KR20190010239A - Composition for preventing, improving or treating skin inflammation comprising Toll-like receptor 3 agonist - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating skin inflammation comprising a toll-like receptor 3 (TLR 3) agonist as an active ingredient. According to the present invention, the TLR 3 agonist increases revelation of PD-L1 having an important role of controlling skin inflammation in a skin horny cell, has an excellent effect to reduce revelation of IL-36γ, and can be variously utilized as a pharmaceutical composition for preventing or treating skin inflammation including psoriasis.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, ameliorating or treating skin inflammation comprising an agonist of tol-like receptor 3 as an active ingredient.

The present invention relates to a pharmaceutical composition for preventing or treating skin inflammation comprising an agonist of toll-like receptor 3 (TLR3) as an active ingredient.

The skin protects the body moisture and acts as a barrier to protect the human body by blocking the invading factors that can enter from the outside. Factors that cause inflammation in the skin include physical stimuli, chemical stimuli, allergens, pathogen infections, and ultraviolet rays. As a result, secondarily produced inflammation-related cytokines or chemokines cause the inflammatory response . The general pathway of skin inflammation reaction is known as an immune response to protect the human body from external stimuli. In order to control inflammation by suppressing such inflammatory reaction, it has recently become a major therapeutic target.

Psoriasis is a chronic inflammatory skin disease, which is covered with silvery white scales, has clear borders, and has various red papules or plate rashes that occur repeatedly in the skin of the whole body. Histologically, epidermal proliferation and inflammation of the dermis . Although the cause of psoriasis has not yet been fully elucidated, the activity of T cells, which are immune cells in the skin, is increased, resulting in the stimulation of the keratinocytes of the skin, resulting in excessive proliferation and inflammation of keratinocytes It is being revealed. In addition, genetic factors, environmental factors, drugs, skin irritation, dryness, inflammation and mental stress are known to cause or exacerbate psoriasis.

Currently, steroids, topical immunosuppressants, topical antibiotics, antihistamines and the like are used as therapeutic agents for skin inflammatory diseases including psoriasis. However, when the drug is used for a long period of time, It is known that side effects such as acne, hirsutism, and palpitation can occur. In particular, when a steroid agent is applied to a wide area, the drug can be systemically absorbed through the skin, which may interfere with the growth of the child, and an adult may have systemic side effects such as osteoporosis.

Accordingly, studies on a therapeutic agent for skin inflammation with minimal side effects have been continued (Korean Patent Laid-Open Publication No. 10-2014-0033610, Korean Patent Publication No. 10-2016-0137035) But it is urgent to develop a therapeutic agent for skin irritation because there are many medicines focused on relieving symptoms that have already occurred.

The present invention aims at providing a pharmaceutical composition for preventing or treating skin inflammation comprising an agonist of toll-like receptor 3 (TLR3) as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating skin inflammation comprising an agonist of toll-like receptor 3 (TLR3) as an active ingredient.

As used herein, the term " Toll-like receptor (TLR) " refers to a pathogen of a bacterium, virus or parasite, Is a receptor that directly recognizes the receptor. Toll receptors were first found in the drosophila, and later in humans, homologous receptors were found and named as toll-like receptors. Among them, Toll-like receptor 3 (TLR3) is known to be expressed in the membrane of B cells, macrophages and dendritic cells.

As used herein, the term " agonist " refers to a substance or medicament that binds to a receptor of a bioactive substance and has the same (or similar) action as the action of the substance, or enhances the activity of the receptor site .

In the present invention, the type of the agonist of the tole-like receptor 3 is not particularly limited, and may include all the agonists which bind to the tole-like receptor 3, preferably poly (I: C).

As used herein, the term "poly (I: C)" is an abbreviation for polyinosinic-polycytidylic acid and is known to interact with toll-like receptor 3. Poly (I: C) is found in some viruses and is known to be structurally similar to double-stranded RNA, a natural stimulant of toll-like receptor 3, and poly (I: C) is thought to be a synthetic analogue of double stranded RNA.

