Extracellular vesicles containing 15-lipoxygenase

Abstract

The present invention provides a method for treating an inflammatory condition or disease comprising administering a pharmaceutically effective amount of extracellular vesicles (EVs), wherein the EVs comprise 15-lipoxygenase (15-LOX) and / or a nucleic acid encoding 15-LOX, and optionally further comprising one or more of 12-lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX), and / or one or more nucleic acids encoding one or more of 12-LOX and 5-LOX. Extracellular vesicles (EVs) for use in treating inflammatory diseases or conditions are also provided, the EVs comprising one or more nucleic acids encoding 15-lipoxygenase (15-LOX) and / or 15-LOX, optionally, in addition, one or more nucleic acids encoding 12-lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX), and / or one or more nucleic acids encoding 15-LOX, 12-LOX, and 5-LOX. Similarly, isolated extracellular vesicles comprising one or more nucleic acids encoding 15-lipoxygenase (15-LOX) and / or 15-LOX, optionally, in addition, one or more nucleic acids encoding 12-lipoxygenase (12-LOX), 5-lipoxygenase (5-LOX), and / or one or more nucleic acids encoding 15-LOX, 12-LOX, and 5-LOX.

Description

[Technical Field]

[0001] The present invention relates to extracellular vesicles (EVs) containing lipoxygenase (LOX), specifically 15-LOX, and optionally 12-LOX and / or 5-LOX, or nucleic acid sequences encoding such LOX, and to the use thereof for treating inflammatory conditions. EVs may be artificial or derived from naturally occurring cells, such as living cells, apoptotic cells, or mesenchymal stem cells.

[0002] Abbreviations used herein: ACdEV - Apoptotic cell-derived extracellular vesicle, COX - Cyclooxygenase, EV - Extracellular vesicle, LM - Lipid mediator of inflammation, LOX - Lipoxygenase, MΦ - Macrophage, LT - Leukotriene, LX - Lipoxin, PD - Protectin, PG - Prostaglandin, PS - Phosphatidylserine, PUFA - Polyunsaturated fatty acid, SPM - Specialized convergence-promoting lipid mediator, RV - Resolvin, VD3-1α,25-Dihydroxyvitamin D3. [Background technology]

[0003] Programmed cell death, also known as apoptosis, is a physiological process in which infected, damaged, or simply unwanted cells are eliminated. For a long time, it was considered an "inactive" process, primarily due to its distinctive morphological characteristics. 1 Today, it is widely recognized that apoptosis plays a central role in regulating homeostasis of the immune response. 2 Therefore, failure to properly perform apoptosis can lead to chronic inflammation and autoimmunity. 3 , and tumor development 4 This poses a risk of significant consequences such as those mentioned above.

[0004] Inflammation is a complex and carefully coordinated process at both the cellular and molecular levels, a protective immune response to acute loads that, if not efficiently controlled, can become chronic and lead to inflammatory diseases. The release of exogenous or endogenous chemical mediators within injured tissue promotes an inflammatory response through local vascular changes, enabling the recruitment of professional phagocytic cells (i.e., neutrophils and macrophages). However, once they encounter and eliminate the immune load, the tissue must return to its pre-inflammatory state through the resolution phase of inflammation. For inflammation to resolve, continuous inflammatory cell recruitment must cease, recruited neutrophils undergo apoptosis, and are phagocytosed by resident and recruited macrophages, escaping the tissue from the lymphatic system. Thus, apoptosis plays a central role in re-establishing tissue homeostasis by interacting with macrophages and promoting the resolution stage of inflammation, although the complete molecular mechanisms are still unclear. 5 .

[0005] Molecular signals that regulate the migration of neutrophils and macrophages to inflammatory tissues include bioactive metabolites known as inflammatory lipid mediators (LMs). These are products of phospholipid catabolism and are released locally by recruited cells, although some may be of microbial origin. The entire convergence process is driven by the overall balance of inflammatory and inflammatory lipid mediators, secreted as a result of the coordinated action of cell receptors and enzymes. 6 During the acute phase of inflammation, phagocytic cell mobilization is performed on leukotrienes (LTs). 7 and prostaglandins (PG) 8is supported by their local release. The increase in their levels promotes driving inflammation. Thus, both metabolite classes are known for their pro-inflammatory properties. However, PGD2 and PGE2 produced from the cyclooxygenase (COX) pathway can act as a convergence-promoting molecular “switch” that has the ability to induce the transcription of the enzyme lipoxygenase (LOX), which is responsible for the synthesis of small but very potent specialized proresolving mediators (SPM) such as lipoxin (LX), resolvin (RV), and protectin (PD). 9 For example, such a “class switch” in neutrophils and macrophages would require upregulation of 15-LOX, which is accompanied by a switch from the use of arachidonic acid (AA) for LTB4 production (via 5-LOX) to the synthesis of LXA4 via 15-LOX. This would actively promote further neutrophil uptake, stimulate non-inflammatory monocyte recruitment, support macrophage uptake of apoptotic neutrophils, and facilitate their escape from the lymphatic system. Thus, neutrophil apoptosis and its communication with the innate immune system represent key events that lay the groundwork for a successful outcome - convergence leading to resolution of inflammation. 10 .

[0006] The nature of the complex cell - cell communication between dying cells and the immune system is not yet fully clear. In addition to lipid mediators, dying cells drive phagocyte recruitment by releasing so - called soluble “find - me” signals (ATP and UTP, fractalkine, lysophosphatidylcholine, sphingosine 1 - phosphate), and present “eat - me” tags on their surface to promote phagocyte uptake. 11 . In recent years, extracellular vesicles have emerged as novel mediators of cell communication. 12 . Considering the nature of their interaction with host “recipient” cells, extracellular vesicles can not only trigger surface receptors on immune cells, but also deliver their numerous components, as well as their cargo of proteins, lipids, small molecules, and genetic material, thereby inducing an immune response. 13While extracellular vesicles from living cells have been studied in more detail, extracellular vesicles derived from apoptotic cells (ACdEVs) and their immunomodulatory properties have received relatively little attention. 14 To date, different research groups have demonstrated the chemical attraction properties of ACdEV. 15,16,17 Fractal caine (CX3CL1) on the surface of ACdEV 16,18 and ICAM-3 17 It has been identified as a "find-me" signal responsible for macrophage migration and ACdEV binding with immune cells, while glycosylated ligands on the ACdEV surface act as an "eat-me" signal. 19 It is highly probable that various other ACdEV-related molecules may possess immunomodulatory properties. [Overview of the Initiative] [Means for solving the problem]

[0007] Focusing on their ability to communicate with the immune system and actively promote the resolution of inflammation, the inventors investigated the release of EVs from dying leukocytes and sought to identify their composition. The inventors demonstrate that during apoptosis, human primary T cells actively release ACdEVs with a “pro-resolution phenotype” characterized by the presence of active lipoxygenase, while carrying a total lipid metabolome with high levels of SPM compared to pro-inflammatory LT and PG. The inventors also demonstrate LOX transport in EVs from various leukocytes, including primary origins of monocytes and B cells, as well as cell lines. These “active EVs” carry active lipoxygenase, with 15-LOX having the highest specific activity, delivering active lipoxygenase to recipient cells to support AA catabolism. Importantly, the inventors reveal that these ACdEVs interact with macrophages, thereby driving the pro-resolution phenotype while significantly reducing their endogenous LT levels. In summary, these data reveal important new metabolic activities in ACdEV.

[0008] Identifying the key elements required to produce convergence-promoting activity will enable the production of EVs that have the ability to reduce inflammatory conditions.

[0009] The present invention provides a method for treating an inflammatory disease or condition comprising administering a pharmaceutically effective amount of extracellular vesicles (EVs), the EVs comprising one or more of 15-lipoxygenase (15-LOX), 12-lipoxygenase (12-LOX), and / or 5-lipoxygenase (5-LOX), and / or one or more nucleic acids encoding one or more of 15-LOX, 12-LOX, and / or 5-LOX. Preferably, the EVs comprise 15-lipoxygenase (15-LOX) and / or nucleic acids encoding 15-LOX, and optionally further comprising one or more of 12-lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX), and / or one or more nucleic acids encoding one or more of 12-LOX and 5-LOX.

[0010] 15-LOX (encoded by ALOX15, i.e., arachidonic acid 15-lipoxygenase) catalyzes the oxygenation of arachidonic acid at carbon 15 to form 15-HpETE. 15-LOX can be used alone.

[0011] 15-LOX may also have the ability to catalyze the oxygenation of arachidonic acid at carbon 12. Alternatively or in addition, 15-LOX may be used in conjunction with arachidonic acid 12-lipoxygenase (12-LOX), which has the ability to oxidize arachidonic acid at position 12.

[0012] Arachidonic acid 5-lipoxygenase, also known as ALOX-5, 5-lipoxygenase, or 5-LOX, is known to convert several essential fatty acids into leukotrienes and a wide range of other bioactive components. It has two catalytic activities. For arachidonic acid, it adds a hydroperoxy to carbon 5 of arachidonic acid. The 5S-HpETE intermediate is then released by the enzyme and can be rapidly reduced to its corresponding alcohol by cellular glutathione peroxidase, or alternatively, further metabolized to its epoxide by the enzyme's epoxidase activity. The enzyme is thought to have both pro-inflammatory and converging activity (i.e., it reduces inflammation). Therefore, the presence of 5-LOX may be beneficial in promoting a converging phenotype and reducing inflammation. One possibility is to inhibit 5-LOX or provide its inhibitors to reduce its pro-inflammatory activity. Lipoxygenases typically have the ability to oxidize both free arachidonic acid (AA) and AA bound to phospholipids and lipoproteins. Nucleic acids may encode one or more of 15-LOX, 12-LOX, or 5-LOX. Each nucleic acid typically encodes an active lipoxygenase. One or both of 15-LOX and 12-LOX can typically be used. Preferably, 15-LOX is used in combination with one or more of 12-LOX and 5-LOX, optionally.

[0013] Nucleic acids can be ribonucleic acid (RNA). Optionally, RNA can be messenger RNA (mRNA).

[0014] Extracellular vesicles are also provided for use in treating inflammatory diseases or conditions, comprising one or more nucleic acids encoding 5-lipoxygenase, 12-lipoxygenase, and 15-lipoxygenase, or one or more nucleic acids encoding one or more of 15-LOX, 12-LOX, and 5-LOX. Preferably, the EV comprises 15-lipoxygenase (15-LOX) and / or nucleic acids encoding 15-LOX, and optionally, in addition, one or more 12-lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX), and / or one or more nucleic acids encoding one or more of 12-LOX and 5-LOX.

[0015] Inflammatory diseases can be chronic inflammatory conditions or states. Inflammatory diseases may be selected from chronic wounds, inflammatory skin diseases, autoimmune diseases, nephritis, aging, and dementia. It has been noted that inflammatory processes are involved in many signs of aging. The ability to use any of 15-LOX, 12-LOX, and 5-LOX, and / or combinations thereof, to promote a convergent, supportive phenotype for reducing inflammation is expected to be able to treat one or more aging-related conditions. Inflammatory diseases may be selected from internal or external wounds, diabetic wounds, and SLE (systemic lupus erythematosus).

[0016] EV may contain 15-lipoxygenase (15-LOX) or nucleic acids encoding 15-LOX.

[0017] EVs can be isolated, naturally occurring EVs or non-naturally occurring EVs. For example, EVs can be cell-derived EVs, such as apoptotic cell-derived extracellular vesicles (ACdEVs), or mesenchymal stem cell EVs. Both types of EVs are generally known in the art.

[0018] EVs can be artificial and do not exist naturally.

