RXR agonists in eye disorders

Abstract

Methods and compositions are provided for the treatment and / or prevention of ocular disorders, including dry eye disorder and any condition that has dry eye as a symptom. [0003] Embodiments of the present disclosure relate to the treatment and / or prevention of ocular disorders, including dry eye disorders and any condition having dry eye as a symptom. In specific embodiments, the treatment and / or prevention can occur by administering a therapeutic composition comprising one or more RXR agonists to at least one eye of an individual.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 305,596, filed February 1, 2022, which is incorporated by reference herein in its entirety.

[0002] This invention was made with Government support under award EY011915 from the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present disclosure relates to at least the fields of cell biology, biochemistry, molecular biology, medicine, and ophthalmology. [Background technology]

[0004] Dry eye disease is widespread, affecting tens of millions of people worldwide (Pflugfelder and de Paiva, 2017). Clinical trial results and animal models have demonstrated that inflammation contributes to the development of ocular surface disease in dry eye (Perez et al., 2020). The ocular surface is an exposed mucosal tissue, exposed to dryness stress, osmotic stress, and microbial danger signals. The conjunctiva harbors immune cells that produce factors that suppress vision-threatening inflammation for homeostasis, but respond to pathogens and environmental danger signals. Indeed, ocular surface dryness has been found to be a potent inflammatory stress that stimulates the activation and production of inflammatory mediators (cytokines, chemokines, and proteases) by ocular surface epithelial cells and inflammatory cells (Pflugfelder and de Paiva, 2017). This may lead to the clinical manifestations of dry eye, such as the breakdown of the corneal barrier and the loss of conjunctival goblet cells (Alam et al., 2020a; Pflugfelder and Stern, 2020).

[0005] The lacrimal functional unit regulates tear production and distribution, including factors that maintain the health of the ocular surface epithelium and suppress ocular surface inflammation (Stern et al., 1998). One such lacrimal secretory factor is vitamin A in the form of retinol, which is metabolized by the ocular surface epithelium, especially the conjunctival goblet cells, to retinoic acid (RA), which can be delivered to immune cells present in the underlying stroma (Xiao et al., 2018; Pflugfelder and de Paiva, 2020). Dry eye with corneal and conjunctival epithelial disease develops in systemic vitamin A deficiency. However, its pathogenic mechanism remains unclear. Vitamin A signals through two nuclear receptor families, the retinoid acid receptor (RAR) and the retinoid X receptor (RXR), which are organized as homo- or heterodimers (partners include RAR, PPAR, vitamin D receptor, etc.) (Alam et al., 2021b). RXRα is expressed in various immune cells, including myeloid and lymphoid lineages (Fritsche et al., 2000;Roszer et al., 2013;Raverdeau and Mills, 2014), and in myeloid cells of the conjunctiva (Alam et al., 2021b). Mice with loss-of-function mutations in the nuclear receptor RXRα have been reported to develop dry eye (Du et al., 2005).

[0006] The disclosure herein describes mechanisms of dry eye pathogenesis associated with loss of function of RXRα, as well as compositions and methods for alleviating, preventing, and / or treating such mechanisms. One such mechanism involves an increase in the population of IL-17-producing γδ T cells (γδ T17 cells) in the dry eye environment with reduced RXRα signaling, which promotes the development of dry eye disease. The present disclosure fulfills a long-standing need in the art of dry eye treatment. Summary of the Invention

[0007] The present disclosure relates to a composition comprising at least one RXR agonist and a method of utilizing such a composition. In some embodiments, the composition, which may be a therapeutic composition, is administered to an individual to treat or prevent eye disorder. In some embodiments, the eye disorder is dry eye disease, Sjogren's syndrome, meibomian gland disease, tear instability, unstable tear film, tear dysfunction, or one or more ocular surface inflammatory conditions. The individual may have or be at risk of having dry eye disease, Sjogren's syndrome, meibomian gland disease, unstable tear film, tear dysfunction, or ocular surface inflammatory conditions, vitamin A deficiency, chemical corneal injury, thermal corneal injury, corneal inflammation after bacterial, fungal or viral infection, corneal neovascularization, or a combination thereof.

[0008] In some embodiments, the RXR agonist includes any type of RXRα agonist. The RXR agonist can include 9-cis retinoic acid, oleic acid, omega-3 docosahexaenoic acid, vitamin D, bexarotene, taxerotene, honokiol, AM80, rosiglitazone, dorupanin, garcinic acid, 4-(ethyl(3-isobutoxy-4-isopropylphenyl)amino)benzoic acid (NEt-3IB) or combinations thereof.

[0009] One or both eyes may be affected by an eye disorder or as a result of having a medical condition that does not primarily target the eye but secondarily affects the eye. The composition may be administered to one or both eyes of an individual. In some embodiments, the composition is administered as an eye drop, which may or may not include microdroplets. In some embodiments, the composition is administered as a suspension, nanoparticles, ointment, cream, or a combination thereof. In some embodiments, the composition is administered by subconjunctival injection.

[0010] The composition may be administered at any dose capable of treating or preventing an eye disorder. In some embodiments, the composition may be administered at approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 1 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133 , 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193 , 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536 , 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569 , 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 300 The composition may comprise 0, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 ng or μg of one or more RXR agonists. In some embodiments, the composition comprises one or more RXR agonists in any range inducible therein, such as approximately 0.01 ng / mL to 50 ng / mL, or 0.03 ng / mL to 30 ng / mL. In some embodiments, such as for topical administration of the RXR agonist, the concentration of the RXR agonist is approximately 0.03 ng / mL to 30 ng / mL in the topical formulation.

[0011] In some embodiments, the individual has a condition that predisposes the individual to an eye disorder or dry eye, which may include Sjogren's syndrome, rheumatoid arthritis, systemic lupus, lupus erythematosus, systemic sclerosis, graft-versus-host disease, and / or Stevens-Johnson syndrome. [Brief description of the drawings]

[0012] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0013] [Figure 1A] Figure 1A-D shows that the Pinkie strain with reduced Rxrα signaling develops dry eye disease. 1A) Dry eye phenotype in the Pinkie strain. Graphs showing representative Oregon Green Dextran (OGD) staining and mean gray level fluorescence of corneas from 8-week-old and 32-week-old C57BL / 6 (B6) and Pinkie strains (left) (n=9-22 / group). Mean ± SD, ***P ≤ 0.01; ****P ≤ 0.0001. [Figure 1B] Representative images of PAS staining of conjunctival goblet cells in paraffin sections prepared from B6 and Pinkie lines (left) and bar graphs of mean goblet cell density (right), n = 5 / group. [Figure 1C] Representative SPPR2 immunostaining of whole-mount conjunctiva stained with SPRR2 polyclonal antibody that recognizes multiple isoforms and nuclei stained with Hoechst 33343 dye. Images captured by confocal microscopy; Relative fold expression of Sprr2g, Sprr2f and Sprr2a genes in Pinkie conjunctiva, n=5 / group. [Figure 1D] Flow cytometry scatter plot showing the increased percentage of CD45+ cells in Pinkie conjunctiva (left) and bar graph comparing CD45+ cells in conjunctiva obtained from B6 and Pinkie (n=7).

[0014] [Figure 2A] Figures 2A-2E show single-cell RNA sequencing (scRNA-seq) that revealed differences in conjunctival immune cell populations between B6 and Pinkie. 2A) UMAP of 19 distinct immune cell clusters in the conjunctiva generated from single-cell transcriptome profiles of CD45+ cells using Seurat package v3. [Figure 2B] UMAP comparing conjunctival immune cell clusters obtained by scRNA-seq of CD45+ cells from eight female C57BL / 6J and eight Pinkie 16 week olds. The percentage of cells in each cluster is shown in brackets, and cell numbers and percentages relative to clusters are provided in Table 1. [Figure 2C] Heatmap of the top 50 differentially expressed genes in C57BL / 6 and Pinkie conjunctival immune cells. Heatmap colors based on the natural log of normalized RNA expression. [Figure 2D] Violin plots showing expression of IL-17a (top) and IL-17f (bottom) with expression in each cluster of the plot to the right. *adj p-value between lines ≦0.05. [Figure 2E] Violin plot of γδT17 signature genes Ltb, Cxcr6, Rorc and IL1rf with significantly higher expression in Pinkie vs. C57BL / 6 (B6). ****p≦0.0001.

[0015] [Figure 3A] Figures 3A-3D show an increase in γδT17 cells in Pinkie conjunctiva. 3A) Flow cytometry of γδ T cell receptor positive cells (γδTCR) left, IL-17A+CD3+γδTCR- middle and IL-17A+CD3+γδTCR+ right [top: C57BL / 6 (B6) strain and bottom: Pinkie strain]. Bar graphs show the mean + / - SD of the percentage of cells in these groups (n=6 / group). [Figure 3B] Confocal microscopy of whole-mount conjunctiva from B6 and Pinkie co-stained with antibodies specific for γδ TCR and conjunctival-specific cytokeratin 13 (CK-13) (top), IL-17A (middle) and γδ T cell transcription factor RORγt (bottom) (n=3 / group). The number of γδ T cells positive for IL-17A and RORγt is shown in the bar graphs on the right. Antibody details are provided in Table 3. [Figure 3C]Confocal microscopy of whole-mount conjunctiva from B6 and Pinkie stained with CXCL16 antibody (left, n=3). Minimal staining was observed in B6 conjunctiva. Comparison of CXCL16 expression levels in conjunctiva measured by real-time PCR (n=6). [Figure 3D] Tear concentrations of the IL-17 signature cytokines IL-12A / IL-23A (top) and TNF-a (middle) and the angiogenic factor VEGF-A (bottom) measured by Luminex multiplex assay (n=5–12 / group).

[0016] [Figure 4A] Figures 4A-D show the inhibitory effect of 9-cis retinoic acid. 4A) IL-17A / F concentrations in the supernatants of cultured γδ T cells isolated with magnetic beads from C57BL / 6 or Pinkie spleens. Cells are stimulated with anti-CD3 / CD28 beads or beads + recombinant IL-23 without or with 100 nM 9-cis retinoic acid (RA). IL-17A / F is measured by ELISA. !p≦0.05 between treatment groups, *p≦0.05 between B6 and Pinkie strains. [Figure 4B] Volcano plots showing expression levels of genes in monocytes cultured in medium alone, medium + LPS or medium + LPS and 100 nM 9-cis RA. Gene expression was assessed using mouse bone marrow innate immune nanostring arrays. Dotted vertical lines indicate less than or greater than 1.5 log2 fold change and horizontal lines indicate genes with adjusted p-values ​​≥ 0.05. Red dots are genes significantly increased by LPS (top) or LPS + 9-cis RA (bottom). γδT17 inducers (TNF-a, IL-1a, IL-1b and IL-23a) are stimulated by LPS and decreased by 9-cis RA. [Figure 4C] ATAC Seq: Principal component analysis of peak sequences identified by ATAC seq in three experimental groups of cultured mouse monocytes: control, cells stimulated with LPS, and cells stimulated with LPS and 100 nM 9-cis RA (n=2 / group). [Figure 4D]Sequence logos of transcription factor binding motifs found to be increased (top) or decreased (bottom) in the second group compared to the first group (top 4-5 motifs are shown for each group, except for control vs. LPS, where no decrease in motifs was observed). Motifs are identified by HOMER peak caller from a database of known motif sequences (Yan et al., 2020).