In the present invention, poly (I: C) can be isolated from viruses or the like, or synthesized and used. In the examples of the present invention, poly (I: C) was purchased from Sigma-Aldrich (Sigma-Aldrich).

The term "skin inflammation" as the target disease of the present invention means a skin inflammatory disease accompanied by an inflammatory reaction occurring in the skin, and a specific disease name is not specifically limited as long as it falls medically within the category of skin inflammation. For example, it may include atopic dermatitis, contact dermatitis, seborrheic dermatitis, allergic dermatitis, sunshine dermatitis, infectious dermatitis, dermatitis due to immune diseases, and the like, including psoriasis, eczema, acne, urticaria, May be included. In the present invention, skin inflammation may more preferably be psoriasis.

The inventors of the present invention have for the first time determined that the agonist of the tole-like receptor 3 has excellent efficacy for preventing and treating skin inflammation.

Specifically, in the examples of the present invention, 8-week-old female C57BL / 6 mice were divided into four groups (normal control group: IMQ (inflammation regulator) treated group (IMQ + DW) (IMQ + poly (I: C) + isoAb) treated with poly (I: C) and anti-PD- (I: C) + anti-PD-L1 Ab), and the severity of skin inflammation was assessed using PASI scores. In the iso Ab group, skin irritation was significantly suppressed in both erythema, sclerosis and thickness, and weight loss was the least in weight change. After 6 days of experiment, body weight was recovered to a similar degree to that of normal control (Figs. 1 and 2).

The agonists of the toll-like receptor 3 of the present invention have the effect of increasing the expression of PD-L1 in keratinocytes.

As used herein, the term " PD-L1 " is abbreviated as Programmed death-ligand 1, PD-L1 is known as a type 1 transmembrane protein of about 40 kDa, and is also called CD274 or B7-H1. PD-L1 is known to play an important role in suppressing and controlling the immune system in the event of pregnancy, tissue allografts, or diseases such as autoimmune diseases or hepatitis.

(I: C) and the isotype control group Ab (IMC) after treatment with IMQ were administered to a group of four 8 week old female C57BL / 6 mice (normal control; IMQ (IMQ + poly (I: C) + isoAb) and IMQ + poly (I: C) and anti-PD- anti-PD-L1 Ab) was prepared, and then PD-L1 was detected by flow cytometry.

Specifically, automatic analysis of FACSCalibur (BD Biosciences, Franklin Lakes NJ, USA) using FlowJo Software (Tree Star, Ashland, OR, USA) was used to detect PD-L1 in the dorsal skin of all experimental mice. The dorsal skin cell suspension of each mouse was suspended in PerCP-conjugated anti-mouse CD4 (GK1.5), APC-conjugated anti-mouse PD-L1 (10F.9G2), FITC-conjugated anti-mouse Gr- 8C5) and PE-conjugated anti-mouse CD11C (N418) antibody (all BioLegend). Cells positive for CD4, Gr-1 or CD11c were each gated to compare PD-L1 expression. Negative-negative cells were gated on CD4, Gr-1 and CD11c, and analyzed by non-immunocompetent cell fraction of the dorsal skin.

IL-17 or IL-22, as a result of flow cytometric analysis, first examined whether IL-17 or IL-22 cytokines essential for the development of psoriasis immunohistochemistry in HaCaT cells affected the expression of PD- -L1 expression, and the expression of PD-L1 was increased by poly (I: C) (Fig. 5).

In contrast, PD-L1 expression in Gr-1, CD4, or CD11c-positive immune cells was increased only in IMQ-treated mice, but poly (I: C) The highest increase was observed when administered together. However, the expression of PD-L1 by non-immune cells was confirmed to be up-regulated only when poly (I: C) was administered together (Fig. 6)

The agonists of the toll-like receptor 3 of the present invention have the effect of reducing the expression of IL-36? In keratinocytes.

The term " IL-36 gamma " used in the present invention is also referred to as interleukin-36 gamma and is known as interleukin-1 family member 9 (IL1F9). IL-36γ is known to be used as a biomarker of skin inflammation, mainly psoriasis.

