WO2024086955A1 - Immunosuppressive effect on cd4+ t lymphocytes of a circulating mitochondria-enriched composition - Google Patents
Immunosuppressive effect on cd4+ t lymphocytes of a circulating mitochondria-enriched composition Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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Definitions
- T lymphocytes Among the great variety of immune cells are T lymphocytes. T lymphocytes begin their development in the bone marrow. They subsequently go to the thymus, where they mature and go through the processes of positive and negative selection. After passage through the thymus, immature T lymphocytes become mature na ⁇ ve T lymphocytes. When na ⁇ ve T cells are presented with an antigen and receive the appropriate co-stimulatory signals from antigen-presenting cells (APCs), they begin the production of IL-2, a cytokine that acts as a growth and survival factor.
- APCs antigen-presenting cells
- CD45 isoforms which are CD45RA and CD45RO, respectively.
- CD8+ T lymphocytes are known as cytotoxic T lymphocytes and participate in adaptive immunity by directly eliminating tumor cells or cells infected by intracellular pathogens.
- CD4+ T cells also participate in the adaptive immune response and are known as T helper (Th) cells, which are classified into four main groups of cells: Thl, Th2, Thl7 and regulatory T cell (Treg). These groups of CD4+ T lymphocytes participate in adaptive immunity with various functions mediated through the release of cytokines. Thl cells mainly produce IFNy and provide protection against intracellular pathogens. Th2 cells secrete IL-4, IL-5 and IL-13 and fight infections against helminthic parasites. Thl7 cells are characterized by the production of IL-17 and are responsible for the elimination of extracellular bacteria. On the other hand, Tregs are cells with an immunosuppressive function. Tregs are responsible for maintaining tolerance against one's own antigens and regulate the immune response. Tregs can be recognized by the expression of the transcription factor Foxp3, which is necessary for developing and maintaining Tregs, and the constitutive expression of CD25.
- Thl cells mainly produce IFNy and provide protection against intracellular pathogens. Th2 cells secrete
- T lymphocytes Although there are mechanisms to control the immune response (induction of apoptosis, expression of inhibitory molecules and action of Tregs), there are times when these mechanisms fail. Failures in the mechanisms of regulation of the immune response, exerted by T lymphocytes, can lead to the development of autoimmunity, or to improper regulation of the magnitude of the immune response exerted against pathogens, which can generate excess inflammation. . Furthermore, during these harmful processes, and due to the failure of the mechanisms that limit the immune response, the development of a hyperactive phenotype in T lymphocytes can be favored. This hyperactive phenotype has been described as a greater expression of activation markers and greater proliferation. This hyperactivity of T lymphocytes causes the development of autoimmunity. In humans, the deregulation of CD4+ T lymphocytes plays a central role in the development of pathologies such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE).
- RA Rheumato
- CD4+ T lymphocytes are essential to maintain physiological homeostasis and prevent the development of autoimmune diseases and harmful inflammatory processes.
- the inventors have found a method that allows regulating the activation and proliferation of CD4+ T cells from human donors in normal and pathological conditions where these cells are hyperactivated, by exposing the CD4+ T lymphocytes to a composition that comprises an isolated fraction of plasma. , enriched in circulating mitochondria, called MitoCir.
- This composition could be applied in all cases where CD4+ T lymphocytes are deregulated, such as in Systemic Lupus Erythematosus (SLE); Rheumatoid arthritis; acute inflammatory processes, Psoriasis; Antiphospholipid syndrome; Multiple sclerosis; Atherosclerosis; Osteoarthritis; Type I diabetes; Sjogren's syndrome; Myasthenia gravis; Crohn's disease ; Ulcerative colitis; Vasculitis; Vasculitis associated with antineutrophil cytoplasmic antibodies; Graft versus host disease; Preeclampsia and/or Celiac Disease DESCRIPTION OF THE FIGURES.
- SLE Systemic Lupus Erythematosus
- Figure 1 Isolated from plasma enriched in extracellular or circulating mitochondria. Image of an isolated plasma enriched in mitochondria obtained by transmission electron microscopy, arrowheads indicate extracellular or circulating mitochondria. The sample was obtained from a healthy donor.
- Figure 2 Effect of plasma isolate containing mitochondria on the activation of CD4+ T lymphocytes cultured under activation and differentiation conditions.
- A) Flow cytometry plots for CD25 expression on CD4+ T lymphocytes. The analysis was performed after 4 days of culture for conditions without mitochondria, with mitochondria isolated from plasma MitoCir and with mitochondria isolated from damaged plasma MitoCir D.
- C Flow cytometry plots for PD-1 expression in CD4+ T lymphocytes. The analysis was performed after 4 days of culture for conditions without mitochondria, with mitochondria isolated from plasma MitoCir with mitochondria isolated from plasma damaged MitoCir D.
- Figure 3 Effect of plasma isolate containing mitochondria on the proliferation of CD4+ T lymphocytes cultured under activation and differentiation conditions.
- Figure 4 Effect of plasma isolate containing mitochondria derived from a healthy donor on the activation and proliferation of CD4+ T lymphocytes from patients with SLE cultured under activation and differentiation conditions.
- A) Flow cytometry plots for Ki-67 expression in CD4+ T lymphocytes from a patient with SLE. The analysis was performed after 4 days of culture for conditions without mitochondria, with mitochondria isolated from MitoCir plasma and with mitochondria isolated from damaged plasma MitoCir D.
- the inventors have evaluated the application of an isolated fraction of plasma, enriched in circulating mitochondria, called MitoCir, to regulate the activation and proliferation of CD4+ T cells from human donors in normal and pathological conditions where these cells are hyperactivated or deregulated.
- MitoCir has an immunosuppressive effect on CD4+ T cells.
- This MitoCir composition contributes to the cellular homeostasis of individuals with pathologies in which these cells are deregulated, and is a powerful tool for long-distance intercellular communication and alleviating the severity of autoimmune diseases.
- the invention relates to a composition for use in a method of treating a patient with an immunological disorder of hyperactivation or deregulation of CD4+ T lymphocytes that comprises administering a composition that comprises a plasma isolate, enriched in extracellular mitochondria, in a subject with hyperactivation or deregulation of CD4+ T lymphocytes.
- This composition is administered to a patient parenterally, for example, intramuscularly or intravenously.
- the immunological disorder of hyperactivation or deregulation of CD4 + T lymphocytes to be treated with the composition of the invention is chosen from any disease or syndrome that involves hyperactivation or deregulation of CD4+ T lymphocytes, such as: Systemic Lupus Erythematosus (SLE); Rheumatoid arthritis; acute inflammatory processes, Psoriasis; Antiphospholipid syndrome; Multiple sclerosis; Atherosclerosis; Osteoarthritis; Type I diabetes; Sjogren's syndrome; Myasthenia gravis; Crohn's disease ; Ulcerative colitis; Vasculitis; Vasculitis associated with antineutrophil cytoplasmic antibodies; Graft versus host disease; Preeclampsia and/or Celiac Disease.
- SLE Systemic Lupus Erythematosus
- Rheumatoid arthritis acute inflammatory processes, Psoriasis
- Antiphospholipid syndrome e.g., Multiple sclerosis
- Atherosclerosis Osteoarthritis
- the method of the invention takes special care to avoid the activation of platelets, since it is described that they release mitochondria when they are activated, in this way we are blocking the release of platelet mitochondria, which are recognized by the surface markers CD42b, CD42A and/or CD41, hallmarks of platelets, so a platelet inactivator prostaglandin El (PGE1) (Focus Biomolecules) is used at a final concentration of preferably 10 pM diluted in an anticoagulant buffer, such as citrate solution A dextrose (0. 1 M trisodium citrate, 0.11 M glucose and 0.08 M citric acid).
