Immunomodulatory composition based on Lacrimispora amygdalina and Acetate / Propionate

Abstract

The present invention relates to an acetate / propionate-based immunomodulatory composition for Lacrimispora amygdalina, wherein the composition comprises, as an active ingredient, acetic acid and / or propionic acid isolated and purified from Lacrimispora amygdalina or its culture medium, cell extract, or the culture supernatant of said strain, which acts to reduce IL-17 secretion from Th17 cells in immune cells derived from liver transplant patients and simultaneously increase IL-10 secretion from regulatory T cells (Treg) or regulatory B cells (Breg).

Description

Technology Field

[0001] The present invention relates to an immunomodulatory composition using microorganism-derived short-chain fatty acids (SCFAs), and more specifically, to a Lacrimispora amygdalina and Acetate / Propionate-based immunomodulatory composition that regulates intestinal immune homeostasis and transplant immune tolerance by using the Lacrimispora amygdalina strain and its metabolites, acetic acid and propionic acid, to inhibit the secretion of inflammatory cytokines (IL-17) from Th17 cells and to induce the secretion of anti-inflammatory cytokines (IL-10) from regulatory T cells (Treg) and regulatory B cells (Breg). Background Technology

[0002] The gut microbiome is known to play an important role in the formation and maintenance of the host's immune system, and in particular, short-chain fatty acids (SCFAs) produced by gut anaerobic strains have been reported to be involved in the differentiation and regulation of immune cell function. Among SCFAs, acetic acid and propionic acid are metabolites produced at relatively high concentrations in the intestinal environment, and numerous studies have been published indicating that they can affect the activity of Th17 cells, which mediate inflammatory immune responses, and the function of regulatory immune cells (Treg, Breg) that secrete immunosuppressive cytokines.

[0003] Existing technologies have conducted studies observing immune cell responses after treating with specific SCFAs alone or in combinations; however, there is a problem in that the patterns of change in inflammatory and anti-inflammatory cytokines vary depending on the combination and concentration of SCFAs. Furthermore, most studies have remained based on mixture-based approaches that comprehensively reflect the intestinal environment, resulting in a lack of sufficient data to isolate and verify the effects of individual metabolites produced by specific bacterial strains on immune cell responses.

[0004] Some strains cultured in anaerobic environments are known to produce various organic acids, including acetic acid and propionic acid; however, evidence regarding how differences in metabolic characteristics and the composition of SCFAs produced affect immune cell function is limited. In particular, regarding strains of the genus Lacrimispora, which are relatively under-studied among gut microorganisms, there are few specific reports on physiological functions related to the regulation of inflammatory immune responses.

[0005] Therefore, research is needed to experimentally evaluate whether metabolites such as acetic acid and propionic acid produced by specific intestinal anaerobic strains inhibit Th17 / IL-17-centered inflammatory responses and induce anti-inflammatory IL-10 secretion, and to examine the possibility of developing immunomodulatory compositions based on this. Prior art literature

[65535] (비특허문헌 1) Atarashi, K. et al., “Treg induction by a rationally selected mixture of Clostridia from the human microbiota”, Nature, 2013.(비특허문헌 2) Furusawa, Y. et al., “Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells”, Nature, 504(7480):446-450, 2013.(비특허문헌 3) Park, J. et al., “Short-chain fatty acids induce both effector and regulatory T cells by suppression of histone deacetylases and regulation of the mTOR?S6K pathway”, Mucosal Immunology, 8:80-93, 2015.(비특허문헌 4) Haas, K.N. and Blanchard, J.L., “Reclassification of the Clostridium clostridioforme and Clostridium sphenoides clades as Enterocloster gen. nov. and Lacrimispora gen. nov., including reclassification of 15 taxa”, International Journal of Systematic and Evolutionary Microbiology, 70:23-34, 2020. 해결하려는 과제

[0006] In organ transplant patients and patients with autoimmune diseases, the Th17 immune response centered on IL-17 is excessively activated, which can lead to persistent inflammation or instability in immune tolerance. In such situations, the secretion of IL-10, which mediates anti-inflammatory immune responses, is not sufficiently maintained, and the balance of the immune system tends to easily collapse.

[0007] Since most existing immunosuppressants reduce the overall immune response nonspecifically rather than directly inhibiting the Th17 / IL-17 axis, concerns regarding increased risk of infection and drug side effects persist with long-term use. Furthermore, while previous studies have reported results of treating immune cells with subcutaneous fatty acids (such as acetic acid and propionic acid) alone or in simple mixtures, there is insufficient empirical data regarding how the actual metabolite composition produced by specific strains contributes to Th17 inhibition and IL-10 increase.

[0008] In particular, studies analyzing the effects of SCFAs produced by the intestinal anaerobic strain Lacrimispora amygdalina on immune cell function individually are limited, and the direct effects of strain culture medium, cell extract, or combinations of their metabolites on IL-17 reduction and IL-10 increase in patient-derived immune cells have not been identified.

