Compositions and uses thereof

Abstract

The present invention relates to a composition comprising heterotrophic skin bacteria and / or a heterotrophic skin bacterial extract for use in modulating cytokine expression in skin cells, which is particularly suitable for use in the prevention and / or treatment of skin damage caused by UV light and in the treatment of skin conditions such as polymorphous light eruption (PLE).

Description

[Technical Field]

[0001] The present invention relates to a composition for use in modulating cytokine expression in skin cells, comprising heterotrophic skin bacteria and / or a heterotrophic skin bacterial extract. In particular, the heterotrophic skin bacteria is Micrococcus luteus (M. luteus), and / or the heterotrophic skin bacterial extract is derived from M. luteus. The composition is well suited for use in mediating cytokine expression in the skin. In particular, the composition is suitable for use in preventing and / or treating skin conditions associated with cytokine expression, such as those caused by skin exposure to UV light. [Background technology]

[0002] The mechanisms used by bacteria to confer beneficial effects on humans are diverse and include suppressing pathogens, modulating immune responses, strengthening epithelial barrier function, etc. However, in general, the underlying nature of symbiosis is not well understood, and relatively little information is available on the molecular mechanisms mediating the observed effects of probiotics.

[0003] Topical application of probiotics to the skin has been investigated in a limited number of studies. In general, the mechanisms underlying these effects are not well understood.

[0004] Current theory suggests that immune regulation involves a homeostasis between T helper 1 (Th1) and T helper 2 (Th2) activity, which direct different immune response pathways. Excessive activation of either pattern can cause disease, and either pathway can downregulate the other. The Th1 and Th2 pathways are generally associated with the secretion of different cytokines.

[0005] Exposure to ultraviolet radiation (UVR) suppresses immune responses. Data from numerous laboratories have demonstrated that one consequence of UVR exposure is the suppression of T helper type 1 (Th1) cell function, accompanied by activation of normal Th2 cells, resulting in a shift toward a Th2-like phenotype. Furthermore, there are numerous conditions that can be exacerbated or precipitated by exposure to UVR. These are collectively known as photodermatoses and include conditions such as urticaria, photoexacerbated eczema, and polymorphous light eruption (PLE or PMLE).

[0006] Polymorphous light eruption (PLE or PMLE) is a skin "irritation" caused by sunlight. The name comes from polymorphic exanthema, which refers to a rash with many forms. PLE is thought to be caused by an immune response to skin compounds that have been altered by exposure to ultraviolet radiation. PLE typically develops after 2-3 days of sun exposure, but can also occur with less exposure (such as direct exposure to incoming light through a window or 15-20 minutes in direct sunlight).

[0007] A delayed, patchy, itchy rash appears on the skin and can take 5 to 10 days to heal. The rash usually consists of small red spots or blisters and can appear on any part of the body exposed to sunlight, generally sparing the face and backs of the hands. It tends to heal without scarring. PLE differs from miliaria, which develops on the torso due to hot weather rather than sun exposure.

[0008] PLE affects 20–30% of the population in Northern Europe and the United States (where frequent sun exposure is uncommon) and 10–20% elsewhere, and is more prevalent in women than men. It typically develops by age 30, with peak incidence occurring between the ages of 20 and 40. The problem is more prevalent in temperate climates, such as Europe and the United States, and is most prevalent from early spring through summer. PLE is likely associated with an immune system response in the skin of certain individuals, more commonly those with fair skin. Evidence of PLE has also been identified in northern China and Australia. Current data suggest that the development of actinic dermatoses such as PLE may be related to the overexpression of TH1 and the suppression of TH2 responses. To date, no proven treatments have been found to help alleviate skin conditions like polymorphous light eruption (PLE), although calamine lotion and topical steroid creams (including corticosteroids, hydrocortisone, and hydroxychloroquine), along with some common anti-itch medications, are currently used with limited efficacy.

[0009] Currently, there is no effective treatment for PLE, and most treatments and compositions are aimed at alleviating the symptoms rather than treating or preventing the condition itself. For at least the reasons set forth above, there is a need to develop compositions, treatments, and methods for preventing and / or treating and / or ameliorating PLE and skin conditions caused by exposure of the skin to UV rays. Summary of the Invention

[0010] The present invention is defined in the appended claims and includes combinations of the embodiments and preferred features described below, except where such combinations are clearly impermissible or explicitly avoided.