[0017] [Figure 5A] 5A-5C show pathway analysis. 5A) A heat map of canonical pathways showing significant differences between lineages and cell clusters was generated by Qiagen™ Ingenuity Pathway Analysis™. This analysis identified pathways from the Ingenuity Pathway Analysis library of canonical pathways that were most associated with the dataset. Molecules from the dataset that had an adjusted p-value ≦0.05 and associated with canonical pathways in the Ingenuity Knowledge Base were considered for analysis. The significance of association between the dataset and canonical pathways was measured in two ways: 1) by displaying the ratio of the number of molecules from the dataset that map to the pathway divided by the total number of molecules that map to the canonical pathway; 2) a right-sided Fisher's exact test was used to calculate a p-value that determines the probability that the association between genes in the dataset and the canonical pathways is explained by chance alone. The IL-17 signaling pathway and the PPARα / RXRα activation pathway are among the pathways identified with significant differences. [Figure 5B]IL-17 signaling pathway network showing molecular relationships generated with Qiagen Ingenuity Pathway Analysis with modifications. All connections are supported by at least one reference from the literature, from textbooks, or from canonical information stored in the Ingenuity Knowledge Base. Lines and arrows between nodes represent direct (solid lines) or indirect (dashed lines) interactions between gene products and are indicated by cellular localization (extracellular space, plasma membrane, cytoplasm, or nucleus). Rectangles are cytokines and cytokine receptors, triangles are phosphatases, concentric circles are groups or complexes, diamonds are enzymes, and ellipses are transcriptional regulators or regulators. P=phosphorylation, U=ubiquitination. [Figure 5C] Heatmap of differentially expressed genes in conjunctival bulk RNAseq between C57BL / 6 (B6) and Pinkie strains, including IL-17 pathway-related genes and other innate inflammatory mediators in red. All genes were passed through the Benjamini-Hochberg procedure to eliminate false discoveries; selected genes had an adjusted p-value ≥ 0.05. Each row represents a specific gene, the right column represents the Pinkie strain, and the left column represents B6.

[0018] [Figure 6A] 6A-6E show a comparison of γδT17 and dry eye symptoms in bone marrow chimeras. 6A) Conjunctival goblet cell counts in C57BL / 6 mice exposed to desiccation stress (DS) for 5 days (DS5) with or without systemic treatment with anti-IL-17 neutralizing antibody or isotype control as described in the methods herein. [Figure 6B] Representative flow cytometry plots of donor (CD45.2+) and recipient (CD45.1+) bone marrow-derived cells (left) and γδTCR high and low CD3+ T cells in the conjunctiva of Pepc / BoyJ recipient (host) chimeric mice reconstituted with B6 or Pinkie bone marrow after 5 days of desiccation stress. The method of chimera generation is provided in FIG. [Figure 6C]Top left: Bar graphs show the percentage of CD45.2+CD3+γδTCR+ in recipient conjunctiva (n=11 / group). Bottom left: Histogram of the percentage of IL-17+ cells from the CD45.2+CD3+γδTCR+ gate in a representative sample. Right: Mean + / - SD percentage (top) and mean fluorescence intensity (bottom) of IL-17A+CD3+γδTCR+ cells in chimera conjunctiva (n=11 / group). [Figure 6D] Confocal microscopy of whole-mount conjunctiva or frozen sections from B6 and Pinkie bone marrow chimeras (n=3 / group) prepared as shown in Figure 3 and exposed to DS for 5 days, stained with an antibody specific for MMP-9 (top), assessed for in situ gelatinase (zymography) activity in frozen sections (middle), or stained with a polyclonal antibody against the cornified membrane precursor SPRR2 (bottom), with or without systemic treatment with anti-IL-17 neutralizing antibody or isotype control as described in Methods. Bar graphs on the right show the mean ± SD fluorescence intensity of the fluorochrome / fluorescent gelatin measured by Nikon Elements software (n=3 / group). [Figure 6E] Conjunctival goblet cell counts in Pinkie donor bone marrow chimeric mice exposed to desiccation stress (DS) for 5 days (DS5) with or without systemic treatment with anti-IL-17 neutralizing antibody or isotype control as described in the methods. Representative photomicrographs of periodic acid stained sections for each treatment group (left) and graphs of mean ± SD of goblet cells / mm (n=5). Some goblet cells in the control group appear to be trapped in the epithelium as previously reported (Corrales et al., 2011).

[0019] [Figure 7A] Figures 7A-7E show corneal neovascularization, opacification and ulceration with age. 7A) Appearance of non-ulcerated (NC) C57BL / 6 (B6) and NC and ulcerated (UC) Pinkie corneas in 40-50 week old mice. [Figure 7B]Volcano plots of differentially expressed genes in corneas of NC B6 and Pinkie (left) and NC and UC Pinkie (right) detected by mouse bone marrow innate immune nanostring array. Dotted vertical lines indicate changes less than or greater than 1.5 log2 fold, horizontal lines indicate genes with adjusted p-values ​​> 0.05. Red dots are genes significantly increased in Pinkie NC vs. B6 NC (left) or in Pinkie UC vs. Pinkie NC (left). Labeled genes in the left plot are found in the IL-17 signaling pathway. [Figure 7C] Heatmap generated from nanostring array of IL-17 pathway genes. [Figure 7D] Fold change in expression levels of factors involved in the pathogenesis of corneal neovascularization (Vegfa, Tnf, Fgf7) or corneal ulceration (Mmp9) as measured by RT-PCR. Values ​​are mean mean ± SD (n=4 / sample). [Figure 7E] Immunostaining for blood (CD31) and lymphatic (LyVE-1) endothelial cell markers in 25- and 60-week-old B6 and Pinkie corneas. Arrows indicate the corneal epithelium. n=4 / sample). *p≦0.01; **p≦0.001; ****p≦0.0001.

[0020] [Figure 8] Figure 8 shows an overview of RXRα-mediated suppression of IL-17 production by gamma delta T cells and IL-17-mediated dry eye disease. RXRα suppresses the production of IL-17 inducers (IL-23, IL-1β and TNF-α) by myeloid cells and directly suppresses IL-17 production by activated gamma delta T cells. IL-17 promotes corneal barrier disruption, reduced epithelial lubrication, increased expression of SPRR2, a cornified membrane precursor that seals the goblet cell opening, and a decrease in mucin-filled conjunctival goblet cells.

[0021] [Figure 9]Figure 9 shows UMAP feature plots of highly expressed genes in each cluster identified by scRNA-seq (cluster identity in brackets), except for cluster 1, where Lcn2 expression is low.

[0022] [Figure 10] FIG. 10 shows violin plots of γδT17 signature genes Ltb, Cxcr6, Rorc and IL1rf in scRNA-seq identified cell clusters in Pinkie and C57BL / 6 strains.

[0023] [Figure 11] Figure 11 shows the generation of bone marrow chimeric mice. Bone marrow ablation in Pepc / BoyJ recipient (host) mice was accomplished by 1300cGy of 137Cs irradiation followed by intraorbital injection of 2x10^6 bone marrow cells from wild-type B6 or Pinkie donors. Two weeks after receiving donor cells, chimeric mice were exposed to desiccation stress for 5 days to induce dry eye, and the presence of donor bone marrow-derived cells was identified by flow cytometry performed on conjunctival samples. Representative scatter plots show endogenous (CD45.1) or transplanted (CD45.2) immune cells in the conjunctiva. The phenotypic characteristics of the CD45.2+ cells indicated in the gate were further investigated.

[0024] [Figure 12] FIG. 12 shows IL-17A / F cytokine concentrations measured in supernatants of magnetic bead-isolated cultured murine γδ T cells from C57BL / 6 spleens.

[0025] [Figure 13] FIG. 13 provides UMAPs of 14 distinct immune cell clusters in corneas from C57B / 6 (B6) and Pinkie, generated from single-cell transcriptome profiles of CD45+ cells using the Seurat package v3.

[0026] [Figure 14]FIG. 14 shows a heat map comparing the percentages of 14 cell types in B6 and Pinkie corneas.

[0027] [Figure 15] FIG. 15 shows a volcano plot comparing the levels of differentially expressed genes in the corneas of B6 and Pinkie strains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] I. Definition Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error in the measuring or quantitating method.

[0029] The use of the word "a" or "an" in conjunction with the term "comprising" may mean "one," but is also consistent with the meaning of "one or more," "at least one," or "one or more than one."

[0030] and / or means "and" or "or." For example, A, B, and / or C includes: A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0031] The words "comprising" (and any form of comprising, such as "comprises", "comprises"), "having" (and any form of having, such as "have", "has"), "including" (and any form of including, such as "includes", "include"), or "containing" (and any form of containing, such as "contains", "contain") are inclusive or open-ended and do not exclude additional, non-reproduced elements or method steps.

[0032] The compositions and methods of use thereof may "comprise," "consist essentially of," or "consist" of any of the components or steps disclosed throughout this specification. Compositions and methods "consist essentially of" any of the disclosed components or steps limit the scope of the claim to those specified materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0033] The term "drop" or "microdrop" as used herein refers to a liquid formulation of less than 0.1 mL. A microdrop may be about 0.001 mL, 0.002 mL, 0.003 mL, 0.004 mL, 0.005 mL, 0.006 mL, 0.007 mL, 0.008 mL, 0.009 mL, 0.01 mL, 0.02 mL, or 0.03 mL. A drop may be about 0.04 mL, 0.05 mL, 0.06 mL, 0.07 mL, 0.08 mL, or 0.09 mL.

[0034] The phrases "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as conventional media or agents are incompatible with the active ingredient, their use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infectives and vaccines, can also be incorporated into the composition.

[0035] The term "prevention" (or grammatical equivalents) as used herein with respect to an eye disorder refers to an intervention that seeks to prevent the eye disorder from occurring or recurring in an individual, or to delay the onset of the eye disorder in an individual.

[0036] As used herein, the term "treatment" (or grammatical equivalents) refers to an intervention that seeks to change the natural course of the disease being treated. Treatment serves to achieve one or more of a variety of desired results, including, for example, alleviation of symptoms, reduction of direct or indirect pathological consequences of the disease, slowing the rate of disease progression, amelioration or alleviation of the disease state, and remission or improvement of prognosis.

[0037] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Further, compositions of the invention can be used to achieve methods of the invention.

