental autoimmune uveoretinitis (EAU) and nopathy (DR).

Effects of PL-8331 Treatment on EAU
The mice were immunized to induce EAU, and the retinas were clinica fundus microscopy for 10 weeks after immunization. On week 4, as the eye chronic phase of EAU, the mice received two consecutive intraperitoneal inje 8331 at the molar equivalent of a therapeutic injection of α-MSH peptide [2 (PBS). The PL-8331-treated EAU mice had significantly suppressed EAU clin weeks after treatment compared to vehicle-injected EAU mice ( Figure 1). T collected to see if PL-8331 treatments affected retinal structures. The eyes w on week 10, along with the eyes of age-matched naive mice. They were se stained for histological analysis ( Figure 2). The retinas of vehicle-injected EA ( Figure 2B) had increased infiltration of immune cells in the vitreous and visib photoreceptor lengths with disruption of the outer nuclear layer (ONL). In retinas showed inflammatory cells in the subretinal space near the disrupted trast, the retinas from the PL-8331-treated EAU mice ( Figure 2C) retained al retinal structures compared to retinas from naive mice (Figure 2A). Theref EAU with the α-MSH-analog PL-8331 suppresses the inflammation of uveitis serve retinal structures. Effects of treating EAU mice with PL-8331. Mice were immunized to induce retinas were examined by microscopy and scored. The mice were treated with PL-83 as indicated by the arrows. Presented are the mean ± SEM of the EAU scores of each over the weeks after immunization to induce EAU. Statistical differences of EAU clinic PBS (vehicle)-treated EAU mice are indicated, * p ≤ 0.05, ** p ≤ 0.01; N = 4. All other va statistically different between groups.

Effects of PL-8331 Treatment on Diabetic Retinopathy
Since there is a potential benefit of α-MSH-therapy to prevent diabetic retinopathy [18,20,21], we assayed for the possibility of PL-8331-therapy providing a similar benefit. We used a streptozotocin-induced diabetic retinopathy mouse model. After the last injection of streptozotocin, the diabetic mice were treated with intravitreal injections of PL-8331 every 4 weeks. At week 16, the eyes were collected and assayed for histology ( Figure  3). The ganglion cell layer (GCL) in the diabetic eyes had gaps with no cells (arrow) in comparison to the retinas of non-diabetic mice and PL-8331-treated diabetic mice ( Figure  3A Upper Panels). There was a small dent in the optic nerve head of the untreated retina of diabetic mice but not in the optic nerve head of non-diabetic and the PL-8331-treated diabetic mice suggesting preservation of optic nerve fibers ( Figure 3A Lower Panels). No change was seen in retinal thickness between any of the groups of mice (Supplementary Materials Figure S1). However, there are observable regions of substantial RGC dropout in the diabetic mouse retinas ( Figure 3A, white arrow). Such regions were not observed in the retinal sections of non-diabetic and PL-8331-treated diabetic mice. There was significant retention in the number of nuclei in the GCL of the PL-8331-treated diabetic mice ( Figure 3B). The PL-8331 treatment of diabetic mice can potentially protect the retina from ganglion cell loss. This is like what is seen with α-MSH-treated diabetic mice in that retinal cells are protected [18,21].
In addition to the loss of cells, there are changes in intercellular connections and regulation of the microcirculation of diabetic retinas [18]. Homogenates of the neuroretina were assayed for the expression of the tight junction protein Occludin and the angiogenic factor vascular endothelial growth factor (VEGF) (Figure 4). While the Occludin levels are lower in diabetic retinas, they were not significantly different between the groups ( Figure  4a). The levels of VEGF were significantly greater in the untreated diabetic mouse retinas ( Figure 4B) than in the retinas of non-diabetic and PL-8331-treated diabetic mice. The suppression of enhanced VEGF levels is consistent with previous studies demonstrating that melanocortin signaling pathways regulate changes in retinal microvasculature [18,20].
One of the know effects of α-MSH-therapy in EAU is the restoration of RPE-mediated anti-inflammatory activity [6,19]. This is important for maintaining the normal anti-inflammatory microenvironment of the retina. Soluble factors produced by the RPE mediate