[0011] According to one aspect of the present invention, a composition for use in regulating cytokine expression in skin cells is provided, comprising heterotrophic skin bacteria and / or a heterotrophic skin bacterial extract. As will be appreciated, in embodiments in which the composition comprises heterotrophic skin bacteria, the composition may comprise whole / intact bacteria. Alternatively, in another embodiment, the heterotrophic skin bacterial extract is in a lysed form. The heterotrophic skin bacteria or heterotrophic skin bacterial extract may be lyophilized, freeze-dried, or lysed. Preferably, the heterotrophic skin bacteria is Micrococcaceae, and / or the heterotrophic skin bacterial extract is derived from Micrococcaceae bacteria. More preferably, the heterotrophic skin bacteria or heterotrophic skin bacterial extract is a saprophytic bacterium or is derived from a saprophytic bacterium. Even more preferably, the heterotrophic skin bacteria is Micrococcus, and / or the heterotrophic skin bacterial extract is derived from a Micrococcus bacterium. Most preferably, the Micrococcus bacterium may be Micrococcus luteus (M. luteus). As will be understood by those skilled in the art, M. luteus is a Gram-positive saprophytic coccus that is part of the normal microflora of mammalian skin. Preferably, the composition comprises M. luteus and / or a bioactive extract of a lytic form of M. luteus. The M. luteus may be equivalent to that deposited under accession number 22110401.

[0012] In a related embodiment, the composition is formulated for topical application.

[0013] In further related embodiments, the modulation of cytokine expression results in modulation of one or more of the following cytokines: IL-6, and / or IL-8, PTX-3, VEGF, and / or MIP-3α. The modulation of cytokine expression in skin cells may be an increase in secretion of one or more of the following cytokines: IL-6 and / or IL-8 and / or PTX-3, and / or a decrease in secretion of one or more of the following cytokines: VEGF and / or MIP-3α.

[0014] In another embodiment, the composition is formulated in the form of a cream, gel, spray, ointment, or oil. In another related embodiment, the composition further comprises one or more pharmaceutically acceptable ingredients or excipients. The composition may suitably be in the form of a liquid, solution (e.g., aqueous, non-aqueous), suspension (e.g., aqueous, non-aqueous), emulsion (e.g., water-in-oil, oil-in-water), elixir, syrup, electuary, mouthwash, gargle, tablet, granule, powder, ampoule, bolus, suppository, pessary, tincture, gel, paste, ointment, cream, lotion, oil, foam, spray, mist, or aerosol. The composition may suitably be provided as part of a patch, adhesive plaster, bandage, dressing, or the like, impregnated with one or more active compounds and, optionally, one or more other pharmaceutically acceptable ingredients (e.g., penetration, permeation, and absorption enhancers, etc.). The compositions may also be suitably provided in the form of a depot or reservoir, or may be provided in the form of a coating for medical devices such as implants, prostheses, surgical instruments, gloves, catheters, valves, pacemakers, etc.

[0015] The compositions according to this aspect of the invention may further comprise one or more pharmaceutically or cosmetically acceptable ingredients or excipients. Pharmaceutically acceptable ingredients are well known to those skilled in the art and include, but are not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, carriers, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, fragrances, and penetrating agents.

[0016] In some cases, the carrier may comprise the medium that was in contact with the bacteria during culturing. The composition of the medium has changed during culturing, for example, due to secretion of substances from the bacteria. The composition may consist of or comprise the culture medium in which the bacteria grew.

[0017] Preferably, the composition is formulated for topical administration, particularly for use or application to the skin or on the skin.The composition can be formulated for topical administration in the form of a patch, bandage, dressing, depot, cement, adhesive, and reservoir, as well as in the form of a gel, paste, ointment, cream, spray, lotion, and oil.Preferably, the composition can be formulated for topical administration in the form of a cream, gel, spray, ointment, or oil.

[0018] An ointment is typically prepared from the composition and a paraffinic or water-miscible ointment base.

[0019] Creams are typically prepared from the extract and an oil-in-water cream base. Optionally, the aqueous phase of the cream base may contain at least about 30% w / w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol, and polyethylene glycol, and mixtures thereof. The topical formulation may desirably contain a compound that enhances absorption or penetration of the active compound into the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogues.

[0020] Emulsions are typically prepared from the heterotrophic skin bacteria and / or a bioactive extract of the heterotrophic skin bacteria and an oily phase, and may contain only an emulsifier (also known as an emulsifying agent) or a mixture of a fat, an oil, or both a fat and an oil with at least one emulsifier. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include both an oil and a fat. Overall, the emulsifier, with or without a stabilizer, constitutes a so-called emulsifying wax, and the wax, together with the oil and / or fat, constitutes a so-called emulsifying ointment base, which forms the oily dispersed phase of a cream formulation.

[0021] Suitable emulsions and emulsion stabilizers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate. Because the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations can be very low, the selection of an oil or fat suitable for the formulation is based on achieving the desired cosmetic properties. Therefore, the cream should preferably be a non-greasy, non-staining, and washable product with the appropriate viscosity to prevent leakage from tubes or other containers. Linear or branched mono- or dibasic alkyl esters such as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a blend of branched esters known as crodamol CAP may be used, the last three being preferred. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used.

