阅读说明：本技术 调节神经炎症 (Modulating neuroinflammation ) 是由 D.F.陈 K-S.乔 季敏 于 2020-03-03 设计创作，主要内容包括：用于治疗神经炎症以及神经变性和/或降低神经炎症以及神经变性的发展或进展的风险的组合物与方法,其包含HSP60,例如用于鼻部施用,和IGFBPL1,例如用于鼻部、全身、或眼部(例如玻璃体内)施用。(Compositions and methods for treating and/or reducing the risk of development or progression of neuroinflammation and neurodegeneration comprising HSP60, e.g., for nasal administration, and IGFBPL1, e.g., for nasal, systemic, or ocular (e.g., intravitreal) administration.)

1. A method for treating and/or reducing the risk of development or progression of neuroinflammation and neurodegeneration, comprising administering to a subject in need thereof a therapeutically effective amount of one or more of: (i) HSP60 or HSP27, or an active fragment thereof; and/or (iii) IGFBPL or an active fragment thereof.

2. The method of claim 1, wherein the therapeutically effective amount is sufficient to reduce inflammation and neuronal cell death in the subject.

3. The method of claim 1 or 2, comprising nasal or subcutaneous administration of HSP60 and systemic or ocular administration of IGFBPL.

4. The method of claim 3, wherein ocular administration of IGFBPL comprises intravitreal injection.

5. The method of claims 1-4, wherein the subject has non-arterial ischemic optic neuropathy (NAION), glaucoma, autism, multiple sclerosis, alzheimer's disease, parkinson's disease, ischemic retinopathy, age-related macular degeneration, stroke, ischemic and traumatic optic neuropathy, or diabetic retinopathy.

6. The method of any one of claims 1-5, wherein the method reduces inflammation and neuronal death in the eye of the subject.

7. A kit, comprising: compositions comprising HSP60 and/or HSP27, and compositions comprising IGFBPL1, for use in the methods described herein.

8. A composition comprising HSP60 and/or HSP27, and/or a composition comprising IGFBPL1 for use in a method of treating and/or reducing the risk of development or progression of neuroinflammation and neurodegeneration.

9. The kit of claim 7 or composition for use of claim 8, wherein the HSP60 and/or HSP27 is formulated for nasal administration and the IGFBPL1 is formulated for ocular administration, e.g., intravitreal administration.

10. The kit of claim 7 or composition for use of claim 8, wherein the HSP60 and/or HSP27 are formulated for nasal or subcutaneous administration and the IGFBPL1 is formulated for systemic administration.

11. The kit of claim 7 or composition for use of claim 8, wherein the HSP60 and/or HSP27 are formulated for nasal or subcutaneous administration and the IGFBPL1 is formulated for nasal administration.

12. The kit for use or composition of claim 11, wherein one, two or all three of the HSP60 and/or HSP27 and IGFBPL1 are formulated together in a single composition for nasal or subcutaneous administration.

Technical Field

Described herein are compositions and methods for treating and/or reducing the risk of development or progression of neuroinflammation and neurodegeneration, including HSP60 and/or HSP27, e.g., for nasal administration, and IGFBPL1, e.g., for nasal, systemic, or ocular, e.g., intravitreal, administration.

Background

Glaucoma is a leading cause of blindness and an unresolved medical challenge worldwide.

Summary of The Invention

Provided herein are methods for treating and/or reducing the risk of development or progression of neuroinflammation and neurodegeneration in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of one or more of: (i) HSP60 and/or HSP27, or an active fragment thereof; and/or (ii) IGFBPL or an active fragment thereof.

In some embodiments, the therapeutically effective amount is sufficient to reduce inflammation and neuronal cell death in the subject.

In some embodiments, the methods include nasal administration of HSP60 and/or HSP27 and systemic (e.g., nasal or oral) or ocular administration of IGFBPL.

In some embodiments, ocular administration of IGFBPL comprises intravitreal injection.

In some embodiments, the subject has glaucoma, autism, multiple sclerosis, alzheimer's disease, parkinson's disease, ischemic retinopathy, age-related macular degeneration, stroke, ischemic and traumatic optic neuropathy, or diabetic retinopathy.

In some embodiments, the method reduces inflammation and neuronal death in the eye of the subject.