[0038] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0039] II. RXR agonists The embodiments herein include compositions and methods of using compositions that include at least one RXR agonist. In some embodiments, the RXR agonist includes a composition that can bind to a heterodimer or homodimer that includes an RXR molecule. The RXR agonist may be an RXRα agonist. In certain aspects, the RXRα agonist is a small molecule. The RXR agonist may include 9-cis retinoic acid, oleic acid, omega-3 docosahexaenoic acid, vitamin D, bexarotene, taxerotene, honokiol, AM80, rosiglitazone, dorupanin, garcinoic acid, NEt-3IB, any analog thereof, or combinations thereof. The RXR agonist may include a steroid ring structure. In some embodiments, the RXR agonist consists of a retinol ring structure (including any vitamin A composition) without or with one or more chemical modifications.

[0040] In some embodiments, RXR agonists, such as 9-cis RA, suppress the production of IL-17 by γδ T cells and / or the production of IL-17-induced cytokines by monocytes. RXR agonists useful in the embodiments herein can be screened from a library of potential RXR agonists. Screening can be any method capable of detecting RXR agonist activity. These include, by way of example, ligand binding assays and / or biological activity assays.

[0041] III. Eye Diseases Certain embodiments herein relate to the treatment or prevention of one or more ocular disorders in an individual, and / or the treatment or prevention of dry eye that is the result of a medical condition that is not an ocular disorder, and / or the treatment or prevention of dry eye that is the result of aging, environment, etc. In some embodiments, the ocular disorder is treated or prevented by administering one or more RXR agonists to the individual. The ocular disorder may be any disorder in which overproduction of IL-17 by γδ T cells and / or overproduction of IL-17-induced cytokines by monocytes occurs. The ocular disorder may be a dry eye disorder, such as aqueous humor deficient dry eye. In some embodiments, the individual has changes in the meibomian gland. In some embodiments, the individual has a meibomian gland disease. The ocular disease may be an ocular surface inflammatory disease, such as scleritis. The individual has dry eye because they have an ocular disease or because they have dry eye as a result of another medical condition that has dry eye as a secondary effect (e.g., not all individuals with a medical condition have dry eye). In a specific embodiment, the individual has decreased tear production for some reason. In a specific embodiment, the individual is older, such as being (or at least being) 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 years old. Decreased tear production may be due to certain medical conditions, such as Sjögren's syndrome, allergic eye disease, rheumatoid arthritis, lupus, scleroderma, graft-versus-host disease, sarcoidosis, thyroid disease, diabetes, scleroderma, Parkinson's disease, Graves' disease, and vitamin A deficiency. Certain medications, such as antihistamines, decongestants, hormone replacement therapy, antidepressants, tranquilizers, high blood pressure medications, acne medications, certain heart medications, diuretics, birth control pills, and ulcer medications, may also decrease tear production. Decreased tear production may be due to corneal nerve sensitization caused by contact lens use, nerve injury, or laser eye surgery. In women, hormonal changes, such as after menopause and during pregnancy, can also decrease tear production.

[0042] In some embodiments, the individual has increased tear evaporation. In some cases, the oil film produced by the small glands at the edge of the eyelid (meibomian glands) can become clogged. Clogged meibomian glands can occur in individuals with rosacea or other skin disorders.

[0043] In some embodiments, an individual may have increased tear evaporation due to posterior blepharitis (meibomian gland dysfunction), ocular allergies, vitamin A deficiency, exposure to preservatives such as topical eye drops, exposure to wind, smoke, or dry air, decreased blinking frequency (such as occurs in certain conditions such as Parkinson's disease), or concentration during certain activities such as reading, driving, or working at a computer; or decreased ability to concentrate during certain activities such as reading, driving, or working at a computer), eyelid problems such as eyelids turning outward (ectropion) or eyelids turning inward (entropion).

[0044] In certain embodiments, the individual has dry eye symptoms such as dry eyes, greasiness or burning, redness, watery or teary eyes, mucus that makes the eyes feel "glued" after sleep, feeling like something is in the eye or eye fatigue, itching, light sensitivity, etc. In certain embodiments, the symptoms are worse later in the day. In certain embodiments, dry eye can be diagnosed based on symptoms. In certain embodiments, one or more tests are utilized for diagnosis, such as measuring tear production, special dyes, and evaluation of tear film constitution. In certain aspects, the tests rule out other potential problems such as conjunctivitis that may produce the same symptoms.

[0045] In some embodiments, the individual has a higher risk of dry eye than the general population, such as being over 50 years of age, being female (e.g., pregnant, using birth control pills, or during menopause), consuming a diet low in vitamin A, consuming a diet low in omega-3 fatty acids, wearing contact lenses, and / or having had refractive surgery.

[0046] IV. Pharmaceutical Compositions In certain embodiments, the composition or agent for use in the method, such as any RXR agonist, is preferably included in a pharma- ceutically acceptable carrier.In some embodiments, the carrier is selected to be non-toxic, biocompatible, and not to adversely affect the biological activity of the agent.The agent in some aspects of the present disclosure can be formulated into a formulation for local delivery (i.e., to a specific location in the body, such as any ocular tissue or other tissue) or systemic delivery, in the form of solid, semi-solid, gel, liquid, or gas, such as tablets, capsules, powders, granules, ointments, solutions, deposits, inhalants, and injections, which allow oral, parenteral, or surgical administration.Some aspects of the present disclosure also contemplate the local administration of the composition by coating medical devices, etc.

[0047] Carriers suitable for parenteral administration by injection, infusion or irrigation and local administration include distilled water, physiological phosphate buffered saline, normal or lactated Ringer's solution, dextrose solution, Hank's solution, or propanediol. In addition, sterile fixed oils can be used as a solvent or suspension medium. For this purpose, any biocompatible oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injections. The carrier and the drug can be formulated as a liquid, suspension, polymerizable or non-polymerizable gel, paste, or ointment.

[0048] Carriers may also include delivery vehicles to sustain (i.e., extend, delay or modulate) delivery of an agent or to enhance delivery, uptake, stability or pharmacokinetics of a therapeutic agent. Such delivery vehicles may include, by way of non-limiting example, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles.

[0049] In certain embodiments, the actual dosage of the composition administered to a patient or subject may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, characteristics of the patient, and route of administration. The physician responsible for administration will, in any event, determine the concentration of active ingredient in the composition and appropriate dose for the individual subject.

[0050] Solutions of the pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, hyaluronic acid, mixtures thereof, and oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0051] In some embodiments, pharmaceutical compositions are advantageously administered in the form of injectable compositions, as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid before injection can also be prepared. These preparations may also be emulsified. A typical composition for such purposes consists of a pharmaceutically acceptable carrier. For example, the composition can contain up to 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per ml of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers, etc.

[0052] Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, dimethylsulfoxide (DMSO), ethanol, vegetable oils, injectable organic esters such as ethylolates, etc. Aqueous carriers include water, alcohol / aqueous solutions, parenteral vehicles such as saline, sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include infusion and nutritional supplements. Preservatives include antibacterial agents, antifungal agents, antioxidants, chelating agents, inert gases, etc. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to well-known parameters.

[0053] Additional formulations are suitable for oral administration. Oral formulations include typical excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0054] In further embodiments, the pharmaceutical composition may include classical pharmaceutical formulations. Administration of the pharmaceutical composition according to certain aspects may be via any common route, so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be via orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions are usually administered as pharma- ceutical acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. Aerosol delivery may be used to treat pulmonary conditions. The volume of the aerosol may be, for example, between about 0.01 ml and 0.5 ml.

[0055] The effective amount of the pharmaceutical composition is determined based on the intended purpose. The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the pharmaceutical composition calculated to produce the desired response as described above in relation to its administration, i.e., the appropriate route and treatment regimen. The amount to be administered depends on the protection or effect desired, according to both the number of treatments and the unit dosage.

[0056] The precise amount of pharmaceutical composition to be administered also depends on the judgment of the skilled artisan and is peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended therapeutic goal (e.g., relief of symptoms versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent.

[0057] A. Combination In some embodiments, at least one of the active compounds disclosed herein is formulated in a sustained release vehicle.The sustained release vehicle can be suitable for eye drops, including microdrops, and / or injections.The active compound can be formulated for parenteral administration, for example, for injection by intravenous, intramuscular, subcutaneous, or intraperitoneal route.Typically, such compositions can be prepared as liquid solutions or suspensions;solid forms can also be prepared that are suitable for adding liquid to prepare solutions or suspensions before injection;the preparation can also be emulsified.

[0058] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; formulations, for example, containing aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the formulation must be sterile and fluid to the extent that easy syringability exists. It must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0059] The pharmaceutical compositions may contain solvents or dispersion media, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), DMSO, suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0060] Sterile injectable solution is prepared by incorporating the required amount of active compound into a suitable solvent, together with various other ingredients as listed above as necessary, and then sterilizing by filtration or equivalent procedures.In general, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients as listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains a powder of active ingredient and any additional desired ingredient from the solution previously sterile filtered.

[0061] The administration of the composition is typically via any common route. Alternatively, administration can be via orthotopic, intraocular, subconjunctival, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. In some embodiments, the administration of the composition is via eye drops, which can be microdrops, drops, creams, or ointments. In some embodiments, the composition occurs via an ointment or cream delivered to the eye or eyelid. Such compositions described herein will usually be administered as a pharma- ceutically acceptable composition, including physiologically acceptable carriers, buffers, or other excipients.

[0062] The formulated solutions can be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically or prophylactically effective. The formulations can be easily administered in a variety of dosage forms, such as the above-mentioned injectable solutions.

[0063] B. Other drugs It is contemplated that other drugs may be used in combination with certain aspects of the present embodiment to improve the therapeutic effect of the treatment. These additional drugs include corticosteroids, cyclosporine A, lifitegrast, tetracyclines (doxycycline, minocycline) and varenicline, and / or drugs that may be useful in the management, prevention, or treatment of any of the disorders disclosed herein.

[0064] V. Administration of Therapeutic Compositions In some embodiments, a therapeutic composition, which may also be referred to as a pharmaceutical composition, is administered to a subject. Different aspects may include administering an effective amount of the composition to a subject. In some embodiments, an antibody or antigen-binding fragment capable of binding to RXR (or any dimer of RXR) may be administered to a subject to protect against or treat a condition (e.g., an eye disorder). Alternatively, one or more such antibodies or expression vectors encoding polypeptides or peptides may be administered to a subject as a prophylactic treatment. In addition, such compositions may be administered in combination with additional therapeutic agents (e.g., chemotherapeutic agents, immunotherapeutic agents, biotherapeutic agents, etc.). Such compositions are generally dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0065] The therapies provided herein may include administration of a combination of therapeutic agents, such as a first therapy and a second therapy. The therapeutic agents may be administered in any suitable manner known in the art. For example, the first therapy and the second therapy may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second therapy are administered in separate compositions. In some embodiments, the first and second therapy are in the same composition.

[0066] In some embodiments, the first and second therapies are administered substantially simultaneously. In some embodiments, the first and second therapies are administered sequentially. In some embodiments, the first, second, and [third] therapies are administered sequentially. In some embodiments, the first therapy is administered prior to administration of the second therapy. In some embodiments, the first therapy is administered after administration of the second therapy.