[0022] The compositions may be administered alone or in combination with other treatments, simultaneously or sequentially. The compositions according to the invention may further comprise other active substances, for example antimicrobial agents such as bactericides and fungicides to prevent spoilage of the composition during storage.

[0023] In some embodiments, the composition may be provided as a suspension in a pharmaceutically or cosmetically acceptable excipient, diluent or carrier.

[0024] The composition of the present invention can be formulated as a medicine, i.e., as a pharmaceutical product, or as a medical device.The pharmaceutical product can contain other pharmaceutically acceptable components known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners.The formulation can also contain other active agents, such as other therapeutic or prophylactic agents.

[0025] Suitable media for culturing bacteria (such as M. luteus) are well known to those skilled in the art. As used herein, the term "media" encompasses any nutrient-containing liquid in which bacteria can grow, survive, develop, and / or multiply. The media may contain the minimum nutrients necessary to support bacterial survival and may optionally contain other nutrients. Exemplary nutrients contained in the media include sugars, magnesium, phosphate, phosphorus, and sulfur. The media may be made or modified with nutrient combinations known in the art, such as tryptic soy agar / medium. Media may be commercially available premixed or may be prepared in-house.

[0026] In some embodiments, the composition is acellular and does not contain any viable bacterial cells. In such embodiments, the entire bacterial cells can be removed from the medium, for example, by centrifugation and / or filtration (or other suitable methods for removing viable bacteria). For example, the bacteria can be removed by centrifugation at 15,000 x g for a time sufficient to precipitate substantially all of the bacteria from the medium. The medium can be filtered using a microporous filter with pores of an appropriate size to remove substantially all of the bacteria from the medium. These methods can remove intact bacteria, and if the extract is obtained by cell lysis, can also remove bacterial debris.

[0027] The composition can be sterilized. That is, the composition has been subjected to a sterilization process, such as irradiation, heat, chemicals, pressure, or filtration, or any combination thereof. For example, the composition can be filter-sterilized (a term understood by those skilled in the art). As understood by those skilled in the art, filter sterilization uses a 0.22 micron filter. The sterilization procedure must be adapted so as not to damage or reduce the efficacy of the heterotrophic skin bacteria and / or heterotrophic skin bacteria extract. In the case of a medium containing an extract, sterilization can be performed before the heterotrophic skin bacteria are introduced and cultured, and also after the bacteria are removed from the medium.

[0028] In some cases, the extract of the composition is substantially free of intact bacteria. The composition may also be substantially free of lysed bacteria or bacterial fragments. The intact bacteria and / or lysed bacteria or bacterial fragments can be separated from the extract. Separation can be performed by any suitable means known in the art, such as centrifugation or filtration. "Substantially free" means that the extract contains no or minimal contamination of non-secreted bacterial components, such as whole bacteria, lysed bacteria, or bacterial fragments. Thus, the composition may contain 100% extract, at least 99% extract, at least 95% extract, at least 90% extract, at least 85% extract, at least 80% extract, at least 75% extract, or at least 70% extract. The extract may contain additional non-bacterial components, such as carrier solutions, other active agents, or preservatives, as described herein.

[0029] The compositions described herein can be prepared by culturing the heterotrophic skin bacteria in a medium, separating the heterotrophic skin bacteria from the medium, and preparing a composition from the medium. The heterotrophic skin bacteria can be cultured under anaerobic conditions. The heterotrophic skin bacteria can be cultured at a temperature higher than normal human body temperature. The heterotrophic skin bacteria can be cultured at 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or 41°C. Preferably, the bacteria are cultured at 37°C. The bacteria can be cultured in the medium for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. The bacteria, or lysed bacteria, or fragments can be separated from the medium by centrifugation, such as at 15,000 x g. The medium can be separated from the heterotrophic skin bacteria, lysed heterotrophic skin bacteria, or fragments of heterotrophic skin bacteria by filtration. The medium can be separated by a combination of filtration and centrifugation. The medium can be subjected to sterilization before or after the heterotrophic skin bacteria are removed. For example, the medium can be subjected to sterilization after separation of the whole bacteria, lysed bacteria, or bacterial fragments from the medium. The medium can be concentrated to increase the ratio of the heterotrophic skin bacterial extract to the total volume of the medium. Concentration can be performed by any method well known in the art, such as evaporation. The heterotrophic skin bacterial extract can be separated from the medium. Any method for separating a substance from a carrier solution can be used. For example, the heterotrophic skin bacterial extract can be separated from the medium by chromatography, crystallization, distillation, drying, electrophoresis, or precipitation. After isolation from the medium or concentration in the medium, the extract can be dissolved or diluted in a carrier, or otherwise formulated into a composition disclosed herein.