In some embodiments, the method reduces inflammation and neuronal death in the brain or spinal cord of the subject.

Also provided herein are kits comprising a composition comprising HSP60 and/or HSP27 and a composition comprising IGFBPL1 for use in the methods described herein.

Further, provided herein are compositions comprising HSP60 and/or HSP27, and/or compositions comprising IGFBPL1, for use in methods of treating and/or reducing the risk of development or progression of neuroinflammation and neurodegeneration.

In some embodiments, HSP60 and/or HSP27 is formulated for nasal administration and IGFBPL1 is formulated for ocular administration, e.g., intravitreal administration. In some embodiments, HSP60 and/or HSP27 is formulated for nasal administration and IGFBPL1 is formulated for systemic administration, e.g., oral or nasal administration. In some embodiments, HSP60 and/or HSP27 is formulated for nasal administration and IGFBPL1 is formulated for nasal administration. In some embodiments, one, two, or all three of HSP60 and/or HSP27 and IGFBPL1 are formulated together in a single composition for nasal administration.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Drawings

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

Figure 1 Microbead (MB) -induced elevation of intraocular pressure (IOP) was not affected by treatment with nasal spray Heat Shock Protein (HSP) 60. Left panel: saline and MB filled anterior chamber of mouse eye. Right panel: IOP levels over time in untreated saline-injected control mice (light grey), saline-treated MB-injected mice (grey) and HSP 60-treated MB-injected mice (black).

Figure 2 Treg cells were induced in mice receiving nasal spray of HSP 60. The upper diagram: CD4 from draining lymph node of eye (upper cervical lymph node)⁺Real-time gating of cells. The following figures: histograms of Treg + counts (CD4+ CD25+ FOXP3+) 2 weeks after MB injection, HSP 60-treated and saline-treated mice were compared.

Figures 3A-c. rescue of vision in MB-induced glaucoma mice by HSP60 nasal spray. A-c. visual contrast sensitivity (CS,3A), visual acuity (VA,3B) and amplitude (3C) of positive scotopic threshold response (pSTR) evaluated at 2, 4 and 6 weeks after IOP elevation in all groups P < 0.001P < 0.01P < 0.05.

Figures 4A-b Retinal Ganglion Cells (RGCs) and axons were rescued in MB-induced glaucoma mice by HSP60 nasal spray. Quantification of retinal ganglion cells (RGCS; immunohistochemistry using Brn3a, 4A) and axons (4B) (. p.0.001. P. < 0.01. P. < 0.05) at 2, 4 and 6 weeks after IOP elevation in all groups.

FIG. 5 is a schematic diagram of the hypothetical mechanism of HSP60 nasal spray.

IGFBPL1 protects RGCs from elevated IOP-induced damage and prevents loss of RGC function and vision in glaucoma models. A. IOP levels in sham surgery (triangles) or in mice injected with microbeads receiving intravitreal saline (squares) or IGFBPL1 (circles). B. InitialAnd RGC counts in sham injected mice and MB injected mice receiving saline or IGFBPL1 treatment. C. Quantification of ERG positive scotopic threshold response (pSTR) in saline and IGFBPL 1-treated mice showed significant improvement in RGC function in IGFBPL 1-treated mice compared to saline-treated mice. The okr test showed a significant improvement in Visual Acuity (VA) and visual Contrast Sensitivity (CS) of IGFBPL 1-treated group compared to saline-treated group, as determined 2-8 weeks after MB injection.

Microglial expression of IGFBPL1, IGF-1 receptor (IGF-1R), and IGF-1. A. Dual immunolabeling of microglia against microglia markers Iba-1 (green) and IGFBPL1 (red) in culture and in retinal whole mount counterstained with nuclear marker DAPI (blue). B. The microglial markers Iba-1 (green) and IGF-1R (red) or IGF-1 (red) were doubly immunolabeled in a flat-packed sheet of adult mouse retina.

Igfbpl1 inhibits microglial activation in the retina of glaucoma mice. Counts of activated microglia in retinal whole-discs of naive (normal), microbead-saline injection (MB + saline) and microbead-IGFBPL 1 injection (MB + IGFBPL1) mice 5 to 14 days after iop elevation. Qpcr results show that induction of activated microglia markers and inhibition of activated microglia markers by IGFBPL1 administration following IOP elevation.