[0067] The embodiments of the present disclosure relate to compositions and methods, including therapeutic compositions. Different therapeutic agents may be administered in one composition, or in two or more compositions, such as two compositions, three compositions, or four compositions. Various combinations of agents may be employed.

[0068] The therapeutic agents of the present disclosure may be administered by the same or different routes of administration. In some embodiments, the therapeutic agents are administered intravenously, intramuscularly, subcutaneously, topically, orally, subconjunctivally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0069] Therapeutic agents include various "unit doses." A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount administered, as well as the particular route and formulation, are within the discretion of those skilled in the art of clinical medicine. A unit dose need not be administered as a single injection, but may include continuous infusions over a period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0070] In some embodiments, the therapy comprises at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 110, 111 2, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 1 39, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267、 268、 269、 270、 271、 272、 273、274、 275、 276、 277、278、 279、 280、 281、 282、283、 284、 285、 286、 287、 288、 289、290、291、 292、 293、 294、 295、 296、 297、 298、299、 300、 301、 302、 303、 304、 305、 306、307、308、 309、 310、 311、 312、 313、 314、315,316,317,318, 319, 320, 321, 322, 323,324,325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348,349,350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397,398,399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,435, 436, 437, 438,439,440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462,463,464, 465, 466, 467, 468, 469, 470, 471,472,473,474, 475, 476, 477, 478, 479,480, 481,482, 483, 484, 485, 486, 487,488,489, 490, 491, 492, 493, 494, 495, 496,497, 498,499, 500, 501, 502, 503, 504,505, 506, 507, 508, 509, 510, 511, 512,513,514, 515,516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, In some embodiments, the treatment is administered at a dose of between 0.14 mg / kg and 0.57 mg / kg.

[0071] In some embodiments, the second therapy is administered in a single dose. In some embodiments, the second therapy is administered in multiple doses. In some embodiments, the second therapy is administered in a dose of 1 mg / kg to 100 mg / kg. In some embodiments, the second therapy is administered in a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 100 mg / kg.

[0072] The dosage depends on the desired therapeutic effect, both in number of treatments and unit dosage. Effective amount is understood to refer to the amount required to achieve a specific effect. In the implementation of certain embodiments, it is contemplated that a dosage ranging from 10 μg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, dosages include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such dosages can be administered multiple times during the day and / or on multiple days, weeks, or months.

[0073] In certain embodiments, an effective amount of the pharmaceutical composition may provide a blood level of about 0.1 nM to 150 μM. In another embodiment, an effective amount provides a blood level of about 4 μM to 100 μM. Or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to about 50 μM; or about 25 μM to about 150 μM; or about 25 μM to about 100 μM; or about 25 μM to about 50 μM; or about 50 μM to about 150 μM; or about 50 μM to about 100 μM (or any range derivable therein). In other embodiments, the dose may provide the following blood levels of drug resulting from the therapeutic agent being administered to a subject: about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derivable therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in the body to become a metabolized therapeutic agent, in which case blood concentration can refer to the amount of that therapeutic agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, blood concentrations discussed herein can refer to the unmetabolized therapeutic agent.

[0074] Precise amounts of therapeutic compositions also depend on the judgment of the practitioner and are peculiar to each individual. Factors influencing the dosage include the physical and clinical condition of the patient, the route of administration, the intended therapeutic goal (palliation of symptoms versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other therapies the subject may be undergoing.

[0075] Those skilled in the art will understand and appreciate that dosage units of ng / kg, μg / kg or mg / kg body weight can be converted and expressed in equivalent concentration units of μg / ml or mM (blood concentration). It is also understood that uptake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions to be made regarding uptake and concentration measurements are well known, and those skilled in the art will be able to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacy and results described herein.

[0076] In certain embodiments, it may be desirable to administer multiple doses of the composition, for example, 2, 3, 4, 5, 6 or more doses, which may be administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 weeks, including all ranges therebetween.

[0077] The phrases "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonicity agents and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as conventional media or agents are incompatible with the active ingredient, their use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the composition.

[0078] Active compound can be formulated for parenteral administration, for example, can be formulated for injection via intravenous, intramuscular, subcutaneous or intraperitoneal route.Typically, such compositions can be prepared as either liquid solution or suspension;solid forms suitable for adding liquid before injection to prepare solution or suspension can also be prepared;preparation can also be emulsified.

[0079] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations, for example, containing aqueous propylene glycol; and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.In some embodiments, the form must be sterile and fluid enough to be easily syringable.It must also be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0080] Proteinaceous compositions can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of protein) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0081] The pharmaceutical compositions may contain solvents or dispersion media, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0082] Sterile injectable solution is prepared by incorporating the required amount of active compound into a suitable solvent, together with various other ingredients as listed above as necessary, and then sterilizing by filtration or equivalent procedures.In general, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients as listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains a powder of active ingredient and any additional desired ingredient from the solution previously sterile filtered.

[0083] The administration of the composition is usually by a common route, including but not limited to oral or intravenous administration. Alternatively, administration may be orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal. Such compositions will usually be administered as a pharma- ceutically acceptable composition, including a physiologically acceptable carrier, buffer, or other excipient.

[0084] The formulated solutions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above. EXAMPLES

[0085] The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. Example 1 - General embodiment

[0086] Embodiments of the disclosure include methods for investigating mechanisms of dry eye disease development, such as in Pinkie mouse strains that carry loss-of-function RXRα mutations.

[0087] In some embodiments, dry eye disease measures were evaluated in the cornea and conjunctiva. To compare gene expression in conjunctival immune cells, expression profiling was performed by single cell RNA sequencing (scRNA-seq). Conjunctival immune cells were immunophenotyped by flow cytometry and confocal microscopy. The activity of the RXRα ligand 9-cis retinoic acid (RA) was evaluated in cultured monocytes and γδ T cells.

[0088] Analysis of specific methods herein revealed that compared to wild-type (WT) C57BL / 6, Pinkie exhibited increased signs of dry eye disease, including corneal barrier disruption, conjunctival keratinization and goblet cell loss, and age-related corneal neovascularization, opacification, and ulceration. scRNA-seq of conjunctival immune cells identified γδ T cells as the predominant IL-17-expressing population in both lineages, with a four-fold increase in the percentage of γδ T cells in Pinkie. Significantly increased expression of IL-17a and IL-17f in conventional T cells, and IL-17f in γδ T cells, was found in Pinkie compared to WT. Flow cytometry and immunostaining demonstrated increased expression of IL-17 in Pinkie. + Increased numbers of γδ T cells were evident. Tear fluid concentrations of the IL-17 inducer IL-23 were significantly higher in Pinkie. 9-cis RA treatment suppressed stimulated IL-17 production by γδ T and the stimulatory activity of monocyte supernatants on γδ T cell IL-17 production. Compared with WT bone marrow chimeras, Pinkie chimeras had significantly higher levels of IL-17 in the conjunctiva after desiccation stress. + γδ T cells are increased, and anti-IL-17 treatment suppresses dry eye-induced corneal MMP-9 production / activity and conjunctival goblet cell loss.

[0089] The findings herein show that RXRα suppresses the development of dry eye disease-inducing γδT17 cells in the conjunctiva, identifying RXRα as a therapeutic target in dry eye. Example 2 - Materials and Methods for Specific Embodiments

[0090] animal

[0091] The animal protocol for this study was designed in accordance with the ARVO Statement for the use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine (Protocol AN-2032). Six- to eight-week-old female C57BL / 6J (B6) mice and Pepc / BoyJ mice were purchased from Jackson Laboratories (Bar Harbor, ME). The RXRα Pinkie mutant strain was purchased from Mutant Mouse Resource and Research Centers (MRRC, University of California, Davis, Sacramento, CA) to establish a genotyped breeder colony that was propagated in the Baylor College of Medicine vivarium and refreshed every eight generations. At the time of the experiment, both B6 and Pinkie strains were 16–60 weeks old and housed in a normal vivarium environment.

[0092] Corneal barrier function evaluation

[0093] Corneal epithelial permeability to dextran conjugated to 70 kDa Oregon-Green (OGD; Invitrogen, Eugene, OR) was assessed as previously described (Alam et al., 2020b). Briefly, 1 μL of OGD (50 mg / mL) was instilled onto the ocular surface 1 min before euthanasia; the eyes were then rinsed with 2 mL of phosphate-buffered saline (PBS) from the temporal and nasal sides and photographed under 470 nm fluorescence excitation with a high-resolution digital camera (Coolsnap HQ2; Photometrics, Tucson, AZ) mounted on a stereo zoom microscope (SMZ 1500; Nikon, Melville, NY). The intensity of corneal OGD staining was graded on digital images using NIS Elements (version 3.0; Nikon) within a 2 mm diameter circle placed on the central cornea by two masked observers. The mean fluorescence intensity measured by the software within this central zone was transferred to a database and the results were averaged within each group.

[0094] Measurement of goblet cell density

[0095] After euthanasia, eyes and ocular adnexa were excised from B6 and Pinkie mice (n=5 / group), and tissues were fixed in 10% formalin, subsequently embedded in paraffin, and 5 μm sections were cut on a microtome (Microm HM 340E; Thermofisher Wilmington, DE) and stained with Periodic Acid Schiff (PAS) reagent. Sections from both eyes of each group were examined and photographed under a microscope (Eclipse E400; Nikon) equipped with a digital camera (DXM1200; Nikon) using NIS Elements software; goblet cells were manually counted. To determine the length of the conjunctival goblet cell area, the first PAS + Final PAS from goblet cells + A line was drawn on the surface of the conjunctival image up to the goblet cells. The results were analyzed using PAS + Shown as goblet cells / mm.

[0096] RNA isolation and quantitative PCR

[0097] After euthanasia, the cornea / conjunctiva was excised and total RNA was extracted using the RNeasy® Plus Mimi Kit (Cat No. 74134, QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions. RNA concentration was measured and cDNA was synthesized using the Ready-To-Go-You-Prime-First-Strand kit (GE Healthcare). Quantitative real-time PCR was performed using specific probes for mouse MGB probes, Cxcl16 (Mm00801778), Sprr2a (Mm00845122_s1), Sprr2f (Mm00448855_s1), Sprr2g (Mm01326062_m1), Vegfa (Mm00437304), Vegfb (Mm00442102), Vegfc (Mm00437310), Tnf (Mm00443260), Fgf7 (Mm00433291), Mmp9 (Mm00442991) and hypoxanthine phosphoribosyltransferase (Hprt1, Mm00446968). Hprt-1 gene was used as an endogenous standard for each reaction. The real-time PCR results were analyzed by comparative CT method, and the CT value of Pinkie was compared with that of B6.

[0098] Tear washings and multiplex immunoassays

[0099] Tear-fluid washings were collected from both mouse strains using capillary tubes as previously described (Zheng et al., 2010) and cytokine concentrations were assayed in tear samples using a commercially available ProcartaPlex Luminex Assay (Thermofisher) following the manufacturer's protocol. Reactions were detected with streptavidin-phycoerythrin using a Luminex LX200 (Austin, TX, USA) (Zaheer et al., 2018). One sample consisted of tear washings (8 μL) from both eyes of four mice pooled in a tube containing 8 μL of PBS + 0.1% BSA and stored at -80 °C until the assay was performed. Results are presented as mean ± standard deviation (picograms / milliliter).