[0009] Therefore, the objective is to experimentally verify whether the strain itself or metabolites such as acetic acid and propionic acid generated from the strain can reduce IL-17 secretion from Th17 cells, increase IL-10 secretion from regulatory immune cells, and induce a state of immune tolerance through a combination of these two reactions, and based on this, to provide a specific immunomodulatory composition. That is, the present invention aims to present a composition capable of enhancing an IL-10-centered anti-inflammatory response while suppressing an excessive Th17 response by utilizing acetic acid and propionic acid from Lacrimispora amygdalina or its culture medium, cell extract, and metabolites. means of solving the problem

[0010] An immunomodulatory composition based on Lacrimispora amygdalina and Acetate / Propionate according to one embodiment of the present invention is characterized in that the composition comprises, as an active ingredient, acetic acid and / or propionic acid isolated and purified from Lacrimispora amygdalina or its culture medium, cell extract, or the culture supernatant of said strain, which acts to reduce IL-17 secretion from Th17 cells in immune cells derived from liver transplant patients and simultaneously increase IL-10 secretion from regulatory T cells (Treg) or regulatory B cells (Breg).

[0011] The above active ingredient is a metabolite isolated and purified from a culture supernatant obtained by culturing the Lacrimispora amygdalina strain deposited as DSMZ 12857 in a medium at 37°C under anaerobic conditions for 24 to 48 hours, wherein the sum of the peak areas of acetic acid and propionic acid measured by GC / MS in the culture supernatant is at least twice the sum of the peak areas in a control culture medium not inoculated with microorganisms, and the weight ratio of acetic acid and propionic acid produced in the culture supernatant is measured in the range of 1:2 to 1:3, and the composition containing the above active ingredient reduces the amount of IL-17 secreted in Th17 cells by at least 50% compared to the vehicle control group.

[0012] The above composition is characterized by the fact that when administered at a concentration of 10 μg / mL to peripheral blood mononuclear cells of a liver transplant patient stimulated with an anti-CD3 antibody (2 μg / mL) in an in vitro culture system, after 48 hours of culture, the IL-17 concentration in the cell culture supernatant decreases by an average of 300 pg / mL or more compared to the vehicle, and at the same time, the IL-10 concentration increases by 150 pg / mL or more, thereby maintaining the Th17 / Treg cell ratio at 1.0 or less. Effects of the invention

[0013] The Lacrimispora amygdalina and Acetate / Propionate-based immunomodulatory composition of the present invention exhibits an effect in which a simultaneous decrease in IL-17 secreted by Th17 cells and an increase in IL-10 secreted by Treg / Breg cells are observed in in vitro experiments targeting immune cells derived from liver transplant patients or patients with autoimmune diseases. According to experimental results conducted under the same conditions, IL-17 levels in the Lacrimispora amygdalina-treated group were consistently maintained at lower levels compared to the vehicle, and a stable trend of IL-17 reduction was also confirmed in the short-chain fatty acid (SCFA) treatment groups, such as propionate and acetate.

[0014] In addition, analysis of metabolites based on the culture medium or supernatant of Lacrimispora amygdalina confirmed through GC / MS analysis that acetic acid and propionic acid are significantly produced during the culture process, and immune cell responses associated with a decrease in IL-17 and an increase in IL-10 were observed even when the short-chain fatty acids were treated alone. These results suggest that the composition of the present invention exerts an action that regulates the secretion patterns of inflammatory cytokines in immune cells through SCFA components derived from the strain itself or its culture.

[0015] The composition of the present invention acted to inhibit IL-17 secretion, which increases under anti-CD3 stimulation under experimental conditions, while simultaneously increasing IL-10 secretion; similar trends were reproduced in multiple patient-derived cell experiments applying the same protocol. This supports the fact that Lacrimispora amygdalina or its metabolites are compositions that help alleviate the abnormal activation of immune cells and regulate the balance of immune responses under conditions where inflammation persists.

[0016] Furthermore, the composition of the present invention can be prepared in various forms, such as live bacteria, dead bacteria, culture supernatants, or SCFA components isolated therefrom, thereby providing a basis for confirming the same experimental efficacy in various formulations, such as oral and enteral administration. The experimental results of the present invention support the potential for the composition to be utilized as an immunomodulator that influences the cytokine secretion patterns of immune cells to alleviate inflammatory immune responses and promote regulatory immune responses. Brief explanation of the drawing

[0017] Figure 1 is a graph showing the results of inhibiting the secretion of IL-17, an inflammatory cytokine of the Th17 axis, by treatment with a component derived from Lacrimispora amygdalina and short-chain fatty acids (acetate / propionate / butyrate) according to one embodiment of the present invention. Figure 2 is a graph showing the results of a quantitative analysis of the production amounts of acetic acid and propionic acid among the metabolites of a Lacrimispora amygdalina strain according to one embodiment of the present invention. Figures 3 to 6 are graphs showing that the frequency of IL-17 expression in Th17 cells is reduced by treatment with Lacrimispora amygdalina and its metabolites according to one embodiment of the present invention. FIGS. 7 to 9 illustrate experimental results confirming that, despite variations in immune cell response among patients, the expression of IL-17 is suppressed and the expression of IL-10 is increased by treatment with Lacrimispora amygdalina and its metabolites according to one embodiment of the present invention, thereby alleviating the inflammatory response of the immune system. Specific details for implementing the invention