Figure 9.IGFBPL1 inhibits pro-inflammatory cytokine production in glaucomatous retina. The results of qPCR show that the induction of activated pro-inflammatory cytokines after IOP elevation and their inhibition by IGFBPL1 administration.

Igfbpl1 deficiency results in microglial activation in adult retinas. A. Quantification of activated microglia in retinal whole-mount. Qpcr results showed increased levels of activated microglia markers in IBKO retinas compared to WT retinas.

FIG. 11 progressive RGC loss in IGFBPL 1-deficient mice. Quantification of RGC density shows gradual loss of RGC from 4 weeks to 7 months old IBKO mice.

FIG. 12 IGFBPL1 inhibits LPS-induced inflammation. Quantification of proinflammatory cytokine levels in isolated microglia cell cultures treated with control, IGFBPL1, LPS, and LPS + IGFBPL 1.

FIG. 13 protection of RGCs from ischemia-reperfusion-induced death and functional impairment. Data are presented as mean ± SD (5 mice per group). By student's t-test, p < 0.05.

Detailed Description

Described herein are methods for treating and reducing the risk of development or progression of neuroinflammation and neurodegeneration. The method includes one or more of: (i) administration of HSP60 or HSP27, or an active fragment thereof; and/or (ii) administration of IGFBPL or an active fragment thereof. The methods can be used to reduce inflammation and neuronal cell death in the eye and other sites, including the CNS and PNS. The methods may include nasal administration of HSP60 or HSP27 and nasal, systemic or ocular administration of IGFBPL, such as intravitreal injection or ocular surface (topocal), e.g. for the treatment of glaucoma.

HSP60(HSPD1, Heat shock protein family D (Hsp60) member 1)

Emerging evidence suggests an autoimmune mechanism in glaucoma, but its relative importance in disease pathogenesis has not been demonstrated. As shown herein, neuronal and vision loss and other immune related conditions in glaucoma are associated with pre-existing memory T cells that are pre-primed early in life by exposure to the bacterium HSP 60. Revealing the immune mechanism and its association with commensal microorganisms in the progressive neurodegeneration in glaucoma, providing a basis for new diagnostic treatments.

The amino acid sequence of human hsp60 is provided in GenBank accession No. NP _002147.2, and the amino acid sequence of bacterial hsp60 is provided in GenBank accession No. WP _000729117.1, incorporated herein by reference. HSP60 is preferably formulated for nasal administration to induce tolerance to HSP 60. Alternatively, oral or subcutaneous administration may be used. See also WO 2012118863.

HSP27 (Heat shock protein family B (small) member 1(HSPB1))

HSP27, also known as HSPB1, is shown herein to directly induce a pro-inflammatory response in HMC3 cells. The amino acid sequence of human HSP27 is provided in GenBank accession No. NP _001531.1, incorporated herein by reference. HSP27 is preferably formulated for nasal administration to induce tolerance to HSP 27. Alternatively, oral or subcutaneous administration may be used. See also WO 2012118863.

IGFBPL (insulin-like growth factor binding protein-like 1)

Insulin growth factor binding protein-like 1(IGFBPL1) promotes survival and neurite outgrowth of neonatal mouse Retinal Ganglion Cells (RGCs), which are regulated via insulin-like growth factor 1-mediated signaling pathways (Guo et al, Sci Rep.2018Feb 1; 8(1): 2054). As shown herein, IGFBPL1 is active in inhibiting neuroinflammatory microglia in adult/post-neonatal animals. The amino acid sequence of human IGFBPL1 is provided in GenBank accession No. NP _001007564, incorporated herein by reference. See also WO 2012118796.

Methods of treatment and prevention

The compositions described herein can be administered to a subject to treat or prevent a disorder associated with an abnormal or unwanted immune response, such as a neuroinflammatory or neurodegenerative disorder associated with excessive or abnormal activation of microglia. Examples of such disorders include, but are not limited to, non-arterial ischemic optic neuropathy (NAION), autism, multiple sclerosis, alzheimer's disease, parkinson's disease, ischemic retinopathy, glaucoma, age-related macular degeneration, stroke, ischemic and traumatic optic neuropathy, and diabetic retinopathy; in some embodiments, the disease is associated with loss of vision and/or elevated intraocular pressure. The methods may be used to treat subjects suffering from these diseases, for example, to reduce the risk of or treat vision and neuronal loss associated with these diseases. See also US8/198,284; WO/2017/213504; WO 2012118863; and WO2012118796, which is incorporated herein by reference in its entirety.