[0100] Flow cytometry and cell sorting

[0101] Conjunctiva was excised, chopped into small pieces with scissors, and incubated with 0.1% type IV collagenase for 1 hour to obtain a single cell suspension. Samples were incubated with anti-CD16 / 32 (2.4G2, Cat. No. 553141, BD Pharmingen™, San Diego, CA) for 5 minutes at room temperature, followed by staining with anti-CD45 (clone 30-F11, Cat. No. 103138, BioLegend) and infrared fluorescent viability dye (Life Technologies, Grand Island, NY). The gating strategy was as follows: forward scatter area (FSC-A) and side scatter area (SSC-A) gates, followed by two singlet gates (FSC-A vs. FSC-W and SSC-A vs. SSC-W), followed by live / dead discrimination using infrared fluorescent viability dye to identify lymphocytes. CD45+ cells were sorted using an Aria-II cell sorter at the Cytometry and Cell Sorting Core at Baylor College of Medicine.

[0102] IL-17 in the conjunctiva +Antibodies for determining cell phenotype included anti-CD45 (clone 30-F11, catalog number 103138, BioLegend), Alexa Fluor® 488 anti-mouse CD45.1 (clone A20, catalog #110718, BioLegend Way San Diego, CA), Brilliant Violet 510™ anti-mouse CD45.2 (clone 104, catalog number 109838, BioLegend Way San Diego, CA), PerCP / Cyanine5.5 anti-mouse CD3ε (clone 500A2, catalog number 152312, BioLegend Way San Diego, CA), PE anti-mouse γδ T cell receptor (Clone GL3, catalog number 553178, BD Pharmingen™, San Diego, CA), Alexa Fluor® 647 anti-mouse IL-17A (clone TC11-18H10, catalog number 560184, BD Pharmingen™, San Diego, CA) was included. To exclude dead cells, violet live / dead fixable dye (Life Technologies) was used. A Canto II flow cytometer (BD Biosciences) and FlowJo 7.6.5 software (TreeStar, Ashland, OR, USA) were used for analysis.

[0103] Library preparation

[0104] Single-cell gene expression libraries were prepared using the Chromium Single Cell Gene Expression 3v3.1 kit (10xGenomics) from the Single Cell Genomics Core at Baylor College of Medicine. Briefly, single cells, reverse transcription (RT) reagents, gel beads containing barcoded oligonucleotides, and oil were loaded into a Chromium controller (10xGenomics) to generate single-cell Gel Beads-In-Emulsions (GEMs), in which full-length cDNA was synthesized and barcoded for each single cell. The GEMs are then broken and the cDNA from all single cells is pooled. After cleanup with Dynabeads MyOne Silane Beads (Thermofisher, Waltham, MA), the cDNA is amplified by PCR. The amplified products are fragmented to optimal size before end repair, A-tailing, and adapter ligation. The final libraries were generated by amplification.

[0105] Sequencing of 10XGEM3'v3.1 single-cell libraries

[0106] The BCM Genomic and RNA Profiling (GARP) Core first performed a quality check of the samples using a NanoDrop spectrophotometer and an Agilent Bioanalyzer 2100. To quantify the adaptor-ligated libraries and confirm successful incorporation of the P5 and P7 adaptors, the Applied Biosystems ViiA7 Real-Time PCR System and the KAPA Illumina / Universal Library Quantification Kit (p / n KK4824) were used. The GARP Core sequenced the libraries on a NovaSeq 6000 sequencing system using an S2 v1.0 flow cell as follows: Cluster generation by Exclusion Amplification (ExAMP): Using concentrations from the ViiA7™ qPCR instrument described above, 150 pM equimolar pooled libraries were loaded onto one lane of a NovaSeq S2 v1.0 flow cell (Illumina kit p / n 20021664) according to the XP workflow protocol (Illumina kit p / n 20012860) and amplified by Exclusion Amplification onto a nanowell designed patterned flow cell using an Illumina NovaSeq 6000 sequencing instrument. PhiX Control v3 adaptor ligated libraries (Illumina p / n FC-110-3001) were added at 1 wt% to ensure balanced diversity and to monitor clustering and sequencing performance. Libraries were sequenced according to the 10XGenomics protocol, 28 cycles for read 1, 10 cycles each for i7 and i5 reads, and 90 cycles for read 2. An average of 251 million read pairs were sequenced per sample. FastQ file generation was performed using bcl2fastq and QC reports were generated using CellRanger v5.0.1 by the BCM Multiomics Core.

[0107] Bioinformatics analysis of scRNA-seq data

[0108] Alignment of raw sequencing data with genomic reference mouse (mm10) 2020-A. Raw sequence reads in FASTQ format were aligned against the mouse reference genome using the Cell Ranger Count v 6.0.1 pipeline (https: / / cloud.10xgenomics.com) with default settings for barcode assignment and UMI counting. The resulting gene expression matrix was subjected to preprocessing according to the guidelines provided by Seurat v 4.0.5. Briefly, single cells with less than 200 genes were filtered to remove empty droplets. Genes expressed in less than 3 cells in the data were filtered. A global scaling normalization method is then employed using the Seurat function "LogNormalize" that normalizes the feature expression.

[0109] Clustering, visualization and cell annotation

[0110] First, the "FindVariableFeatures" function was used to identify a set of 2000 genes that were highly variable in the two datasets, and the "FindIntegrationAnchors" and "IntegrateData" functions combined the two datasets for downstream analysis such as dimensionality reduction and clustering. Principal component analysis (PCA) was then performed to construct a linear dimensionality reduction of the datasets and identify 19 PCs that contained most of the complexity of the dataset. Cells were clustered in the PCA space in a graph-based approach and then nonlinear dimensionality reduction was applied using UMAP for further visualization purposes. A set of canonical cell type markers was used to assign annotations to each cluster using the Cluster Identity Predictor (CIPR) web-based tool (https: / / aekiz.shinyapps.io / CIPR / ). Finally, differential expression was performed using the "FindAllMarkers" function in Seurat to find cluster-specific marker genes.

[0111] Purification and in vitro stimulation of monocytes

[0112] Monocytes were purified from mouse bone marrow cells cultured for 3 days using a monocyte isolation kit according to the manufacturer's instructions (BM, Miltenyi Biotec, Bergisch Gladbach, Germany). 5x10 5 Monocytes were preincubated with 100 nM 9-cisRA for 1 h, followed by stimulation with 0.5 μg / ml LPS for 4 h for RNA or overnight for cytokines. Total RNA was extracted using the RNeasy® Plus Mimi Kit (Cat No. 74134, QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions. RNA and collected supernatants were stored at -80°C until further use.

[0113] Isolation of γδT17 cells and in vitro experiments

[0114] Pooled γ / δT17 cells from spleens of 8-10 week old B6 and Pinkie mice were isolated using a TCR γ / δ T cell isolation kit according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). To determine the effect of 9CisRA and monocyte conditioned medium on IL17 cytokine production, purified γδT17 cells were stimulated with anti-CD3 / CD28 Dynabeads (catalog no. 11452D, Life Technologies AS, Norway) alone or in combination with IL-23 (10 ng / ml, eBioscience), 9-cisRA (100 nM) or monocyte conditioned medium for 96 h for cytokine measurements.

[0115] IL-17 ELISA

[0116] Mouse IL17 was measured from cell culture supernatants after 96 h of incubation using a mouse IL-17 DuoSet enzyme-linked immunosorbent assay (ELISA) (R&D systems, Minneapolis, USA).

[0117] NanoString nCounter Gene Expression Analysis

[0118] This was performed by the Genomic and RNA Profiling Core at Baylor College of Medicine using the NanoString Technologies nCounter Gene Expression Mouse Myeloid Innate Immunity V2 Panel code set (NS_MM_Myeloid_V2.0), which contains 770 unique pairs of 35-50 bp reporter probes and biotin-labeled capture probes, including an internal reference control (NanoString, Seattle, WA), as previously described (Alam et al., 2021a). Data were analyzed using HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA) using ROSALIND® (https: / / rosalind.bio / ).

[0119] Bulk RNAseq and data analysis

[0120] Conjunctival epithelium was dissected from B6 and Pinkie lines, and total RNA was extracted using the QIAGEN RNeasy Plus Micro RNA Isolation Kit (Qiagen) according to the manufacturer's instructions. RNA concentration and purity were assessed using a NanoDrop 1000 (ThermoFisher Scientific, Waltham, MA). RNA-Seq was performed by Beijing Genomics Institute (BGI) using BGISEQ500RS to generate 100 bp paired-end reads. Raw data were cleaned by removing reads containing adapters or polyN sequences and low-quality reads using SOAPnuke (version 1.5.2, parameters: -l 15-q 0.2-n 0.05). The expression levels of the resulting genes and transcripts were determined using RSEM (version 2.2.5, default parameters). Detection of DEGs (differentially expressed genes) was performed using DEseq2 (parameters: fold change ≥ 2.00 and adjusted P value ≤ 0.05). A total of 19,511 genes were obtained as raw data. The genes were passed through the Benjamini-Hochberg procedure to obtain the critical value for false discovery, and a total of 1375 genes were passed with a P value > 0.0006. The selected genes of the IL-17 signaling pathway were clustered in a heat map.

[0121] ATAC Seq

[0122] Cultured bone marrow-derived monocytes were harvested and frozen in culture medium containing FBS and 5% DMSO. Cryopreserved cells were shipped to Active Motif (Carlsbad, CA) for ATAC-seq assays. Cells were then thawed in a 37°C water bath, pelleted, washed with cold PBS, and tagmented as previously described (Buenrostro et al., 2013) with some modifications. (Corces et al., 2017) Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzymes and buffers provided in the Nextera Library Prep Kit (Illumina, San Diego, CA). Tagmented DNA was then purified using the MinElute PCR Purification Kit (Qiagen, Germantown, MD), amplified by 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter, Brea, CA). The resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems, St Louis, MO) and sequenced by PE42 sequencing on a NextSeq 500 sequencer (Illumina).

[0123] Analysis of ATAC-seq data was similar to that of ChIP-Seq data. Reads were aligned using the BWA algorithm (mem mode; default settings). Duplicate reads were removed and only reads mapping as matched pairs and uniquely mapped reads (mapping quality >=1) were used for further analysis. Alignments were extended in silico at their 3' ends to a length of 200 bp and assigned to 32 nt bins along the genome. The resulting histogram (genomic "signal map") was saved in a bigWig file. Peaks (accessible regions) were identified using the MACS 2.1.0 algorithm with a cutoff of p-value 1e-7, without a control file, and with the -nomodel option. Peaks that were on the ENCODE blacklist of known false ChIP-Seq peaks were removed. Signal maps and peak positions were used as input data for Active Motifs' proprietary analysis program, and Excel tables were generated containing detailed information on sample comparisons, peak metrics, peak positions and gene annotations. For differential analysis, reads were counted in all combined peak regions (using Subread) and replicates for each condition were compared using DESeq2. The location and frequency of motif sequences in each peak region were identified using the search tool HOMER or known sequences in databases (Yan et al., 2020).