[0019] Extracellular vesicles (EVs) are particles defined by a lipid bilayer and are naturally released from almost all types of cells, but unlike cells, they cannot replicate. Naturally occurring EVs typically carry cargo such as proteins, nucleic acids, lipids, metabolites, or other components from the parent cell. It is also possible to produce artificial EVs that contain a lipid bilayer and one or more additional components.

[0020] For example, EVOX Therapeutics has several unique EV systems for transporting mRNA or proteins, such as active enzymes. See, for example, International Publication Nos. 2020 / 225392, 2022 / 229220, and 2018 / 153581, which are incorporated herein by reference in their entirety.

[0021] Therefore, EVs can be obtained from naturally occurring or non-naturally occurring sources. EVs can be targeted to be absorbed by pro-inflammatory macrophages. Therefore, they may contain one or more markers that can target pro-inflammatory macrophages. These may be one or more "eat-me" markers, e.g., annexin A1, callecticulin, and phosphatidylserine. One or more adhesion molecules, e.g., IgSF members, most typically ICAM-3 (intercellular adhesion molecule 3), CD44, integrin α-1, and integrin β-2 may be provided on the EVs.

[0022] One or more "find-me" molecules, such as fractalkine (CX3CL1) and ICAM-3, may also be present on the EV surface.

[0023] It is desirable to eliminate pro-inflammatory compounds. These include, for example, leukotrienes, such as arachidonic acid 5-lipoxygenase activating protein (AL5AP, also known as FLAP) and leukotriene A-4 hydrolase (LKHA4). Flap may be present when 5-LOX is used in EV because it plays a role in 5-LOX activity.

[0024] EV typically has a size range of 10–1,000 nm, and more typically 70–700 nm.

[0025] Typically, EVs produced from naturally occurring sources (e.g., ACdEVs or mesenchymal stem cell EVs) have a diameter in the range of approximately 90 nm to 130 nm, more typically 100 nm to 200 nm. Optionally, EVs produced from naturally occurring sources (e.g., ACdEVs or mesenchymal stem cell EVs) have a mode size in the range of 90 nm to 130 nm, more typically 100 nm to 200 nm.

[0026] EV can be administered to the subject, or can be administered intravenously, intraperitoneally, topically, intranasally, or orally by inhalation, and can be administered by nebulizer or inhaler.

[0027] The present invention also provides isolated EVs comprising one or more nucleic acids containing either 15-LOX or 12-LOX, or either or both of 15-LOX or 12-LOX. Preferably, the isolated EVs contain 15-LOX in combination with 12-LOX as an option.

[0028] As discussed above, these may also include 5-LOX. Furthermore, as previously discussed, EVs can be isolated, naturally occurring EVs or non-naturally occurring EVs, as described above.

[0029] EVs may be supplied with pharmaceutically acceptable excipients.

[0030] Naturally occurring extracellular proteins (EVs) can be modified to remove one or more unwanted or undesirable pro-inflammatory molecules, such as those mentioned above, or to incorporate inhibitors of such pro-inflammatory molecules.

[0031] EV can optionally consist of 15-LOX and / or 12-LOX, with or without 5-LOX. Optionally, EV can essentially consist of 15-LOX. EV can essentially consist of 15-LOX in combination with one or more of 12-LOX and 5-LOX.

[0032] EVs may, optionally, be naturally occurring EVs, such as ACdEVs or mesenchymal stem cell (MSC) EVs. While not constrained by theory, the inventors believe that EVs derived from apoptotic cells may have different uptake mechanisms depending on their membrane lipid composition. MSC-derived EVs may also exert different effects through the transport of other immunomodulatory molecules, such as INDO.

[0033] One or more markers capable of targeting pro-inflammatory macrophages, such as one or more "eat-me" markers, such as annexin A1, calreticulin, or phosphatidylserine, may be provided.

[0034] One or more "find-me" molecules, such as fractalkine (CX3CL1) and ICAM-3, may also be present on the EV surface.

[0035] Typically, EVs have a diameter of approximately 10 nm to 1,000 nm, more typically 70 nm to 700 nm. EVs may have a diameter of approximately 90 nm to 130 nm, more typically 100 nm to 200 nm. Optionally, EVs may have mode sizes of 10 nm to 1,000 nm or 70 nm to 700 nm. EVs may have mode sizes of 90 nm to 130 nm or 100 nm to 200 nm.

[0036] EV can be administered to the subject intraperitoneally, topically, intranasally, orally by inhalation, more typically via nebulizer or inhaler.

[0037] The inventors' in vivo studies show beneficial effects with an EV (specified by protein concentration) dose of just 10 μg per animal (approximately 10 e from approximately 50 million cells). 9(Equivalent to EV). Typically, up to 100 μg, and more typically 30–50 μg, are used in the art.

[0038] The present invention is illustrated here by reference to the following figures. [Brief explanation of the drawing]

[0039] [Figure 1A] Characterization of EVs derived from T cells during apoptosis. Primary human T cells were induced to apoptosis by anti-Fas in the presence of cycloheximide. AcdEVs were isolated and analyzed for size, concentration, PS exposure, and protein composition. TRPS measurements of size and concentration of EVs isolated from apoptotic T cell supernatant by size exclusion chromatography. Western blot of inset:TSG-101 indicates that some of the isolated EVs are of exosome origin. The data shown are representative of similar experiments. [Figure 1B] Characterization of EVs derived from T cells during apoptosis. Primary human T cells were induced to apoptosis by anti-Fas in the presence of cycloheximide. AcdEVs were isolated and analyzed for size, concentration, PS exposure, and protein composition. Representative flow cytometry histograms of collected EVs stained with the thiol-reactive dye Bodipy show that approximately 89% of the measured particles were stain-positive and of vesicle origin. Unstained AcdEVs were used as a negative control. The data shown are representative of similar experiments. [Figure 1C]Characterization of extracellular proteins (EVs) derived from T cells during apoptosis. Primary human T cells were induced to apoptosis by anti-Fas in the presence of cycloheximide. AcdEVs were isolated and analyzed for size, concentration, PS exposure, and protein composition. Representative flow cytometry histograms of EVs stained with annexin V (AnxV) show that 69% of EVs had exposed PS. Ca2+ EDTA depletion was used as a negative control to eliminate PS recognition by AnxV. The data shown are representative of similar experiments. [Figure 1D] Characterization of T cell-derived EVs during apoptosis. Primary human T cells were induced to apoptosis by anti-Fas in the presence of cycloheximide. AcdEVs were isolated and analyzed for size, concentration, PS exposure, and protein composition. A Venn diagram visualizes the proteomic overlap between AcdEVs, the entire Vesiclepedia, and all T cell-derived EVs. The data shown are representative of similar experiments. [Figure 1E] Characterization of EVs derived from T cells during apoptosis. Primary human T cells were induced to apoptosis by anti-Fas in the presence of cycloheximide. AcdEVs were isolated and analyzed for size, concentration, PS exposure, and protein composition. Volcano plot showing significant protein enrichment in ACdEVs (right) compared to apoptotic T cells. The data shown are representative of similar experiments. [Figure 1F] Characterization of EVs derived from T cells during apoptosis. Primary human T cells were induced to apoptosis by anti-Fas in the presence of cycloheximide. AcdEVs were isolated and analyzed for size, concentration, PS exposure, and protein composition. Gene ontology enrichment (simplified graph) of biological processes (green), cellular components (purple), and protein molecular function (orange) was found to be more than twice as high in ACdEVs compared to T cells. The data shown are representative of similar experiments. [Figure 1G]Characterization of EVs derived from T cells during apoptosis. Primary human T cells were induced to apoptosis by anti-Fas in the presence of cycloheximide. AcdEVs were isolated and analyzed for size, concentration, PS exposure, and protein composition. Schematic diagram of ACdEVs with the major protein classes enriched in ACdEVs in this study. The data shown are representative of similar experiments. [Figure 2A] Lipid metabolome profiling of EVs derived from apoptotic primary human T cells. Dual-acting SPM (green) and pro-inflammatory LM (red) are endogenously produced from arachidone (AA), eicosapentaene (EPA), and docosahexaenoic acid (DHA) via the LOX and COX pathways. [Figure 2B] Lipid metabolome profiling of extracellular genes (EVs) derived from apoptotic primary human T cells. Targeted LC-MS / MS (MRM) analysis revealed that EVs were loaded with: PUFAs (AA, EPA, and DHA), n=4. All results are expressed as mean ± SEM. Statistical analysis: t-test, two-tailed, no pairing. [Figure 2C] Lipid metabolome profiling of extracellular viable cells (EVs) derived from apoptotic primary human T cells. Targeted LC-MS / MS (MRM) analysis revealed that EVs were loaded with: LOX-oxidized PUFAs as major LOX metabolites, n=5. All results are expressed as mean ± SEM. Statistics: t-test, two-tailed, no pairing. [Figure 2D] Lipid metabolome profiling of extracellular viable cells (EVs) derived from apoptotic primary human T cells. Targeted LC-MS / MS (MRM) analysis revealed that EVs were loaded with: LOX-oxidized PUFAs as pro-inflammatory metabolites, n=5. All results are expressed as mean ± SEM. Statistical analysis: t-test, two-tailed, no pairing. [Figure 2E]Lipid metabolome profiling of extracellular viable cells (EVs) derived from apoptotic primary human T cells. Targeted LC-MS / MS (MRM) analysis revealed that EVs were loaded with: low-inflammation, resolving lipid mediators, LOX-oxidized PUFAs as SIs, n=5. All results are expressed as mean ± SEM. Statistical analysis: t-test, two-sided, no pairing. [Figure 2F] Lipid metabolome profiling of EVs derived from apoptotic primary human T cells. ACdEVs carry high levels of both AA-derived and total SPM compared to pro-inflammatory metabolites. All results are expressed as mean ± SEM. Statistical analysis: t-test, two-sided, no pairing. [Figure 3A] ACdEV is an "active" metabolic compartment. Typical Western blots show the presence of 5-LOX, 15-LOX, 12-LOX, and sPLA2 in ACdEV, but not COX-2. [Figure 3B] ACdEV is an "active" metabolic compartment. Fluorescence analysis lipoxygenase activity assays have shown that ACdEV carries active LOX. [Figure 3C] ACdEV is an "active" metabolic compartment. The measured decrease in LOX-related fluorescence in the presence of specific LOX inhibitors was used to calculate the specific activities of 5-LOX, 12-LOX, and 15-LOX in ACdEV. All results are expressed as mean ± SEM, n=3. Statistical analysis: t-test, two-tailed, no pairing. [Figure 3D] ACdEV is an "active" metabolic compartment. LOX activity (cells + EV, blue bars) in ALOX12ko cells incubated with EV for 2 hours was 41% higher than in cells without EV (cells, pink bars), indicating that EV can deliver active enzymes after internalization by host cells. Data were further normalized to total EV protein levels. All results are expressed as mean ± SEM, n=3. Statistical analysis: t-test, two-tailed, no pairing. [Figure 3E]ACdEV is the “active” metabolic compartment. Incubation of ALOX5ko cells with 5-LOX-filled ACdEV for 18 hours (incubated and annotated; blue bars) resulted in a significant increase in the 5-LOX metabolites 5(S)-HETE and 5(S)-HEPE compared to their cumulative levels in ACdEV and ALOX5ko cells (EV+ cells, yellow / pink bars). Individual contributions of the metabolites quantified in ACdEV (EV, yellow bars) and ALOX5ko cells (cells, pink bars) are also shown as separate bars. The levels of metabolites shown by incubation and EV+ cells were statistically compared. All results are expressed as mean ± SEM, n=3. Statistics: t-test, two-tailed, no pairing. [Figure 4A] ACdEV modulates the macrophage response. THP-1-derived macrophages (M0, unpolarized, clear bars) were incubated with ACdEV (blue bars), and indicated lipid mediators were assayed. The data suggest upregulation of 15-lipoxygenase (15-LOX) that switches the macrophage phenotype, with a significant increase in anti-inflammatory and pro-converging lipid metabolites (15(S)-hydroxy PUFA and SPM produced by dual 15-LOX oxygenation of DHA) derived from the 15-LOX oxygenation pathway (green boxes). This is accompanied by the complete disappearance of pro-inflammatory 5-lipoxygenase (5-LOX)-derived leukotrienes (LT) after vesicle treatment (red boxes). No changes in the levels of metabolites produced by 12-lipoxygenase and cyclooxygenase were observed. Results are expressed as mean ± SEM, n=3, statistics: t-test, paired, two-sided, *P<0.05. [Figure 4B1]ACdEV modulates the macrophage response. Primary human monocyte-derived macrophages were analyzed by flow cytometry after immunofluorescence staining, either as unpolarized macrophages (M0, clear bars), after polarization to M1 macrophages (red bars), M2 macrophages (green bars), or after 24 hours of treatment with ACdEV (M+EV, blue bars). Surface expression of CD11b, CD14, CD16, CD40, CD64, CD80, CD86, CD163, CD206, and CD209 was monitored using flow cytometry, enabling the identification of phenotypic differences in three macrophage families: M0-CD14+, M1-CD40+CD64+CD86+, and M2-CD11b+CD206+CD209+. Data were normalized to the mean fluorescence intensity observed for M0 within each replicate and set to 100%. PCA analysis revealed that EV-treated macrophages (M0+EV) exhibited a phenotype similar to that of convergence-promoting M2 macrophages. Flow cytometry histograms and PCA biplots are single biological replicates representative of at least three independent experiments. All results are expressed as mean ± SEM. Statistics: One-way ANOVA with Tukey's multiple comparison test. [Figure 4B2]ACdEV modulates the macrophage response. Primary human monocyte-derived macrophages were analyzed by flow cytometry after immunofluorescence staining, either as unpolarized macrophages (M0, clear bars), after polarization to M1 macrophages (red bars), M2 macrophages (green bars), or after 24 hours of treatment with ACdEV (M+EV, blue bars). Surface expression of CD11b, CD14, CD16, CD40, CD64, CD80, CD86, CD163, CD206, and CD209 was monitored using flow cytometry, enabling the identification of phenotypic differences in three macrophage families: M0-CD14+, M1-CD40+CD64+CD86+, and M2-CD11b+CD206+CD209+. Data were normalized to the mean fluorescence intensity observed for M0 within each replicate and set to 100%. PCA analysis revealed that EV-treated macrophages (M0+EV) exhibited a phenotype similar to that of convergence-promoting M2 macrophages. Flow cytometry histograms and PCA biplots are single biological replicates representative of at least three independent experiments. All results are expressed as mean ± SEM. Statistics: One-way ANOVA with Tukey's multiple comparison test. [Figure 4B3]ACdEV modulates the macrophage response. Primary human monocyte-derived macrophages were analyzed by flow cytometry after immunofluorescence staining, either as unpolarized macrophages (M0, clear bars), after polarization to M1 macrophages (red bars), M2 macrophages (green bars), or after 24 hours of treatment with ACdEV (M+EV, blue bars). Surface expression of CD11b, CD14, CD16, CD40, CD64, CD80, CD86, CD163, CD206, and CD209 was monitored using flow cytometry, enabling the identification of phenotypic differences in three macrophage families: M0-CD14+, M1-CD40+CD64+CD86+, and M2-CD11b+CD206+CD209+. Data were normalized to the mean fluorescence intensity observed for M0 within each replicate and set to 100%. PCA analysis revealed that EV-treated macrophages (M0+EV) exhibited a phenotype similar to that of convergence-promoting M2 macrophages. Flow cytometry histograms and PCA biplots are single biological replicates representative of at least three independent experiments. All results are expressed as mean ± SEM. Statistics: One-way ANOVA with Tukey's multiple comparison test. [Figure 4B4]ACdEV modulates the macrophage response. Primary human monocyte-derived macrophages were analyzed by flow cytometry after immunofluorescence staining, either as unpolarized macrophages (M0, clear bars), after polarization to M1 macrophages (red bars), M2 macrophages (green bars), or after 24 hours of treatment with ACdEV (M+EV, blue bars). Surface expression of CD11b, CD14, CD16, CD40, CD64, CD80, CD86, CD163, CD206, and CD209 was monitored using flow cytometry, enabling the identification of phenotypic differences in three macrophage families: M0-CD14+, M1-CD40+CD64+CD86+, and M2-CD11b+CD206+CD209+. Data were normalized to the mean fluorescence intensity observed for M0 within each replicate and set to 100%. PCA analysis revealed that EV-treated macrophages (M0+EV) exhibited a phenotype similar to that of convergence-promoting M2 macrophages. Flow cytometry histograms and PCA biplots are single biological replicates representative of at least three independent experiments. All results are expressed as mean ± SEM. Statistics: One-way ANOVA with Tukey's multiple comparison test. [Figure 5] Administration of EVs to mice with experimentally induced allergic airway inflammation reduces the presence of eosinophils and inflammatory macrophages. Mice were exposed to either PBS as a control (blue bars) or dust mite (HDM) antigen (red bars) to induce pneumonia. Three days after exposure, primary human T cell-derived EVs were administered topically (in, intranasally) or systemically (ip, intraperitoneally). Eighteen hours later, the mice were sacrificed, their lungs dissected, and immune cells were stained for multicolor flow cytometry. (A) Neutrophils (CD11b+ / Ly6G+), (B) Eosinophils (SiglecF+), and (C) Inflammatory macrophages MΦ (Ly6C+) are shown. Six mice were used in two independent experiments for each treatment. *P<0.05, **P<0.01. EVs did not exert any detectable significant effect in PBS-treated mice. [Modes for carrying out the invention]