The methods of treatment or prevention described herein may comprise nasal or subcutaneous administration of an HSP60 or HSP27 composition to a subject, e.g., sufficient to stimulate the mucosal immune system. In some embodiments, the methods comprise administering a nasal HSP60 or HSP27 composition sufficient to increase regulatory T cell levels, e.g., by about 50%, 75%, 100%, 200%, 300% or more from baseline.

In some embodiments, the methods comprise administering nasal or subcutaneous HSP60 or HSP27 composition and/or IGFBPL1 composition in an amount sufficient to produce an improvement in one or more clinical markers of vision loss (e.g., decreased visual acuity) or disability; for example, in multiple sclerosis, such markers may include gadolinium-enhanced lesions visualized by MRI, or MRI criteria of patey, Fazekas or Barkhof, or diagnostic criteria of McDonald. IGFBPL1 compositions can be administered nasally, systemically, or ocularly, for example, by eye drops or intravitreally.

In some embodiments, the treatment is administered to a subject who has been diagnosed with a disorder associated with microglial activation; such diagnosis can be made by a skilled practitioner using known methods and ordinary techniques. In some embodiments, the method comprises the step of diagnosing or identifying or selecting a subject having a disorder associated with microglia activation, or identifying or selecting a subject based on the presence or diagnosis of a disorder associated with microglia activation. In some embodiments, the subject is an adult, e.g., a human at least 18 years of age, or a human after a newborn, e.g., a human at least 6 months or 1 year of age.

Pharmaceutical compositions and methods of administration

The methods described herein include the use of pharmaceutical compositions comprising one or more of HSP60, HSP2, or IGFBPL1 as an active ingredient.

The pharmaceutical composition typically includes a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

The pharmaceutical compositions are generally formulated to be compatible with their intended route of administration.

Examples of routes of administration include systemic parenteral, e.g., intravenous, intraperitoneal, intradermal, or subcutaneous; ocular local, e.g., topical (topocal), intravitreal, intraocular, intraorbital, periorbital, subconjunctival, subretinal, sub-tenon's space (subcornens), or transscleral; and systemic oral administration. In some embodiments, intraocular administration may be used or administration via eye drops, ointments, creams, gels, lotions, or the like. In some embodiments, the composition is administered systemically, e.g., orally; in preferred embodiments, the composition is administered to the eye, for example via topical (eye drops, lotions or ointments) or by local injection, for example periocular or intravitreal injection; see, e.g., Gaudana et al, AAPS J.12(3): 348-; fischer et al, Eur J Ophthalmol.21suppl 6: S20-6 (2011). Administration can be provided as periodic bolus injections (e.g., intravitreal or intravenous) or continuous infusions from internal storage (reservoir) (e.g., from implants disposed in intraocular or external locations (see, U.S. Pat. nos. 5,443,505 and 5,766,242)) or from external storage (e.g., from intravenous bags or contact lens slow release formulation systems). The compositions may be administered topically, for example, by continuous release from a sustained release drug delivery device affixed to the inner wall of the eye or via targeted transscleral controlled release into the choroid (see, e.g., PCT/US00/00207, PCT/US02/14279, PCT/US2004/004625, Ambat et al (2000) invest.Ophthalmol. Vis.Sci.41: 1181. and Ambat et al (2000) invest.Ophthalmol. Vis.Sci.41: 1186. 1191). A variety of devices suitable for topical administration of agents to the interior of the eye are known in the art. See, for example, U.S. Pat. Nos. 6,251,090, 6,299,895, 6,416,777, 6,413,540, and 6,375,972, and PCT/US 00/28187.