- PGE1 platelet inactivator prostaglandin El
- the invention aims at a method of obtaining a composition enriched in circulating mitochondria which includes the steps of: a) add a composition of prostaglandin El (PGE1) in 0.08 to 0.2 M trisodium citrate, 0.8 to 0.20 M of glucose and 0.05 to 0.1M of citric acid, to reach a concentration final between 8 to 15 pM of PGE1 to blood plasma from healthy donors b) centrifuge 150g for 5 to 20 minutes, and collect the supernatant; c) centrifuge the supernatant from b) at 2500 g for 20 to 60 minutes; d) concentrate the supernatant from c) by centrifuging at 13000 g for 5 to 20 minutes at least once; e) resuspend the pellet from step d) in a buffer solution.
- PGE1 prostaglandin El
- the buffer comprises: 0.2 M sucrose, 0.01 M TRIS, 0.001 M EGTA, protease inhibitor IX.
- the buffer comprises: 0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, protease inhibitor IX.
- composition enriched in circulating mitochondria that comprises a high concentration of circulating mitochondria and at the same time comprises a low concentration of mitochondria positive for the CD42b, CD42A and/or CD41 markers.
- the composition enriched in circulating mitochondria of the invention serves to prepare a drug useful to counteract the hyperactivation of CD4+ T lymphocytes. Where these CD4+ T lymphocytes are hyperactive in acute inflammatory processes or in autoimmune diseases.
- autoimmune diseases are chosen from: Systemic Lupus Erythematosus (SLE); Rheumatoid arthritis; Psoriasis; Antiphospholipid syndrome; Multiple sclerosis; Atherosclerosis; Osteoarthritis; Type I diabetes; Sjogren's syndrome; Myasthenia gravis; Crohn's disease ; Ulcerative colitis; Vasculitis; Vasculitis associated with antineutrophil cytoplasmic antibodies; Graft versus host disease; Preeclampsia and/or Celiac Disease.
- SLE Systemic Lupus Erythematosus
- the composition of the invention serves to prepare a medicament for parenteral administration.
- Example 1 Obtaining an isolate rich in mitochondria, MitoCir. Using centrifugations at different speeds, an isolate of human blood plasma containing extracellular mitochondria, known as circulating mitochondria, was obtained. Figure 1 shows a photograph of the composition obtained, where the high concentration of mitochondria can be seen.
- Isolation of mitochondria from plasma was performed avoiding platelet activation.
- Fresh blood was collected from healthy human donors using blood collection tubes with 3.2% sodium citrate as anticoagulant (BD Vacutainer®). The blood was centrifuged at 150 g for 20 min at room temperature (RT) without brakes and without acceleration.
- Plasma was collected and prostaglandin El (PGE1) (Focus Biomolecules) was added to a final concentration of 10 pM diluted in anticoagulant buffer citrate solution A dextrose (0.1 M trisodium citrate, 0.11 M glucose and 0.08 M citric acid) (ACD-A) to prevent the activation of platelets, since it has been described that they release mitochondria when they are activated, in this way we are blocking the release of platelet mitochondria.
- PGE1 prostaglandin El
- the plasma was centrifuged at 150g for 10 min at 4°C without brake and without acceleration to remove red blood cells and leukocytes.
- the plasma was collected and centrifuged at 2500g for 30 min at 4°C without brake and without acceleration to remove the platelets and obtain the supernatant with circulating mitochondria.
- Mitochondria were concentrated through two successive centrifugations at 13,000g for 10 min at 4°C, washing with mitochondrial isolation buffer (MIB) (0.2 M sucrose, 0.01 M TRIS, 0.001 M EGTA, protease inhibitor IX).
- MIB mitochondrial isolation buffer
- the pellet obtained after the last differential centrifugation at 13000g is the circulating mitochondria, MitoCir.
- the circulating mitochondrial pellet was resuspended in MIB (Mitochondrial Isolation Buffer 0.2 M sucrose, 0.01 M TRIS, 0.001 M EGTA, protease inhibitor IX)) or in mitochondrial respiration buffer 05 (MIR05) (0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, protease inhibitor IX), depending on the experiment.
- MIB Mitochondrial Isolation Buffer 0.2 M sucrose, 0.01 M TRIS, 0.001 M EGTA, protease inhibitor IX
- MIR05 mitochondrial respiration buffer 05
- PBMCs Peripheral Blood Mononuclear Cell
- the stained PBMCs were washed with PBS-1X + 2% FBS by centrifuging at 600g for 5 min at 4°C. They were resuspended in PBS-1X + 10% filtered FBS and the CD4+ (CD4+CD8-) T lymphocyte population was selected using the fluorescence-activated cell sorting technique, for which a BD FacsAriaTM Fusion cell sorter was used.
- the CD4+ T lymphocytes were centrifuged at 700g for 5 min at 4°C and, in the case of subsequently analyzing cell proliferation, they were stained with 5 pM CellTraceTM Violet (CTV) for 20 min at 37°C prior to culture.
- CD4+ T cells were resuspended in lymphocyte medium and 1x10 5 cells were seeded in a round-bottom 96-well plate with anti-CD3 (1 pg/mL), anti-CD28 (2 pg/mL), TGF-01 ( 10 ng/mL) and IL-2 (50 U/mL). Stimulation with anti-CD3 and anti-CD28 emulates stimulation by APCs and allows T lymphocytes to activate and proliferate.
- the cytokine TGF-01 stimulates the differentiation of CD4+ T lymphocytes into Tregs (Regulatory T cells). 20 pg of circulating mitochondrial protein was added to the wells with CD4+ T cells.
- Mitochondria were stained with MitoTracker, as mentioned in section 3.8, to subsequently visualize the cells that incorporated them.
- CD4+ T cells were incubated at 37°C with 5% CO 2 .
- CD4+ T lymphocytes under activation conditions plus TGF-01 and with circulating mitochondria were evaluated on day 1 by confocal microscopy, to visualize the internalization of circulating mitochondria.
- CD4+ T lymphocytes were analyzed on day 4 by flow cytometry, to evaluate the effect of circulating mitochondria.
- As a control cultured CD4+ T lymphocytes with cytokines, but without circulating mitochondria, were used. To corroborate that the observed effects were dependent on viable mitochondria, as a control, 20 pg of protein from circulating mitochondria sonicated for 5 min at 60% intensity in an ultrasonic cleaner equipment and exposed to UV light for 30 min were added.
- MitoCir has an immunosuppressive effect on the activation and proliferation processes of CD4+ T lymphocytes from healthy donors.
- SLE Systemic Lupus Erythematosus
- the effect of the plasma isolate of the invention that contains mitochondria on the CD4+ T lymphocytes from donors with SLE shows a remarkable and interesting result, given that in these CD4+ T lymphocytes from patients with SLE, they have a very proliferative and activated profile, which It is regulated with the composition of the invention, isolated from plasma containing mitochondria derived from a healthy donor, decreasing proliferation and activation.
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a method for obtaining a circulating mitochondria-enriched composition, avoiding platelet activation. This method obtains a circulating mitochondria-enriched composition comprising a high concentration of circulating mitochondria and at the same time comprising a low concentration of positive mitochondria for the CD42b, CD42A and/or CD41 markers. This composition is used to prepare a drug useful for counteracting CD4+ T lymphocyte hyperactivation. These CD4+ T lymphocytes are hyperactive in acute inflammatory processes or in autoimmune diseases. These autoimmune diseases include: Systemic Lupus Erythematosus (SLE); Rheumatoid arthritis; Psoriasis; Antiphospholipid syndrome; Multiple sclerosis; Atherosclerosis; Osteoarthritis; Type I diabetes; Sjögren's syndrome; Myasthenia gravis; Crohn's disease; Ulcerative colitis; Vasculitis; Vasculitis associated with anti-neutrophil cytoplasmic antibodies; Graft-versus-host disease; Preeclampsia and/or Celiac disease.