[0040] material RPMI-1640 cell culture medium (with and without phenol red), Iskov modified Dulbecco's medium (IMDM), L-glutamine, penicillin / streptomycin, cycloheximide, Dulbecco's phosphate-buffered saline, EDTA, goat-produced anti-rabbit IgG (total molecule)-peroxidase antibody (catalog number A0545-1ML), rabbit-produced anti-mouse IgG (total molecule)-peroxidase antibody (catalog number A9044-2ML), and docosahexaenoic acid were purchased from Sigma Aldrich (Irvine, UK). South American-origin fetal bovine serum and ACK (ammonium chloride-potassium) lysis buffer were purchased from Gibco (Thermo Fisher). The anti-Fas antibody (human activated, CH11 clone) and Amicon® Ultra-15 centrifugal filter unit 10kDa were from Millipore (Watford, UK). 1α,25-dihydroxyvitamin D3 was from Enzo Life Sciences (Exter, UK). Megamix Plus FSC and SSC beads were manufactured by Biocytex (Marseille, France). Acetonitrile and formic acid (both ULC-MS grade) were from Biosolve (Valkenswaard, Netherlands). HPLC-grade methanol and ethanol, as well as MS-grade water, were purchased from Fisher Scientific (Loughborough, UK). Trypsin gold and sequencing were from Promega (Southempton, UK). Recombinant human granulocyte / macrophage colony-stimulating factor (GM-CSF) Catalog number 215-GM-010), Interleukin-4 (IL-4, catalog number 204-IL-010), Interferon-gamma (IFNγ, catalog number 285-IF-100), BW-B 70C (catalog number 1304), and PD146176 (catalog number 2850) are from R&D systems (Abingdon,The Western blotting ECL substrate kit (catalog number 32109) and the Prestein protein ladder (catalog number 26619) were from Pierce (Thermo Fisher Scientific, UK). The apoptosis detection kit (catalog number BMS500FI / 300), mouse anti-human CD11b-PE (catalog number 12-0118-42), mouse anti-human CD209-PE [eB-h209] (catalog number 17-2099-42), mouse anti-human CD64-PE [10.1] (catalog number 12-0649-42), mouse anti-human CD206-PE [19.2] (catalog number 12-2069-42), mouse IgG1 kappa isotype control (P3.6.2.8.1) PE (catalog number 12-4714-42), and lipopolysaccharide (LPS) were all from eBioscience (Thermo Fisher Scientific, UK). Mouse anti-human CD86-PE[BU63] (catalog number MHCD8604), mouse anti-human CD80-PE[MEM-233] (catalog number MHCD8004), mouse anti-human CD40-PE[HB14] (catalog number CD4004), mouse anti-human CD16-PE[3G8] (catalog number MHCD1604), mouse anti-human CD14-PE[Tuk4] (catalog number MHCD1404), mouse IgG1 isotype control PE (catalog number MG104), mouse IgG2 alpha isotype control PE (catalog number MG2A04), and BODIPY™ FL N-(2-aminoethyl)maleimide (catalog number B10250) were from Invitrogen (Thermo Fisher Scientific, UK). Remley buffer (6x reduction) was from Alfa Aesar (Thermo Fisher Scientific, UK). Urea, thiourea, Tris base, and Coomassie Brilliant Blue G250 (proteomics grade) were obtained from VWR (Lutterworth, UK). Tween-20 was obtained from Bio-Rad (Watford, UK).The mouse anti-human TSG101 antibody (ab83), rabbit anti-human 5-lipoxygenase (catalog no. ab169755), mouse anti-human 15-lipoxygenase antibody (catalog no. ab119774), rabbit anti-human 12-lipoxygenase (catalog no. ab168384) antibody, rabbit anti-human cyclooxygenase 2 (catalog no. ab151571) antibody, and rabbit anti-human phospholipase A2 antibody (catalog no. ab139692) were from Abcam (Cambridge, UK). RosetteSep® total T cell enrichment cocktail (catalog no. 15061), RosetteSep® monocyte enrichment cocktail (catalog no. 55018), EasySep® buffer, and Lymphoprep were purchased from Stemcell (Cambridge, UK). Resolvin E1 (catalog number 10007848), 5(S)-HEPE (catalog number 32210), 12(S)-HEPE (catalog number 32550), 15(S)-HEPE (catalog number 32710), (±)18-HEPE (catalog number 32840), 5(S)-HETE (catalog number 34230), 12(S)-HETE (catalog number 34570), 15(S)-HETE (catalog number 34720), Lipoxin A4 (catalog number 90410), Lipoxin A4-d5 (catalog number 10007737), Lipoxin B4 (catalog number Catalog No. 90420), 5(S)-HETE-d8 (Catalog No. 334230), Leukotriene B4 (Catalog No. 20110), Leukotriene B4-d4 (Catalog No. 320110), Prostaglandin E2 (Catalog No. 14010), Prostaglandin E2-d4 (Catalog No. 314010), Prostaglandin D2 (Catalog No. 12010), 15(R)-Lipoxin A4 (Catalog No. 90415), Prostaglandin F2α (Catalog No. 16010), Prostaglandin F2α-d4 (Catalog No. 316010), 5(S),15(S)-DiHETE (catalog number 35280), 6-trans-12-epi-leukotriene B4 (catalog number 10012554), resolvin D1 (catalog number 10012554), resolvin D2 (catalog number 10007279), resolvin D2-d5 (catalog number 11184), resolvin D3 (catalog number 13834), resolvin D4 (catalog number 13835), malesin 1 (catalog number 10878), 7-epi-malesin 1 (catalog number 13161), (±)4 -HDHA (catalog no. 33200), (±)7-HDHA (catalog no. 33300), 14(S)-HDHA (catalog no. 15253), 17(S)-HDHA (catalog no. 10009799), 10(S),17(S)-DiHDHA (catalog no. 10008128), arachidonic acid (catalog no. 10007268), arachidonic acid-d8 (catalog no. 10007277), eicosapentaenoic acid (catalog no. 90110), and ML355 (catalog no. 18537) were from Cayman Chemical Company (Ann Arbor, Michigan, USA). The lipoxygenase activity assay kit (catalog no. K978-100) was from BioVision (Milpitas, California, USA). The THP-1 cell line was purchased from ATCC (LGC Standards, Teddington, UK). Lipoxygenase CRISPR-Cas9 knockout HAP1 ALOX12, ko and ALOX5 ko , and double knockout ALOX 5, 15 ko The cells were prepared by Horizon Discovery (Cambridge, UK). The leukocyte blood cones were purchased from NHS Blood and Transplant (Birmingham, UK).