The pharmaceutical compositions are generally formulated to be compatible with their intended route of administration. Examples of routes of administration include systemic (e.g., parenteral, nasal, subcutaneous, and oral) and topical (ocular, e.g., intravitreal or ocular surface) administration. Thus also within the scope of the present disclosure are compositions, e.g., eye drops, lotions, creams, e.g., comprising microcapsules, microemulsions or nanoparticles, comprising the compositions described herein in a formulation for ocular administration. Methods of formulating suitable Pharmaceutical compositions for ocular delivery are known in the art, see, e.g., Losa et al, Pharmaceutical Research 10:1(80-87 (1993); Gasco et al, J.Pharma Biomed anal, 7(4):433-439 (1989); Fischer et al, Eur J Ophthalmol.21suppl 6: S20-6 (2011); and Tangri and Khurana, Intl J Res Pharma Biomed Sci, 2(4):1541-1442 (2011).

General methods of formulating suitable pharmaceutical compositions are known in The art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and Series books Drugs and the Pharmaceutical Sciences a Series of Textbooks and monograms (Dekker, NY). For example, a solution or suspension for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents such as water for injection, saline solution, fixed oils (fixed oils), polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral formulations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^TM(BASF, Parsippany, NJ) or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should have a degree of fluidity that allows for easy syringability. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be obtained by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Can be prepared by including in the composition an agent that delays absorption (e.g., a mono-ester Aluminum stearate and gelatin) to achieve prolonged absorption of the injectable composition.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any other desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions typically include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compounds can be mixed with excipients and used in the form of tablets, dragees or capsules (e.g., gelatin capsules). Fluid carriers can also be used to prepare oral compositions for use as mouthwashes. Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds may be delivered in the form of an aerosol spray from a pressurized container or a dispenser or nebulizer containing a suitable propellant, e.g., a gas such as carbon dioxide. Such methods include those described in U.S. patent No. 6,468,798.

Systemic administration of the therapeutic compounds described herein may also be performed by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as is well known in the art.

Pharmaceutical compositions may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the therapeutic compound is prepared with a carrier that will protect the therapeutic compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Such formulations can be prepared using standard techniques, or obtained from commercial sources (e.g., from Alza Corporation and Nova Pharmaceuticals, inc.). Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

The pharmaceutical composition may be contained in a container, package or dispenser together with instructions for administration.

Reagent kit

Also provided herein are kits for use in the methods described herein. For example, a kit can include a composition comprising HSP60 or HSP27, e.g., for nasal administration, and a composition comprising IGFBPL1, e.g., for nasal, systemic (e.g., oral) or ocular (e.g., ocular surface or intravitreal) administration. Instructions for use may also be included in the kit.

Examples

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1 immune tolerance to HSP60 attenuates neurodegeneration in a mouse model of glaucoma

Primary Open Angle Glaucoma (POAG), a disease that damages the optic nerve, is a leading cause of blindness worldwide. Although POAG is primarily associated with high intraocular pressure (IOP), therapies affecting IOP do not completely prevent vision loss and blindness.^1,2

Elevated intraocular pressure (IOP) induces a T cell-mediated autoimmune response to HSP60, and HSP-specific T cell responses and neuronal loss are eliminated following elevated intraocular pressure in mice raised in the absence of a microbiota. ^3-9This example tests whether induction of tolerance to HSP60 would attenuate glaucomatous damage.

Immune tolerance to HSP60 was induced in male and female C57BL/6J mice 6-8 weeks old by administration of low doses of HSP60 (2 uM daily HSP60 for 7 days) into the nostrils. Control mice were treated with saline. Glaucoma is induced by injecting Microbeads (MB) twice into the anterior chamber, thereby maintaining IOP elevated for 8 weeks. IOP was monitored weekly. Visual function was assessed by Optokinetic Motor Response (OMR) and electroretinogram scotopic threshold response (pSTR). Mice were sacrificed at 2, 4 and 8 weeks. Immune responses and tolerance of T cells to HSP60 were analyzed by Fluorescence Activated Cell Sorting (FACS). Glaucomatous nerve damage was quantified by Retinal Ganglion Cell (RGC) and axon counts.

By 4 weeks, MB was injected into the eye to reveal IOP of 20.1 + -0.56 mmHg or higher, compared to 11.6 + -0.21 mmHg in the contralateral uninjected eye (FIG. 1). As shown by FACS analysis, nasal administration of low doses of HSP60 induced immune tolerance and increased Treg levels (fig. 2). We observed no significant difference in IOP levels between HSP60 or saline treated mice. Treatment with HSP60 did not change Visual Acuity (VA), Contrast Sensitivity (CS) or pSTR prior to MB injection compared to saline-treated mice. However, at all time points after MB injection, HSP60 treated mice exhibited significantly higher VA and CS than saline treated mice as assessed by OMR (fig. 3A-C). Consistently, RGC function assessed by pSTR was also significantly improved in HSP 60-treated mice compared to saline-treated non-immune-tolerant mice at all time points after MB injection (fig. 4A-B).