Description
EFECTO INMUNOSUPRESOR SOBRE LINFOCITOS T CD4+ DE UNA COMPOSICIÓN ENRIQUECIDA EN MITOCON DRIAS CIRCULANTES IMMUNOSUPPRESSIVE EFFECT ON CD4+ T LYMPHOCYTES OF A COMPOSITION ENRICHED IN CIRCULATING MITOCON DRIAS
MEMORIA DESCRIPTIVA DESCRIPTIVE MEMORY
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
Dentro de la gran variedad de células inmunes se encuentran los linfocitos T. Los linfocitos T comienzan su desarrollo en la medula ósea. Posteriormente se dirigen al timo, donde maduran y pasan por los procesos de selección positiva y negativa. Luego del paso por el timo, los linfocitos T inmaduros pasan a ser linfocitos T naive maduros. Cuando a los linfocitos T naive se les presenta un antígeno y reciben las señales co-estimuladoras apropiadas desde las células presentadoras de antígenos (APC), comienzan la producción de IL-2, una citoquina que actúa como factor de crecimiento y sobrevida. Among the great variety of immune cells are T lymphocytes. T lymphocytes begin their development in the bone marrow. They subsequently go to the thymus, where they mature and go through the processes of positive and negative selection. After passage through the thymus, immature T lymphocytes become mature naïve T lymphocytes. When naïve T cells are presented with an antigen and receive the appropriate co-stimulatory signals from antigen-presenting cells (APCs), they begin the production of IL-2, a cytokine that acts as a growth and survival factor.
Asimismo inician los procesos de proliferación y diferenciación de los linfocitos T naive. Lo anterior da lugar a los linfocitos T con funciones específicas denominados linfocitos T efectores. De la población de linfocitos T efectores una pequeña proporción se diferencia a linfocitos T de memoria. Los estados de diferenciación de los linfocitos T naive y memoria pueden reconocerse según la expresión de las ¡soformas de CD45, las que son CD45RA y CD45RO, respectivamente. Por otro lado, dentro de la población de linfocitos T es posible diferenciar entre aquellos que expresan las moléculas correceptoras CD8 y CD4 en la membrana plasmática. Los linfocitos T CD8+ son conocidos como linfocitos T citotóxicos y participan en la inmunidad adaptativa eliminando directamente células tumorales o infectadas por patógenos intracelulares. Los linfocitos T CD4+ también participan en la respuesta inmune adaptativa y se conocen como linfocitos T cooperadores (Th), que se clasifican en cuatro grupos principales de células: Thl, Th2, Thl7 y célula T reguladora (Treg). Estos grupos de linfocitos T CD4+ participan en la inmunidad adaptativa con diversas funciones mediadas a través de la liberación de citoquinas. Las células Thl producen principalmente IFNy y entregan protección contra patógenos intracelulares. Las células Th2 secretan IL-4, IL-5 e IL-13 y combaten infecciones contra parásitos helmínticos. Las células Thl7 están caracterizadas por la producción de IL-17 y se encargan de la eliminación de bacterias extracelulares. Por otra parte, los Tregs son células con función inmunosupresora. Los Tregs se encargan de mantener la tolerancia
contra los antígenos propios y de regular la respuesta inmune. Los Tregs se pueden reconocer por la expresión del factor de transcripción Foxp3, que es necesario para desarrollar y mantener los Tregs, y la expresión constitutiva de CD25. They also initiate the proliferation and differentiation processes of naïve T lymphocytes. This gives rise to T lymphocytes with specific functions called effector T lymphocytes. Of the population of effector T lymphocytes, a small proportion differentiates into memory T lymphocytes. The differentiation states of naïve and memory T lymphocytes can be recognized according to the expression of the CD45 isoforms, which are CD45RA and CD45RO, respectively. On the other hand, within the T lymphocyte population it is possible to differentiate between those that express the CD8 and CD4 coreceptor molecules on the plasma membrane. CD8+ T lymphocytes are known as cytotoxic T lymphocytes and participate in adaptive immunity by directly eliminating tumor cells or cells infected by intracellular pathogens. CD4+ T cells also participate in the adaptive immune response and are known as T helper (Th) cells, which are classified into four main groups of cells: Thl, Th2, Thl7 and regulatory T cell (Treg). These groups of CD4+ T lymphocytes participate in adaptive immunity with various functions mediated through the release of cytokines. Thl cells mainly produce IFNy and provide protection against intracellular pathogens. Th2 cells secrete IL-4, IL-5 and IL-13 and fight infections against helminthic parasites. Thl7 cells are characterized by the production of IL-17 and are responsible for the elimination of extracellular bacteria. On the other hand, Tregs are cells with an immunosuppressive function. Tregs are responsible for maintaining tolerance against one's own antigens and regulate the immune response. Tregs can be recognized by the expression of the transcription factor Foxp3, which is necessary for developing and maintaining Tregs, and the constitutive expression of CD25.
A pesar de que existen mecanismos de control de la respuesta inmune (inducción de apoptosis, expresión de moléculas inhibitorias y acción de los Tregs), hay ocasiones en que estos mecanismos fallan. Las fallas en los mecanismos de regulación de la respuesta inmune, ejercida por los linfocitos T, puede conllevar al desarrollo de autoinmunidad, o, a una indebida regulación de la magnitud de la respuesta inmune ejercida contra patógenos, lo que puede generar un exceso de inflamación. Además, durante estos procesos nocivos, y debido a la falla de los mecanismos que limitan la respuesta inmune, se puede favorecer el desarrollo de un fenotipo hiperactivo en los linfocitos T. Este fenotipo hiperactivo ha sido descrito como una mayor expresión de marcadores de activación y una mayor proliferación. Esta hiperactividad de los linfocitos T provoca el desarrollo de autoinmunidad. En humanos la desregulación de los linfocitos T CD4+ tiene un rol central en el desarrollo de patologías como la Artritis Reumatoide (AR) y el Lupus Eritematoso Sistémico (LES).Although there are mechanisms to control the immune response (induction of apoptosis, expression of inhibitory molecules and action of Tregs), there are times when these mechanisms fail. Failures in the mechanisms of regulation of the immune response, exerted by T lymphocytes, can lead to the development of autoimmunity, or to improper regulation of the magnitude of the immune response exerted against pathogens, which can generate excess inflammation. . Furthermore, during these harmful processes, and due to the failure of the mechanisms that limit the immune response, the development of a hyperactive phenotype in T lymphocytes can be favored. This hyperactive phenotype has been described as a greater expression of activation markers and greater proliferation. This hyperactivity of T lymphocytes causes the development of autoimmunity. In humans, the deregulation of CD4+ T lymphocytes plays a central role in the development of pathologies such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE).
Por lo que regular la actividad de los linfocitos T CD4+ es fundamental para mantener la homeostasis fisiológica y evitar el desarrollo de enfermedades autoinmunes y de procesos inflamatorios nocivos.Therefore, regulating the activity of CD4+ T lymphocytes is essential to maintain physiological homeostasis and prevent the development of autoimmune diseases and harmful inflammatory processes.