[0041] Isolation of primary T cells and induction of apoptosis Primary human CD3+ T cells were negatively isolated from a leukocyte cone (healthy donor). Briefly, the contents of one cone (approximately 7 mL) were mixed with 3 mL of sterile PBS and incubated with 0.5 mL of antibody cocktail (room temperature, 20 minutes). The volume was adjusted to 20 mL with PBS and layered on 20 mL of Lymphoprep. T cells were separated by centrifugation at 1,200 × g for 20 minutes. The isolated T cells were washed with PBS. The cell pellet was resuspended in ACK lysis buffer (5 mL, room temperature, 5 minutes) to remove erythrocytes. The cells were washed twice with PBS and 4 × 10⁶ cells were removed. 6 The blood was resuspended in serum-free RPMI 1640 medium (phenol red-free, antibiotic-added) at a density of cells / mL. From one blood cone, 2.5–3 × 10⁶ cells were obtained. 8 It was possible to isolate all T cells.

[0042] Induction of apoptosis Apoptosis occurs in primary T cells and HAP1 ALOX5,15 ko Cellular cells were induced by anti-Fas antibody (1:10000 dilution) and cycloheximide (20 μg / mL) under serum-free conditions for 15 and 12 hours, respectively. The degree of cell death (apoptosis vs. secondary necrosis) was evaluated using flow cytometry (Cytoflex S, Beckman Coulter, High Wycombe, UK), and staining was performed using Annexin V-FITC and propidium iodide.

[0043] Isolation of EVs Extracellular vesicles (EVs) were isolated from the supernatant of serum-free cell cultures in which apoptosis had been induced. Briefly, apoptotic cells and cell debris were pelleted (2,000 × g for 20 minutes), and the remaining apoptotic secretome was concentrated to a final volume of less than 1.5 mL by spin column. EVs were separated from soluble proteins using a pre-packed size exclusion chromatography column (qEV original, Izon Science, Oxford, UK). The column was pre-conditioned and eluted with PBS. The first 3 mL of eluate was discarded, but the subsequent 3.5 mL of eluate was rich in EVs and had little to no soluble protein contamination.

[0044] For Western blotting and proteomic profiling of extracellular vesicles (EVs), 3.5 mL of EV isolate was further concentrated to a volume of less than 150 μL (equivalent to one blood cone isolate) to ensure high EV density. For lipoxygenase activity assays, the final EV volume was kept below 100 μL, and the EVs were used immediately for downstream analysis. When EVs were to be used for further co-incubation with monocyte-derived macrophages, EV separation was performed by serum-free RPMI using a qEV column.

[0045] EV protein concentrations were estimated using the Bradford assay. Isolated EVs were then stored at -20°C until analysis.

[0046] Measurement of EV size and concentration The size and concentration of extracellular vesicles (EVs) were measured from final EV isolates before storage or for use in different assays. The EV size distribution and their concentrations were measured in PBS using adjustable resistance pulse sensing (TRPS) technology with a 150 nm nanopore qNano Gold (Izon Science, Oxford, UK) particle analyzer. The instrument was calibrated with 200 nm silica beads. For optimal measurement accuracy, 500 events were recorded for each measurement.

[0047] EV flow cytometry Bodipy dye (1 μM final concentration) linked to a maleimide probe was added to 2000 × g apoptotic T cell supernatant, incubated on ice for 4 hours, and then stained with EV. For annexin V staining of EV cells, 50 μL of 2000 × g supernatant was diluted 10-fold with ice-cold annexin V binding buffer, and the probe was treated with 5 μL of annexin V-FITC before measurement.

[0048] Using MegamixPlus beads, the cytometer settings were adjusted to enable reliable detection of particles larger than 150 nm. Detection of smaller particles was triggered by Violet SSC (manual cutoff for area 149713) and manual cutoff, with the following gains applied: FSC-101, SSC-74, VSSC-1801, FITC-141). Samples were injected at a flow rate of 10 μL / min until 50,000 events were collected.

[0049] Relative quantification of unlabeled proteins by LC-MS / MS Proteins (5 replicates, 30 μg each) from apoptotic T cell lysates and ACdEV isolates were reduced in Remley buffer at 65°C for 15 minutes, separated by molecular weight on 10% SDS-PAGE, and stained with Coomassie G250 blue (0.5% w / v in 40% methanol aqueous solution and 10% glacial acetic acid) for 4 hours. After destaining, each sample lane was divided into five bands of the same size for all samples on the gel. Gel sections were cut, transferred to polypropylene tubes, and shredded. Proteins were then destained in 50% acetonitrile in 50 mM ammonium bicarbonate. After complete destaining, the shredded gel was dehydrated with pure acetonitrile and vacuum-dried in a vacuum concentrator (Eppendorf, UK) for at least 30 minutes. The gel was rehydrated with trypsin solution in 6 mM ammonium bicarbonate (25:1 protein-to-trypsin ratio) and the protein was digested overnight with shaking (700 × g, 37°C). Peptides were sequentially extracted from shredded gels in an ultrasonic bath using 30%, 50%, and pure acetonitrile for 15 minutes. Extracts from single sample sections were combined into one tube, vacuum-dried, and stored at -20°C before analysis.

[0050] The samples were reconstituted with 100 μL of 3% acetonitrile aqueous solution and 0.1% formic acid for tandem mass spectrometry (LC-MS / MS) analysis coupled with liquid chromatography. The peptides were separated and analyzed using an nUPLC system (Acquity M class, Waters, UK) coupled to a 5600 TripleTof (AB Sciex, UK) operating in information-dependent (IDA) mode. Using a single-pump trap mode (5 μL / min, 1 minute, 1% acetonitrile in 0.1% formic acid aqueous solution), a peptide solution (5 μL, approximately 15 pmol) was injected into a trap column (nanoEase M / Z symmetric C18 trap column, 100 Å, 5 μm, 180 μm × 20 mm, Waters, UK), and the peptide was separated using an analytical column (viper-fitting PepMap®, C18, 5 μm, 100 Å, 300 μm × 1 mm, ThermoScientific, UK) with 1% eluent B (acetonitrile in 0.1% formic acid aqueous solution) at a flow rate of 15 μL / min. Next, the peptides were separated using an analytical column (Acclaim®, PepMap® C18, 3 μm, 100 Å, 75 μm × 150 mm, ThermoScientific, UK) with the following gradient: 1-45% B from 0–45 mins, 45–49 mins, 45–90% B from 49–52 mins, and 1% B from 52–67 mins. A stable electrospray was formed at 2200 V using a PicoTip® emitter (New Objective, Germany). Ten of the highest intensity ions from each high-resolution MS survey scan were selected for high-sensitivity MS / MS, and the acquired ions were temporarily excluded from MS / MS acquisition for 30 seconds. The mass spectrometer was calibrated before acquisition to ensure high mass accuracy at both MS and tandem mass spectrometry (MS / MS) levels.

[0051] Relative quantification was performed using the Progenesis QI software (version 4, Nonlinear Dynamics, UK) multi-fraction setup for proteomics. Only protein-specific peptides were used for relative quantification. MS / MS data were searched against the curated SwissProt database using Mascot Daemon (version 2.5) with the following search restriction parameters: a mass tolerance of 0.1 Da in MS and 0.6 Da in MS / MS spectra, a maximum of two trypsin cleavage failures, Homo sapiens taxa, variable modifications of methionine oxidation, and cysteine ​​carbamide methylation.

[0052] Gene ontology (GO) data analysis was performed on the Homo sapiens whole genome list, available from March 23, 2020, using the PANTHER (Protein Analysis Through Evolutionary Relationships) classification system. The analysis was performed using statistical overrepresentation tests with Bonferroni correction for multiple testing.

[0053] Detection of lipid metabolites and PUFAs by targeted mass spectrometry For metabolome analysis, the EV should be approximately 1 × 10⁻⁶. 9Lipid metabolites were collected and purified from primary T cells. The sample was spiked with 1 ng of isotope-labeled internal standard, kept on ice for 10 minutes, then treated with 5 volumes of ice-cold methanol, and kept on ice for 10 minutes with occasional vortexing to extract lipid metabolites. Lipid metabolites were concentrated in a solid-phase extraction cartridge (Oasis HLB, 10 mg, Waters, Manchester, UK) pre-wetted with methanol (200 μL) and equilibrated with a 15% methanol aqueous solution (200 μL) acidified with 0.1% formic acid. The sample (acidified in 15% methanol) was loaded, and the cartridge was washed with equilibration buffer and hexane (600 μL). Lipid metabolites were eluted with 20% methanol in butyl acetate (2 × 1 mL) and vacuum-dried. Samples were stored at -20°C before analysis.

[0054] MS acquisition conditions were optimized using an ESI-QqLIT-MS (QTRAP 5500, AB Sciex, Warrington, UK) in negative ion mode, with an ionization voltage of -4.5kV, an inlet potential of -10V, and an ion source temperature of 400°C. Standard materials and internal standard solutions (500 pg / μL in 0.1% formic acid in 50% methanol aqueous solution) were used to optimize the normalized collision energy (CE), declustering potential (DP), and quadrupole exit potential (CXP) for each Q1 / Q3 transition. Standard solutions were directly injected into the mass spectrometer for optimization of Q1 / Q3 transition pairs using a syringe pump (Harvard Apparatus GmbH, March-Hugstetten, Germany) at a flow rate of 20 μL / min. The final time-scheduled MRM included 88 Q1 / Q3 transition pairs with up to four of the highest intensity structure-specific transitions for each analyte.

[0055] LC-MS / MS (MRM) was performed using a Dionex Ultimate 3000 RS UPLC (Thermo Fisher Scientific, UK) ESI-QqLIT-MS system. Samples were separated using an XSelect HSS T3 C18 column (particle size 2.5 μm, 2.1 × 10 mm, pore size 100 Å) at a column temperature of 50°C and a flow rate of 200 μL / min. A sample (50 μL) was injected using 50% methanol in a 0.1% formic acid aqueous solution. Analytes were chromatographically separated, and eluents A (0.1% formic acid in water) and B (0.1% formic acid in methanol) were mixed as follows: 50% B for 5 minutes, 50–90% B for 40 minutes, 90–100% B for 5 minutes, 100% B for 4 minutes, 100–50% B for 1 minute, and 50% B for 11 minutes. The quantification of analytes in the samples was achieved for the same Q1 / Q3 transition pairs against the corresponding calibration curves after normalization of their values ​​to an internal standard. All standards were measured at various concentrations from 500 fg to 750 pg using the final acquisition method to enable the design of individual calibration curves with the best linear fit. Data were processed using Analyst software (version 1.6.2, AB Sciex).