These results indicate that immune tolerance to HSP60 attenuates glaucomatous RGC loss and loss of function in mice, as shown in figure 5.

Example 2 Effect of insulin-like growth factor binding protein-like protein 1 in microglia in adult mice

Insulin-like growth factor binding protein-like protein 1(IGFBPL1) plays a crucial role in promoting axonal growth and survival of Retinal Ganglion Cells (RGCs) during development (Guo et al, Sci Rep.2018Feb 1; 8(1): 2054). This requires the presence of insulin-like growth factor 1(IGF1) and is mediated through IGF1 receptor (IGF 1R). Since adult RGCs are known to express low or undetectable levels of IGF1R, this example discusses whether IGFBPL1 supports RGC survival following injury in the adult retina.

The expression of IGF1, IGFBPL1 and IGF1R in adult retinas was examined in retinal whole-tablets by using immunohistochemistry. Double immuno-labeling was performed with a primary anti-retinal whole-mount against the RGC marker Brn-3 or the microglia marker Iba-1 to identify cell type specific expression of IGF1, IGF1R and IGFBPL 1.

Purified microglia cells isolated from post-natal day 5 Cx3CR1/GFP mice and RGCs were co-cultured in the presence or absence of LPS and/or IGFBPL1 and IGF 1. Neuronal survival was determined by the live/dead viability kit and the percentage of live RGCs was quantified using Image J software.

The results showed that IGF1, IGF1R and IGFBPL1 were expressed by microglia rather than RGCs in adult mouse retinas (FIGS. 6A-B). Addition of IGF1 and/or IGFBPL1 to purified RGC cultures did not promote neuronal survival, but LPS stimulated microglial activation and resulted in significant RGC death compared to control cultures (P < 0.05). IGF1 and/or IGFBPL1 significantly reduced neuronal cell death in LPS-treated microglia-induced RGC death in microglia-RGC co-cultures (see fig. 12).

Intravitreal injection of IGFBPL1 3 and 10 days after IOP elevation protected RGCs from IOP elevation-induced damage and prevented loss of RGC function and vision in the glaucoma model (FIGS. 7A-D). IGFBPL1 inhibited microglial activation in glaucomatous retinas (fig. 8A-B), and also inhibited microglial activation, reactive gliosis, and proinflammatory cytokine production (fig. 9). As shown in fig. 10A-B, IGFBPL1 deficiency results in microglial activation and elevated levels of proinflammatory cytokines in the adult retina. FIG. 11 shows RGC loss and functional defects in IGFBPL 1-deficient mice, which were rescued by intravitreal injection of IGFBPL 1.

This study revealed that IGFBPL1 is expressed by microglia in adult mice, rather than RGCs. It exerts a neuroprotective effect by acting on microglia. These results indicate that IGFBPL1 protects neurons from death by modulating neuroinflammation.

Example 3 IGFBPL1 in IGFBPL1 with ocular hypertension^-/-Protection against neuronal and visual loss in mice

Insulin growth factor binding protein-like 1(IGFBPL1) promotes survival and neurite outgrowth of neonatal mouse Retinal Ganglion Cells (RGCs) that are regulated via insulin-like growth factor 1-mediated signaling pathways (Guo et al, Sci Rep.2018Feb 1; 8(1): 2054). Neonatal IGFBPL1 deficiency (IGFBPL1) compared to wild-type control mice^-/-) Mice have approximately 20% less RGCs. This study investigated IGFBPL1 protein in adult IGFBPL1 with or without elevated intraocular pressure (IOP)^-/-Neuronal survival and visual performance effects in mice.