En este contexto los inventores han encontrado un método que permite regular la activación y proliferación de células T CD4+ de donantes humanos en condiciones normales y patológicas donde estas células están hiperactivadas, al exponer los linfocitos T CD4+ a una composición que comprende una fracción aislada de plasma, enriquecida en mitocondrias circulantes, llamada MitoCir. Esta composición podría aplicarse en todos los casos donde los linfocitos T CD4+ se encuentren desregulados, como por ejemplo en Lupus Eritematoso Sistémico (LES); Artritis reumatoide; procesos inflamatorios agudos, Psoriasis; Síndrome antifosfolipídico; Esclerosis múltiple; Aterosclerosis; Osteoartritis; Diabetes tipo I; Síndrome de Sjogren; Miastenia gravis; Enfermedad de Crohn ; Colitis ulcerosa; Vasculitis; Vasculitis asociadas a anticuerpos anticitoplasma de neutrófilos; Enfermedad injerto contra huésped; Preeclamsia y/o Enfermedad celíaca
DESCRIPCIÓN DE LAS FIGURAS. In this context, the inventors have found a method that allows regulating the activation and proliferation of CD4+ T cells from human donors in normal and pathological conditions where these cells are hyperactivated, by exposing the CD4+ T lymphocytes to a composition that comprises an isolated fraction of plasma. , enriched in circulating mitochondria, called MitoCir. This composition could be applied in all cases where CD4+ T lymphocytes are deregulated, such as in Systemic Lupus Erythematosus (SLE); Rheumatoid arthritis; acute inflammatory processes, Psoriasis; Antiphospholipid syndrome; Multiple sclerosis; Atherosclerosis; Osteoarthritis; Type I diabetes; Sjogren's syndrome; Myasthenia gravis; Crohn's disease ; Ulcerative colitis; Vasculitis; Vasculitis associated with antineutrophil cytoplasmic antibodies; Graft versus host disease; Preeclampsia and/or Celiac Disease DESCRIPTION OF THE FIGURES.
Figura 1. Aislado de plasma enriquecido en mitocondrias extracelulares o circulantes. Imagen de un aislado plasma enriquecido en mitocondrias obtenida por microscopía electrónica de transmisión, las puntas de flechas señalan mitocondrias extracelulares o circulantes. La muestra se obtuvo de un donante sano. Figure 1. Isolated from plasma enriched in extracellular or circulating mitochondria. Image of an isolated plasma enriched in mitochondria obtained by transmission electron microscopy, arrowheads indicate extracellular or circulating mitochondria. The sample was obtained from a healthy donor.
Figura 2. Efecto del aislado de plasma que contiene mitocondrias sobre la activación de linfocitos T CD4+ cultivados en condiciones de activación y diferenciación. A) Gráficos de citometría de flujo para la expresión de CD25 en linfocitos T CD4+. El análisis se realizó después de 4 días de cultivo para las condiciones sin mitocondrias, con mitocondrias aisladas desde plasma MitoCir y con mitocondrias aisladas desde plasma dañadas MitoCir D. B) Gráfico resumen de la expresión de CD25 en linfocitos T CD4+ para los experimentos realizados. C) Gráficos de citometría de flujo para la expresión de PD-1 en linfocitos T CD4+. El análisis se realizó después de 4 días de cultivo para las condiciones sin mitocondrias, con mitocondrias aisladas desde plasma MitoCir con mitocondrias aisladas desde plasma dañadas MitoCir D. D) Gráfico resumen de la expresión de PD-1 en linfocitos T CD4+ para los experimentos realizados. Figure 2. Effect of plasma isolate containing mitochondria on the activation of CD4+ T lymphocytes cultured under activation and differentiation conditions. A) Flow cytometry plots for CD25 expression on CD4+ T lymphocytes. The analysis was performed after 4 days of culture for conditions without mitochondria, with mitochondria isolated from plasma MitoCir and with mitochondria isolated from damaged plasma MitoCir D. B) Summary graph of CD25 expression in CD4+ T lymphocytes for the experiments carried out. C) Flow cytometry plots for PD-1 expression in CD4+ T lymphocytes. The analysis was performed after 4 days of culture for conditions without mitochondria, with mitochondria isolated from plasma MitoCir with mitochondria isolated from plasma damaged MitoCir D. D) Summary graph of PD-1 expression in CD4+ T lymphocytes for the experiments carried out .
Figura 3. Efecto del aislado de plasma que contiene mitocondrias sobre la proliferación de linfocitos T CD4+ cultivados en condiciones de activación y diferenciación. A) Imágenes de microscopía óptica de los clústeres formados por linfocitos T CD4+. Las imágenes se tomaron después de 4 días de cultivo para las condiciones sin mitocondrias, con mitocondrias aisladas desde plasma MitoCir con mitocondrias aisladas desde plasma dañadas MitoCir D. Escala = 500 pm. B) Gráfico resumen de la cuantificación del área de los clústeres formados por linfocitos T CD4+ para los experimentos realizados. C) Gráficos de citometría de flujo para la proliferación de linfocitos T CD4+. La proliferación se evaluó mediante la dilución de la sonda CTV. El análisis se realizó después de 4 días de cultivo para las condiciones sin mitocondrias, con mitocondrias aisladas desde plasma MitoCir y con mitocondrias aisladas desde plasma dañadas MitoCir D. D) Gráfico resumen de la proliferación de linfocitos T CD4+ teñidos con CTV para los experimentos realizados. Figure 3. Effect of plasma isolate containing mitochondria on the proliferation of CD4+ T lymphocytes cultured under activation and differentiation conditions. A) Optical microscopy images of the clusters formed by CD4+ T lymphocytes. Images were taken after 4 days of culture for conditions without mitochondria, with mitochondria isolated from plasma MitoCir with mitochondria isolated from damaged plasma MitoCir D. Scale = 500 pm. B) Summary graph of the quantification of the area of the clusters formed by CD4+ T lymphocytes for the experiments carried out. C) Flow cytometry plots for CD4+ T lymphocyte proliferation. Proliferation was assessed by dilution of the CTV probe. The analysis was performed after 4 days of culture for conditions without mitochondria, with mitochondria isolated from plasma MitoCir and with mitochondria isolated from damaged plasma MitoCir D. D) Summary graph of the proliferation of CD4+ T lymphocytes stained with CTV for the experiments carried out .
Figura 4. Efecto del aislado de plasma que contiene mitocondrias derivado de un donante sano sobre la activación y proliferación de linfocitos T CD4+ de paciente con LES cultivados en condiciones de activación y diferenciación. A) Gráficos de citometría de flujo para la expresión de Ki-67 en linfocitos T CD4+ de un paciente con LES. El análisis se realizó después de 4 días de cultivo para las condiciones sin mitocondrias, con mitocondrias aisladas desde plasma MitoCir y con
mitocondrias aisladas desde plasma dañadas MitoCir D. B) Gráfico resumen de la expresión de Ki- 67 en linfocitos T CD4+ de un paciente con LES. C) Gráficos de citometría de flujo para la expresión de CD25 en linfocitos T CD4+ de un paciente con LES. El análisis se realizó después de 4 días de cultivo para las condiciones sin mitocondrias, con mitocondnas aisladas desde plasma MitoCir y con mitocondnas aisladas desde plasma dañadas MitoCi D. D) Gráfico resumen de la expresión de CD25 en linfocitos T CD4+ de un paciente con LES. Figure 4. Effect of plasma isolate containing mitochondria derived from a healthy donor on the activation and proliferation of CD4+ T lymphocytes from patients with SLE cultured under activation and differentiation conditions. A) Flow cytometry plots for Ki-67 expression in CD4+ T lymphocytes from a patient with SLE. The analysis was performed after 4 days of culture for conditions without mitochondria, with mitochondria isolated from MitoCir plasma and with mitochondria isolated from damaged plasma MitoCir D. B) Summary graph of Ki-67 expression in CD4+ T lymphocytes from a patient with SLE. C) Flow cytometry plots for CD25 expression on CD4+ T lymphocytes from a patient with SLE. The analysis was performed after 4 days of culture for conditions without mitochondria, with mitochondria isolated from plasma MitoCir and with mitochondria isolated from damaged plasma MitoCi D. D) Summary graph of CD25 expression in CD4+ T lymphocytes from a patient with SLE .
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
Los inventores han evaluado la aplicación de una fracción aislada de plasma, enriquecida en mitocondrias circulantes, llamada MitoCir, para regular la activación y proliferación de células T CD4+ de donantes humanos en condiciones normales y patológicas donde estas células están hiperactivadas o desreguladas. The inventors have evaluated the application of an isolated fraction of plasma, enriched in circulating mitochondria, called MitoCir, to regulate the activation and proliferation of CD4+ T cells from human donors in normal and pathological conditions where these cells are hyperactivated or deregulated.