(I: C) and the isotype control group Ab (IMC) after treatment with IMQ were administered to a group of four 8 week old female C57BL / 6 mice (normal control; IMQ (IMQ + poly (I: C) + isoAb) and IMQ + poly (I: C) and anti-PD- anti-PD-L1 Ab) were prepared, and experiments were conducted to detect the levels of PD-L1 and IL-36γ through Western blotting.

Specifically, in order to detect levels of PD-L1 and IL-36γ in the experimental animals, mouse skin was homogenized and proteins were separated and loaded with 20 μg protein per lane. After blotting, the membranes were incubated with primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) for PD-L1 (sc-50298), IL-36γ (sc- And cultured together overnight. The membrane was then washed and then incubated with the corresponding secondary antibody and finally tested using ECL (enhanced chemiluminescence; ThermoFisher Scientific).

As a result of the above experiment, it was confirmed that the expression of IL-36γ induced by IMQ treatment was significantly inhibited by the administration of poly (I: C), and the expression of PD-L1 by poly (I: And strongly inhibited the expression of IL-36γ (FIG. 3).

In another embodiment of the present invention, real-time PCR was performed to measure the expression level of IL-36γ in HaCaT cells in the presence of IL-17, IL-22 or poly (I: C) Lt; RTI ID = 0.0 > (I: C) < / RTI >

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier in addition to containing an agonist of tole-like receptor 3 as an active ingredient.

The kind of carrier which can be used in the present invention is not particularly limited and any carrier commonly used in the technical field can be used. Examples of the carrier include, but are not limited to, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, . These may be used alone or in combination of two or more.

In addition, the pharmaceutical composition of the present invention may be supplemented with other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostats, and may be used as fillers, extenders, wetting agents, disintegrants, , A binder, a lubricant, and the like may be additionally used.

In the pharmaceutical composition of the present invention, the agonist of the tole-like receptor 3 may be contained in an amount of 0.00001% by weight to 99.99% by weight, preferably 0.0001% by weight to 90% by weight, based on the total weight of the pharmaceutical composition, Preferably from 0.0001% by weight to 50% by weight, more preferably from 0.0001% by weight to 30% by weight, and it may be variously changed depending on the condition of the subject to be administered, have. If desired, it may also be included in the total content of the pharmaceutical composition.

The pharmaceutical composition of the present invention may be formulated into various formulations suitable for oral administration or parenteral administration.

Non-limiting examples of oral dosage forms using the pharmaceutical compositions of the present invention include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft Capsules, syrups, and elixirs.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; Excipients such as dicalcium phosphate and the like; Disintegrating agents such as corn starch or sweet potato starch; Magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax, and the like. Sweetening agents, fragrances, syrups, and the like may also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, liquid carriers such as fatty oils can be further used.

Non-limiting examples of the parenteral preparation using the pharmaceutical composition of the present invention include injections, suppositories, respiratory inhalation powders, aerosol preparations for spraying, ointments, powder for application, oils and creams.

In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and external preparations may be used. Examples of the non-aqueous solutions and suspensions include propylene glycol, polyethylene Glycerol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like.

When the pharmaceutical composition of the present invention is formulated into an injection, the pharmaceutical composition of the present invention is mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which is used for unit administration of an ampoule or a vial Can be formulated.

When the pharmaceutical composition of the present invention is formulated into an aerosol formulation, a propellant or the like may be added together with the additive such that the water-dispersed concentrate or the wet powder is dispersed.

When the pharmaceutical composition of the present invention is formulated into an ointment, a cream, a powder for application, an oil, an external preparation for skin or the like, an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, a polyethylene glycol, , Silica, talc, zinc oxide or the like as a carrier.

The pharmaceutically effective amount and the effective dose of the pharmaceutical composition of the present invention can be varied depending on the formulation method, administration method, administration time and / or administration route of the pharmaceutical composition, and the kind of reaction to be achieved by administration of the pharmaceutical composition Including the age, weight, general health condition, degree and severity of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously with the subject, and other compositional components, etc. May be varied according to factors well known in the art and in the pharmaceutical arts, and those of ordinary skill in the art can readily determine and prescribe dosages that are effective for the desired treatment.