[0018] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. In the drawings, identical reference numbers or symbols refer to components that perform substantially the same function, and the size of each component in the drawings may be exaggerated for clarity and convenience of explanation. However, the technical concept of the present invention and its core components and operations are not limited only to the components or operations described in the following embodiments. In describing the present invention, if it is determined that a detailed description of known technologies or components related to the present invention may unnecessarily obscure the essence of the present invention, such detailed description will be omitted.

[0019] In embodiments of the present invention, terms including ordinal numbers, such as first, second, etc., are used solely for the purpose of distinguishing one component from another, and singular expressions include plural expressions unless the context clearly indicates otherwise. Furthermore, in embodiments of the present invention, terms such as 'composed of,' 'include,' 'have,' etc., should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0020] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. This description is intended to be detailed enough for a person skilled in the art to easily practice the invention, and it should be noted that the technical scope and concept of the present invention are not limited thereby.

[0021] Acetate / Propionate-based immunomodulation according to one embodiment of the present invention refers to a physiological response in which acetate and propionate, which belong to short-chain fatty acids (SCFAs), influence the host immune system to regulate the intensity and balance of the immune response. This regulatory action may occur naturally when SCFAs generated through microbial fermentation, dietary fiber degradation, or direct external administration reach immune cells or related tissues.

[0022] Acetate and propionate exist relatively stably in vivo and can contribute to alleviating the excessive activation of inflammation-related signals in various immune cell environments, or conversely, regulating suppressed immune responses. It is widely known that the influence of these SCFAs is not limited to specific diseases, immune cells, or tissues, but is involved in the host's overall immune balance (homeostasis).

[0023] These short-chain fatty acids exist in the body as water-soluble organic acids and can induce changes in immune-related responses at different levels depending on concentration, duration of exposure, and application environment. For instance, in environments where SCFAs are present, modulation of immune responses can be observed, such as the suppression of excessive secretion of inflammatory mediators or a relative increase in the expression of regulatory cytokines; this is a response that naturally occurs under various physiological conditions.

[0024] Additionally, acetate and propionate may exist alone or in a mixed form, and may be prepared in the form of microbial culture supernatants, extracts, chemical compounds, or their salts. These SCFAs may be utilized alone or together with other components in the composition, and may be prepared in various forms, such as liquids, freeze-dried powders, or soluble formulations.

[0025] Acetate / propionate-based immunomodulation in this specification is defined as a general and comprehensive concept not limited to specific mechanisms, pathways, or conditions. That is, it encompasses a wide range of immunomodulatory phenomena that appear under various physiological and environmental conditions, including the immune response modulating effects of SCFAs naturally observed in vivo.

[0026] Therefore, in the present invention, Acetate / Propionate is used as a broad concept encompassing microbial origins, chemical synthesis, biological conversion reactions, and salt, ester, and precursor forms, and is not limited to a specific production pathway or specific mechanism of action.

[0027] By defining it in this way, the present invention can broadly include immunomodulatory effects applicable in various compositions or formulations based on the general biological activity of SCFAs.

[0028] Lacrimispora amygdalina according to one embodiment of the present invention is an anaerobic Gram-positive bacterium belonging to the phylum Firmicutes and is a strain generally included in the microbial group of the Clostridia lineage. This strain is known to form spores and to produce short-chain fatty acids (SCFAs) using various carbon sources. According to the disclosed strain information, Lacrimispora amygdalina can grow under anaerobic conditions at 30–40°C and within a pH range of 6–7, and SCFAs such as acetic acid and propionic acid can be detected in the culture medium in general culture media.

[0029] This strain may contain substances (e.g., cell wall components, metabolites, etc.) that can affect immune cell responses not only in the form of live cells but also in the form of dead cells or cell extracts, and these characteristics can be maintained even when prepared in various formulations (freeze-dried products, concentrated supernatants, etc.). The 16S rRNA gene sequence of Lacrimispora amygdalina has been reported to have high similarity to bacteria of the Clostridium family, and there are strains isolated and preserved from strains registered at accredited depositary institutions such as DSMZ.

[0030] Acetate and propionate in this specification are representative short-chain fatty acids (SCFAs) widely produced during microbial fermentation processes, represented by the chemical formulas CH₃COOH and CH₃CH₂COOH, respectively. These are weakly acidic organic compounds that exist primarily in an ionized state under physiological conditions (pH 6–7) and can be produced in the intestinal environment through the fermentation of carbohydrates or organic acids by various microorganisms. Acetate and propionate may exist individually or in a mixed form, and these organic acids can be extracted and quantified from the supernatant of live cultures and, if necessary, prepared in the form of a salt or a concentrate.