[0030] In a further embodiment of the present invention, there is provided a composition comprising heterotrophic skin bacteria and / or a heterotrophic skin bacteria extract for use in the prevention and / or treatment of skin damage caused by UV light.

[0031] In a further embodiment of the present invention, there is provided the use of a composition comprising heterotrophic skin bacteria and / or a heterotrophic skin bacteria extract for use in the manufacture of a medicament for the prevention, management or treatment of skin damage caused by UV light.

[0032] In yet a further embodiment of the present invention, there is provided a composition comprising heterotrophic skin bacteria and / or a heterotrophic skin bacterial extract for use in the prevention, management, or treatment of a skin condition caused by exposure to UV light, hi a related embodiment, the skin condition is PLE.

[0033] In some embodiments, the composition can be for preventing skin damage caused by UV light and skin conditions caused by exposure to UV light. In such embodiments, the composition can be applied or administered before / prior to exposure to UV light. As will be understood by those skilled in the art, before / prior to exposure to UV light refers to an amount of UV light sufficient to cause skin damage or skin conditions, such as PLE.

[0034] In a further related embodiment, the composition is formulated for topical application to the skin. In another related embodiment, the skin condition is a VEGF- and / or MIP-3α-mediated skin condition. In a further related embodiment, the skin condition is an IL-6- and / or IL-8- and / or PTX-3-mediated skin condition.

[0035] In another embodiment of the present invention, a composition comprising heterotrophic skin bacteria and / or a heterotrophic skin bacterial extract for use in a cosmetic formulation for application to skin damage caused by UV light is provided. Typically, such cosmetic formulations are marketed as "after-sun" formulations and are intended for application to the skin after prolonged exposure to the sun.

[0036] Compositions and formulations according to the present invention may further comprise other active agents, for example other antimicrobial agents such as disinfectants.

[0037] In some embodiments, the composition contains at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, The composition may contain about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, or about 50.0% of the heterotrophic skin bacteria and / or heterotrophic skin bacterial extract.

[0038] In some embodiments, the composition may comprise at least about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 5%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 1% to about 5% by weight of one of the heterotrophic skin bacteria and / or heterotrophic skin bacterial extracts.

[0039] In another embodiment, the present invention provides a method for preventing, managing, or treating a skin condition caused by exposure to UV light, the method comprising applying a composition as defined in any other embodiment to the skin. The skin condition may be PLE. Also provided is a method for preventing and / or treating skin damage caused by UV light, the method comprising applying a composition as defined in any other embodiment to the skin. In some embodiments, the composition may be for the prevention of skin damage caused by UV light or a skin condition caused by exposure to UV light. In such embodiments, the composition may be applied or administered before / prior to exposure to UV light. As will be understood by those skilled in the art, before / prior to exposure to UV light refers to an amount of UV light sufficient to cause skin damage or a skin condition, such as PLE.

[0040] In yet another embodiment, the present invention provides a method of modulating cytokine expression in skin cells, the method comprising application of a composition as defined in any other embodiment.

[0041] The heterotrophic skin bacteria preparations according to the present invention may be formulated as pharmaceutical compositions for clinical use and may include pharmaceutically acceptable carriers, diluents, or adjuvants. They may be formulated for topical administration.

[0042] The dosage is preferably a preventively or therapeutically effective amount, which is an amount sufficient to bring about benefit to an individual.The actual amount administered, as well as the rate and time course of administration, depend on the nature and severity of the disease being treated.Prescribing treatment, such as determining dosage, is the responsibility of general practitioners and other medical professionals, and typically takes into account the disorder to be treated or prevented, the condition of each individual patient, the site of administration, the method of administration, and other factors known to general practitioners.Examples of the above-mentioned techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20 th Edition, 2000, pub. Lippincott, Williams & Wilkins. Those skilled in the art will recognize that appropriate dosages of the active compounds and compositions containing the active compounds may vary from patient to patient.

[0043] The composition of the present invention can be formulated as a medicine, that is, as a pharmaceutical product or medical device.The pharmaceutical product can contain other pharmaceutically acceptable components known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, coloring agents, flavoring agents, and sweeteners.The formulation can also contain other active agents, such as other therapeutic or prophylactic agents.

[0044] The compositions of the present invention can be formulated cosmetically, i.e., formulated as a cosmetic product, which can contain other cosmetically acceptable ingredients known to those skilled in the art, including, but not limited to, cosmetically acceptable carriers, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., humectants), masking agents, colorants, and fragrances.