The administration of the pharmaceutical composition of the present invention may be administered once a day or divided into several doses. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking into consideration all of the above factors, in such an amount as to obtain the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The route of administration and the mode of administration of the pharmaceutical composition of the present invention may be independent of each other, and may be arbitrarily selected depending on the route of administration and the mode of administration as long as the pharmaceutical composition can reach the desired site. The pharmaceutical composition may be administered orally or parenterally.

Examples of the parenteral administration method of the pharmaceutical composition of the present invention include intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, and the method of applying, spraying or inhalation the above composition But are not limited thereto.

The pharmaceutical composition of the present invention may exert an excellent effect even when it is used alone, but it can be used in combination with various methods such as hormone therapy and drug therapy to increase the therapeutic efficiency.

The agonists of the Toll-like receptor 3 (TLR 3) of the present invention increase the expression of PD-L1, which plays an important role in the control of skin inflammation in dermal keratinocytes, and reduce the expression of IL-36γ And can be used as a pharmaceutical composition for the prevention and treatment of skin inflammation including psoriasis.

FIG. 1 shows the results of immunohistochemical staining of normal control mice, IMQ + distilled water (DW) mice, IMQ + poly (I: C) + iso Ab mice, and IMQ + poly (I: + < / RTI > anti-PD-L1 blocking Ab mice, and H &
Figure 2 (a) shows the effect of the anti-PD-L1 blocked Ab mouse on the normal control mice, IMQ + distilled water (DW) mice, IMQ + poly (I: C) + iso Ab mice, (B) is a graph showing the cumulative scores of the erythema, scars and thickness, and (c) is a graph showing the cumulative score of the erythema, The graph of the mouse body weight is shown. In the graphs, n = 6; * P < 0.05, and all data are expressed as mean + standard deviation (SD).
FIG. 3 is a graph showing the effect of IMQ + poly (I: C) + iso Ab mouse and IMQ + poly (I: C) + anti-PD-L1 blocking Ab mice on the dorsal skin of normal control mice, IMQ + distilled water PD-L1 and IL-36γ protein levels were measured by Western blotting (* P <0.05).
4 (a) is a graph showing the results of real-time PCR measurement of the expression level of IL-36γ in HaCaT cells in the presence of poly (I: C) (n = 3; P <0.05) (b) is a graph showing the results of immunohistochemical staining in the dorsal skin of normal control mice, IMQ + distilled water (DW) mice, IMQ + poly (I: C) + iso Ab mice, and IMQ + poly (N = 6; * P < 0.05).
5 is a graph (n = 4; * P &lt; 0.05) showing PD-L1 surface expression level in HaCaT cells exposed to IL-17, IL-22 or poly (I: C) through flow cytometry.
Figure 6 (a) shows normal control mice gated with Gr-1 positive, CD4 positive, CD11c positive or negative, IMQ + distilled water (DW) mice, IMQ + poly (I: C) + iso Ab mice, and IMQ + poly (N = 4), (b) is a graph comparing the expression level of PD-L1 in the dorsal skin of the anti-PD-L1 blocking antibody (I: ± standard deviation) Fluorescence intensity (MFI) (n = 4; * P <0.05).

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Statistical analysis

The data herein are expressed as mean SD (SD). Statistical significance was analyzed using one-way or two-way ANOVA using standard software (Prism 6; GraphPad Software Inc., San Diego, CA, USA) and setting statistical significance at p <0.05.

Example 1: Cell culture

(Invitrogen [ThermoFisher Scientific]) supplemented with 10% fetal bovine serum (Invitrogen), streptomycin (100 / / mL) and penicillin (100 U / mL) was added to HaCaT human keratinocyte , Waltham, MA, USA). The cells were cultured until the cell density reached 70-80%, and IL-17 (100 ng / ml; R & D Systems, Minneapolis, MN, USA) I: C) (20 [mu] g / ml; Sigma-Aldrich). The cells were then subjected to flow cytometry or quantitative real-time PCR (PCR).