[0031] Acetate and propionate are universal subcutaneous fatty acids (SCFAs) that can be produced by diverse intestinal microbial communities without being dependent on specific microorganisms; their production amounts may vary depending on the microbial culture environment, the composition of the medium used, and the type of carbon source. Because these SCFAs are highly biochemically water-soluble and exhibit stable characteristics under general experimental conditions, they can be utilized as components of various biological compositions.

[0032] Acetate / Propionate as defined in this specification is not limited to specific production pathways or specific microbial species, and is used as a broad concept that includes naturally occurring SCFAs produced during microbial culture, SCFAs produced by chemical or enzymatic methods, and their salts, esters, and precursor forms.

[0033] Accordingly, the present invention may comprehensively include the strain itself, the strain culture medium, or various forms of compositions containing SCFA components, and each component may be used individually or in combination.

[0034] The composition according to one embodiment of the present invention comprises short-chain fatty acids (acetate and propionate) or components derived from Lacrimispora amygdalina, in which a response was observed in which IL-17 secretion from Th17 cells decreased and IL-10 secretion from regulatory T cells or regulatory B cells increased in immune cells derived from liver transplant patients or patients with autoimmune diseases. This composition is based on the results of confirming the immune response regulating effect through the decrease in IL-17 and the increase in IL-10 measured in experiments, as specified in claims 1 to 3.

[0035] Lacrimispora amygdalina used in this example is a strain capable of being cultured under anaerobic conditions and possesses the characteristic of producing short-chain fatty acids during the fermentation process. Culture was performed in an anaerobic environment at 37°C with an oxygen concentration of 0.1% or less, and PYG medium containing 10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, 0.5 g / L cysteine ​​hydrochloride, and 1 mg / L vitamin B₁₂ was used as the medium. After 24 hours of culture, when the OD600 value reached the range of 0.8 to 1.0, the culture medium was harvested, and the supernatant from which the cells were removed by centrifugation (10,000×g, 10 min) was purified by 0.22 μm filtration. The purified supernatant was freeze-dried to obtain a composition derived from the culture medium in powder form.

[0036] The acetate and propionate content in this supernatant was quantified through GC / MS analysis. In the analysis, the relative concentration of short-chain fatty acids was calculated based on the instrument's correction factors (k₁, k₂) and sample injection volume (V₁, V₂), and reproducibility was confirmed within a deviation of ±5% in repeated measurements. The ratio of acetate to propionate obtained in this example was measured to be approximately 1:2 to 1:3, which is a level that can be reproduced by a person of ordinary skill under the same culture conditions.

[0037] The obtained acetate and propionate can each be used alone or included in a composition together with powder derived from Lacrimispora amygdalina culture medium. No immunocytotoxicity was observed for either component in the range of 0.1 to 1.0 mM, and a decrease in IL-17 secretion and an increase in IL-10 in Th17 cells were detected in the same range.

[0038] The immune cell experiment used 2×10 peripheral blood mononuclear cells (PBMCs) derived from liver transplant patients. 6 The study was performed after suspending the cells in RPMI1640 medium at a concentration of cells / mL and inducing Th17 differentiation using an anti-CD3 antibody (2 μg / mL). The composition was treated at a concentration of 10 μg / mL, and after 48 hours of incubation, IL-17 and IL-10 concentrations in the supernatant were quantified by ELISA. Experimental results showed that compared to the vehicle, IL-17 concentration decreased by an average of more than 300 pg / mL, while IL-10 concentration increased by an average of more than 150 pg / mL. These changes were consistently confirmed in at least three independent experiments.

[0039] The composition of the present embodiment may include Lacrimispora amygdalina culture medium, cell extract, or acetate and propionate obtained therefrom, and each component may be formulated in the form of a freeze-dried powder, a liquid extract, or a salt (sodium acetate, calcium propionate, etc.). A buffer solution in the pH range of 6.5 to 7.0 may be used during formulation, which is a suitable condition for maintaining the storage stability of the composition.

[0040] As such, the configuration and numerical values ​​of this embodiment are based on results confirmed in actual experiments and are specified to a level reproducible under the same conditions by a person skilled in the art using basic culture equipment and analytical instruments.

[0041] The active ingredient according to one embodiment of the present invention comprises short-chain fatty acids (acetate and propionate) isolated and purified from the supernatant obtained by culturing the Lacrimispora amygdalina strain deposited as DSMZ 12857. The strain is used as a seed for main culture after thawing the original strain distributed in a freeze-dried state and undergoing pre-culture at 37°C for 24 hours.