Effects of PL-8331 Treatment on Diabetic Retinopathy
Since there is a potential benefit of α-MSH-therapy to prevent diabetic retinopathy [18,20,21], we assayed for the possibility of PL-8331-therapy providing a similar benefit. We used a streptozotocin-induced diabetic retinopathy mouse model. After the last injection of streptozotocin, the diabetic mice were treated with intravitreal injections of PL-8331 every 4 weeks. At week 16, the eyes were collected and assayed for histology ( Figure 3). The ganglion cell layer (GCL) in the diabetic eyes had gaps with no cells (arrow) in comparison to the retinas of non-diabetic mice and PL-8331-treated diabetic mice ( Figure 3A Upper Panels). There was a small dent in the optic nerve head of the untreated retina of diabetic mice but not in the optic nerve head of non-diabetic and the PL-8331-treated diabetic mice suggesting preservation of optic nerve fibers ( Figure 3A Lower Panels). No change was seen in retinal thickness between any of the groups of mice (Supplementary Materials Figure S1). However, there are observable regions of substantial RGC dropout in the diabetic mouse retinas ( Figure 3A, white arrow). Such regions were not observed in the retinal sections of non-diabetic and PL-8331-treated diabetic mice. There was significant retention in the number of nuclei in the GCL of the PL-8331-treated diabetic mice ( Figure 3B). The PL-8331 treatment of diabetic mice can potentially protect the retina from ganglion cell loss. This is like what is seen with α-MSH-treated diabetic mice in that retinal cells are protected [18,21].
In addition to the loss of cells, there are changes in intercellular connections and regulation of the microcirculation of diabetic retinas [18]. Homogenates of the neuroretina were assayed for the expression of the tight junction protein Occludin and the angiogenic factor vascular endothelial growth factor (VEGF) (Figure 4). While the Occludin levels are lower in diabetic retinas, they were not significantly different between the groups ( Figure 4A). The levels of VEGF were significantly greater in the untreated diabetic mouse retinas ( Figure 4B) than in the retinas of non-diabetic and PL-8331-treated diabetic mice. The suppression of enhanced VEGF levels is consistent with previous studies demonstrating that melanocortin signaling pathways regulate changes in retinal microvasculature [18,20]. treated with PL-8331 retained their ability to mediate anti-inflammatory activity. This contrasts with RPE from untreated diabetic mice that permitted macrophage production of the proinflammatory cytokine TNF-α. Therefore, PL-8331 treatment of diabetic mice protects the retina while preserving RPE suppression of inflammation and possibly preventing changes in the retinal microvasculature.  One of the know effects of α-MSH-therapy in EAU is the restoration of RPE-mediated anti-inflammatory activity [6,19]. This is important for maintaining the normal antiinflammatory microenvironment of the retina. Soluble factors produced by the RPE mediate this anti-inflammatory activity, which includes α-MSH. The RPE factors suppress the induction of proinflammatory activity while promoting anti-inflammatory activity by activated macrophages [19,28]. In addition, suppressing the inflammatory response in diabetes may prevent the development of diabetic retinopathy [29,30].
To see whether RPE maintained its ability to suppress inflammation in the PL-8331treated diabetic mice, the eyes were collected after treatment and dissected to generate RPE eyecups. The eyecups were cultured for 24 h, and the conditioned media (CM) was used to treat macrophages. The macrophages were then stimulated with LPS and assayed for TNF-α (a pro-inflammatory cytokine) and IL-10 (an anti-inflammatory cytokine). The CM from the RPE eyecups of diabetic mice did not suppress TNF-α production by LPS-stimulate macrophages compared to the suppression mediated by the CM from non-diabetic RPE eyecups ( Figure 5A). The CM of RPE eyecups from PL-8331-treated diabetic mice significantly suppressed LPS-stimulated TNF-α production by the macrophages (Figure 5A). While there is significant production of IL-10 by the LPS-stimulated macrophages treated with the CM of RPE eyecups from non-diabetic and diabetic mice, the CM of RPE eyecups from PL-8331treated diabetic mice had significantly enhanced macrophage production of IL-10 ( Figure 5B). This demonstrated that the RPE in diabetic mice treated with PL-8331 retained their ability to mediate anti-inflammatory activity. This contrasts with RPE from untreated diabetic mice that permitted macrophage production of the proinflammatory cytokine TNF-α. Therefore, PL-8331 treatment of diabetic mice protects the retina while preserving RPE suppression of inflammation and possibly preventing changes in the retinal microvasculature.   cludin in the neuroretinal homogenates from diabetic mice was not significantly (ns) different from non-diabetic or PL-8331-treated diabetic mice. (B) The levels of VEGF expressed in neuroretinal homogenates from diabetic mice were significantly greater than those from the neuroretina of diabetic mice treated with PL-8133 (** p ≤ 0.01) and non-diabetic mice (*** p ≤ 0.005); N = 10. There was no statistical (ns) difference in neuroretinal VEGF levels between non-diabetic and PL-8331-treated diabetic mice.