[0045] Aspects and embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0046] BRIEF DESCRIPTION OF THE DRAWINGS Examples and experiments illustrating the principles of the present invention will now be described with reference to the accompanying figures, in which: [Brief explanation of the drawings]

[0047] [Figure 1] Figure 1 is a graph showing that the cytotoxic effect of UVB is dose-dependent. NHEK (p3-5) cells cultured to 80% confluence were irradiated with 5-30 mJ / cm2. Cell viability (n=3) was assessed with trypan blue 24 hours after irradiation. Data are shown as mean ± SD. **P<0.01, *P<0.05, determined by one-way analysis of variance followed by Tukey's test. [Figure 2] Figure 2 shows two graphs demonstrating that UVB has no significant effect on IL-10 / IL-12 secretion. Supernatants collected 24 hours after irradiation (5-30 mJ / cm2, p3-5) were analyzed for (A) IL-10 (n = 3) and (B) IL-12 (n = 2). Data are shown as mean ± SD. Significance was assessed by one-way analysis of variance followed by Dunnett's test. [Figure 3]Figure 3 is a collection of array blot images and graphs showing that UVB exerts a dose-dependent effect on inflammatory mediators. Figure 3A shows an image of an array blot after analysis of cytokine release from pooled supernatants (n = 3, p3-5) collected 24 h after irradiation with 0 (i), 10 (ii), 20 (iii), and 30 mJ / cm2 (iv). Figure 3B shows a graph depicting changes in protein secretion relative to non-irradiated controls. Figure 3C shows ImageJ quantification of dose-dependent changes in inflammatory mediators (I, ii, and iii): IL-6 (i), IL-8 (ii), and MIP-3α (iii), angiogenic markers (iv and v): VEGF (iv), MMP-9 (v), and acute-phase protein PTX3 (vi). [Figure 4] Figure 4 is a set of graphs showing the validation of cytokine array analysis by ELISA. Supernatants collected 24 hours after irradiation (5-30 mJ / cm², n = 3, p3-5) were analyzed for (A) IL-6, (B) IL-8, (C) MIP-3α (D), and PTX-3 (E). Data are shown as mean ± SD. ****P < 0.0001, **P < 0.01, *P < 0.05, as determined by one-way analysis of variance followed by Dunnett's test. [Figure 5] Figure 5 is a graph showing that the protective effect of Micrococcus luteus is dose-dependent. NHEK (p3-5) cells were cultured to 80% confluence, treated with medium ± M. luteus 104 / 102 CFU / ml for 90 minutes, and then irradiated at 5-30 mJ / cm2. Cell viability was assessed 24 hours after irradiation using trypan blue. Data are shown as mean ± SD. ****P<0.0001, *P<0.05, determined by two-way ANOVA followed by Tukey's test. [Figure 6]Figure 6 shows two graphs demonstrating that UVB promotes M. luteus adhesion to keratinocytes. NHEK (p3-5) cells were cultured to 80% confluence, treated with medium ± M. luteus 104 / 102 CFU / ml for 90 minutes, and then irradiated at 5-30 mJ / cm2. M. luteus adhesion at 1 × 104 CFU / ml (A, n = 3) and 1 × 102 CFU / ml (B, n = 4) was assessed 24 hours after irradiation. Data are shown as mean ± SD. *P < 0.05, determined by one-way analysis of variance followed by Dunnett's test. [Figure 7] Figure 7 shows three graphs demonstrating that M. luteus exacerbates UVB-induced inflammatory cytokine production. Supernatants collected 24 hours after irradiation, ± M. luteus application (5-30 mJ / cm², n = 3, p3-5), were analyzed for (A) IL-6, (B) IL-8, and (C) PTX-3. Data are shown as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, as determined by two-way ANOVA followed by Tukey's test. [Figure 8] Figure 8 shows two graphs demonstrating that M. luteus modulates UVB-induced VEGF secretion. Supernatants collected 24 hours after irradiation, ± M. luteus application (5-30 mJ / cm², n = 3, p3-5), were analyzed for (A) VEGF and (B) MIP-3α. Data are shown as mean ± SD. **P < 0.01, *P < 0.05, determined by two-way ANOVA followed by Tukey's test. [Figure 9] Figure 9 is a graph showing that Micrococcus luteus exerts a protective effect on differentiated keratinocytes treated with UVR. Differentiated NHEKs (p2-3) were treated with medium ± M. luteus 104 / 102 CFU / mL for 1 hour and then irradiated at 5-30 mJ / cm2. 24 hours after irradiation, cell viability was assessed with trypan blue. Data are shown as mean ± SD. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, as determined by two-way ANOVA followed by Tukey's test. [Figure 10] Figure 10 shows two graphs showing the adhesion of M. luteus to keratinocytes in response to UVB exposure. NHEK (p2-3) cells were treated with medium ± M. luteus 104 / 102 CFU / mL for 1 hour and then irradiated at 5-30 mJ / cm2. M. luteus adhesion at 1 x 104 CFU / mL (A, n = 3) and 1 x 102 CFU / mL (B, n = 3) was assessed 24 hours after irradiation. Data are shown as mean ± SD. Significance was assessed by one-way analysis of variance followed by Dunnett's test. [Example]