[0042] Pre-culture is performed in 15 mL of PYG (peptone-yeast extract-glucose) medium, and then the culture medium is inoculated into a 500 mL anaerobic Erlenmeyer flask at a ratio of approximately 1:20. To maintain anaerobic conditions, nitrogen gas is injected throughout the culture process to keep the oxygen concentration inside the flask below 0.1%, and resoajurin (0.001%) is added as a redox indicator to visually check the anaerobic status.

[0043] The medium composition consists of 20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, and 0.5 g / L cysteine ​​hydrochloride, and includes biotin (0.1 mg / L), vitamin B12 (0.05 mg / L), FeSO₄ (0.01 g / L), and MgSO₄ (0.05 g / L) as vitamins and trace metals. When GIFU medium is used instead of PYG, 15 g / L sodium pyruvate is used instead of glucose, and the protein source is adjusted to a peptone:yeast extract ratio of 2:1 to allow verification of differences in metabolite composition according to culture characteristics.

[0044] Strain culture was carried out at 37°C for 24 to 48 hours, and the culture was terminated when the OD600 value reached the range of 1.0 to 1.2. Subsequently, the cells and supernatant were separated by centrifugation (10,000 × g, 10 min), and only the supernatant was recovered to remove residual cells by 0.22 μm filtration. The filtered supernatant was frozen at -80°C and freeze-dried to obtain it in powder form.

[0045] The obtained freeze-dried material was redissolved using distilled water and prepared as a sample for GC / MS analysis. GC / MS analysis was performed using an HP-INNOWAX (30 m × 0.25 mm × 0.25 μm) column, with a sample injection temperature of 250°C and helium (1 mL / min) as the carrier gas. The oven temperature was maintained at 60°C for 2 minutes and then increased to 180°C at a rate of 10°C / min.

[0046] As a result of the analysis, acetic acid and propionic acid were separated at their respective retention times, with peaks detected at approximately 4.2 minutes for acetic acid and approximately 6.8 minutes for propionic acid. The areas of each peak (A₁, A₂) were converted into concentration values ​​using a calibration equation (C = (A / S) × F) prepared from standard solutions prepared under the same conditions. Here, C is the concentration (mM), A is the sample peak area, S is the standard solution peak area, and F is the correction factor.

[0047] The concentrations of acetic acid and propionic acid measured under the culture conditions were found to be in the average ranges of 1.2 ± 0.1 mM and 2.4 ± 0.2 mM, respectively, and showed reproducibility within ±10% in experiments repeated independently at least three times. In addition, the relative production ratio between the two media (PYG, GIFU) maintained a similar range (approx. 1:2 to 1:3).

[0048] Purification was performed using an ion exchange resin (Dowex 50W × 8-200, H type). The freeze-dried material was redissolved and passed through a column, then washed with distilled water to remove residual salts, followed by elution with 0.1 M ammonia solution (NH₄OH). The eluent was vacuum concentrated at 40°C or below to obtain a concentrate, which was then filtered through a 0.22 μm filter to secure a sterile final active ingredient sample. The pH of the final composition was adjusted to approximately 6.8 ± 0.2.

[0049] The purified acetic acid and propionic acid mentioned above can be used alone or in combination, and the composition ratio was maintained within the range naturally formed under culture conditions (approximately 1:2 or 1:3 level). This mixed composition is one in which a decrease in IL-17 and an increase in IL-10 were confirmed in experiments using immune cells derived from liver transplant patients.

[0050] The active ingredient is presented under conditions reproducible using conventional microbial culture equipment and analytical instruments (GC / MS, basic centrifuges, etc.), and is not dependent on specific equipment or reagents during the culture, separation, and purification stages. Therefore, by following the same culture conditions and purification procedures, a person of ordinary skill can obtain metabolites with substantially the same composition when repeating the experiment.

[0051] In one embodiment of the present invention, a composition containing the active ingredient can be applied within a concentration range in which the effects of reducing IL-17 and increasing IL-10 are confirmed, and can be used stably without cytotoxicity. All numerical values ​​in this embodiment are values ​​confirmed in actual experiments or describe conditions within a range reproducible by the experimenter.

[0052] According to one embodiment of the present invention, the fact that the total peak area of ​​the metabolite is detected to be more than twice that of the control culture means that the sum of short-chain fatty acid peaks (A_total), calculated by comparing the GC / MS analysis results of a control culture not inoculated with Lacrimispora amygdalina and a test culture inoculated under the same culture conditions, significantly increases in the test group. To confirm this, the culture supernatant obtained in the previous example was prepared as a sample for GC / MS analysis. An equal volume (1 mL) of each sample was taken, an internal standard (isobutyric acid 0.5 mM) was added, and the organic layer was recovered by extracting three times with ethyl acetate in a 1:1 ratio. The recovered solution was concentrated under a nitrogen stream and then diluted to 100 μL for analysis.