[0056] Western blotting The following protein amounts were used for EV protein detection: 30 μg for TSG101, and 50 μg for LOX-5, LOX-15, LOX-12, COX-2, and PLA2. EV samples were reduced in Remley buffer (65°C for 15 minutes), and proteins were separated using 10% SDS-PAGE. After transfer to a nitrocellulose membrane (Amersham® Protran® Premium NC Membrane, GE Healthcare), the membrane was blocked with 5% milk in TBS-T on an orbital shaker (4°C for 1 hour or overnight) and probed with a primary monoclonal antibody (1:1000 dilution). After three washes with TBS-T, the membrane was incubated with HRP conjugate polyclonal antibody (anti-mouse or anti-rabbit, 1:5000 dilution). Labeled proteins were detected on a G:BOX XT4 (Syngene, Cambridge, UK) using an ECL substrate.

[0057] LOX activity assay For the LOX activity assay, extracellular viable cells (EVs) were collected from apoptotic cultures of primary human T cells as described above. The LOX activity assay was performed using a fluorescence analysis assay kit according to the manufacturer's guidelines. Fluorescence was recorded in kinetic mode using a SpectraMax Gemini EM microplate reader (Molecular Devices, Reading, UK), and fluorescence was measured at 500 / 536 nm (excitation / emission) every 5 minutes for up to 90 minutes. The individual activity contributions of 5-LOX, 12-LOX, and 15-LOX were investigated using their corresponding inhibitors, BW-B 70C, ML355, and PD146176, respectively. The inhibitors were prepared daily in DMSO to their maximum soluble concentrations.

[0058] Transfer of LOX activity Transfer of LOX activity from EVs to cells using CRISPR / Cas9 ALOX12 ko Newly isolated EV (7×10) cells 7The synthesis of 5(S)-hydroxy-PUFA was measured after 2 hours of incubation of T cell-derived 170 μg protein in CRISPR / Cas9 ALOX5 knockout cells (2 × 10¹⁶) after 18 hours of incubation with newly prepared EVs. 6 Measurements were taken in cells. Cells were plated in a 6-well plate under full confluence. Newly prepared EV (4 × 10⁶) 7 Cells corresponding to T cells were incubated with adherent cells in IMDM serum-free medium. As a control, cells without EV were plated and treated under the same conditions. Cells were collected by scraping and centrifugation (5 minutes, 300 × g).

[0059] For the LOX activity assay, pelleted cells were lysed in LOX buffer (provided by the LOX assay kit). Protein concentrations were estimated using the Bradford assay. Samples were immediately used in the LOX assay. For lipid metabolomics profiling of 5-LOX-derived LM, enrichment and detection of LM in EV, cells, and corresponding supernatants were performed in parallel as described above.

[0060] Human macrophage phenotypic analysis Human THP-1 monocytes were cultured using a standard method in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin. Differentiation into THP-1-derived macrophages (M0) was performed as needed, with 5 × 10⁻⁶ cells cultured. 5 Cells were induced with 100 nM VD3 for 48 hours at an initial cell density of cells / mL. Differentiated cells were washed and 1 × 10⁶ cells were extracted. 6 Resuspend cells / ml in serum-free medium, and add freshly prepared ACdEV(4×10) as needed. 7 Cells collected from apoptotic T cells were incubated at 37°C for 24 hours on a 6-well plate. As a control, differentiated cells were incubated under the same conditions without ACdEV. Cells were collected from the plates, pelletized at 300 × g, and lipid metabolites were concentrated and analyzed as described above.

[0061] For primary human monocyte-derived macrophages, a single leukocyte cone (healthy donor) was volume-adjusted to 10 ml in PBS in a sterile polypropylene tube containing 1 mM EDTA. Blood samples were incubated with 0.5 mL of monocyte-concentrated cocktail (20 minutes, room temperature), the blood was diluted with 10 mL of EasySep buffer, and human monocytes were negatively isolated by separating them using a Lymphoprep gradient (1200 × g, 20 minutes). Isolated monocytes were washed with EasySep buffer, and any remaining erythrocytes were removed by resuspending in RCK lysis buffer (5 mL, room temperature, 5 minutes). The cells were washed twice with EasySep buffer and 3 × 10⁶ cells were placed in serum-free RPMI 1640 medium (phenol red-free, antibiotic-added). 6 Resuspend at a density of cells / mL and 75cm³ 2 Seeds were sown in a flask. From one leukocyte cone, 1 × 10⁶ cells were produced. 8 It was possible to isolate super monocytes.

[0062] Human monocytes were differentiated into monocyte-derived macrophages (2.5 × 10⁶) using 20 ng / ml GM-CSF for 7 days. The cells were carefully collected and washed with PBS. 6 Cells ( / ml) were stimulated with 20 ng / mL IFN-γ and 1 ng / mL LPS for 48 hours to promote the M1 phenotype, and the M2 phenotype was stimulated with 20 ng / mL IL-4. The ability of EVs to induce monocyte-derived M0 macrophage polarization to the M1 or M2 phenotype was evaluated at 3 × 10⁻⁶. 8 The phenotypic fingerprinting of human monocyte-derived macrophages was tested by 24-hour incubation with EVs derived from apoptotic T cells. Phenotypic fingerprinting of human monocyte-derived macrophages was evaluated by flow cytometry using a panel of fluorescent antibodies: CD14, CD16, CD40, CD80, CD86, CD11b, CD64, CD206, CD209, and corresponding isotype controls.

[0063] result ACdEV is released from apoptotic cells. The focus of this study was to investigate how immune cells during early apoptosis communicate their presence to the immune system via ACdEV release, and whether these vesicles possess immunomodulatory properties. Our previous research has shown that small ACdEVs (<1 μm) in 2000 × g supernatant promote macrophage recruitment. 17 This study focused on these smaller EVs, which, due to their size, may be able to transduce over longer distances in vivo. For this purpose, ACdEVs were immediately confined to apoptosis by a combination of agonist anti-Fas antibody in the presence of cycloheximide, along with newly isolated primary human CD3 + The cells were prepared from T cells. Flow cytometry monitoring of apoptosis using annexin V / propidium iodide staining revealed that the maximum level of apoptosis with minimal secondary necrosis was reached 15 hours after induction. It is noteworthy that this timeframe for primary cells was longer than typical in cell lines. 20 In accordance with established guidelines in this field. 21ACdEVs were isolated from the serum-free supernatant after fractional centrifugation to remove apoptotic cells, cell debris, and larger apoptotic bodies. Finally, high-purity ACdEVs were isolated using size exclusion chromatography. Measurement of ACdEV size and concentration revealed that during the process of apoptosis, T cells release ACdEVs with a mode size of 110 nm, equivalent to an average of 400–600 ACdEVs per cell (Figure 1A). Healthy cells released significantly lower EV numbers, which did not reach the TRPS count number (>500 events per sample) required for quality data processing. The wide size range of 60–720 nm indicates that apoptotic T cells produce a heterogeneous population of ACdEVs, likely consisting of a mixture of exosomes and microvesicles. Indeed, Western blotting of the exosome marker TSG101 showed that at least some of the isolated ACdEVs were likely of exosome origin (Figure 1A, inset). In addition, the inventors confirmed the presence of vesicles by flow cytometry after fluorescent labeling with a thiol-reactive Bodipy dye (Figure 1B).

[0064] Exposure of phosphatidylserine (PS) on the outer layer of the plasma membrane during apoptosis promotes the recognition and uptake of apoptotic cells by phagocytic cells. 22,23 Therefore, it was considered highly likely that ACdEVs also possess exposed PS on their surfaces. Labeling of ACdEVs with annexin V revealed that approximately 69% of ACdEVs carry and possess exposed PS on their surfaces (Figure 1C). This suggests that PS on apoptotic cells modulates the immune response by promoting the uptake of apoptotic cells. 24 , and ACdEV drives TGF-β1 production. 25 Considering this, it is possible that PS can be an "active" component of ACdEV, independent of the type of cell from which they arise.

[0065] To capture the overall composition of ACdEVs, the inventors investigated whether there is a preferential enrichment of proteins into ACdEVs during apoptosis and what proteomic fingerprints ACdEVs may possess. Using a bottom-up proteomics approach and label-free quantification, the inventors compared the proteome of apoptotic T cells and the ACdEVs they release during apoptosis. The inventors were able to identify a total of 1114 proteins, of which 865 qualified for relative quantification based on selection / exclusion criteria. At the time of publication, the extracellular vesicle database Vesiclepedia was used. 26 This study identified 2,676 proteins associated with all types of extracellular vesicles (EVs) derived from T cells. Our ACdEV proteome overlapped with 608 proteins, and 260 proteins were identified for the first time in our T cell-derived extracellular vesicles, of which 19 proteins were associated with extracellular vesicles of any cell origin (Figure 1D).

[0066] Here, the inventors compared the proteomes of apoptotic T cells and ACdEVs and found that ACdEVs were significantly enriched with 406 proteins (P<0.01) (Figure 1E). Of these, 10 proteins were exclusively present on ACdEVs. These 406 proteins were further classified according to their biological processes, cellular components, and molecular functions using gene ontology (GO) enrichment (Figure 1F). As expected, the highest protein enrichment in ACdEVs was attributed to those related to the gene ontology processes "extracellular exosomes" and "vesicles," further confirming efficient ACdEV isolation. Specifically, the inventors identified enrichment of proteins from T cell-specific receptors, including the annexin family, HSP90, 14-3-3 member proteins, histone H1, Rab, GTPase, fluorotilin 1 and 2, tetraspanin, and members of HLA I and HLA II, all of which are generally associated with extracellular vesicles. 27(Figure 1G). Importantly, the biological processes were involved not only in "vesicle-mediated transport," "exocytosis," and "mRNA metabolism," but also in adhesion, leukocyte activation, immune response, MHC I class antigen presentation, upregulation of cell migration, and wound healing.

[0067] Detailed examination of the identified proteins revealed that ACdEV preferentially enriches a number of proteins that enable them to communicate their presence to the immune system and ensure their uptake, such as well-known "eat-me" signals, e.g., annexin A1 28 and calreticulin 29 It was found on ACdEV cells, with annexin A1 being 5.2 times higher in ACdEV cells, and calreticulin being found mainly on apoptotic T cells (8.1 times). Interestingly, CD47 30 (3.1x) and CD31 31 "Do-not-eat-me" signals, such as (2.5 times), were also more abundant in ACdEV. In addition, clathrin heavy chain 1 32 (CLH1, 7.0x) is a transforming protein, RhoA, which controls vesicle uptake via clathrin-dependent endocytosis. 33 (2.3 times) and Ras-related C3 botulinum toxin substrate 1 33 Clathrin-independent endocytosis such as (Rac1, 2.2x), and ICAM-3 17 Adhesion molecules containing (5.4 times), such as CD44 34 Integrin alpha-L (3.8x), integrin beta-2 (4.9x), and integrin beta-2 (3.0x) were also enriched in ACdEV cells compared to apoptotic T cells.

[0068] In addition to the uptake mechanism, ACdEV is enriched with proteins such as CD48 (2.8 times), CD266, and SLAMF6 (both limited to ACdEV). These may correspond to the mechanism by which ACdEV communicates with lymphocytes. It has been shown that CD48, CD266, and SLAMF6 interact with T cells and NK cells, leading to their activation, through ligation or binding to their receptors CD244 and LFA-1, or through self-ligation. 35,36,37 It should be noted that ACdEV showed significant enrichment of cellular receptors such as GP183 (also known as EBI2, with a 458-fold increase) and SLAMF6 (limited to ACdEV). However, the roles of these proteins on EVs have not been investigated until now.

[0069] Furthermore, the inventors have identified proteins with previously described immunomodulatory properties, such as interleukin-16 (IL-16). 38 and macrophage migration inhibitors (MIFs) 39 However, we found that it is present on ACdEV. Finally, arachidonic acid 5-lipoxygenase activating protein (AL5AP, also known as FLAP) and leukotriene A-4 hydrolase (LKHA4), both involved in the synthesis of pro-inflammatory leukotrienes, were found on ACdEV.