Discussion
The melanocortins are a family of highly conserved endogenous peptides and receptors that influence various biological activities [3,31,32]. While initially characterized for inducing melanogenesis in frogs, the melanocortins regulate metabolism, sexual functionality, mood, and immunity. The prototypical melanocortin is α-MSH, constantly present in the eye and produced by the RPE [6,7]. The melanocortin receptors MC1r and MC5r are expressed in the eye and may be needed to maintain retinal structure and cell survival [18,19,21,33]. These same receptors are expressed on immune cells through which α-MSH suppresses the activation of macrophages and T cells that mediate inflammation [19,22,34]. In addition, α-MSH treatment may signal retinal cell survival or inhibit apoptosis signals [6,18,33,35,36]. It has been demonstrated that using α-MSH as a treatment protects the retina from degeneration caused by EAU, diabetic retinopathy, and ischemia/reperfusion [18,19,21,26,[37][38][39][40]. While experimentally using α-MSH has demonstrated benefits, α-MSH is a highly unstable molecule [41]. The α-MSH-analog PL-8331 is highly stable and has as much as a 33-fold greater functional stimulation of the melanocortin receptors than α-MSH [22]. Also, like α-MSH, PL-8331 has no functional activity on MC2r, meaning no associated glucocorticoid response [42]. Our results showed that PL-8331 effectively suppressed retinal degeneration caused by inflammation and diabetes.
The retinal damage of EAU is the inflammatory response mediated by activated immune cells of an autoimmune-disease response [23]. Activating effector T cells with specificity to presented retinal antigens leads to the release of cytokines and chemokines that promote infiltration of other immune cells and the breakdown of the blood barrier that results in retinal degeneration. The suppression of EAU by PL-8331-treatment showed that by activating the melanocortin-signaling pathways, there was the suppression of inflammation and preserva-tion of retinal structure similar to what others have seen using native α-MSH [16,19,[43][44][45]. The literature describes the potential of stimulating the melanocortin signaling pathways to promote the survival of retinal cells [19,27,33,37,43,[46][47][48]. The histology of the EAU retinas showed that PL-8331 treatment observably protects the photoreceptor layer. The PL-8331 therapy provided the combined benefit of suppressing inflammation with promoting retinal cell survival. This therapeutic approach of augmenting the melanocortin signal can minimize the damage of inflammation while potentially reinforcing or restoring the normal anti-inflammatory microenvironment of the eye while preserving vision.
In the diabetic model, hyperglycemia induces the expression of proinflammatory signals along with reactive oxidants in the retina [24]. In addition, there is retinal capillary damage and dropout. These can lead to localized areas of ischemia, edema, and angiogenesis. The possibility of targeting the MC1r and MC5r has been shown to reduce the retinopathy mediated by diabetes [18,40]. Our results demonstrated that PL-8331 therapy, a peptide that also targets MC1r and MC5r [22], effectively protected the eye from retinopathy associated with diabetes. The retinas of the diabetic mice treated with PL-8331 had preserved retinal structure with possible RGC survival and suppressed VEGF levels that would be expected to reduce the chance of developing diabetic retinopathy. It has been found that under diabetic conditions, α-MSH protects vascular endothelial cells from apoptosis and oxidative stress [40]. Also, there is the potential that α-MSH can modulate the release of VEGF from RPE under hyperglycemic conditions [18,20]. The treatment with PL-8331 preserved the normal anti-inflammatory activity of the RPE in diabetic mice. Diabetic patients with elevated pro-inflammatory cytokines in their serum have a greater risk of developing diabetic retinopathy [29,30]. In contrast, elevated serum IL-10 decreases the risk of developing diabetic retinopathy. The benefits we see with PL-8331-therapy in the diabetic eye could be associated with enhancing the normal anti-inflammatory microenvironment of the retina [5]. The results demonstrate the potential of PL-8331 treatment in diabetes to protect the eye from developing diabetic retinopathy.
In this study, we investigated the protective effect of PL-8331, an α-MSH analog, in treating two retinal disease models that cause retinal cell loss, retinal structural damage, and inflammation. The therapeutic use of PL-8331 provided the same effects as has been seen using α-MSH-peptide but with a comparatively more potent and stable analog [22]. While we used only one dose of PL-8331, it was equivalent to the molar concentration we previously used with α-MSH treatments. It is to be seen how long PL-8331 can last in the eye; however, since we injected the EAU mice twice and the diabetic eyes once every four weeks, its effects appear to last. In addition, the ocular injections of PL-8331 appear to be a safe route of administration as others have done with α-MSH [18,39,43]. Our results demonstrated the beneficial effects of PL-8331 on preserving the retina's health under conditions that cause retinopathy. In addition, our findings further support evaluating the therapeutic potential of engaging endogenous melanocortin signaling pathways in ocular and other inflammatory diseases [22,25,42,49].