[0053] The peak area corresponding to acetic acid (A₁) and propionic acid (A₂) in the GC / MS chromatogram was calculated by integrating them, and the total peak area (A_total) was defined as A_total = A₁ + A₂. The ratio R = A_total_sample / A_total_control between the control group (A_total_control) and the experimental group (A_total_sample) was calculated, and the average R was measured to be 2.3 ± 0.2. This value was calculated as the average of three independent replicates, and the range of variation between experiments was maintained at ±8% or less.

[0054] The above pattern of increasing total peak area is a quantitative indicator showing that the production of short-chain fatty acids increases compared to the control group when the strain is inoculated and cultured. This showed a clear difference compared to the control group, where the production of organic acids is extremely low and A_total_control remains in a low range. The acetic acid and propionic acid peaks on GC / MS were analyzed under the same conditions at each time point and were reproducibly confirmed based on the analytical equipment and standard calibration curve.

[0055] The weight ratio of acetic acid to propionic acid was calculated by converting each peak area into a standard calibration curve to quantify the molar concentration (C₁, C₂), and then multiplying it by the molecular weight (M₁ = 60.05 g / mol, M₂ = 74.08 g / mol) to convert it to a mass basis. The formula is (C₁×M₁):(C₂×M₂), and in this example, the average ratio was measured to be 1:2.1 in PYG medium and 1:3.4 in GIFU medium. This ratio showed a variation within ±10% under the same culture conditions and did not show significant differences with changes in culture time (24–48 hours) and pH range of 6.0–6.5.

[0056] This ratio range (approximately 1:2 to 1:3) is a composition naturally formed during the culture of Lacrimispora amygdalina and was confirmed to be the same under both media conditions (PYG, GIFU). The ratio of the two substances was reproduced identically in GC / MS analysis after purifying and concentrating the culture medium, and serves as a standard for the quantitative determination of active ingredients for the consistent production of the composition.

[0057] It was confirmed in experiments using human peripheral blood-derived mononuclear cells (PBMCs) that the composition of the present invention reduces IL-17 secretion in Th17 cells by more than 50% compared to the vehicle. PBMCs are 2×10 6Th17 induction culture was performed for 5 days under conditions containing IL-6, IL-1β, IL-23, TGF-β, and anti-CD3 / CD28 antibodies prepared at a concentration of cells / mL. Subsequently, a composition containing the metabolites was treated at a concentration of 10 μg / mL and cultured for 48 hours. The culture supernatant was collected, and IL-17 concentration was measured by ELISA. As a result, the composition-treated group showed a decrease to 290 ± 30 pg / mL compared to the vehicle control group's average of 620 ± 45 pg / mL. The inhibition rate was calculated as [(IL-17_vehicle - IL-17_sample) / IL-17_vehicle] × 100, with an average of 53.2%. This value was consistent across three independent replicates and was statistically significant at the p < 0.01 level.

[0058] The IL-17 inhibitory effect showed a similar decreasing trend within the total SCFA concentration range of 2 mM even when the ratio of the two components was varied, and in particular, both compositions obtained in PYG medium (approx. 1:2 ratio) and GIFU medium (approx. 1:3 ratio) exhibited a consistent inhibitory effect relative to the vehicle. This result experimentally supports the fact that a mixture of the two components within the range of 1:1 to 1:5 specified in the present invention can be applied to the regulation of IL-17 secretion in Th17 cells.

[0059] The culture conditions, GC / MS analysis conditions, extraction and quantification procedures, Th17 differentiation, and ELISA verification processes of this example were all performed within a range reproducible by a person of ordinary skill using standardized experimental equipment and reagents. The presented conditions are not dependent on specific equipment or specific reagents, and substantially identical peak area, weight ratio, and IL-17 reduction values ​​can be obtained by applying the same method.

[0060] Consequently, the increase in the total peak area of ​​the above metabolites and the ratio of acetic acid to propionic acid serve as indicators of the composition of the active ingredient that contributes to the quantitative reduction of IL-17 secretion in Th17 cells by the composition of the present invention, and these figures and procedures are values ​​clearly derived based on experiments.

[0061] The immunomodulatory activity of the composition according to one embodiment of the present invention was evaluated in an in vitro culture system using peripheral blood mononuclear cells from liver transplant patients. PBMCs were isolated by density gradient centrifugation (400 × g, 30 min, room temperature) of 10 mL of whole blood collected in a heparin-treated tube using Ficoll-Paque PLUS (1.077 g / mL), and after washing twice with PBS, the cell concentration was set to 2 × 10⁶ 6 The concentration was adjusted to cells / mL. Culture was performed in a 24-well culture plate, and 1 mL of cell suspension was dispensed into each well. The culture medium used was based on RPMI1640 and contained 10% heat-inactivated FBS, penicillin 100 U / mL, streptomycin 100 μg / mL, and L-glutamine 2 mM.

[0062] To induce Th17, anti-CD3 monoclonal antibody (2 μg / mL) was coated onto a plate, and after cell inoculation, IL-6 (20 ng / mL), TGF-β (5 ng / mL), IL-1β (10 ng / mL), and IL-23 (10 ng / mL) were added, and the cells were cultured for an initial 24 hours at 37°C under 5% CO₂ conditions. Subsequently, the above composition was added at a final concentration of 10 μg / mL, and a control group treated with the same volume of vehicle (PBS) was included. Culture was maintained for 48 hours under the same conditions.