[0070] Interestingly, ACdEVs were highly enriched with multidrug resistance protein 1 (MDR1, 13.3-fold) and multidrug resistance-associated protein 1 (MRP1, limited to ACdEVs), also known as ATP-binding cassette proteins (ABC transporters), such as ABCB1 and ABCC1 transporters. Although data on ABC transporters and EVs are limited, they have been reported to act as efflux pumps on EVs derived from cancer cells loaded with drugs that support drug resistance in cancer. 40,41 .

[0071] ACdEV transports lipid metabolites. Inflammatory lipid mediators (LMs) are actively produced and released by immune cells during inflammatory responses. The immunomodulatory properties of ACdEV have been previously observed. 15,16,17,42,43,44,45,46 Only a small amount of information is known about their functionally active components, particularly their bioactive lipid factors. To investigate this in particular, we performed lipid metabolome profiling of ACdEV to identify their LM signatures. We fine-tuned our lipid metabolomics MRM (multiple reaction monitoring)-based platform to target 32 ​​different pro-inflammatory and dual-acting SPMs derived from AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) via the LOX and COX pathways (Figure 2A). For each analyte, a structure-specific set of Q1 / Q3 (parent-child ion-to-daughter ion) "transition pairs" and characteristic elution times were determined using standards. For positive identification, it was necessary for the spectral characteristics (parent-child and daughter ions) and retention times of the analyte to match those of its standards. Using established criteria, the inventors identified and quantified the presence of 16 LMs and their polyunsaturated fatty acid (PUFA) precursors in the ACdEV sample (Figure 2). Furthermore, the inventors detected 11 additional compounds with mismatched retention times in LM-specific Q1 / Q3 transition pairs, indicating the detection of isobaric analytes with spectral characteristics overlapping with LMs. These unknown analytes are likely to be either positional or stereoisomers of the target LM. Therefore, the inventors annotated them with asterisks (e.g., LMs). * , LM ** These, among others, were included in the inventors' report (Figure S2).

[0072] Our results show that ACdEV, loaded with free ω-6 and ω-3 PUFAs, mainly AA and DHA, has a 35- to 45-fold lower level of EPA (Figure 2B). Lipoxygenase catalyzes the addition of PUFA oxygen to PUFA hydroperoxides, which are rapidly reduced to form stable hydroxy-PUFAs. 47,48Subsequently, these initial LOX metabolites can act as substrates for further LOX-mediated oxygenation to form various LMs, including SPMs. We show that during T cell apoptosis, all PUFAs (AA, DHA, EPA) are used as substrates to produce hydroxy-PUFAs by the action of 5-, 12-, and 15-LOX, which are then packed into ACdEVs (Figure 2C). Furthermore, 12-LOX metabolites, such as 12(S)-HETE, 12(S)-HEPE, and 14(S)-HDHA, were found to be the major hydroxy derivatives of AA, EPA, and DHA, respectively.

[0073] In defining LM transport within ACdEV, the inventors have identified pro-inflammatory prostaglandins, such as PGE2, PGD2, and PGF. 2α Significant transport of LTB4 and its aspirin analog 6-trans-12-epi-LTB4 was identified, and it is noteworthy that PGE2 is the most abundant among them (Figure 2D). In addition, one PGF 2α Analog-PGF 2α * It was possible to detect the presence of the four analogs, including LTB4. In fact, PGF 2α * It appears to be the most abundant pro-inflammatory LM in ACdEV, and LTB4 *** and LTB4 **** Both levels were higher than LTB4.

[0074] Dual-acting SPMs (i.e., those with pro-inflammatory and anti-inflammatory functions), such as lipoxin, resolvin, malecin, and protectin, were not detected in ACdEV. However, the inventors have identified five SPM analogs (SPM * The detection of ) suggested that the diversity of SPMs coupled to ACdEV was different from what was expected (Figure 2E). The unidentified SPM analog was AA-derived lipoxin (LXB4 * LXB4 ** ), and DHA-derived resolvin (RvD3 *, RvD4 * ) and protectin (PDX * It is most likely that it corresponds to the family of ). SPMs are a relatively young class of biomolecules, and it is reasonable to assume that the biodiversity of SPMs is higher than what scientific understanding currently identifies.

[0075] The temporal biosynthesis of SPM, as well as the overall balance of convergence-promoting LM and inflammatory LM relative to SPM, drives inflammation or contributes to its resolution. Therefore, the inventors of PG * and LX * The combined level of all inflammatory LMs, including AA-derived SPM, was evaluated and these levels were then used to assess the overall SPM. * and all SPM * This was compared with (Figure 2F). Our findings indicate that during T cell apoptosis, ACdEV with a unique convergence-promoting LM signature is secreted, mainly contributed by AA-derived SPM from the lipoxin family.

[0076] ACdEV corresponds to the active metabolic compartment. Following our identification of ACdEV as a bioactive LM-rich extracellular compartment with a convergence-promoting phenotype, it was a priority to determine the enzymatic activity of ACdEV. Recent reported enzyme transport in EV is 49,50,51 The inventors hypothesize that ACdEVs are "active EVs" in that they also transport the enzymes responsible for LM production. 52 This further supports the finding. In fact, Western blot analysis of 5-LOX, 15-LOX, 12-LOX, and secretory phospholipase A2 (PLA2) showed that ACdEV carries these components of the mechanism for leukotriene and SPM production, but COX-2, which is responsible for the synthesis of pro-inflammatory prostaglandins, was not detected by Western blotting (Figure 3A).

[0077] From these initial observations, the inventors further investigated whether the ACdEV-associated LOX enzymes are in their active forms. To test whether LOX is present in ACdEV as an active enzyme, the inventors used a fluorescence analysis assay. Here, the LOX action on the substrate would form an intermediate sensitive to the presence of the LOX probe, resulting in a fluorescence signal (excited at 500 nm and measured at 536 nm). Measuring the LOX activity in ACdEV over time produced a signal significantly exceeding that from the positive control, and the signal specificity for LOX was confirmed by the disappearance of the signal to the baseline level in the presence of the enzyme inhibitor (Figure 3B). Using the oxidized probe standard, the inventors designed a calibration curve covering 0 and 10 pmol / well. This enabled the inventors to calculate the LOX activity in ACdEV (Figure 3C). The inventors found that the overall LOX specific activity in ACdEV is 2.44 m units mg -1 mL -1 . The specific activities of the 5-LOX, 12-LOX, and 15-LOX isoforms were confirmed by the incorporation of isoform-specific inhibitors. The inventors showed that the measured LOX activity in ACdEV from primary human T cells was dominated by 15-LOX (2.34 m units mg -1 mL -1 ), while the 12-LOX (1.43 m units mg -1 mL -1 ) and 5-LOX (1.21 m units mg -1 mL -1 ) activities were similar but lower values.

[0078] The immunomodulatory properties of EVs are often attributed to the ligation and activation of currently undefined surface receptors on recipient immune cells 53However, since extracellular vesicles are often internalized by recipient cells, the delivery of cargo bound to EVs may also have a significant impact on recipient cells and their phenotypes. Therefore, the inventors investigated whether ACdEVs containing LOX can deliver active enzymes to recipient cells. To specifically analyze this, cells lacking LOX were incubated with ACdEVs, and after incubation, the enzyme activity and the production of LOX-specific metabolites in the cells were evaluated. Recipient single cells endogenously express only low levels of the ALOX5 (0.21 transcripts per million, TPM) and ALOX15 (0.06 TPM) genes, as well as very low levels of ALOX12 (1.07 TPM). However, to control this borderline LOX activity in recipient cells, the inventors used ALOX12 ko (knockout) cells and incubated them with freshly isolated ACdEVs for 2 hours. This time frame was chosen because it should allow for a significant amount of ACdEV uptake without significant loss of ACdEV-related enzyme activity. As a control, ALOX12 cells not treated with ACdEVs were measured to evaluate any limited endogenous LOX activity. The inventors' results showed 41% higher LOX activity in cells incubated with ACdEVs, indicating that LOX remains active in host cells after ACdEV uptake (Figure 3D).

[0079] The most studied human LOX is 5-lipoxygenase (5-LOX). It preferentially oxygenates ω-6 and ω-3 PUFAs on the "S" face of the C-7 carbon, converting them to their 5(S)-hydroperoxy analogs, which are further reduced to 5(S)-hydroxy-PUFAs 54 In fact, the discovery of 5-LOX dates back to 1976 when the novel AA metabolite 5(S)-HETE was detected in neutrophils 55 To examine the biosynthesis of 5-LOX-specific lM from ACdEV-derived enzymes, the inventors incubated ACdEVs with ALOX5​​ko Cells were incubated for 18 hours, and levels of 5-hydroxy-PUFA were monitored under different experimental conditions. The inventors had already established that 5(S)-HETE and 5(S)-HEPE are some of the most abundant LMs present in ACdEVs (Figure 2C). This presented a technical challenge for the reliable detection of newly synthesized 5-LOX products in cells after enzymatic translocation from ACdEVs, as opposed to the detection of pre-formed LMs transported in EVs. To control this, the inventors used a significantly reduced vesicle dose (1 / 25th lower than that used for LM profiling). This resulted in a low level of 5(S)-hETE in ACdEVs of 1.24 pg, and 5(S)-HEPE was below the detection limit. ALOX5 ko Incubation of ACdEV (5-LOX-filled) cells resulted in a significant increase in 5-LOX products: 5(S)-HETE (-EV: 1.98 pg, +EV: 9.62 pg) and 5(S)-HEPE (-EV: 0.53 pg, +EV: 3.19 pg). These levels in knockout cells (annotated as "incubation" in Figure 3E) were significantly higher than the combined levels of these metabolites in ACdEV and control cells (annotated as EV+ cells in Figure 3E).

[0080] In the absence of a suitable analytical standard containing an "S" stereocenter, the inventors measured the DHA metabolites (±)4-HDHA and (±)7-HDHA. The inventors did not observe a significant increase in the level of (±)4-HDHA in cells (-EV: 1.29 pg, +EV: 3.22 pg) compared to ACdEV (1.24 pg), but (±)7-HDHA showed a significant increase in ALOX5 levels compared to ACdEV. koIt was detected only after cell incubation (-EV: not detected, +EV: 7.68 pg). Although both are major 5-LOX metabolites, these data suggest that 7-HDHA is a more potent product of DHA's 5-LOX metabolism. Overall, our results demonstrate that ACdEV can deliver active LOX to cells, leading to the endogenous synthesis of 5-LOX-derived metabolites. Thus, the transfer of active enzymes by ACdEV during leukocyte apoptosis may correspond to a pathway in which ACdEV modulates the immune response. These results also raise the possibility that EV may facilitate the modulation of activity in non-immune (possibly non-professional phagocytic) recipient cells.

[0081] ACdEV controls the macrophage phenotype. Macrophages (MΦ) are considered elite innate immune effector cells due to their professional phagocytic, regulatory, and tissue repair functions. In response to intrinsic and extrinsic stimuli, macrophages exhibit remarkable plasticity, enabling physiological changes including surface protein expression, gene signatures, and the production of inflammatory mediators. 56 This plasticity is central to their function in controlling inflammation. Here, we present the findings that ACdEV released during T cell apoptosis is a dual-acting analog SPM compared to inflammatory leukotrienes (LT) and prostaglandins (PG). * It exhibits a unique convergence-promoting phenotype with a high level of activity and the transport of active lipoxygenase, and shows the highest specific activity of 15-LOX. Furthermore, the inventors demonstrate that ACdEV can deliver active enzymes to host cells. These are continuously oxygenated in non-esterified PUFAs. To investigate whether the delivery of these ACdEVs can induce changes in the macrophage phenotype, the inventors monitored changes in the production of inflammatory lipid mediators (LMs) and surface markers in monocyte-derived macrophages incubated with ACdEV.