Example 2 Preparation of Experimental Animals and IMQ Treatment

8 weeks old female C57BL / 6 mice were purchased (OrientBio, Emsung, Korea). The mice were free to ingest food and water and were maintained under pathogen-free conditions with a 12-h light / dark cycle. All procedures were approved by Ewha Womans University College of Medicine Animal Care and Use Committee (ESM 15-0309). IMQ (inflammation regulator) was applied to 8-week-old mice in the form of 5% cream (Aldara; Osaka City, Japan) for 6 consecutive days (83 mg / day) to induce skin inflammation. A control vehicle (Vaseline / Lanette cream; Fagron, Rotterdam, The Netherlands) was applied in the same manner to the normal control mice. Poly (I: C) mice were administered subcutaneously (100 μg / mouse) with poly (I: C) on days 1 and 3 during 6-day IMQ treatment. Control mice received the same amount of distilled water. (5 mg / kg, 10F.9G2; BioLegend, San Diego, CA, USA) or an isotype control Ab (5 mg / kg, RTK4530, BioLegend) Poly (I: C) was administered subcutaneously before injection.

Example 3: Evaluation of severity of skin inflammation

PASI (Psoriasis Area and Severity Index) score was used to measure skin inflammation objectively. Erythema, scaling and thickening were independently assessed on the scale of 0-4 as follows: 0, none (none); 1, slight; 2, moderate; 3, marked; 4, extreme. Cumulative scores were used as a measure of degree of inflammation (scale, 0-12). We also monitored daily changes in body weight during IMQ application.

As a result, IMQ + poly (I: C) and isotype control Ab groups (IMQ + poly (I)) were observed after IMQ treatment compared with control group (IMQ + DW) I: C) + iso Ab), skin irritation was significantly inhibited in both erythema, sclerosis and thickness. Weight loss was the least in weight change, and normal control (normal control) It was confirmed that the body weight was restored to a similar degree (Figs. 1 and 2).

On the other hand, in the case of IMQ + poly (I: C) + anti-PD-L1 Ab group treated with anti-PD-L1 blocking Ab instead of iso Ab, the poly (I: C) (I: C) inhibits skin inflammation induced by IMQ in a manner dependent on PD-L1 (FIGS. 1 and 2).

Example 4: Histopathological observation

Were fixed in 4% formaldehyde and added to paraffin. Sections (5 μm) were placed on glass slides for microscopic examination and stained with hematoxylin and eosin (H & E).

Example 5: Western blot

To detect levels of PD-L1 and IL-36γ in experimental animals, mouse skin was homogenized and the protein separated and loaded with 20 μg protein per lane. After blotting, the membranes were incubated with primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) for PD-L1 (sc-50298), IL-36γ (sc- And cultured together overnight. The membrane was then washed and then incubated with the corresponding secondary antibody and finally tested using ECL (enhanced chemiluminescence; ThermoFisher Scientific).

As a result of the above experiment, it was confirmed that the expression of IL-36γ induced by IMQ treatment was significantly inhibited by the administration of poly (I: C), and the expression of PD-L1 by poly (I: And strongly inhibited the expression of IL-36γ (FIG. 3).

Example 6: Quantitative reverse transcription PCR (RT-qPCR)