[0048] Example 1: Evaluation of the cytotoxic effect of UVB on epidermal keratinocytes material and method Keratinocyte culture Pooled normal human epidermal keratinocytes (NHEK, n = 3, immature foreskin, Promocell C-12005, passages 1-5) were routinely cultured in keratinocyte growth medium (KGM-2, Promocell). Cells were seeded (2-5 × 10) in 12-well plates. 4 cells / well) and cultured (37°C, 5% CO2) until they reached 80% confluence. Cells were washed with phosphate-buffered saline (PBS, Gibco) and either resuspended in PBS for UV irradiation or inoculated with bacteria as described below.

[0049] UV irradiation A broadband UVB irradiator, TL-12 lamp (Philips), was used, emitting a maximum output of 20 W and wavelengths of 270–400 nm (peak: 313 nm). A UVX radiometer (UV products, California) connected to a UVX-31 detector was used, and the irradiance was maintained at 0.55 ± 0.01 mW / cm for at least 5 min prior to irradiation. 2 The irradiance output was evaluated until it stabilized at 1000 mJ / cm. The irradiation time was then measured as a function of the radiant exposure (mJ / cm). 2 ) / (illuminance × calibration factor), the calibration factor is 0.63, and the 2The radiation exposure dose was used in the experiment. Cell monolayers resuspended in PBS were placed 16 cm from the light source, and half of the plate was covered with aluminum foil to serve as a non-irradiated control. Cells were irradiated at the doses mentioned above, and the medium was replaced with KGM-2 and cultured for 24 hours (37°C, 5% CO2).

[0050] Cell viability and bacterial attachment At 24 hours post-irradiation, cells were washed three times with PBS and trypsin (0.04%) ethylenediaminetetraacetic acid (EDTA, 0.03%, Promocell) was applied for 7 minutes at 37°C and 5% CO2. An equal volume of trypsin neutralizing solution (TNS) was then added to the cells, and the sample was mixed with an equal volume of trypan blue (Gibco). Cell viability was then recorded using a hemocytometer (modified Neubauer). Alternatively, to confirm microbial adhesion to NHEK, the cell suspension was diluted to 1 mL with PBS and diluted to 10 mL with TSB. -5 Serial dilutions were made to 100 μL and 20 μL was applied in triplicate to TSB agar.

[0051] UV-regulated inflammatory cytokine levels 10-30mJ / cm 2 The relative expression of 105 inflammatory cytokines in pooled supernatant samples (n = 3, NHEK p3-5) treated with UVB at doses of 100-1500 was assessed using the Human XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Supernatants were analyzed for human total interleukin-6 (IL-6), IL-8, IL-10, IL-12, pentraxin-3 (PTX-3), macrophage inflammatory protein-3α (MIP-3α), and vascular endothelial growth factor (VEGF) by ELISA according to the manufacturer's protocol (Duoset, R and D systems, Biotechne).

[0052] statistical analysis Data were processed using Excel software (Microsoft) and analyzed using Prism (GraphPad Software, California, US). All experiments were performed in triplicate unless otherwise noted, and data were analyzed using Student's t-test (when comparing two groups) or general linear model (GLM) followed by one-way or two-way ANOVA and Tukey / Dunnett test (when comparing three or more groups).

[0053] result Previous studies have provided evidence for a dose-dependent cytotoxic effect of UVB on epidermal keratinocytes. Our results confirm these findings and demonstrate that UVB doses >20 mJ / cm 2 was sufficient to induce a 34% decrease in cell viability compared to non-irradiated controls, as shown in Figure 1. These cytotoxic effects are thought to result from an imbalance between immunosuppressive cytokines (IL-10) and pro-inflammatory cytokines (IL-12). However, Figure 2 shows that UVB had no significant effect on the secretion of IL-10 or IL-12 (Figure 2). Therefore, UVB-dependent cytokine secretion was characterized using a cytokine array.

[0054] Example 2: Evaluation of dose-dependent changes in cytokine secretion material and method The method was the same as in Example 1 above.