Los resultados mostraron que las células T CD4+ cultivadas en presencia MitoCir redujeron significativamente su activación y diferenciación. Esto se correlacionó con una reducción de la proliferación y la detención de las fases G0/G1 del ciclo celular. The results showed that CD4+ T cells cultured in the presence of MitoCir significantly reduced their activation and differentiation. This was correlated with a reduction in proliferation and arrest of the G0/G1 phases of the cell cycle.
De esta forma, la composición desarrollada por los inventores, MitoCir tiene un efecto inmunosupresor en las célulasT CD4+. Esta composición MitoCir permite contribuir á la homeostasis celular de los individuos con patologías en que estas células están desreguladas, y es una poderosa herramienta para la comunicación intercelular a larga distancia y el alivio de la gravedad de las enfermedades autoinmunes. In this way, the composition developed by the inventors, MitoCir, has an immunosuppressive effect on CD4+ T cells. This MitoCir composition contributes to the cellular homeostasis of individuals with pathologies in which these cells are deregulated, and is a powerful tool for long-distance intercellular communication and alleviating the severity of autoimmune diseases.
De esta forma la invención se refiere a una composición para usar en un método de tratamiento de un paciente con un desorden inmunológico de hiperactivación o desregulación de los linfocitos T CD4+ que comprende administrar una composición que comprende un aislado de plasma, enriquecido en mitocondrias extracelulares, en un sujeto con hiperactivación o desregulación de los linfocitos T CD4+. Esta composición se administra en un paciente vía parenteral, por ejemplo, intramuscular o intravenoso. In this way, the invention relates to a composition for use in a method of treating a patient with an immunological disorder of hyperactivation or deregulation of CD4+ T lymphocytes that comprises administering a composition that comprises a plasma isolate, enriched in extracellular mitochondria, in a subject with hyperactivation or deregulation of CD4+ T lymphocytes. This composition is administered to a patient parenterally, for example, intramuscularly or intravenously.
Donde el desorden inmunológico de hiperactivación o desregulación de los linfocitos T CD4+ a tratar con la composición de la invención se escoge de cualquier enfermedad o síndrome que involucre
hiperactivación o desregulación de los linfocitos T CD4+ , tales como: Lupus Eritematoso Sistémico (LES); Artritis reumatoide; procesos inflamatorios agudos, Psoriasis; Síndrome antifosfolipídico; Esclerosis múltiple; Aterosclerosis; Osteoartritis; Diabetes tipo I; Síndrome de Sjógren; Miastenia gravis; Enfermedad de Crohn ; Colitis ulcerosa; Vasculitis; Vasculitis asociadas a anticuerpos anticitoplasma de neutrófilos; Enfermedad injerto contra huésped; Preeclamsia y/o Enfermedad celíaca. Where the immunological disorder of hyperactivation or deregulation of CD4 + T lymphocytes to be treated with the composition of the invention is chosen from any disease or syndrome that involves hyperactivation or deregulation of CD4+ T lymphocytes, such as: Systemic Lupus Erythematosus (SLE); Rheumatoid arthritis; acute inflammatory processes, Psoriasis; Antiphospholipid syndrome; Multiple sclerosis; Atherosclerosis; Osteoarthritis; Type I diabetes; Sjogren's syndrome; Myasthenia gravis; Crohn's disease ; Ulcerative colitis; Vasculitis; Vasculitis associated with antineutrophil cytoplasmic antibodies; Graft versus host disease; Preeclampsia and/or Celiac Disease.
Los resultados muestran que la sangre de individuos sanos contiene mitocondrias, las que pueden encontrarse en el interior de vesículas o como organelos libres. Estas mitocondrias circulantes mantienen el potencial de membrana y producen ATP. Al evaluar el rol biológico de las mitocondrias circulantes, en relación con el efecto que podrían tener estas mitocondrias en linfocitos T CD4+, se observó que los linfocitos T CD4+ de donantes sanos incorporan mitocondrias circulantes, y estas mitocondrias tienen un efecto inmunosupresor sobre la activación, proliferación y diferenciación sobre este tipo celular. Un resultado similar se obtuvo al usar linfocitos T CD4+ provenientes de pacientes con LES (Lupus Eritematoso Sistémico). A partir de lo resultados se puede concluir que el tratamiento con mitocondrias aisladas permite una disminución de la proliferación y activación de estos linfocitos. Los inventores demostraron que mitocondrias aisladas de plasma de individuos sanos, son bioenergéticamente competentes y tienen efectos inmunoreguladores sobre los linfocitos T CD4+ de donantes sanos y de donantes con LES, dicho hallazgo, modifica el uso tradicional energético de las mitocondrias cuando son mitoceptadas y las posiciona como reguladores de células inmune. The results show that the blood of healthy individuals contains mitochondria, which can be found inside vesicles or as free organelles. These circulating mitochondria maintain membrane potential and produce ATP. When evaluating the biological role of circulating mitochondria, in relation to the effect that these mitochondria could have on CD4+ T lymphocytes, it was observed that CD4+ T lymphocytes from healthy donors incorporate circulating mitochondria, and these mitochondria have an immunosuppressive effect on activation, proliferation and differentiation on this cell type. A similar result was obtained when using CD4+ T lymphocytes from patients with SLE (Systemic Lupus Erythematosus). From the results it can be concluded that treatment with isolated mitochondria allows a decrease in the proliferation and activation of these lymphocytes. The inventors demonstrated that mitochondria isolated from plasma of healthy individuals are bioenergetically competent and have immunoregulatory effects on CD4+ T lymphocytes from healthy donors and donors with SLE. This finding modifies the traditional energy use of mitochondria when they are mitocepted and positions them as regulators of immune cells.
El método de la invención tiene especial cuidado en evitar la activación de plaquetas, dado que está descrito que éstas liberan mitocondrias cuando se activan, de este modo estamos bloqueando la liberación de mitocondrias plaquetarias, las que se reconocen por los marcadores de superficie CD42b, CD42A y/o CD41, distintivos de plaquetas, por lo que se emplea un inactivador de plaquetas prostaglandina El (PGE1) (Focus Biomolecules) a una concentración final de preferentemente 10 pM diluida en un tampón anticoagulante, tal como citrato solución A dextrosa (0,1 M citrato trisódico, 0,11 M glucosa y 0,08 M ácido cítrico). The method of the invention takes special care to avoid the activation of platelets, since it is described that they release mitochondria when they are activated, in this way we are blocking the release of platelet mitochondria, which are recognized by the surface markers CD42b, CD42A and/or CD41, hallmarks of platelets, so a platelet inactivator prostaglandin El (PGE1) (Focus Biomolecules) is used at a final concentration of preferably 10 pM diluted in an anticoagulant buffer, such as citrate solution A dextrose (0. 1 M trisodium citrate, 0.11 M glucose and 0.08 M citric acid).
De este modo la invención apunta a un método de obtención de una composición enriquecida en mitocondrias circulantes el que comprende las etapas de:
a) agregar una composición de prostaglandina El (PGE1) en citrato trisódico 0,08 a 0,2 M , 0,8 a 0,20 M de glucosa y de 0,05 a 0,lM de ácido cítrico, para alcanzar una concentración final entre 8 a 15 pM de PGE1 a plasma sanguíneo de donantes sanos b) centrifugar 150g por 5 a 20 minutos, y recoger el sobrenadante; c) centrifugar el sobrenadante de b) a 2500 g por 20 a 60 minutos; d) concentrar el sobrenadante de c) centrifugando a 13000 g por 5 a 20 minutos al menos una vez; e) resuspender la pella de la etapa d) en una solución tampón. Donde en una realización el tampón comprende: 0,2 M sacarosa, 0,01 M TRIS, 0,001 M EGTA, IX inhibidor de proteasas. En una segunda realización el tampón comprende: EGTA 0,5 mM, MgCI2 3 mM, K-lactobionato 60 mM, taurina 20 mM, KH2PO4 10 mM, HEPES 20 mM, sacarosa 110 mM, inhibidor de proteasas IX. In this way, the invention aims at a method of obtaining a composition enriched in circulating mitochondria which includes the steps of: a) add a composition of prostaglandin El (PGE1) in 0.08 to 0.2 M trisodium citrate, 0.8 to 0.20 M of glucose and 0.05 to 0.1M of citric acid, to reach a concentration final between 8 to 15 pM of PGE1 to blood plasma from healthy donors b) centrifuge 150g for 5 to 20 minutes, and collect the supernatant; c) centrifuge the supernatant from b) at 2500 g for 20 to 60 minutes; d) concentrate the supernatant from c) by centrifuging at 13000 g for 5 to 20 minutes at least once; e) resuspend the pellet from step d) in a buffer solution. Where in one embodiment the buffer comprises: 0.2 M sucrose, 0.01 M TRIS, 0.001 M EGTA, protease inhibitor IX. In a second embodiment, the buffer comprises: 0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, protease inhibitor IX.