To evaluate mouse skin tissue and human HaCaT cells, complementary DNA was synthesized using First Strand cDNA Synthesis Kit (TOYOBO, Osaka, Japan) as instructed by the manufacturer. Next, real-time PCR was performed on a StepOnePlus instrument (Applied Biosystems [ThermoFisher Scientific]) using SYBR Green Reagent (TOYOBO). For amplification, the following primers were used for skin samples: mouse PD-L1 (297 bp), 5'-ATG TCA GGC CGA GGG TTA TC-3 '(forward) and 5'- AGG ATG GAT CCC AGA AGC AC-3 '(reverse); Mouse PD-L2 (189 bp), 5'-GCA GAA GTG TCC TGG CAA AA-3 '(forward) and 5'-CAG AGG GTC AAT GAT GGC TG-3' (reverse); Mouse BD-2 (104 bp), 5'-AAC TTG ACC ACT GCC ACA CC-3 '(forward) and 5'-ACA GGG GTT CTT CTC TGG GA-3' (reverse); Mouse IL-6 (131 bp), 5'-CTG CAA GAG ACT TCC ATC CAG-3 '(forward) and 5'-AGT GGT ATA GAC AGG TCT GTT GG-3' (reverse); Mouse IL-36? (107 bp), 5'-TCC TGA CTT TGG GGA GGT TTT-3 '(forward) and 5'-TCA CGC TGA CTG GGG TTA CT-3' (reverse); Mouse CCL20 (146 bp) 5'-GCC TCT CGT ACA TAC AGC CGC-3 '(forward) and 5'-CCA GTT CTG CTT TGG ATC AGC-3' (reverse); Mouse IL-17A (165 bp), 5'-TTT AAC TCC CTT GGC GCA AAA-3 '(forward) and 5'-CTT TCC CTC CGC ATT GAC AC-3' (reverse); Mouse IL-23 (213 bp), 5'-ATG CTG GAT TGC AGA GCA GTA-3 '(forward) and 5'-ACG GGG CAC ATT ATT TTT AGT CT-3' (reverse); Mouse IFN-? (182 bp), 5'-ATG AAC GCT ACA CAC TGC ATC-3 '(forward) and 5'-CCA TCC TTT TGC CAG TTC CTC-3' (reverse); And mouse GAPDH (173 bp), 5'-GGT AAA GTG GAT ATT GTT GCC ATC AATG-3 '(forward) and 5'-GGA GGG ATC TCG CTC CTG GAA GAT GGT G-3' (reverse). In order to measure the expression of IL-36γ in HaCaT cells, the following primers were used: human IL-36 (232 bp), 5'-TTC CAC GAA GTG ACA GTG TGA-3 ' GCA CGG TAG AAA AGG AAG GGT-3 '(reverse); And human GAPDH (192 bp), 5'-GGT AAA GTG GAT ATT GTT GCC ATC AAT G-3 '(forward) and 5'-GGA GGG ATC TCG CTC CTG GAA GAT GGT G-3' (reverse).

As a result of the above experiment, the degree of expression of IL-36γ in HaCaT cells in the presence of IL-17, IL-22 or poly (I: C) Lt; RTI ID = 0.0 &gt; (FIG. 4A). &Lt; / RTI &gt;

Example 7: Preparation of skin sample

Shaved skin was collected from all experimental mice. After removal of fat, the samples were treated with collagenase type I (6 mg / ml, Roche, Basal, Switzerland) in RPMI 1640 medium supplemented with 10% FBS, streptomycin (100 ug / mL) and penicillin ) And DNase I (10 μg / ml, Stemcell Technologies, Vancouver, BC, Canada) (10 min, 37 ° C). The tissue was then chopped and disassembled on a stirrer (60 min, 37 ° C). For analysis via flow cytometry, all of the degraded tissue was filtered through a 70 um pore cell strainer and washed twice with PBS.

Example 8: Flow cytometry

Surface expression of PD-L1 (CD274) in HaCaT cells was quantified by labeling with phycoerythrin (PE) -binding anti-human CD274 antibody (29E.2A3; BioLegend). The level of nonspecific staining with PE-conjugated mouse IgG _2b isotype antibody (MPC-11; BioLegend) was used as a control measure.

For the detection of PD-L1 in the dorsal skin of all experimental mice, automatic analysis of FACSCalibur (BD Biosciences, Franklin Lakes NJ, USA) using FlowJo Software (Tree Star, Ashland, OR, USA) Mouse CD4 (GK1.5), APC-coupled anti-mouse PD-L1 (10F.9G2), FITC-conjugated anti-mouse Gr-1 (RB6-8C5) And a PE-conjugated anti-mouse CD11C (N418) antibody (entirely BioLegend). Cells positive for CD4, Gr-1 or CD11c were respectively gated and compared with PD-L1 expression. Negative-negative cells were gated on CD4, Gr-1 and CD11c, and analyzed by non-immunocompetent cell fraction of the dorsal skin.