[0055] result A cytokine array was performed to confirm the dose-dependent effect of UVB on the secretion of 107 cytokines. Evaluation of the changes in relative pixel density revealed basal expression of 45 inflammatory proteins. Figure 3B shows that at 10 mJ / cm 2 , 20 mJ / cm 2 , or 30 mJ / cm 2The results show that UVB irradiation resulted in increased secretion of 15, 27, and 15 proteins, respectively, compared with non-irradiated controls. Quantification of the relative pixel density of the blots shown in Figure 3A revealed dose-dependent changes in various inflammatory mediators, as shown in Figure 3C. These changes were quantitatively verified by ELISA in Figure 4. As demonstrated in Figures 4A–C, a dose-dependent increase in pro-inflammatory mediators, including IL-6, IL-8, and MIP-3α, was observed. Concurrently, a dose-dependent increase in the secretion of PTX-3, a sensor and regulator of innate immunity, was also observed, as shown in Figure 4E. In contrast, the dose-dependent decrease in VEGF secretion, as shown in Figure 3C (iv), was not confirmed by ELISA in Figure 4D, as this trend was due to UVB-induced cytotoxicity. Next, we characterized the mediated effect of the skin microbiota on UVB-dependent cytotoxicity and inflammation.

[0056] Example 3: M. luteus exerts a protective effect against cytotoxic doses of UVB material and method Bacterial co-culture Micrococcus luteus was routinely cultured in tryptic soy broth (TSB, Oxoid) at 37°C for 48 hours, and the cells were washed twice with PBS and diluted to 1 × 10 4 ~1×10 6 The cells were resuspended to a concentration of CFU / mL. To confirm viable cell counts, the inoculum was serially diluted, and 20 μL was added in triplicate to TSB / WCB agar. The medium ± M. luteus was then incubated for 90 minutes (37°C, 5% CO), and nonadherent cells were washed twice with PBS. The cell monolayers were then UV-irradiated as described below.

[0057] All other procedures were the same as in Example 1 above.

[0058] result Previous studies have demonstrated that M. luteus can repair DNA damage caused by UV rays. However, whether it provides this protection to the skin remains to be elucidated. The photoprotective effect of M. luteus was dependent on bacterial load. Figure 5 shows that the photoprotective effect of M. luteus was observed in 1 × 10 2 The inoculum of CFU / mL is 30 mJ / cm 2 This shows that 1 × 10 induced a 53% increase in cell viability compared to the non-bacterial treated control. 4 Inoculation of 1 × 10 CFU / mL did not have a significant effect on cell viability. Interestingly, this photoprotective effect was also achieved by increasing microbial adhesion to NHEK. 4 , or 1 × 10 2 30 mJ / cm against NHEK inoculated with M. luteus at CFU / mL 2 Application of UVB to M. luteus resulted in a 97% and 174% increase in adhesion, respectively, compared to unirradiated controls, as shown in Figure 6. Without wishing to be bound by theory, the inventors hypothesized that this increased adhesion rate may modulate UVB-associated inflammation, thus providing photoprotection. Therefore, the effect of M. luteus on inflammatory proteins regulated by UVB was evaluated.

[0059] The inflammatory response to M. luteus is thought to be dependent on toll-like receptor-4 and results in increased production of cytokines, including IL-8. We observed that this response was M. luteus dose-dependent. Figure 7B shows that the response was significantly increased after a high dose (1 × 10 4 CFU / mL) of M. luteus increased IL-8 secretion and cytotoxic doses of UVB (30 mJ / cm 2 This tendency was also observed for IL-6 and PTX-3, and as shown in Figure 7, cytokine secretion increased at 30 mJ / cm 2 UVB±1×10 2 30 mJ / cm compared to cultures treated with M. luteus at CFU / mL. 2 UVB+1×10 4In comparison, the UVB (30 mJ / cm) inoculation at any microbial density significantly increased the UVB radiation intensity in cultures treated with M. luteus at 10 CFU / mL. 2 ) resulted in a significant decrease in the secretion of VEGF (shown in Figure 8a) and MIP-3α (shown in Figure 8b) by M. luteus. These factors may play an important role in the associated photoprotection of M. luteus.

[0060] Example 4: M. luteus has a photoprotective effect on differentiated keratinocytes material and method The materials and methods used were the same as those used in Examples 1 and 3 above.

[0061] result The epidermis is made up of a multi-layered structure of keratinocytes at various stages of differentiation. Actively proliferating keratinocytes form the basal layer, while differentiated keratinocytes function at the surface of the skin, orchestrating the initial response to both UVR and pathogens. To ensure that the model used in this study was more physiologically relevant, the protective effect of M. luteus on differentiated keratinocytes treated with UVR was evaluated. As with undifferentiated keratinocytes, Figure 9 shows that at 30 mJ / cm compared to untreated controls, 2 Following irradiation with 100 mJ / cm, a 52% decrease in cell viability was observed, indicating that UVR reduced cell viability in a dose-dependent manner. 2 ) effectively alleviates the cytotoxic effect of 30 mJ / cm 2 compared with the non-bacterial challenge control at a high dose (1 × 10 4 CFU / mL) and low dose (1 × 10 2 CFU / mL) resulted in a 63% and 49% increase in viability, respectively. Furthermore, Figure 10 shows that this response was accompanied by a dose-dependent trend toward increased adherence of M. luteus to keratinocytes.