1. IGFBPL1 at 2 and 7 months of age^-/-The RGC density of the mice was determined by immunolabeling Brn3a on retinal flat-packs. To induce an increase in IOP, two microliters of polystyrene microbeads (MB; 5X 10) were used⁶/ml) into adult male and female IGFBPL1^-/-Anterior chamber of unilateral eye of mouse. At 3, 7 and 17 days after MB injection, IGFBPL1 recombinant protein or sterile saline as a control was administered by intravitreal injection. At 4 weeks after MB injection, two investigators recorded the visual motor response of the mice in a masked fashion for contrast sensitivity and visual acuity. Mice were sacrificed and retinas were mounted and subjected to Brn3a immunolabeling to uncover Indicating surviving RGCs. Student t-test was used for statistical analysis.

The data show that deletion of IGFBPL1 results in IGFBPL1^-/-Gradual loss of RGC in mice. Transient elevation of IOP leads to adult IGFBPL1^-/-A significant decrease in visual performance and loss of RGC in mice. IGFBPL1 treatment significantly improved both wild type and IGFBPL1 with ocular hypertension^-/-Visual manifestations in mice (P)<0.05) and RGC survival (P)<0.05)。

In addition, retinal ischemia reperfusion injury was induced unilaterally in mice, followed by intravitreal injections of saline (isotype) or IGFBPL1(BPL1) on day 1 (early) or day 10 (late) post injury. Mice were sacrificed 4 weeks after injury and RGC density was quantified. RGC function was assessed by pSTR amplitude at 2 and 4 weeks post-injury (pre-sacrifice) while visual Contrast Sensitivity (CS) and Visual Acuity (VA) were measured using the optokinetic reflex (OKR) assay. As shown in figure 13, RGC density was significantly increased in IGFBPL 1-treated mice after ischemic injury, and pSTR amplitude as well as CS and VA values were improved compared to the saline-treated group.

IGFBPL1 is known to be strongly expressed in embryonic retinas, but barely detectable in adult retinas. These results indicate that the absence of IGFBPL1 during the embryonic stage induces IGFBPL1 ^-/-Progressive denaturation of RGCs in mice. Administration of IGFBPL1 protects mice with ocular hypertension from RGC and vision loss. In general, IGFBPL1 is an important neuroprotective agent in the retina that undergoes progressive degeneration, as in glaucoma.

Example 4 direct activation of human microglia by Heat shock proteins 27 and 60

Glaucoma has an autoimmune component caused by commensal bacteria sensitized CD4+ T cells that enter the retina and cross-react with Heat Shock Protein (HSP) expressing neurons via molecular mimicry mechanisms. As shown herein, microglial activation is responsible for immune responses and retinal degeneration in glaucoma. Given the limited availability of primary human microglia, an immortalized human microglia clone 3 cell line (HMC3) may be used to examine microglial behavior in pathological conditions. To test whether HSP27 and HSP60 can induce activation of microglia, cytokine expression and morphological changes were examined in HMC 3. Other known inflammatory stimuli were used as positive controls.

The method comprises the following steps: HMC3 cell line (ATCC) was cultured in EMEM medium and challenged with 10ug/ml HSP27, 10ug/ml HSP60, 200ng/ml LPS or 100ng/ml LPS (with or without 5mM ATP) for an additional 30 minutes. Cells receiving medium alone served as controls. After 24 hours, the RNA from HMC3 cells was collected by ZYMO Research Quick-RNA Microprep kit and purified by PrimeScript ^TMRT Master Mix RNA reverse transcription. PCR was performed on a Sybr green RT-PCR mixture containing different primers and cDNA samples using the EP realplex real-time PCR system. Relative fold changes of mRNA transcripts are presented and compared to control groups. In addition, low density HMC3 cell cultures were established and images of cell morphology were captured 24 hours after LPS, HSP27 or HSP60 treatment and morphological changes were quantified.

As a result: the data show that although LPS with or without ATP induces increased expression of pro-inflammatory cytokines such as IFNF in HMC3, HSP27 and HSP60 can also activate HMC3 to express higher levels of IFN-rich and TNF-rich. Quantification of cell morphology revealed a shortened dendritic process and an increased round cell body size (P <0.05) in the LPS, HSP27 and HSP60 stimulated groups compared to vehicle controls.

And (4) conclusion: the present study revealed that HMC3 cells respond similarly to known inflammatory stimuli to primary microglia. HSP27 and HSP60 can directly induce the pro-inflammatory response of HMC3 cells, supporting the notion that HSP can induce microglial activation as an early cause of glaucoma-related immune responses.