IL-17 or IL-22, as a result of flow cytometric analysis, first examined whether IL-17 or IL-22 cytokines essential for the development of psoriasis immunohistochemistry in HaCaT cells affected the expression of PD- -L1 expression, and the expression of PD-L1 was increased by poly (I: C) (Fig. 5).

In contrast, PD-L1 expression in Gr-1, CD4, or CD11c-positive immune cells was increased only in IMQ-treated mice, but poly (I: C) The highest increase was observed when administered together. However, the expression of PD-L1 by non-immune cells was confirmed to be up-regulated only when poly (I: C) was administered together (Fig. 6)

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. More variations are possible within a range that does not. Accordingly, the present invention may be embodied in other forms without departing from the spirit or scope of the inventive concept as defined by the appended claims and their equivalents.

<110> EWHA UNIVERSITY - INDUSTRY COLLABORATION FOUNDATION <120> Composition for prevention, improving or treating skin          inflammation comprising Toll-like receptor 3 agonist <130> P17-077-EWHA <160> 24 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> Mouse PD-L1 forward primer <400> 1 atgtcaggcc gagggttatc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> Mouse PD-L1 reverse primer <400> 2 aggatggatc ccagaagcac 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse PD-L2 forward primer <400> 3 gcagaagtgt cctggcaaaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> Mouse PD-L2 reverse primer <400> 4 cagagggtca atgatggctg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse BD-2 forward primer <400> 5 aacttgacca ctgccacacc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse BD-2 reverse primer <400> 6 acaggggttc ttctctggga 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> Mouse IL-6 forward primer <400> 7 ctgcaagaga cttccatcca g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> Mouse IL-6 reverse primer <400> 8 tcctgacttt ggggaggttt t 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> Mouse IL-36 gamma forward primer <400> 9 tcctgacttt ggggaggttt t 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse IL-36 gamma reverse primer <400> 10 tcacgctgac tggggttact 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse CCL20 forward primer <400> 11 gcctctcgta catacagacg c 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse CCL20 reverse primer <400> 12 ccagttctgc tttggatcag c 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> Mouse IL-17A forward primer <400> 13 tttaactccc ttggcgcaaa a 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> Mouse IL-17A reverse primer <400> 14 ctttccctcc gcattgacac 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> Mouse IL-23 forward primer <400> 15 atgctggatt gcagagcagt a 21 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> Mouse IL-23 reverse primer <400> 16 acggggcaca ttatttttag tct 23 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse IFN-gamma forward primer <400> 17 atgaacgcta cacactgcat c 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> Mouse IFN-gamma reverse primer <400> 18 ccatcctttt gccagttcct c 21 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Mouse GAPDH forward primer <400> 19 ggtaaagtgg atattgttgc catcaatg 28 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Mouse GAPDH reverse primer <400> 20 ggagggatct cgctcctgga agatggtg 28 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human IL-36 forward primer <400> 21 ttccacgaag tgacagtgtg a 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Human IL-36 reverse primer <400> 22 gcacggtaga aaaggaaggg t 21 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Human GAPDH forward primer <400> 23 ggtaaagtgg atattgttgc catcaatg 28 <210> 24 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Human GAPDH reverse primer <400> 24 ggagggatct cgctcctgga agatggtg 28

Claims (6)

A pharmaceutical composition for preventing or treating skin inflammation comprising an agonist of toll-like receptor 3 (TLR3) as an active ingredient. The pharmaceutical composition according to claim 1, wherein the agonist of the tole-like receptor 3 is poly (I: C). The pharmaceutical composition according to claim 1, wherein the agonist of the tole-like receptor 3 has an effect of increasing the expression of PD-L1 in keratinocytes. The pharmaceutical composition according to claim 1, wherein the agonist of the tole-like receptor 3 has an effect of reducing the expression of IL-36? In keratinocytes. The composition according to claim 1, wherein the skin inflammation is caused by atopic dermatitis, contact dermatitis, seborrheic dermatitis, allergic dermatitis, sun dermatitis, dermatitis due to infection, dermatitis due to immune diseases, psoriasis, eczema, acne, urticaria and rash &Lt; / RTI &gt; 6. The pharmaceutical composition according to claim 5, wherein the skin inflammation is psoriasis.

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