[0082] Using our established lipid metabolomics platform, we monitored the LM profiles in 1α,25-dihydroxyvitamin D3 (VD3)THP-1-derived macrophages (considered here to be M0 macrophages) before and after 24-hour incubation with ACdEV. In response to vesicle uptake, unpolarized macrophages (M0) can change their phenotype to pro-inflammatory (M1) or tissue-repairing (M2). This will alter their LM profiles. Unique LM signatures are described for these macrophage polarizations. 57 Inflammatory M1 macrophages produce higher levels of PG and LT, while higher levels of SPM-like resolvin (RV) and lipoxin (LX) were characteristic of tissue-repairing M2 macrophages. 57 .

[0083] Overall, the inventors quantified 16 lipid metabolites and 3 PUFAs in THP-1-derived macrophages (see Figure S3 for individual levels). ACdEV incubation with M0 macrophages did not induce changes in the cumulative levels of hydroxy-PUFAs formed from the 5-LOX pathway (MΦ: 598.7 pg, MΦ+EV: 647.3 pg) or the 12-LOX pathway (MΦ: 26.5 pg, MΦ+EV: 50.7 pg), but the level of hydroxy-PUFAs derived from 15-LOX (MΦ: 1.8 pg, MΦ+EV: 4.0 pg) was significantly increased (Figure 4A). The total levels of pro-inflammatory prostaglandins (MΦ: 245.0 pg, MΦ+EV: 191.7 g) did not differ significantly, but suggested a decrease in levels after vesicle uptake, and pro-inflammatory leukotrienes derived from 5-LOX (MΦ: 7.0 pg, MΦ+EV: nd) were no longer detected. Finally, treatment of macrophages with ACdEV did not affect the total levels of 15- / 5-LOX-derived SPMs (MΦ: 613.0 pg, MΦ+EV: 607.7 pg), but notably, ACdEV supported the de novo synthesis of 15- / 15-LOX SPMs (MΦ: nd, MΦ+EV: 68.1 pg). When combined, our results show that incubation of ACdEV with THP-1-derived macrophages leads to the upregulation of macrophage synthesis of 15-LOX, an anti-inflammatory convergence-promoting lipid mediator such as 15(S)-hydroxy-PUFA and protectin D1, but also to the upregulation of inflammatory leukotrienes (LTB4 / LTB4). * This indicates that 5-LOX synthesis is "switched off". These observed changes in the LM profile due to ACdEV exposure are characteristic of LM "class switching" to convergence-promoting (M2) and macrophage phenotypic changes.

[0084] To evaluate the ability of these ACdEVs to alter the phenotype of primary human macrophages, surface immunophenotypic analysis was performed on human monocyte-derived macrophages (MΦ) in the presence or absence of ACdEV treatment. Human primary monocytes were isolated from leukocyte cones and stimulated with GM-CSF to produce monocyte-derived macrophages (unpolarized, M0). The cells were further stimulated with either LPS or IFNγ to produce "classically activated" macrophages (inflammatory, M1), or with IL4 to produce "alternatively activated" macrophages (tissue repair type, M2). Surface expression of CD11b, CD14, CD16, CD40, CD64, CD80, CD86, CD163, CD206, and CD209 in M0, M1, and M2 macrophages was monitored by flow cytometry. Monocyte differentiation antigen CD14 was highest in M0 MΦ, which was significantly downregulated after polarization to M1 or M2 and by treatment with ACdEV, clearly demonstrating ACdEV activity in this assay (Figure 4B). Classically activated M1 macrophages were most characterized by high surface expression of CD40, CD86, and CD64, with CD40 showing the strongest upregulation. Alternatively activated M2 macrophages showed high surface expression of CD11b, CD206 (macrophage mannose receptor 1), and CD209 (dendritic cell-specific ICAM-3 binding nonintegrin 1), with CD206 showing the strongest difference between the M1 and M2 phenotypes.

[0085] The effect of ACdEV on the MΦ phenotype is highlighted by the significantly decreased expression of CD40, CD64, and CD86 (compared to M1). This is further demonstrated by the increased expression of CD11b and CD206 (compared to M1). These results demonstrate the clear ability of ACdEV to transform the phenotype of M0 macrophages (M0+EV) into the convergence-promoting M2. Confirmation of this phenotypic change is provided by our PCA analysis. Here, the mean fluorescence intensity of each antigen shows the highest similarity between M2 MΦ and ACdEV-treated M0 MΦ, with CD206 being the strongest alignment (PCA biplot, Figure 4B).

[0086] Overall, our results provide evidence that extracellular vesicles secreted from leukocytes during apoptosis carry a panel of lipid mediators and biosynthetic enzymes that can control the lipid metabolome profile in macrophages and the expression of surface markers supporting the macrophage M2 tissue repair phenotype. These immunomodulatory properties of ACdEV highlight their importance, particularly when released during specific stages of acute inflammation.

[0087] In Vivo Data method Invivo Dust Mite Model All animal handling was carried out in strict accordance with the approved protocols and recommendations for the proper use and care of laboratory animals (Animals (Scientific Procedures) Act 1986). All animal experiments were conducted in accordance with United Kingdom Home Office regulations (Project License P75A73BEB). All animal handling was performed by qualified personnel. All studies were conducted and reported in accordance with the revised ARRIVE guidelines.

[0088] Thirty female C57Bl / 6 mice (6-8 weeks old) were purchased from Charles River and housed at the Central Animal Facility of Aston University under specific pathogen-free conditions with a 12-hour light-dark cycle. Mice were provided with ad libitum for feed and water. Allergic respiratory disease was induced using a previously described protocol [PMID:15528378]. Briefly, mice (n=36 in three independently conducted experiments) were anesthetized with isoflurane (Sigma-Aldrich) for three consecutive days prior to administration of mites allergen (HDM). HDM extract (Citeq, The Netherlands) was suspended in sterile phosphate-buffered saline (PBS) at a final concentration of 2.5 mg / ml. 10 μl of the solution was administered intranasally. Control mice (n=36) received 10 μl of sterile PBS using the same protocol.

[0089] On the third day of the experiment, mice were also administered human T cell-derived extracellular viable cells (EVs) (prepared as described above) either systemically via intraperitoneal injection (ip, n=48 in three independently conducted experiments) or locally via an intranasal route (in, n=48). The experiment concluded 18 hours after EV delivery (day 4).

[0090] Preparation of single-cell suspension from mouse lung The lungs were removed and placed in an Eppendorf tube with 0.5 ml of DMEM pen / strep (1%). The lungs were finely chopped with scissors, and 0.5 ml of DMEM pen / strep containing collagenase / dispase (Sigma-Aldrich) was added, followed by incubation at 37°C for 60 minutes. The reaction was stopped using 1 ml of FBS with EDTA (5 mM), and the samples were then kept on ice. The digested tissue was mechanically dissociated by passing it through a 70 μm cell strainer (Miltenyi) using a syringe plunger, and then washed twice with RPMI buffer (RPMI, pen / strep, HEPES (25 mM), EDTA (5 mM), and FBS (10%). Centrifuged at 1200 rpm for 5 minutes at 4°C) to prepare a single-cell suspension.

[0091] Staining of lung cells for flow cytometry Cells are stained using a staining buffer (PBS, 10% FBS, EDTA (5 mM)) to a size of 10-50 × 10 6 The cells were diluted to cells / ml and the Fc receptor was blocked with anti-CD16 / 32 (1:100, Biolegend) on ice for 10 minutes. The cells were dispensed into Eppendorf tubes, centrifuged (1200 rpm, 5 minutes, 4°C), and resuspended on ice in the dark for 30 minutes in staining buffer (see below) containing fluorophores and pre-conjugated antibodies. Before resuspending in 200 μl of PBS for flow cytometry, the cells were washed twice to remove unbound antibodies.

[0092] Flow cytometry Monochromatic stained compensation controls using VersaComp antibody capture beads (Beckman Coulter) and fluorescein-minus-one (FMO) controls using cells were prepared at the time of staining according to the same protocol. Stained samples were stored in the dark at 4°C. All flow cytometry analyses were performed using a Cytoflex flow cytometer equipped with 405 nm (BV421), 488 nm (530 / 30-FITC, 695 / 40-PerCp-Cy5.5), 561 nm (585 / 15-PE), and 640 nm (670 / 14-APC, APC / Cy7, AlexaFluor700) lasers and filters. The following markers were used to identify specific immune cell types: SiglecF-BV421, Ly6C-FITC, CD45-PerCP Cy5.5, CD64-PE, Ly6G-APC, CD11b-APC / Cy7, and CD11c-AlexaFluor700 (all from Biolegend). Data were analyzed using FlowJo (BD Biosciences) software.

[0093] result: Following lung dissection, multicolor staining of key immune cells in the lung preparation was performed to assess the presence of neutrophils, eosinophils, and inflammatory macrophages using a Beckman-Coulter Cytoflex S. The results are shown in Figure 6. Neutrophils were detected as CD11b+ / Ly6G+ cells and expressed as a percentage of CD45+ cells. Eosinophils were detected as SiglecF+ cells and expressed as a percentage of CD45+ cells. Inflammatory macrophages were detected as Ly6C+ cells and expressed as a percentage of CD11b+ / CD45+ cells.

[0094] The experiment was conducted in two independent replicates, with six animals provided to each treatment group. Analysis of these data shows a significant decrease in inflammatory macrophages in the lungs 18 hours after EV delivery. This occurred regardless of whether the delivery was local (intranasal) or systemic (intraperitoneal). EV also reduced eosinophil counts in the lungs, but this was noted only with local intranasal delivery. EV delivery did not show any effect on neutrophil presence, which is perhaps expected given the low neutrophil count at this point in time in the inflammatory lung model.

[0095] These data suggest that EV has a beneficial effect in an in vivo model of this pneumonia.

[0096] Consideration Phagocytic cell clearance of apoptotic cells is a highly efficient in vivo process crucial for tissue homeostasis and is known to actively support anti-inflammatory responses [see Fadok and subsequent in vivo studies]. Careful regulation by dying cells enables successful intercellular communication with the innate immune system, but very little is known about the specific molecular details of the communication events that drive phagocytic cell (macrophage) recruitment, nor about the potential effects on macrophage phenotypes, which are known to change with the removal of apoptotic cell debris. 58 It has been established that immediately after the initiation of apoptosis, dying cells actively modify their plasma membranes to promote phagocytic cell recognition and uptake. 24,59,60,61 However, during apoptosis, the cell rapidly releases a significantly increased number of EVs of various sizes. 62,63 Large apoptotic bodies are rapidly removed by local cells (both professional and amateur phagocytes), however 1Smaller ACdEVs (e.g., exosomes and microvesicles / microparticles) can persist and travel longer distances, transmitting apoptosis and attracting more distant immune cells both within and outside the local tissue microenvironment. Previously, we showed that ACdEV populations depleted of smaller apoptotic bodies released from apoptotic B cells induce macrophage chemotaxis to the site of leukocyte cell death, and that ICAM-3 on their surface promotes macrophage chemotaxis and tethering. 17 This study provides some molecular mechanisms for the process of macrophage recruitment, including the release of membrane fragments. 15 and CX3CL1 related to EVs 16 We are strengthening early research that demonstrates its role.

[0097] Here, the inventors further investigated ACdEV released from apoptotic lymphocytes with the aim of identifying their composition more broadly, while maintaining a clear focus on the ability of ACdEV to modulate immune responses and actively promote the resolution of inflammation.