[0062] Overall conclusion Previously, published data on the immune system's response to UVR have been generated primarily in humans, and the specific cell types that produce the immune mediators have not been characterized. This study specifically investigated the response of keratinocytes to UVR. Keratinocytes were chosen because they are a key component of the skin's innate immune system and their response to UVR has not previously been fully understood. Second, keratinocytes are likely to be the primary cell type that interacts with the microbiota, as this is thought to reside primarily on the surface of the skin.

[0063] To date, the list of cytokines produced by keratinocytes has been shown to be vast. Furthermore, data contained herein demonstrate that the cytokine response can be altered by at least one bacterium, M. luteus, which could be used to alter the balance between Th1 and Th2 cytokines, potentially useful in treating conditions such as PLE, which currently affects approximately 18% of Europeans.

[0064] Surprisingly, it has been found that heterotrophic skin bacteria can regulate the response of skin cells to UV exposure. The present inventors have found that inoculating skin cells with M. luteus causes an increase in the survival rate of skin cells after exposure to UV light. Surprisingly, this is the result of M. luteus' ability to regulate the immune response of skin cells by reducing the expression of certain cytokines and increasing the expression of a different set of cytokines.

[0065] The above-described embodiments are not intended to limit the scope of protection provided by the claims, but rather to illustrate examples of how the invention may be practiced.

[0066] biological deposit This application refers to the following designations of deposited biological material: Name: National Collection of Type Cultures Address: UK Health Security Agency, Porton Down, Salisbury, SP4 0JG United Kingdom Entrustment date: November 4, 2022 Accession number: 22110401 Species: Micrococcus luteus Depositor: The University of Manchester, Oxford Road, Manchester, M13 9PL United Kingdom

Claims

1. A composition for use in modulating cytokine expression in skin cells, comprising heterotrophic skin bacteria and / or an extract of heterotrophic skin bacteria.

2. The composition of claim 1 , wherein the heterotrophic skin bacterial extract is in a lysed form.

3. 10. The composition of any of the preceding claims, wherein the heterotrophic skin bacteria are derived from Micrococcus bacteria.

4. 4. The composition of claim 3, wherein the Micrococcus bacterium is Micrococcus luteus.

5. 10. The composition of any preceding claim, wherein the composition is formulated for topical application.

6. 10. The composition of any of the preceding claims, wherein in said skin cells, said modulation of cytokine expression causes modulation in the secretion of one or more of the following cytokines: IL-6, and / or IL-8, PTX-3, VEGF, and / or MIP-3α.

7. 10. The composition of any of the preceding claims, wherein in said skin cells, said modulation of cytokine expression causes an increase in the secretion of one or more of the following cytokines: IL-6, and / or IL-8, and / or PTX-3.

8. 10. The composition of any of the preceding claims, wherein said modulation of cytokine expression in said skin cells causes a decrease in the secretion of one or more of the following cytokines: VEGF, and / or MIP-3α.

9. 10. The composition of any preceding claim, wherein said composition further comprises one or more pharmaceutically acceptable ingredients or excipients.

10. 10. The composition of claim 9, wherein the composition is formulated in the form of a cream, gel, spray, ointment, or oil.

11. 11. A composition according to any one of claims 1 to 10 for use in the prevention and / or treatment of skin damage caused by UV light.

12. 11. A composition according to any one of claims 1 to 10 for use as a cosmetic preparation for application to skin damage caused by UV light.

13. 11. A composition according to any one of claims 1 to 10 for use in the prevention, management or treatment of a skin condition caused by exposure to UV light.

14. The composition of claim 13, wherein the skin condition is PLE.

15. 15. The composition of claim 13 or 14, wherein the composition is formulated to be applied topically to the skin.

16. 16. The composition of any of claims 11 to 15, wherein the composition is administered before exposure to UV light.

17. 16. The composition of any of claims 13 to 15, wherein the skin condition is a VEGF and / or MIP-3α mediated skin condition.

18. The composition of any of claims 13 to 15, wherein the skin condition is an IL-6, and / or IL-8, and / or PTX-3 mediated skin condition.

19. 10. A composition according to any preceding claim, wherein the heterotrophic skin bacteria and / or heterotrophic skin bacteria extract comprises or is derived from Micrococcus luteus (22110401).

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