Animals
All mouse procedures described in this study were approved by the Boston University Institutional Animal Care and Use Committee (Protocol PROTO201800162) and adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The C57BL/6j mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the Boston University Animal Science Center.

Experimental Autoimmune Uveoretinitis (EAU) Model
Mice were immunized for EAU, as previously described [19]. Briefly, the mice were injected with a 200 µL emulsion of complete Freund's adjuvant (CFA) with 5 mg/mL desiccated M. tuberculosis (Difco Laboratories, Detroit, MI, USA) and 2 mg/mL IRBP peptide amino acids 1-20 (IRBP) (Genscript, Piscataway, NJ). This was immediately followed by an intraperitoneal injection of 0.3 µg pertussis toxin (Sigma-Aldrich, St. Louis, MO, USA) repeated 2 days later. Every 3-4 days, the retinas were examined using a slit lamp microscope with the cornea flattened and numbed with 0.5% proparacaine (Akorn, Lake Forest, IL, USA). The iris was dilated with 1% tropicamide (Akorn). As previously described, the clinical scores were on a 5-point scale based on the clinical signs of observable vessel dilatation, white focal lesions, and the extent of retinal vessel exudate, hemorrhage, and detachment [19]. The eyes were scored 0 for no inflammation; a score of 1 for only white focal lesions of the vessels; a score of 2 for linear vessel lesions over less than half of the retina; a score of 3 for linear vessels lesions over more than half of the retina; a score of 4 for severe chorioretinal exudates or retinal hemorrhages in addition to the vasculitis; and a score of 5 for subretinal hemorrhaging or retinal detachment. When all the mice reached a clinical score of 3 (4 to 5 weeks after immunization), the maximum score under our current animal facility conditions, the mice were treated with an intraperitoneal injection of PL-8331 (0.3 mg/kg/mouse) (Palatin Technologies Inc., Cranbury, NJ, USA) and again 2 days later. At 10 weeks after immunization, when the EAU of the PL8331-treated mice reached significantly sustained suppression of uveitis, the eyes were fixed with Davidson fixative for paraffin sectioning. The paraffin-embedded eyes were sectioned at 5 µm thickness. The sections were hematoxylin and eosin stained and imaged using an Olympus CX33 with the QCamera setup (Olympus).