[0098] The inventors have shown that induction of T cell apoptosis by anti-Fas / CHX promotes the release of a heterogeneous ACdEV population containing a mixture of exosomes and microvesicles. Careful analysis of the ACdEV proteomic fingerprint revealed that ACdEVs are enriched with the major protein components of established EVs, such as annexin, tetraspanin, Rab, heat shock proteins, GTPase, 14-3-3, and HLA proteins. 27 Importantly, ACdEV presents "flags" such as annexin A1 and calreticulin, which contain phosphatidylserine (PS), all of which have been previously noted as "eat-me" signals transported on the surface of apoptotic cell debris. Recognition, binding, and uptake of apoptotic cells stimulate macrophage production of anti-inflammatory TGF-β1 and suppression of pro-inflammatory TNF-α, IL-1β, IL-8, IL-10, LtC4, and thromboxane B2, making ACdEV a key pathway through which it can modulate the immune response.64 Indeed, the preferential enrichment of protein mechanisms responsible for ACdEV uptake via clathrin-dependent (CLH1) and clathrin-independent endocytosis (RhoA, Rac1), including various adhesion proteins (e.g., CD44, αL integrin, beta-2 integrin, and IgSF members including ICAM-3), suggests that ACdEV has sufficient capacity to ensure their efficient interaction and internalization with recipient cells. However, ACdEV also exhibits various proteins such as CD48, ICAM-3, CD226, and SLAMF6, which can induce diverse immune responses while enabling them to interact with monocytes / macrophages, T cells, and NK cells. 17,35,36,37 Similarly, ACdEV carries AL5AP (also known as FLAP) and LKHA4, both of which are involved in the synthesis of leukotrienes, inflammatory lipid mediators that can immunomodulate even when present in exosomes. 49 .

[0099] While leukocyte production of ACdEV during acute inflammatory episodes may act to propagate the inflammatory state, our research suggests that ACdEV may actively resist its potential and facilitate the convergence phase. Perhaps notably, we have detected ACdEV-related ABC transporters (MDR1, MRP1). This expands the possibility that ACdEV promotes lipid mediator (LM) signaling for longitudinal control of inflammation. The ABC transporters, specifically MRP1, may act as efflux pumps that actively release bioactive LM into the extracellular space. This would further activate LM-specific GPCRs on surrounding immune cells, thereby modulating the immune response. 65 MRP1 is associated with the release of leukotriene C4, but it is highly probable that it may correspond to a channel for the release of structurally similar LMs, which possess both pro-inflammatory and dual-acting properties. Such activity of EVs highlights a crucial unresolved question in the art: do EVs change after release, or are they immunologically and metabolically active?

[0100] Here, our research emphasizes that EV ligation by recipient immune cell receptors to activate recipient cell signaling or uptake may not be the only pathway through which ACdEV exerts its immunomodulatory properties. While it is known that the delivery of ACdEV-bound cargo plays a crucial role in determining the fate of recipient immune cells, our research focused on the independent bioactivity carried and delivered by vesicles. Firstly, we investigated the bioactive properties of ACdEV by targeted lipid metabolomics LC-MRM analysis and found that ACdEV is involved in the transport of free PUFAs, intermediates of LM biosynthesis (e.g., hydroxyl-PUFAs), and pro-inflammatory and dual-acting SPMs. * It was identified that both analog versions of PGE2, PGD2, and PGF are carried (Figure 2). 2α The presence of pro-inflammatory mediators such as LTB4 is perhaps surprising, given the well-established assertion that apoptotic cells are inherently non-inflammatory and convergent. However, PGD2 / PGE2 also acts as a convergent-promoting molecule "switch," potentially shifting the immune response from pro-inflammatory to convergent. 9 This increases the complexity of interpreting the overall LM signature of ACdEV. Therefore, rather than attempting to assign contributions to individual LMs, the inventors examine the LM signature of ACdEV as an overall balance of bioactive LMs and convergence-promoting SPM * This showed dominant and strong LM expression (Figure 2F).

[0101] Secondly, after identifying the existence of transporters for LM, the inventors hypothesized that EV may encompass all the necessary metabolic mechanisms for LM production. 52This study is the first to identify ACdEV as an active metabolic compartment loaded with LM-rich, immunomodulatory lipoxygenases. The inventors have revealed that ACdEV carries PLA2 and active LOX enzymes, with the highest specific activity for 15-LOX and lower specific activities for 12-LOX and 5-LOX. Furthermore, the inventors have shown that once ACdEV is loaded with LOX enzymes... ko Importantly, upon internalization by recipient cells, ACdEV bioactivity manifests as elevated recipient cell levels of LOX-specific lipid metabolites without stimulation in the presence of increased levels of the enzyme substrate. SPM * Taking into account the higher levels of and the highest activity of 15-LOX, it is reasonable to assume that ACdEV is a convergent-promoting phenotype and, once internalized by immune cells, can stimulate SPM biosynthesis and drive the convergent-promoting phenotype. Indeed, we have shown that ACdEV drives the convergent-promoting (M2) macrophage phenotype by stimulating 15-LOX / 15-LOX SPM macrophage biosynthesis while eliminating LTB4 levels (Figure 4A). The phenotypic change to the M2 phenotype was further confirmed by surface immunophenotypic analysis of macrophages treated with ACdEV (Figure 4B).

[0102] While the transfer of LOX activity was demonstrated here with an immunological focus, it could well offer opportunities for immunomodulation in cells that do not express LOX at significant levels (e.g., nonprofessional phagocytic cells, surrounding tissue cells). Enzyme transport within EVs has been shown in recent studies. 49,50,51 The specific relevance of biosynthetic capacity to the immunomodulatory function of extracellular granulocytes (EVs) remains unclear. A pioneering study by Esser et al. reported the presence of LKHA4 in macrophage-derived exosomes and showed that biostimulation with excess LTA4 led to increased levels of LTA4 metabolites in EV granulocytes, suggesting the activity of LKHA4 bound to EVs. 49 Recent research by Fafian-Labora et al. 51This further supports the hypothesis that EV uptake, rather than surface receptor activation, may mediate downstream effects caused by the delivery of cargo bound to EVs. Specifically, the group showed that glutathione-S-transferase is active in senescent cell-derived EVs and can ameliorate aging.

[0103] Nevertheless, the potential of ACdEV to exert these effects more broadly must be considered. Future research should consider the dynamic changes that may occur in ACdEV from release from donor cells to possibly distant interactions with recipient cells. This research challenges the view that ACdEV can be studied as a "snapshot" and that temporal studies of EV over time are essential to provide a more complete picture of the ACdEV composition, which can change over time, and raises the possibility that ACdEV may modulate the function of many cells, not just recipient cells, in those processes.

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Claims

1. A method for treating an inflammatory condition or disease comprising administering a pharmaceutically effective amount of extracellular vesicles (EVs), wherein the EVs comprise 15-lipoxygenase (15-LOX) and / or a nucleic acid encoding 15-LOX, and optionally further comprising one or more 12-lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX), and / or one or more nucleic acids encoding one or more 12-LOX and 5-LOX.

2. Extracellular vesicles (EVs) for use in treating inflammatory diseases or conditions, wherein the EVs comprise 15-lipoxygenase (15-LOX) and / or a nucleic acid encoding 15-LOX, and optionally further comprising one or more of 12-lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX), and / or one or more nucleic acids encoding one or more of 12-LOX and 5-LOX.

3. The method or EV according to claim 1 or 2, wherein the inflammatory disease is a chronic inflammatory disease.

4. The method or EV according to any one of claims 1 to 3, wherein the inflammatory disease is selected from chronic wounds, inflammatory skin diseases, autoimmune diseases, nephritis, aging, lung diseases, liver diseases, and dementia.

5. The method or EV according to claim 4, wherein the inflammatory disease is selected from internal or external wounds, diabetic wounds, and systemic lupus erythematosus (SLE).

6. The method or EV according to any one of claims 1 to 5, wherein the EV does not include 5-lipoxygenase (5-LOX) or nucleic acid encoding 5-LOX.

7. The method or EV according to any one of claims 1 to 6, wherein the EV is an isolated, naturally occurring EV or an EV that does not exist naturally.

8. The method or EV according to claim 7, wherein the EV is an apoptotic cell-derived extracellular vesicle (ACdEV) or a living cell-derived EV, most typically a mesenchymal stem cell-derived EV.

9. In addition, the method or EV according to any one of claims 1 to 8, comprising one or more markers capable of targeting MΦ macrophages, typically pro-inflammatory macrophages.

10. The method or EV according to any one of claims 1 to 9, wherein the EV comprises one or more eat me markers, preferably selected from annexin A1, callecticulin, and phosphatidylserine.

11. The method or EV according to any one of claims 1 to 10, wherein the EV preferably comprises one or more adhesion molecules selected from IgSF member (most typically ICAM-3), CD44, integrin alpha-1, and integrin beta-2.

12. The method or EV according to any one of claims 1 to 11, wherein the EV does not contain one or more leukotrienes, most typically arachidonic acid 5-lipoxygenase-activating protein (AL5AP) or leukotriene A-4 hydrolase (LKHA4).

13. The method or EV according to any one of claims 1 to 12, wherein the EV has a size range of at least 1 nm, or 10 to 1000 nm, more typically 70 to 700 nm.

14. The method or EV according to any one of claims 1 to 13, wherein the EV has a mode size of 100 to 200 nm, more typically 90 to 130 nm.

15. The method or EV according to any one of claims 1 to 14, wherein the EV can be administered to a subject, or administered to a subject intravenously, intraperitoneally, topically, intranasally, orally by inhalation, preferably by nebulizer or inhaler.

16. An isolated extracellular vesicle (EV) wherein the EV comprises 15-lipoxygenase (15-LOX) and / or a nucleic acid encoding 15-LOX, and optionally further comprises one or more of 12-lipoxygenase (12-LOX), 5-lipoxygenase (5-LOX), and / or one or more nucleic acids encoding one or more of 15-LOX, 12-LOX, and 5-LOX.

17. The EV according to claim 16, wherein the EV does not contain 5-lipoxygenase (5-LOX) or nucleic acid encoding 5-LOX.

18. The EV according to claim 16 or 17, wherein the EV is an isolated, naturally occurring EV or an EV that does not exist naturally.

19. The EV according to claim 18, wherein the EV is an apoptotic cell-derived extracellular vesicle (ACdEV) or a mesenchymal stem cell EV.

20. The EV according to claims 16 to 19, comprising a pharmaceutically acceptable excipient.

21. In addition, the EV according to claims 16 to 20 comprises one or more markers capable of targeting pro-inflammatory macrophages.

22. The EV according to claims 16 to 21, wherein the EV comprises one or more eat me markers, preferably selected from annexin A1, callecticulin, and phosphatidylserine.

23. The EV according to claims 16 to 22, wherein the EV preferably comprises one or more adhesion molecules selected from IgSF member (most typically ICAM-3), CD44, integrin alpha-1, and integrin beta-2.

24. The EV according to claims 16 to 23, wherein the EV does not contain one or more leukotrienes, most typically arachidonic acid 5-lipoxygenase activating protein (AL5AP, also known as FLAP), nor leukotriene A-4 hydrolase (LKHA4).

25. The EV according to claims 16 to 24, wherein the EV has a size range of at least 1 nm, typically 10 to 1000 nm, and more typically 70 to 700 nm.

26. The EV according to claims 16 to 25, wherein the EV has a mode size of 100 to 200 nm, more typically 90 to 130 nm.

27. The EV according to claims 16 to 26, wherein the EV can be administered to a subject intraperitoneally, externally, intranasally, orally by inhalation, preferably by nebulizer or inhaler.

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