[0124] Qiagen Gene Pathway Analysis

[0125] Briefly, differentially expressed genes from single-cell RNAseq data were first uploaded to Qiagen's Ingenuity Pathway Analysis (IPA) system for core analysis. Analysis was performed with an experimental false discovery rate of ≥ 0.05. Comparative analysis tools were used to identify the most relevant canonical pathways that were enriched in Pinkie and presented as a heatmap. The IL17 signaling pathway was adopted from IPA with some modifications.

[0126] Generation of bone marrow chimeras

[0127] CD45.2 using bone marrow cells obtained from 12- to 16-week-old B6 and Pinkie strains + Bone marrow chimeras were generated using 6- to 8-week-old CD45.1 mice as previously reported. + The mice were generated in the Pepc / BoyJ strain (Gibson et al., 2015; Alam et al., 2021a). Ten days after bone marrow reconstitution, mice were subjected to 5 days of desiccation stress (DS5) and T cell populations in the conjunctiva were analyzed by flow cytometry.

[0128] Drought stress and IL-17 neutralization

[0129] DS was induced by inhibiting tear secretion with scopolamine hydrobromide (Greenpark, Houston) in drinking water (0.5 mg / mL) and housing mice in cages with perforated plastic screens on one side and exposed to airflow from a fan placed 6 inches in front of them for 16 h / day for 5 consecutive days. Room humidity was maintained at 20–30%. Control mice were not exposed to airflow and were maintained in a nonstressed (NS) environment at 50–75% relative humidity. Mice were treated i.p. every 2 days with 100 μg / mouse anti-IL-17A (Clone 17F3; BioXcell) or mouse IgG1 isotype control (Clone MOPC-21; BioXcell) beginning on day -2 for the duration of DS. Five days after DS, mice were euthanized and immune cells were harvested from the conjunctiva for flow cytometry (n=11), eyes were embedded in paraffin for sectioning (n=5) or embedded in optimum cutting temperature (OCT) compound (Thermofisher) for frozen sectioning (n=3), or corneas were prepared for whole mount immunostaining (n=3).

[0130] Immunofluorescence staining and confocal microscopy

[0131] Conjunctival and corneal tissue samples were dissected from female C57BL / 6J mice (16 weeks old) and fixed in 100% methanol for 20 min at -20°C, followed by washing with Hank's buffered saline (HBSS) for 3x5 min at room temperature (RT) with gentle shaking. Tissues were permeabilized with 0.4% Triton X-100 in HBSS for 30 min at RT with gentle shaking. 20% goat serum (Sigma, USA) diluted in HBSS was used for blocking for 1 h at RT. Conjunctival tissue samples were then incubated with the stated concentrations of primary antibodies (Table 2) diluted in 5% goat serum in HBSS overnight at 4°C in the dark with gentle shaking. The samples were then washed with 0.4% Triton X-100 for 3x6 min at RT with gentle shaking, followed by incubation with secondary antibodies (Table 2) diluted in 5% goat serum / HBSS for 1 h at RT with gentle shaking and light protection. The samples were then washed with 0.4% Triton X-100 in HBSS for 3x10 min, and Hoechst (1:500 in HBSS) was added for nuclear staining (30 min at RT and in the dark with gentle shaking). The samples were washed with HBSS for 3x5 min, mounted on slides, and flattened with a coverslip. Immunofluorescent staining in whole-mount conjunctival tissue samples was visualized using a laser-scanning Nikon confocal microscope (Nikon A1 RMP, Nikon, Melville, NY, USA) and a 0.5 μm Z-step. Images were processed using NIS Elements Advanced Research (AR) software version 4.20 (Nikon).

[0132] In situ zymography

[0133] In situ zymography was performed to investigate the localization of gelatinase activity in corneal cryosections using a method reported previously (De Paiva et al., 2006b). Sections were thawed and incubated overnight with reaction buffer, 0.05 M Tris HCl, 0.15 M NaCl, 5 mM CaCl2 and 0.2 mM NaN3, pH 7.6, containing 40 mg / ml FITC-labeled DQ gelatin, which was available in a gelatinase / collagenase assay kit (EnzChek, Thermofisher). As a negative control, FITC-labeled DQ gelatin was applied to the cryosections after adding 50 mM 1,10 phenanthroline, a metalloproteinase inhibitor, to the reaction buffer. Proteolysis of the FITC-labeled DQ gelatin substrate yields cleaved gelatin-FITC peptides that are fluorescent at the sites of net gelatinolytic activity. After incubation, sections were washed 3 times for 5 min in PBS, counterstained with Hoechst 33342 dye, and coverslipped. Areas of MMP gelatinolytic activity were viewed and imaged.

[0134] statistical analysis

[0135] Based on normality, parametric Student's T test or nonparametric Mann-Whitney U test was performed for statistical comparisons with an alpha of 0.05 using GraphPad Prism 9.0 software (GraphPad Software, Inc., San Diego, CA, USA). Example 3 - Keratoconjunctivitis develops in Pinkie strains with reduced Rxrα signaling

[0136] reported a Pinkie mouse strain carrying a loss-of-function RXRα mutation (I273N) that alters ligand binding and heterodimerization, resulting in a 90% reduction in ligand-induced transactivation, and develops dry eye symptoms with age (Du et al., 2005), but this study did not evaluate ocular surface disease and immunopathology (Du et al., 2005). Disruption of the corneal epithelial barrier, loss of conjunctival goblet cells, and increased expression of cornified membrane precursors by the surface epithelium are well-characterized pathological hallmarks of dry eye disease (De Paiva et al., 2006a; Corrales et al., 2011).

[0137] Corneal staining following topical application of 70 kDa Oregon Green Dextran (OGD) increases with corneal barrier disruption in dry eye. There is no difference in corneal OGD permeability between young (8 weeks old) Pinkies and wild type (WT) C57BL / 6 (B6), but OGD staining is significantly increased in 32 week old Pinkies (Figure 1A). Decreased conjunctival goblet cell numbers are another marker of dry eye. Pinkies have a significantly reduced number of PAS-positive conjunctival goblet cells at 8 weeks of age compared to the WT strain (Figure 1B). Enhanced immunoreactivity for the cornified membrane precursor SPRR2 in Pinkie conjunctival epithelium (Figure 1C, left) is accompanied by elevated expression of several Sprr isoform genes in the conjunctiva [Sprr2g (>20-fold), Sprr2f (>10-fold) and Sprr2a (>4-fold)] compared to B6 (Figure 1C, bottom right). Dryness of the ocular surface stimulates the recruitment of immune cells to the conjunctiva, and Pinkie found significantly higher numbers of CD45 + These findings indicate that Pinkie has dry eye-related pathological changes in the corneal and conjunctival epithelium and that corneal epithelial disease worsens with age. Example 4 - Pinkie has increased γδT17 cells in the conjunctiva

[0138] Inflammation is known to drive ocular surface epithelial disease in dry eye. Droplet-based single-cell RNA sequencing (scRNA-seq) was used as an unbiased approach to compare immune cell types in the conjunctiva of WT and Pinkie strains. CD45 + We constructed scRNA-seq libraries from immune cells (n=8 biological replicates / strain) and obtained transcriptome profiles of these cells using the 10x Genomics platform. Seurat v3 was used for scRNA-seq data analysis. After quality assessment, standard pre-processing filtering and doublet removal, a total of 11165 cells from B6 and 7096 cells from Pinkie with 2000 various characteristics were analyzed. Graph-based clustering using Seurat divided the cells into 19 clusters (Figure 2A) identified based on the expression of signature marker genes listed in Table 1 and shown in Figure 9. The top 20 differentially expressed genes in each cluster are listed in Table 3. The main difference between the two strains is the lower percentage of macrophages in cluster 0 and the elevated percentage of γδ T cells in cluster 2 (Figure 2B, Table 1). A heatmap of the top 50 differentially expressed genes between the strains is shown in Figure 2C. Il17a is the top differentially expressed gene. The violin plot in Figure 2D shows that gamma delta T cells and conventional T cells had the highest expression of both Il17a and IL17f in both lineages. Pinkie elevated the expression of IL17a and IL17f in conventional T cells and elevated the expression of IL17f in gamma delta T cells.

[0139] Significant differences between lineages were also observed in the expression of γδT17 signature genes, including Ltb ( Powolny-Budnicka et al., 2011 ), Cxcr6 ( Butcher et al., 2016 ), Rorc ( Powolny-Budnicka et al., 2011 ; Malhotra et al., 2013 ; Zuberbuehler et al., 2019 ), and Il1r1 ( Duan et al., 2010 ) ( Figure 2E and Figure 10 ).

[0140] Flow cytometry identifies γδ T cell receptor (TCR) negative and positive CD3 cells in Pinkie conjunctiva. + T cells and IL-17a + γδ TCR - and γδ TCR + γδ T cells show an increased percentage of IL-17 T cells in both lineages (Figure 3A). + Immunostaining of whole-mount conjunctiva revealed that total IL-17a + and RORγt + γδ TCR + We show an increase in the number of CXCL16 cells (Figure 3B). Minimal immunostaining for the chemokine CXCL16, a ligand for CXCR6 expressed by γδT17 cells (Butcher et al., 2016), is seen in B6 corneal epithelium, whereas strong staining is seen in Pinkie conjunctival epithelium, accompanied by elevated mRNA expression in the conjunctival epithelium (Figure 3C).

[0141] Increased concentrations of the γδT17 inducers IL-23 ( Mohn et al., 2020 ) and TNF-α, ( Lahn et al., 1998 ; Wu et al., 2014 ), as well as VEGF, a pro-angiogenic cytokine that promotes corneal neovascularization ( Suryawanshi et al., 2012 ) ( Li et al., 2011 ), are found in Pinkie tears ( Figure 3D ). Example 5-9-cisRA inhibits γδT17 activation and IL-23 production by monocytes.

[0142] Based on the finding of increased γδ T17 in Pinkie conjunctiva, 9-cis RA was evaluated to determine whether it suppresses IL-17 production by activated γδ T cells in culture. γδ T cells isolated from the spleen were stimulated with anti-CD3 / CD28 beads with or without IL-23 and / or 9-cis RA. IL-17A / F in the supernatants was measured by ELISA. IL-17 release was higher in Pinkie γδ T cells stimulated with beads or beads + IL-12 (Figure 4A). 9-cis RA significantly reduced supernatant IL-17 concentrations in cells from both lineages, but the suppressive effect was greater in B6 cells (74% vs. 46% in beads + IL-23 stimulated cells). The majority of myeloid cells in the conjunctiva express RXRα, which, when stimulated with LPS, produces factors known to stimulate IL-17 production by γδ T cells. (Alam et al., 2021b) compared the stimulatory activity of conditioned medium from LPS-treated monocytes with recombinant IL-23 in IL-17 production by γδ T cells and found them to be comparable (Figure 4A). Furthermore, treatment of LPS-stimulated cultured monocytes with 9-cis RA significantly reduced the stimulatory activity of their conditioned medium (Figure 4). Consistent with these findings, both genes encoding IL-23 heterodimers (Il23a and Il12b), as well as other γδ T17 inducing cytokines Il1α, Il1β and TNF-α, were significantly upregulated in LPS-stimulated cultured monocytes as measured by nanostring array (Figure 4B top), and these were found to be suppressed by adding 9-cis RA to the culture medium (Figure 4B bottom).