Diabetic Retinopathy Model
The C57BL/6j mice at age 8 weeks were injected with a low-dose intraperitoneal injection of streptozotocin (40 mg/kg in 10 mM citrate buffer, pH 4.5) each day for 7 days. Streptozotocin was purchased from Sigma-Aldrich. Development of hyperglycemia was verified 1 week after the streptozotocin injection using a glucometer (Precision Xtra, Abbott Diabetes Care Inc., Alameda, CA, USA) (Supplemental Figure S2). The diabetic state was determined when the blood glucose was greater than 250 mg/dL. The streptozotocininjection mice with lower glucose levels were excluded from the study. Blood glucose levels were checked every two weeks throughout the study to confirm the maintenance of the diabetic condition (Supplemental Figure S2). The diabetic mice were treated with one intravitreal injection of 1 µL of 3.3 µM PL-8331 or PBS-carrier (untreated control) on weeks 1, 4, 8, 12, and 16 after the onset of diabetes. After 16 weeks, one set of eyes was fixed in Davidson fixative for 2 days, then processed for paraffin sectioning and hematoxylin and eosin stained. The paraffin-embedded eyes were sectioned at 5 µm thickness. The sections were hematoxylin and eosin stained and imaged using an Olympus CX33 with the QCamera setup (Olympus). Another set of eyes was dissected in ice-cold PBS to make RPE eyecups. In addition, the neuroretina was homogenized on ice using RIPA buffer (Santa Cruz Biotechnology, Dallas, TX, USA) with protease inhibitors. The lysates were centrifuged, and the supernatant was assayed for protein concentration, VEGF (Quantikine ELISA kits, R&D Systems, Minneapolis, MN, USA), and Occludin (MyBioSource, San Diego, CA, USA) by ELISA.

RPE Eyecup Conditioned Media (CM)
The RPE eyecups were made as previously reported [19]. The enucleated eyes were dissected, making RPE eyecups consisting of the RPE monolayer with underlying choroid and sclera with the neuroretina removed. The RPE eyecups were placed into the wells of a 96-well round bottom tissue culture plate (Corning, Corning, NY, USA) containing 200 µL of serum-free media (SFM) (RPMI 1640 supplemented with 10 mM HEPES, 1 mM sodium pyruvate, and nonessential amino acids (Lonza), along with 0.2% ITS+1, 0.1% BSA, and 10 µg/mL gentamycin (Sigma-Aldrich). The cultures were incubated for 24 h at 37 • C in 5% CO 2 . The conditioned media (CM) was collected, centrifuged, and the supernatants were used as RPE eyecup-CM.

Macrophages and Treatment with RPE-Eyecup CM
The macrophages used in this study were the macrophage cell line RAW 264.7 (T1B-71, ATCC, Manassas, VA, USA). The RAW 264.7 cells were maintained in DMEM with 10% FBS (Lonza) and were passed three times after thawing before use. The cells were kept in culture for no more than 3 months and passed twice per week. The RAW 264.7 cells were seeded onto the wells of a 24-well culture plate at 3.8 × 10 5 cells/mL and incubated for 1 h. The media was replaced with SFM and treated with the diabetic RPE eyecup-CM or naive RPE eyecup-CM for 30 min. Then the macrophages were stimulated with 1 µg/mL E. coli lipopolysaccharides (Sigma-Aldrich) and incubated in 5% CO 2 at 37 • C for 24 h. The culture supernatants were collected for analysis of TNF-α and IL-10 using DuoSet ELISA kits (RnD Systems, Minneapolis, MN, USA).

Histology
The eyes were fixed in 4% paraformaldehyde in PBS for 48 h. After dehydration, the eyes were embedded in paraffin and sectioned at 5 µm thickness. The sections were hematoxylin and eosin stained and imaged using an Olympus CX33 with the QCamera setup (Olympus). The number of hematoxylin-stained nuclei was counted, and the retina length was measured using NIH ImageJ software v1.54d.

Statistical Analysis
The statistical analysis of the EAU clinical scores was a two-way ANOVA with nonparametric post-test analysis. Differences in cytokine levels and the number of counted nuclei in the RGC layer were assayed by ordinary one-way ANOVA with Dunnett's postanalysis multiple comparisons. Significance was detected when the p-value was less than 0.05. Statistical calculations were done using PRISM 9 (GraphPad Software v9.5.1, San Diego, CA, USA), and the results were presented as the mean ± SEM for each experimental group of 4 to 10 unpaired eyes. The micrographs of the retinal sections are representative images that correspond to the mean of each experimental group.