Example 5 exploration of spatiotemporal dynamics of microglia/macrophage polarization after ischemia/reperfusion in retina

Background: microglia/macrophages exhibit different functional phenotypes under various microenvironment stimuli and disease processes. The phenotypic dynamic changes of microglia in ischemia/reperfusion (I/R) remain ambiguous. Identification of spatiotemporal patterns of post-I/R microglia/macrophage polarization may improve our understanding of post-I/R damage and recovery.

The method comprises the following steps: I/R in rats was induced by connection to saline storage with a 30 gauge needle cannula to maintain intraocular pressure of 110mm Hg for 60 min. Retinas from rats were collected on days 1, 2, 7 and 14 post-surgery. Flow cytometry, reverse transcriptase polymerase chain reaction, Western blot and immunohistochemical staining were performed for M1 and M2 markers to characterize phenotypic changes in retinal cells, including microglia and infiltrating macrophages.

As a result: flow cytometry results showed CD11b as early as 12 hours after I/R⁺CD45^{Height of}A significant increase in lymphocytes, presumably macrophages and/or activated microglia, followed by CD11b on day 1^-CD45^{Height of}Lymphocytes increased significantly, both peaking at day 7. CD16 was found in the retina at days 1 and 2 after I/R⁺Iba1⁺(M1 marker) cells and Ym-1⁺Iba1⁺(M2 marker) rapid increase in both cells. These cells displayed round bodies with rare short dendrites from day 1 to day 7 and distributed from the inner nuclear layer to the ganglion cell layer.

And (4) conclusion: I/R induces an early response by microglia/macrophages that are differentially activated to both M1 and M2 types, resulting in a gradual increase in lymphocytes. Thus, microglia/macrophages may play a dominant role in the recruitment of infiltrating lymphocytes after I/R.

Example 6 targeting HSP: immune tolerance to HSP60 attenuates neurodegeneration in glaucoma

The purpose is as follows: previous studies have shown that bacterially sensitized T cell mediated autoimmune mechanisms underlie the pathogenesis of glaucoma, and Heat Shock Proteins (HSPs) are considered to be pathogenic autoantigens. We hypothesized that induction of immune tolerance to the bacterium HSP60 may prevent such pathogenic immune responses and attenuate neuronal loss in glaucoma.

The method comprises the following steps: adult C57BL/6J mice were given a low dose of recombinant bacterial HSP60, Ovalbumin (OVA) or saline (both as controls) daily in the nostrils for 7 days. Unilateral induction of IOP elevation by intracameral injection of polystyrene Microbeads (MB). Visual and retinal function was assessed by visual motor response (OMR) and electroretinogram positive scotopic threshold response (pSTR). Mice were sacrificed at 2, 4 and 8 weeks after MB injection. T cell responses to bacterial HSP60 were analyzed by the otic DTH (delayed type hypersensitivity) test and flow cytometry. Glaucomatous nerve damage was quantified by Retinal Ganglion Cells (RGCs) and axon counts.

As a result: as seen by flow cytometry analysis, nasal administration of low doses of HSP60 induced immune tolerance as indicated by decreased DTH responses and increased levels of T regulatory cells. The MB injected eyes maintained IOP levels of 25 + -3 mmHg compared to 12 + -2 mmHg in contralateral uninjected eyes. We observed no significant differences in IOP levels between HSP60, OVA and saline-treated mice. Treatment with HSP60 or OVA did not alter the basal level of Visual Acuity (VA), Contrast Sensitivity (CS) or pSTR prior to MB injection compared to naive or saline-treated mice. However, VA and CS assessed by OMR were significantly better in HSP60 treated mice than saline or OVA treated mice at all time points after MB injection. Consistently, both RGC function and RGC counts assessed by pSTR amplitude were significantly higher in HSP 60-treated mice compared to saline or OVA-treated mice after MB injection.

And (4) conclusion: intranasal administration of multiple low doses of bacterial HSP60 induced immune tolerance and attenuated RGC loss and functional deterioration in a mouse model of MB-induced glaucoma, and these results suggest an attractive antigen-specific therapeutic strategy for the prevention of visual loss in glaucoma. This study may also help us to understand the pathogenesis of cerebral neurodegenerative disorders and to provide innovative interventions for treating neurodegeneration affecting other parts of the central nervous system.

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Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.