[0143] Retinoic acid is known to induce epigenetic changes that may affect transcription factor binding and gene transcription. (Bar-El Dadon and Reifen, 2017) ATAC seq was performed on cultured monocytes to determine whether 9-cis RA treatment alters the number of open transcription factor (TF) binding motifs in LPS-stimulated cultured monocytes. The PCA plot in Figure 4C shows a striking difference in the peak region sequences in the area of ​​open chromatin between control, LPS-treated and LPS + 9-cis RA-treated cells. LPS treatment significantly increased the number of TF motifs that regulate the transcription of inflammatory cytokines, including NFkB and Jun-AP-1 (Figure 4D, top left), but did not decrease the number of any known motifs (Figure 4D, bottom left). Compared to LPS treatment alone, 9-cis RA + LPS increased the number of five known motifs, including RAR:RXR Figure 4D (top right), and decreased the number of four motifs, including AP-1 Figure 4D (bottom right). AP-1 is a key transcription factor for Il23a and other inflammatory mediators (Liu et al., 2009).

[0144] Together, these findings indicate that RXRα suppresses the production of monocyte cytokines known to stimulate IL-17 production by activated γδ T cells and IL-17 production by γδ T17 cells. Example 6 - Differential pathway analysis reveals improved IL-17 signaling in Pinkie

[0145] The RXRα nuclear receptor regulates the expression of a series of inflammatory mediators. Using the QIAGEN Ingenuity Pathway Analysis (IPA) tool, we identified significant differences (p ≤ 0.05) between B6 and Pinkie in inflammatory signaling pathways generated from scRNAseq data. These pathways, grouped by lineage and cell type, are displayed in the heatmap shown in Figure 5A. The most significant differences were seen in neutrophils, myeloid (macrophages and monocytes) and cDC2 cells, including the IL-6, LPS-stimulated MAPK, NFkB, IL-17 and PPARα / RXRα signaling pathways, which contain mediators relevant to dry eye pathogenesis. PPARα / RXRα signaling was significantly reduced in MHCII-low macrophages and monocytes. Two other pathways, CDC42 and CDk5, are involved in NLRP3 inflammasome activation. (Muller et al., 2010; Cheng et al., 2020) The annotated IL-17 signaling pathway generated by IPA (Figure 5B) contains downstream signaling pathways (MAPK and NFkB) that stimulate the expression of γδT17-induced cytokines and IL-17-induced mediators (e.g., matrix metalloproteinases, SPRR2) involved in the development of corneal and conjunctival epithelial disease in dry eye.

[0146] The conjunctiva is a mucosal tissue composed of epithelial, stromal and immune cells that express IL-17 receptors and are potential IL-17 targets (McGeachy et al., 2019). To determine whether IL-17-related genes / pathways were increased throughout Pinkie's conjunctiva, we compared expression profiles generated from bulk RNA-seq performed on whole conjunctival lysates from B6 and Pinkie. Similar to scSeq performed on immune cells, IL-17f was found to be one of the top differentially expressed genes with increased expression in Pinkie, along with IL-17 receptors (IL-17rc, IL-17re) (Figure 4C). There is also increased expression of other IL-17 signaling pathway-related genes, including the cornified membrane precursor genes Sprr2g and Sprr2h (Lambert et al., 2017; Tian et al., 2021), p38 Mapks [Mapk12 (p38 gamma) and Mapk13 (p38 delta)] and the chemokine CCL6. A variety of other inflammatory mediators and signaling molecules (e.g., TLR3, TLR5) are also increased. Collectively, this data indicates that RXRα suppresses IL-17 production by γδ T cells and that IL-17 may have morphological effects on epithelial and immune cells of the ocular surface. Example 7 - IL-17 neutralization suppresses the development of ocular surface disease in Pinkie bone marrow chimeras exposed to desiccation stress

[0147] It was previously reported that IL-17 causes corneal barrier disruption by stimulating the expression of metalloproteinases (MMP-3 and MMP-9) that dissolve tight junction proteins in the apical corneal epithelium in mice subjected to experimental dry stress (DS) (De Paiva et al., 2009). In this study, mice treated with anti-IL-17 had significantly less barrier disruption and reduced MMP-9 expression, MMP-9 immunostaining, and gelatinase activity. In previously unpublished experiments, we also found that IL-17 neutralization prevented DS-induced conjunctival goblet cell loss (Figure 6A).

[0148] Bone marrow chimeras generated with Pinkie donor cells may result in greater ocular surface disease than those generated with B6 donor cells, because reduced RXRα signaling in Pinkie leads to increased infiltration of the conjunctiva with donor γδ T17 cells. Bone chimeras generated by previously reported methods (Alam et al., 2021a) and summarized in Figure 11 were exposed to DS for 5 days. (Alam et al., 2021a) Chimeric mice were treated with either anti-IL-17 or isotype control antibodies every 2 days starting 2 days before initiating DS. Five days after DS, the percentage of γδ T cells and IL-17+ cells in the conjunctiva was assessed by flow cytometry and measurements of dry eye disease including corneal MMP-9 immunoreactivity and gelatinase activity (in situ zymography) and conjunctival goblet cell counts. Pinkie donor chimeras had a higher percentage of γδ T cells and IL-17 + The percentage and MFI of γδ T cells were found to be higher. MMP-9 immunoreactivity was significantly lower in anti-IL-17 treated than in control treated Pinkie chimeras, and in situ gelatinase activity was lower in anti-IL17 treated Pinkie and B6 chimeras (Figure 6C). Anti-IL-17 treatment also reduced MMP-9 and SPPR2 immunostaining in Pinkie corneal epithelium and gelatinase activity in corneal epithelium of both strains (Figure 6C). Conjunctival goblet cell density was significantly higher in anti-IL-17 treated chimeras (Figure 6E).

[0149] Taken together, these data indicate that reduced RXRα signaling promotes the migration of γδT17 cells to the conjunctiva in dry eye, and that IL-17 produced by these cells drives corneal and conjunctival epithelial disease. Example 8 - Pinkie develops corneal neovascularization, opacification and ulceration with age.

[0150] The Pinkie line develops corneal opacity, neovascularization, and ulceration with age (Figure 7A). Corneal opacity and neovascularization were noted in 14% of 144 Pinkie eyes compared to only 2% of 100 B6 eyes. Gene expression profiles were compared in nanostring bone marrow innate immune arrays performed on whole corneal lysates prepared from 45-60 week old B6 or Pinkie with normal appearing corneas (NC) or from Pinkie with ulcerated corneas (UC). Violin plots show four genes with significantly elevated expression in Pinke NC compared to B6 NC (Figure 7B, top left). Significantly elevated expression of numerous genes is noted when comparing normal and ulcerated Pinkie corneas (Figure 7B, right). Among the genes significantly differentially expressed in Pinkie ulcerated corneas are IL-17 signaling pathway genes shown in the heatmap (Figure 7C). These include γδT17 inducers (i.e., Ltb, Tnf, Nfkb2, Relb) and Il17ra.

[0151] The expression levels of several factors that promote corneal angiogenesis (Vegfa, Fgf7) and ulceration (Mmp9) as measured by PCR are significantly elevated in Pinkie UC (Figure 7D). Interestingly, expression of Vegfb, which has trophic activity for corneal nerves, was decreased in Pinkie UC (Guaiquil et al., 2014). Consistent with these findings, immunoreactivity of blood and lymphatic endothelial markers CD31 / LYVE-1 and MMP-9 is elevated in the corneal epithelium of aged Pinkies compared to similarly aged B6s (Figure 7E). These findings, coupled with the chronic elevation of proangiogenic and proteolytic factors in Pinkies, suggest that dry eye promotes corneal angiogenesis, opacification, and ulceration. Example 9 - Mechanism of RXRα in dry eye disease

[0152] In this study, we investigated the mechanism of dry eye disease development in the Pinkie strain, which carries a loss-of-function RXRα gene mutation. Using scRNA-seq as an unbiased approach to investigate conjunctival immune cell populations, we found a four-fold higher percentage of conjunctival γδ T cells with higher expression of IL-17f and other γδ T17 signature genes. Sequencing findings are confirmed by flow cytometry and confocal microscopy showing that these cells are located in the stroma beneath the conjunctival epithelium. The Pinkie strain developed accelerated signs of dry eye disease in the cornea and conjunctiva. To determine the pathogenicity of Pinkie γδ T17 cells, we generated bone marrow chimeras using Pinkie donor cells and found significant reduction in corneal and conjunctival disease in the group that received an IL-17 neutralizing antibody.

[0153] IL-17 is involved in the pathogenesis of corneal epithelial disease in dry eye. IL-17 stimulates MMP expression by the corneal epithelium, as well as the recruitment and activation of neutrophils. (De Paiva et al., 2009; Marzano et al., 2019) MMP-9 disrupts the corneal epithelial barrier through dissolution of tight junction proteins in the apical epithelium, resulting in accelerated desquamation. (Pflugfelder et al., 2005) Loss of conjunctival goblet cells in dry eye may result from cytokine-mediated apoptosis or altered differentiation with capture of goblet cells by abnormally differentiated epithelium with elevated expression of keratinocyte precursors such as SPRR2 induced by IL-17.

[0154] Previously reported studies found that antibody neutralization of IL-17 significantly attenuated corneal barrier disruption as measured by OGD permeability in a desiccation stress model of dry eye. Concurrent with these studies, it was also found that anti-IL-17 prevented desiccation-induced conjunctival goblet cell loss. Studies reported by others have also shown that IL-17 produced by Th17 cells leads to corneal and conjunctival disease. (Chen J Immunol; Chauhan J Immunol 2009). IL-17 is primarily expressed in CD4+ cells, which are involved in the regulation of corneal and conjunctival disease. +In a previous study using a DS dry eye model, CD4 + Although IL-17 was detected in T cells, few have evaluated IL-17 production by conjunctival γδ T cells. Elevated expression of IL-17 was found in the conjunctival epithelium of patients with Sjögren's syndrome keratoconjunctivitis sicca, but the cellular source was not determined (Pflugfelder et al., 2015). γδ T cells are the second most abundant population of intraepithelial lymphocytes in the mouse conjunctiva (Zhang et al., 2012), and Coursey et al. reported that IL-17 is produced by γδ T cells in the lacrimal glands of the NOD mouse strain, which develops KCS and is used as a model for SS (Coursey et al., 2016). This study suggests that conjunctival γδ T cells are another source of IL-17 and that IL-17 expression in these cells is regulated by the RXRα nuclear receptor. γδ T cells are found at many mucosal surfaces and can be activated in a non-antigen-specific manner by a variety of PAMPs and in response to desiccation stress, presumably activating the same signaling pathways as microbial products ( Hedges et al., 2005 ).