De este método se obtiene una composición enriquecida en mitocondrias circulantes que comprende una alta concentración de mitocondrias circulantes y al mismo tiempo comprende una baja concentración de mitocondrias positivas para los marcadores CD42b, CD42A y/o CD41.From this method, a composition enriched in circulating mitochondria is obtained that comprises a high concentration of circulating mitochondria and at the same time comprises a low concentration of mitochondria positive for the CD42b, CD42A and/or CD41 markers.
En una realización de la invención la composición enriquecida en mitocondrias circulantes de la de la invención sirve para preparar un medicamento útil para contrarrestar la hiperactivación de linfocitos T CD4+. Donde estos linfocitos T CD4+ están hiperactivos en procesos inflamatorios agudos o en enfermedades autoinmunes. Donde las enfermedades autoinmunes se escogen entre: Lupus Eritematoso Sistémico (LES); Artritis reumatoide; Psoriasis; Síndrome antifosfolipídico; Esclerosis múltiple; Aterosclerosis; Osteoartritis; Diabetes tipo I; Síndrome de Sjógren; Miastenia gravis; Enfermedad de Crohn ; Colitis ulcerosa; Vasculitis; Vasculitis asociadas a anticuerpos anticitoplasma de neutrófilos; Enfermedad injerto contra huésped; Preeclamsia y/o Enfermedad celíaca. In one embodiment of the invention, the composition enriched in circulating mitochondria of the invention serves to prepare a drug useful to counteract the hyperactivation of CD4+ T lymphocytes. Where these CD4+ T lymphocytes are hyperactive in acute inflammatory processes or in autoimmune diseases. Where autoimmune diseases are chosen from: Systemic Lupus Erythematosus (SLE); Rheumatoid arthritis; Psoriasis; Antiphospholipid syndrome; Multiple sclerosis; Atherosclerosis; Osteoarthritis; Type I diabetes; Sjogren's syndrome; Myasthenia gravis; Crohn's disease ; Ulcerative colitis; Vasculitis; Vasculitis associated with antineutrophil cytoplasmic antibodies; Graft versus host disease; Preeclampsia and/or Celiac Disease.
En una realización la composición de la invención sirve para preparar un medicamento para administración parenteral. In one embodiment, the composition of the invention serves to prepare a medicament for parenteral administration.
EJEMPLOS EXAMPLES
Ejemplo 1. Obtención de aislado rico en mitocondrias, MitoCir.
Utilizando centrifugaciones a diferentes velocidades, se obtuvo un aislado de plasma sanguíneo humano que contiene mitocondrias extracelulares, conocidas como mitocondrias circulantes, la Figura 1 muestra una fotografía de la composición obtenida, donde se aprecia la gran concentración de mitocondrias. Example 1. Obtaining an isolate rich in mitochondria, MitoCir. Using centrifugations at different speeds, an isolate of human blood plasma containing extracellular mitochondria, known as circulating mitochondria, was obtained. Figure 1 shows a photograph of the composition obtained, where the high concentration of mitochondria can be seen.
El aislamiento de mitocondrias desde plasma se realizó evitando la activación de plaquetas. Sangre fresca se recolectó desde donantes humanos sanos utilizando tubos de recolección de sangre con citrato de sodio 3.2% como anticoagulante (BD Vacutainer®). La sangre se centrifugó a 150g por 20 min a temperatura ambiente (TA) sin freno y sin aceleración. Se recolectó el plasma y se le añadió prostaglandina El (PGE1) (Focus Biomolecules) a una concentración final de 10 pM diluida en tampón anticoagulante citrato solución A dextrosa (0,1 M citrato trisódico, 0,11 M glucosa y 0,08 M ácido cítrico) (ACD-A) para evitar la activación de plaquetas, dado que está descrito que éstas liberan mitocondrias cuando se activan, de este modo estamos bloqueando la liberación de mitocondrias plaquetarias. El plasma se centrifugó a 150g por 10 min a 4°C sin freno y sin aceleración para remover los glóbulos rojos y leucocitos. El plasma se recolectó y centrifugó a 2500g por 30 min a 4°C sin freno y sin aceleración para remover las plaquetas y obtener el sobrenadante con mitocondrias circulantes. Las mitocondrias se concentraron a través de dos centrifugaciones sucesivas a 13000g por 10 min a 4°C lavando con tampón para aislamiento de mitocondrias (MIB) (0.2 M sacarosa, 0.01 M TRIS, 0.001 M EGTA, IX inhibidor de proteasas). El pellet obtenido luego de la última centrifugación diferencial a 13000g son las mitocondrias circulantes, MitoCir. La pella de mitocondrias circulantes se resuspendió en MIB (Mitochondrial Isolation Buffer 0.2 M sacarosa, 0.01 M TRIS, 0.001 M EGTA, IX inhibidor de proteasas)) o en tampón de respiración mitocondrial 05 (MIR05) (EGTA 0.5 mM, MgCI2 3 mM, K-lactobionato 60 mM, taurina 20 mM, KH2PO4 10 mM, HEPES 20 mM, sacarosa 110 mM, inhibidor de proteasas IX), dependiendo del experimento. Isolation of mitochondria from plasma was performed avoiding platelet activation. Fresh blood was collected from healthy human donors using blood collection tubes with 3.2% sodium citrate as anticoagulant (BD Vacutainer®). The blood was centrifuged at 150 g for 20 min at room temperature (RT) without brakes and without acceleration. Plasma was collected and prostaglandin El (PGE1) (Focus Biomolecules) was added to a final concentration of 10 pM diluted in anticoagulant buffer citrate solution A dextrose (0.1 M trisodium citrate, 0.11 M glucose and 0.08 M citric acid) (ACD-A) to prevent the activation of platelets, since it has been described that they release mitochondria when they are activated, in this way we are blocking the release of platelet mitochondria. The plasma was centrifuged at 150g for 10 min at 4°C without brake and without acceleration to remove red blood cells and leukocytes. The plasma was collected and centrifuged at 2500g for 30 min at 4°C without brake and without acceleration to remove the platelets and obtain the supernatant with circulating mitochondria. Mitochondria were concentrated through two successive centrifugations at 13,000g for 10 min at 4°C, washing with mitochondrial isolation buffer (MIB) (0.2 M sucrose, 0.01 M TRIS, 0.001 M EGTA, protease inhibitor IX). The pellet obtained after the last differential centrifugation at 13000g is the circulating mitochondria, MitoCir. The circulating mitochondrial pellet was resuspended in MIB (Mitochondrial Isolation Buffer 0.2 M sucrose, 0.01 M TRIS, 0.001 M EGTA, protease inhibitor IX)) or in mitochondrial respiration buffer 05 (MIR05) (0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, protease inhibitor IX), depending on the experiment.
Ejemplo 2. Efecto del aislado MitoCir sobre linfocitos aislados. Example 2. Effect of the isolated MitoCir on isolated lymphocytes.