[0063] After the culture was completed, the supernatant was collected, cells were removed by centrifugation at 300 × g for 5 minutes, and the concentrations of each cytokine were quantified using Human IL-17A and IL-10 ELISA kits (commercial products). The ELISA was performed by measuring absorbance at 450 nm according to the manufacturer's protocol, and standard curves were constructed using each cytokine standard solution.

[0064] As a result of the experiment, the IL-17 concentration in the vehicle treatment group was measured at 615 ± 40 pg / mL, and upon treatment with the composition, it decreased to 295 ± 35 pg / mL, confirming an average reduction of more than 300 pg / mL. The reduction rate was approximately 52% when calculated using the formula [(IL-17_vehicle - IL-17_sample) / IL-17_vehicle] × 100. Under the same conditions, the IL-10 concentration increased from 180 ± 25 pg / mL in the vehicle treatment group to 330 ± 30 pg / mL in the composition treatment group, confirming an increase of more than 150 pg / mL. These changes were consistently observed in all three independent replicate experiments and showed a statistically significant difference (p < 0.01).

[0065] The Th17 / Treg cell ratio was measured via flow cytometry. Cells recovered after culture were treated with permeabilization buffer, stained with anti-IL-17A-PE and anti-FOXP3-FITC antibodies, and analyzed using FACSVerse (BD Biosciences). In the analysis using FlowJo software, the Th17 / Treg ratio of the vehicle group was measured as 1.45 ± 0.10, whereas that of the group treated with the composition was calculated as 0.93 ± 0.08. This result indicates that a decrease in the Th17 cell ratio and an increase in the Treg cell ratio were observed together, and this was confirmed to be repeatable under the same experimental conditions.

[0066] It was confirmed that the treatment concentration (10 μg / mL) of the above composition was within a non-toxic range in the evaluation of PBMC viability. As a result of the MTT assay, at a concentration of 10 μg / mL, the absorbance (570 nm) was approximately 0.98 times that of the vehicle, and the cell viability was maintained at 95% or higher.

[0067] The decrease in IL-17 (average 300 pg / mL or more), the increase in IL-10 (150 pg / mL or more), and the Th17 / Treg ratio (maintained at 1.0 or less) confirmed in this example are all results derived from actual measurements and are based on figures repeatedly verified in actual culture experiments, without relying on specific mechanisms or presumed pathways. In addition, the experimental procedure is configured under conditions that can be performed identically by a person of ordinary skill using general cell culture equipment, flow cytometers, and commercial ELISA reagents, thereby ensuring reproducibility.

[0068] Figure 1 is a graph showing the trend of decreasing IL-17 secretion under Th17 differentiation conditions by treatment with a component derived from Lacrimispora amygdalina and short-chain fatty acids (acetate, propionate, butyrate) according to one embodiment of the present invention. Peripheral blood mononuclear cells derived from liver transplant patients were stimulated with an anti-CD3 antibody (2 μg / mL) to induce Th17 differentiation, and then treated with vehicle, Lacrimispora amygdalina (10 μg / mL), acetate (1 mM), propionate (1 mM), and butyrate (1 mM) for 48 hours each. The vertical axis represents the IL-17 concentration (pg / mL) in the culture supernatant, and the horizontal axis represents each treatment condition. While high levels of IL-17 were detected in the vehicle treatment group, lower values ​​compared to vehicle were repeatedly observed in the L. amygdalina treatment group, and a trend of further decreasing IL-17 levels was confirmed in the propionate and butyrate treatment groups. This figure illustrates the experimental trend of L. amygdalina or its metabolites and short-chain fatty acids reducing IL-17 secretion from Th17 cells.

[0069] Figure 2 is a graph showing the results of quantitative analysis of the relative production amounts of acetic acid and propionic acid in the culture supernatant of the Lacrimispora amygdalina (DSMZ 12857) strain according to one embodiment of the present invention using GC / MS peak areas. The strain was cultured in GIFU medium and PYG medium at 37°C under anaerobic conditions for 24 to 48 hours, and the peak areas of each short-chain fatty acid were confirmed by analyzing the supernatant after culture using GC / MS. The horizontal axis indicates the control group and the strain treatment group, and the vertical axis represents the peak area based on a log scale. In the case of acetic acid, a clear increase in the peak was observed compared to the control group, and higher peak values ​​than the control group were also detected for propionic acid under both medium conditions. This result experimentally confirms that the strain produces acetic acid and propionic acid.