[0155] The RXR nuclear receptor family regulates the transcription of numerous genes involved in immune function, cell differentiation and homeostasis. RXRα can function as a homodimer or heterodimer with partner receptors (PPARγ and vitamin D receptor) that have been found on the ocular surface. (Nien et al., 2010; Panigrahi et al., 2021) The ocular surface is a retinoid-rich environment. (Alam et al., 2021b) In addition to the retinol form of vitamin A in tears, which is converted to the natural ligand 9-cis RA by aldehyde dehydrogenase in bone marrow and epithelial cells on the ocular surface (Xiao et al., 2018), nutritional ligands such as vitamin D, the omega-3 fatty acid DHA in fish oil and oleic acid in olive oil can bind to specific RXR dimer partners. (Alam et al., 2020a)

[0156] The majority of CD11b+ myeloid cells are RXRα positive and respond to retinoic acid. (Alam et al., 2021b) The finding of increased IL-17-producing γδ T cells in the Pinkie lineage indicates that RXRα is also a key regulator of IL-17 production by γδ T cells. The synthetic retinoid AM80 was found to suppress IL-17 production by γδ T cells stimulated with anti-CD28 antibodies and a cytokine cocktail of IL-23 and IL-1β. (Mohn et al., 2020) 9-cis RA suppresses IL-17 production by >70% in cultured γδ T cells stimulated with CD28 beads or beads + IL-23. In addition to direct suppression of γδ T cells, 9-cis RA has also been found to suppress the expression of γδ T17 inducers (IL-23, IL-1, TNF-α) by cultured monocytes, and it was previously reported that the levels of IL-1β and IL-23β in the supernatants of 9-cis RA-treated monocytes were reduced (Alam et al., 2021b). Monocyte-conditioned medium has comparable stimulatory activity to recombinant IL-23, but this was significantly reduced in monocytes cultured with 9-cis RA. 9-cis RA was also found to reduce the number of open AP-1 transcription factor binding motifs detected by ATAC-seq. Both the AP-1 and NFkB pathways are involved in stimulated IL-17 expression by γδ T cells. (Powolny-Budnicka et al., 2011; Criado et al., 2014) Figure 8 summarizes the primary and secondary suppressive activity of 9-cis RA on IL-17 production by γδ T cells.

[0157] Because it is difficult to obtain sufficient numbers of donor cells from the cornea, we performed single-cell profiling on conjunctival immune cells. Corneal pathology may be due to IL-17 produced by conjunctival γδ T cells, which have been shown to infiltrate the cornea after epithelial trauma (Li et al., 2007; Li et al., 2011). We performed ATAC-seq on monocytes, which demonstrated the epigenetic effect that 9-cis RA has on these cells. The finding that RXRα suppresses IL-17 production by γδ T cells provides a rationale for evaluating the epigenetic activity of 9-cis RA on these cells in the future.

[0158] Findings indicate that RXRα retinoid signaling suppresses activation and IL-17 production by moused conjunctival γδ T cells under homeostatic conditions. This signaling may, in some embodiments, be reduced in aqueous deficient dry eye due to reduced secretion of retinol into tears by dysfunctional lacrimal glands. Furthermore, in some embodiments, there may be reduced expression of aldehyde dehydrogenase in the conjunctiva of dry eye, which may result in reduced synthesis of RA. Strategies that maintain the ocular surface retinoid axis in dry eye may, in some embodiments, prevent IL-17-induced epithelial lesions. [Table 1] [Table 2] [Table 3] JPEG2025504988000005.jpg100157 Example 10 - Example of the effect of RXRa agonists

[0159] This example shows the inhibitory effect of an example RXRa agonist on IL-17 production by cultured γδ T cells, as well as differences in immune cell and gene expression between wild-type C57BL / 6 and Pinkie mice. As in the conjunctiva, there was an increase in IL-17-producing γδ T cells, indicating the importance of RXRa in suppressing this important proinflammatory cytokine.

[0160] Figure 12 shows IL-17A / F cytokine concentrations measured in the supernatants of cultured murine γδ T cells isolated with magnetic beads from C57BL / 6 spleens. Cells are stimulated with anti-CD3 / CD28 beads + recombinant IL-23 without or with the addition of RXRα agonist 9-cisRA (9-cis), bexarotene (Bexa), omega-3 fatty acid DHA, fatty acid oleic acid (OA) and honokiol (Hono) at concentrations of 10 nM and 100 nM. The RXRα antagonist HX531 blocked the inhibitory effect of all agonists but had no effect on the inhibitory activity of the RAR agonist all trans retinoic acid (ATRA). Figure 13 shows the CD45 T cell cytoplasmic level measured using Seurat package v3. + We provide UMAPs of 14 distinct immune cell clusters in corneas from C57B / 6 (B6) and Pinkie, generated from single-cell transcriptome profiles of cells. Figure 14 shows a heatmap comparing the percentages of 14 cell types in B6 and Pinkie corneas. The largest changes are an increase in γδ T cells and neutrophils, and a decrease in macrophages (MPs) in Pinkie corneas. Figure 15 shows a volcano plot comparing the levels of differentially expressed genes in corneas of B6 and Pinkie strains. Gene expression was assessed using mouse bone marrow innate immune nanostring arrays. Solid vertical lines indicate changes less than or greater than 0.5 log2 fold. Red dots are cells that were significantly increased (right) or decreased (left). IL-17a and IL-17f are among the significantly increased genes.

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[0162] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods described herein and in the steps of the methods or in the sequence of the steps of the present invention without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that the agents described herein can be replaced with specific agents that are chemically related and physiologically related while still achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. 1. A therapeutic composition for use in a method for treating or preventing an eye disorder or reducing the risk of having an eye disorder in an individual, the therapeutic composition comprising an effective amount of one or more retinoid X receptor (RXR) agonists.

2. The therapeutic composition of claim 1 , wherein the RXR agonist is an RXRα agonist.

3. 3. The therapeutic composition of claim 1 or 2, wherein the RXR agonist comprises 9-cis retinoic acid, oleic acid, omega-3 docosahexaenoic acid, vitamin D, bexarotene, taxerotene, honokiol, AM80, rosiglitazone, dorupanin, garcinic acid, NEt-3IB, or a combination thereof.

4. The therapeutic composition of any one of claims 1 to 3, which is administered to one or both eyes of the individual.

5. The therapeutic composition of claim 4 administered as eye drops.

6. The therapeutic composition of claim 5 , wherein the eye drops comprise microdrops.

7. The therapeutic composition of any one of claims 1 to 4, which is administered as an ointment or cream.

8. The therapeutic composition of any one of claims 1 to 4, which is administered by subconjunctival injection.

9. 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 9, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 , 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 2 03, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 23 4, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,２６６、２６７、２６８、２６９、２７０、２７１、２７２、２７３、２７４、２７５、２７６、２７７、２７８、２７９、２８０、２８１、２８２、２８３、２８４、２８５、２８６、２８７、２８８、２８９、２９０、２９１、２９２、２９３、２９４、２９５、２９６、２９７、２９８、２９９、３００、３０１、３０２、３０３、３０４、３０５、３０６、３０７、３０８、３０９、３１０、３１１、３１２、３１３、３１４、３１５、３１６、３１７、３１８、３１９、３２０、３２１、３２２、３２３、３２４、３２５、３２６、３２７、３２８、３２９、３３０、３３１、３３２、３３３、３３４、３３５、３３６、３３７、３３８、３３９、３４０、３４１、３４２、３４３、３４４、３４５、３４６、３４７、３４８、３４９、３５０、３５１、３５２、３５３、３５４、３５５、３５６、３５７、３５８、３５９、３６０、３６１、３６２、３６３、３６４、３６５、３６６、３６７、３６８、３６９、３７０、３７１、３７２、３７３、３７４、３７５、３７６、３７７、３７８、３７９、３８０、３８１、３８２、３８３、３８４、３８５、３８６、３８７、３８８、３８９、３９０、３９１、３９２、３９３、３９４、３９５、３９６、３９７、３９８、３９９、４００、４０１、４０２、４０３、４０４、４０５、４０６、４０７、４０８、４０９、４１０、４１１、４１２、４１３、４１４、４１５、４１６、４１７、４１８、４１９、４２０、４２１、４２２、４２３、４２４、４２５、４２６、４２７、４２８、４２９、４３０、４３１、４３２、４３３、４３４、４３５、４３６、４３７、４３８、４３９、４４０、４４１、４４２、４４３、４４４、４４５、４４６、４４７、４４８、４４９、４５０、４５１、４５２、４５３、４５４、４５５、４５６、４５７、４５８、４５９、４６０、４６１、４６２、４６３、４６４、４６５、４６６、４６７、４６８、４６９、４７０、４７１、４７２、４７３、４７４、４７５、４７６、４７７、４７８、４７９、４８０、４８１、４８２、４８３、４８４、４８５、４８６、４８７、４８８、４８９、４９０、４９１、４９２、４９３、４９４、４９５、４９６、４９７、４９８、４９９、５００、５０１、５０２、５０３、５０４、５０５、５０６、５０７、５０８、５０９、５１０、５１１、５１２、５１３、５１４、５１５、516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546 , 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 100 9. The therapeutic composition of any one of claims 1 to 8, comprising 0, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900 or 5000 ng or μg of an RXR agonist.

10. 10. The therapeutic composition of any one of claims 1 to 9, wherein the individual has dry eye disease, Sjogren's syndrome, meibomian gland disease, an unstable tear film, tear dysfunction or an ocular surface inflammatory condition, vitamin A deficiency, chemical corneal injury, thermal corneal injury, corneal inflammation following bacterial, fungal or viral infection, corneal neovascularization, or a combination thereof.

11. The therapeutic composition of any one of claims 1 to 10, wherein the composition is administered once daily.

12. The therapeutic composition of any one of claims 1 to 10, wherein the composition is administered multiple times.

13. 13. The therapeutic composition of claim 12, wherein the composition is administered 1, 2, 3, 4 or more times daily.

14. The therapeutic composition of any one of claims 1 to 13, wherein the composition is administered in conjunction with another treatment.

15. 15. The therapeutic composition of claim 14, wherein the composition is administered in conjunction with a corticosteroid, cyclosporin A, tetracycline, lifitegrast, or a combination thereof.

16. 16. The therapeutic composition of claim 15, wherein the tetracycline comprises minocycline and / or doxycycline.

17. The therapeutic composition of any one of claims 1 to 16, wherein the individual has a condition that predisposes them to the eye disorder.

18. 18. The therapeutic composition of claim 17, wherein the condition comprises corneal inflammation, Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, lupus erythematosus, systemic sclerosis, graft-versus-host disease, and / or Stevens-Johnson syndrome.

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