Con esta preparación obtenida en el ejemplo 1 se desarrollaron experimentos in vitro en los que se depositaron mitocondrias aisladas desde plasma sanguíneo humano junto con linfocitos T CD4+ en condiciones de activación y diferenciación. Los linfocitos T CD4+ en condiciones de activación y diferenciación en presencia de MitoCir, mitocondrias aisladas de plasma, presentaron una menor activación y proliferación al compararlo con los linfocitos T CD4+ en condiciones de activación y diferenciación, pero sin MitoCir, como se muestra en la figura 2 y 3.
Obtención de linfocitos With this preparation obtained in example 1, in vitro experiments were developed in which mitochondria isolated from human blood plasma were deposited together with CD4+ T lymphocytes under conditions of activation and differentiation. CD4+ T lymphocytes under conditions of activation and differentiation in the presence of MitoCir, mitochondria isolated from plasma, presented lower activation and proliferation when compared to CD4+ T lymphocytes under conditions of activation and differentiation, but without MitoCir, as shown in the figure. 2 and 3. Obtaining lymphocytes
Se recolectó la sangre de donantes sanos o con LES, y las PBMCs (Peripheral Blood Mononuclear Cell)se aislaron utilizando un gradiente de densidad Ficoll-Paque PLUS centrifugando a 400g por 30 min a TA sin freno y aceleración media. Las PBMCs se recolectaron y se lavaron con PBS-1X. Las células se resuspendieron en medio para linfocitos y se realizó el recuento celular. Luego se centrifugaron a 600g por 5 min a 4°C. Se resuspendieron en PBS-1X + 2% FBS y se tiñeron con CD4 - APC-H7 y CD8-PE a una concentración de 20x6 células/mL para ser sorteadas. Las PBMCs teñidas se lavaron con PBS-1X + 2% FBS centrifugando a 600g por 5 min a 4°C. Se resuspendieron en PBS-1X + 10% FBS filtrado y se seleccionó la población de linfocitos T CD4+(CD4+CD8-) utilizando la técnica de clasificación de células activadas por fluorescencia, para lo que usó un cell sorter BD FacsAria™ Fusion. Los linfocitos T CD4+ se centrifugaron a 700g por 5 min a 4°C y, en el caso de analizar posteriormente la proliferación celular, se tiñeron con CellTrace™ Violet (CTV) 5 pM por 20 min a 37°C previo al cultivo. Los linfocitos T CD4+ se resuspendieron en medio para linfocitos y se sembraron lxlO5 células en una placa de 96 pocilios de fondo redondo con anti-CD3 (1 pg/mL), anti- CD28 (2 pg/mL), TGF-01 (10 ng/mL) e IL-2 (50 U/mL). La estimulación con anti-CD3 y anti-CD28 emula la estimulación por las APCs y permite que los linfocitos T se activen y proliferen. La citoquina TGF-01 estimula la diferenciación de los linfocitos T CD4+ a Tregs (Regulatory T cell). Se añadió 20 pg de proteína de mitocondrias circulantes a los pozos con linfocitos T CD4+. Las mitocondrias se tiñeron con MitoTracker, como se mencionó en la sección 3.8, para posteriormente visualizar las células que las incorporaron. Los linfocitos T CD4+ se incubaron a 37°C con 5% CO2. Los linfocitos T CD4+ bajo condiciones de activación más TGF-01 y con mitocondrias circulantes se evaluaron al día 1 por microscopía confocal, para visualizar la internalización de mitocondrias circulantes. Los linfocitos T CD4+ se analizaron al día 4 por citometría de flujo, para evaluar el efecto de las mitocondrias circulantes. Como control se utilizó linfocitos T CD4+ cultivados con citoquinas, pero sin mitocondrias circulantes. Para corroborar que los efectos observados eran dependientes de mitocondrias viables, como control se añadieron 20 pg de proteína de mitocondrias circulantes sonicadas por 5 min a 60% de intensidad en un equipo ultrasonic cleaner y expuestas a luz UV por 30 min. Blood was collected from healthy donors or those with SLE, and PBMCs (Peripheral Blood Mononuclear Cell) were isolated using a Ficoll-Paque PLUS density gradient by centrifuging at 400g for 30 min at RT without brake and medium acceleration. PBMCs were collected and washed with PBS-1X. The cells were resuspended in lymphocyte medium and cell counting was performed. They were then centrifuged at 600g for 5 min at 4°C. They were resuspended in PBS-1X + 2% FBS and stained with CD4 - APC-H7 and CD8-PE at a concentration of 20x6 cells/mL to be sorted. The stained PBMCs were washed with PBS-1X + 2% FBS by centrifuging at 600g for 5 min at 4°C. They were resuspended in PBS-1X + 10% filtered FBS and the CD4+ (CD4+CD8-) T lymphocyte population was selected using the fluorescence-activated cell sorting technique, for which a BD FacsAria™ Fusion cell sorter was used. The CD4+ T lymphocytes were centrifuged at 700g for 5 min at 4°C and, in the case of subsequently analyzing cell proliferation, they were stained with 5 pM CellTrace™ Violet (CTV) for 20 min at 37°C prior to culture. CD4+ T cells were resuspended in lymphocyte medium and 1x10 5 cells were seeded in a round-bottom 96-well plate with anti-CD3 (1 pg/mL), anti-CD28 (2 pg/mL), TGF-01 ( 10 ng/mL) and IL-2 (50 U/mL). Stimulation with anti-CD3 and anti-CD28 emulates stimulation by APCs and allows T lymphocytes to activate and proliferate. The cytokine TGF-01 stimulates the differentiation of CD4+ T lymphocytes into Tregs (Regulatory T cells). 20 pg of circulating mitochondrial protein was added to the wells with CD4+ T cells. Mitochondria were stained with MitoTracker, as mentioned in section 3.8, to subsequently visualize the cells that incorporated them. CD4+ T cells were incubated at 37°C with 5% CO 2 . CD4+ T lymphocytes under activation conditions plus TGF-01 and with circulating mitochondria were evaluated on day 1 by confocal microscopy, to visualize the internalization of circulating mitochondria. CD4+ T lymphocytes were analyzed on day 4 by flow cytometry, to evaluate the effect of circulating mitochondria. As a control, cultured CD4+ T lymphocytes with cytokines, but without circulating mitochondria, were used. To corroborate that the observed effects were dependent on viable mitochondria, as a control, 20 pg of protein from circulating mitochondria sonicated for 5 min at 60% intensity in an ultrasonic cleaner equipment and exposed to UV light for 30 min were added.
Además, se incorporó un control experimental en el que las mitocondrias aisladas desde plasma se dañaron utilizando procesos de sonicación y exposición a luz UV, MitoCir D. El análisis de los linfocitos T CD4+ se realizó mediante citometría de flujo, evaluando marcadores de activación como
CD25 y PD-1 (Programmed cell death protein 1) y la dilución de una sonda específica para evaluar proliferación (CTV, CellTrace™ Violet). In addition, an experimental control was incorporated in which mitochondria isolated from plasma were damaged using sonication processes and exposure to UV light, MitoCir D. The analysis of CD4+ T lymphocytes was performed by flow cytometry, evaluating activation markers such as CD25 and PD-1 (Programmed cell death protein 1) and the dilution of a specific probe to evaluate proliferation (CTV, CellTrace™ Violet).
Efecto sobre Linfocitos de donantes sanos. Effect on Lymphocytes from healthy donors.
Se evaluó el efecto de las mitocondrias circulantes sobre linfocitos T CD4+ cultivados en condiciones de activación más TGF-01 para evaluar el rol inmunoregulador de las mitocondrias circulantes Mito Cir. Se observó que existe una disminución en la activación de los linfocitos (figura 2) y proliferación (figura 3) de los linfocitos T CD4+ cultivados con Mito Cir. The effect of circulating mitochondria on CD4+ T lymphocytes cultured under activation conditions plus TGF-01 was evaluated to evaluate the immunoregulatory role of circulating mitochondria Mito Cir. It was observed that there is a decrease in lymphocyte activation (figure 2) and proliferation (figure 3) of CD4+ T lymphocytes cultured with Mito Cir.