[0070] Figure 3 is a graph showing the change in the proportion of IL-17 positive cells decreasing under Th17 differentiation conditions by treatment with Lacrimispora amygdalina and short-chain fatty acids according to an embodiment of the present invention. Under the same Th17 induction conditions, the vehicle, Lacrimispora amygdalina, acetate, propionate, butyrate, and a control probiotic (Control GG) were treated, respectively, and the vertical axis represents the proportion of IL-17 positive cells (%). A lower cell proportion was observed in the L. amygdalina treatment group compared to the vehicle, and a pattern of further decreasing IL-17 positive cell proportions was repeatedly confirmed under propionate and butyrate conditions. This figure shows the tendency of L. amygdalina-based compositions and short-chain fatty acids to inhibit the Th17 response.

[0071] Figure 4 illustrates the results of measuring changes in the proportion of IL-17 positive cells in FACS experiments independently repeated under the same conditions as Figure 3. All experiments were performed under the same stimulation conditions, the same concentrations, and the same incubation times, and a lower IL-17 positive ratio compared to the vehicle was reproduced in the L. amygdalina, propionate, and butyrate treatment groups. This figure demonstrates that the composition of the present invention exhibits a stable IL-17 inhibition pattern even between repeated experiments.

[0072] Figure 5 is a graph showing the results of a decrease in the proportion of IL-17-positive cells in the L. amygdalina and short-chain fatty acid treatment groups compared to the vehicle in independent repeated experiments. While the IL-17 ratio in the vehicle treatment group remained within a high range, consistently low values ​​were observed in the L. amygdalina, propionate, and butyrate treatment groups. The lowest IL-17 ratios were confirmed with propionate and butyrate treatment, and the inhibitory trend of the L. amygdalina treatment group was also reconfirmed under the same conditions.

[0073] Figure 6 shows the results of another replicate experiment performed under the same Th17 induction conditions, illustrating that a lower proportion of IL-17 positive cells was observed in the L. amygdalina and short-chain fatty acid treatment group compared to the vehicle. Since no IL-17 inhibition was confirmed for the control probiotic (Control GG), the immune response inhibition pattern unique to the L. amygdalina and short-chain fatty acid combination is clearly distinguished.

[0074] FIG. 7 is a graph showing experimental data in which a decrease in IL-17 and an increase in IL-10 were simultaneously observed upon treatment of immune cells derived from liver transplant patients with L. amygdalina and short-chain fatty acids according to one embodiment of the present invention. The upper graph represents the IL-17 concentration (pg / mL), and the lower graph represents the IL-10 concentration (pg / mL). A trend of decreasing IL-17 values ​​and increasing IL-10 values ​​was repeatedly observed upon treatment with L. amygdalina, acetate, propionate, and butyrate compared to the vehicle, supporting the immunomodulatory effect of the composition of the present invention.

[0075] Figure 8 illustrates the results of an experiment using peripheral blood mononuclear cells obtained from multiple liver transplant patients, showing that when treated with L. amygdalina and short-chain fatty acids, a decrease in IL-17 and an increase in IL-10 were observed in common, transcending individual differences among patients. When treated with propionate and butyrate, the decrease in IL-17 was significant, and IL-10 increased under the same conditions, maintaining a high IL-10 / IL-17 ratio.

[0076] Figure 9 shows the results of an extended analysis of multiple samples from liver transplant patients, in which a decrease in IL-17 concentration and an increase in IL-10 are repeatedly observed under conditions treated with L. amygdalina and short-chain fatty acids compared to the vehicle. The lowest IL-17 values ​​were observed in the propionate and butyrate treatment groups, and IL-10 concentrations remained in a high range under the same conditions, allowing for visual confirmation of the immunomodulatory combination effect.

Claims

Claim 1 A Lacrimispora amygdalina and Acetate / Propionate-based immunomodulatory composition used in an in vitro culture system in which peripheral blood mononuclear cells of a liver transplant patient are stimulated with an anti-CD3 antibody (2 μg / mL), wherein the composition comprises, as active ingredients, both acetic acid and propionic acid isolated and purified from the culture supernatant obtained by culturing the Lacrimispora amygdalina strain deposited as DSMZ 12857 in a medium at 37°C under anaerobic conditions for 24 to 48 hours, wherein the sum of the peak areas of acetic acid and propionic acid measured by GC / MS in the culture supernatant is at least twice the sum of the peak areas in a control culture medium not inoculated with microorganisms, and the weight ratio of acetic acid and propionic acid produced in the culture supernatant is measured to be in the range of 1:2 to 1:3, and the composition is used for the liver transplant patient An immunomodulatory composition characterized by acting such that when administered at a concentration of 10 μg / mL to an in vitro culture system in which peripheral blood mononuclear cells are stimulated with an anti-CD3 antibody (2 μg / mL), after 48 hours of culture, the IL-17 concentration in the cell culture supernatant decreases by an average of 300 pg / mL or more compared to the vehicle, and simultaneously the IL-10 concentration increases by 150 pg / mL or more, thereby maintaining a Th17 / Treg cell ratio of 1.0 or less. Claim 2 delete Claim 3 delete

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