Para evidenciar que los efectos observados eran por mitocondrias circulantes intactas se agregó un control experimental, en el que las mitocondrias circulantes se sonicaron y se expusieron a luz UV. Cuando se realizó el tratamiento con MitoCir D, los efectos sobre los linfocitos T CD4+ se vieron disminuidos, lo que indica que la mitocondria intacta es la que estaría ejerciendo el efecto inmunoregulador. To show that the observed effects were due to intact circulating mitochondria, an experimental control was added, in which circulating mitochondria were sonicated and exposed to UV light. When treatment with MitoCir D was performed, the effects on CD4+ T lymphocytes were diminished, indicating that intact mitochondria are what would be exerting the immunoregulatory effect.
Considerando los resultados expuestos, se puede concluir que MitoCir presenta un efecto inmunosupresor sobre los procesos de activación y proliferación de los linfocitos T CD4+ de donantes sanos. Considering the results presented, it can be concluded that MitoCir has an immunosuppressive effect on the activation and proliferation processes of CD4+ T lymphocytes from healthy donors.
Efecto sobre Linfocitos de donantes con enfermedades autoinmunes. Effect on Lymphocytes from donors with autoimmune diseases.
Por otra parte, el aislado de plasma sanguíneo humano que contiene mitocondrias se evaluó sobre linfocitos T CD4+ provenientes de donantes con Lupus Eritematoso Sistémico (LES). El LES es una enfermedad donde los linfocitos T CD4+ están desregulados y presentan un fenotipo de hiperactivación que contribuye al desarrollo y daño provocado por la enfermedad. Al evaluar el efecto sobre linfocitos T CD4+ de donantes con LES se observó, al igual que en el caso de los linfocitos T CD4+ de donantes sanos, una disminución de la activación y proliferación, como se presenta en la figura 4. On the other hand, the human blood plasma isolate containing mitochondria was evaluated on CD4+ T lymphocytes from donors with Systemic Lupus Erythematosus (SLE). SLE is a disease where CD4+ T lymphocytes are deregulated and present a hyperactivation phenotype that contributes to the development and damage caused by the disease. When evaluating the effect on CD4+ T lymphocytes from donors with SLE, a decrease in activation and proliferation was observed, as in the case of CD4+ T lymphocytes from healthy donors, as shown in Figure 4.
Al cultivar linfocitos T CD4+ provenientes de donantes con LES, con MitoCir, en condiciones de activación más TGF-01, se evidenció un efecto similar, aunque en menor medida, que cuando se cultivó linfocitos T CD4+ de donantes sanos con MitoCir. Se observó una disminución de la activación (figura 4A y 4B) y de la proliferación (4C y 4D) de los linfocitos T CD4+ provenientes de donantes con LES. La proliferación se evaluó mediante la expresión de Ki-67, una proteína intracelular que se
expresa más en células proliferativas. Mientras que la activación se midió mediante la expresión de CD25. When culturing CD4+ T lymphocytes from donors with SLE, with MitoCir, under conditions of activation plus TGF-01, a similar effect was evident, although to a lesser extent, than when culturing CD4+ T lymphocytes from healthy donors with MitoCir. A decrease in the activation (Figure 4A and 4B) and proliferation (4C and 4D) of CD4+ T lymphocytes from donors with SLE was observed. Proliferation was evaluated by the expression of Ki-67, an intracellular protein that is expresses more in proliferating cells. While activation was measured by CD25 expression.
El efecto del aislado de plasma de la invención que contiene mitocondrias sobre los linfocitosT CD4+ de donantes con LES, muestra un resultado destacadle e interesante, dado que en estos linfocitos T CD4+ de pacientes con LES, tienen un perfil muy proliferativo y activado, lo que se regula con la composición de la invención, aislado de plasma que contiene mitocondrias derivado de un donante sano, disminuyendo la proliferación y activación.
The effect of the plasma isolate of the invention that contains mitochondria on the CD4+ T lymphocytes from donors with SLE, shows a remarkable and interesting result, given that in these CD4+ T lymphocytes from patients with SLE, they have a very proliferative and activated profile, which It is regulated with the composition of the invention, isolated from plasma containing mitochondria derived from a healthy donor, decreasing proliferation and activation.
Claims
1.- Método de obtención de una composición enriquecida en mitocondrias circulantes CARACTERIZADO porque comprende las etapas de: a) agregar una composición de prostaglandina El (PGE1) en un tampón anticoagulante que comprende citrato trisódico 0,08 a 0,2 M , 0,8 a 0,20 M de glucosa y de 0,05 a 0,lM de ácido cítrico, para alcanzar una concentración final entre 8 a 15 pM de PGE1 a plasma sanguíneo de donantes sanos b) centrifugar 150g por 5 a 20 minutos, y recoger el sobrenadante; c) centrifugar el sobrenadante de b) a 2500 g por 20 a 60 minutos; d) concentrar el sobrenadante de c) centrifugando a 13000 g por 5 a 20 minutos al menos una vez; e) resuspender la pella de la etapa d) en una solución tampón. 1.- Method of obtaining a composition enriched in circulating mitochondria CHARACTERIZED because it comprises the steps of: a) adding a composition of prostaglandin El (PGE1) in an anticoagulant buffer that comprises trisodium citrate 0.08 to 0.2 M, 0. 8 to 0.20 M of glucose and 0.05 to 0.1M of citric acid, to reach a final concentration between 8 to 15 pM of PGE1 to blood plasma from healthy donors b) centrifuge 150g for 5 to 20 minutes, and collect the supernatant; c) centrifuge the supernatant from b) at 2500 g for 20 to 60 minutes; d) concentrate the supernatant from c) by centrifuging at 13000 g for 5 to 20 minutes at least once; e) resuspend the pellet from step d) in a buffer solution.
2.- Método de acuerdo con la reivindicación 1 CARACTERIZADO porque en la etapa e) el tampón comprende: 0,2 M sacarosa, 0,01 M TRIS, 0,001 M EGTA, IX inhibidor de proteasas. 2.- Method according to claim 1 CHARACTERIZED because in step e) the buffer comprises: 0.2 M sucrose, 0.01 M TRIS, 0.001 M EGTA, protease inhibitor IX.
3.- Método de acuerdo con la reivindicación 1 CARACTERIZADO porque en la etapa e) el tampón comprende: EGTA 0,5 mM, MgCI2 3 mM, K-lactobionato 60 mM, taurina 20 mM, KH2PO4 10 mM, HEPES 20 mM, sacarosa 110 mM, inhibidor de proteasas IX. 3.- Method according to claim 1 CHARACTERIZED because in step e) the buffer comprises: 0.5 mM EGTA, 3 mM MgCI2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH 2 PO 4 , HEPES 20 mM, 110 mM sucrose, protease inhibitor IX.
4.- Composición enriquecida en mitocondrias circulantes CARACTERIZADA porque comprende una alta concentración de mitocondrias circulantes y se obtiene de acuerdo con el método de la cláusula 1. 4.- Composition enriched in circulating mitochondria CHARACTERIZED because it comprises a high concentration of circulating mitochondria and is obtained according to the method of clause 1.
5.- Composición enriquecida en mitocondrias circulantes de acuerdo a la cláusula 4 CARACTERIZADA porque comprende una baja concentración de mitocondrias positivas para los marcadores CD42b, CD42A y/o CD41. 5.- Composition enriched in circulating mitochondria according to clause 4 CHARACTERIZED because it comprises a low concentration of mitochondria positive for the markers CD42b, CD42A and/or CD41.
6.- Uso de la composición enriquecida en mitocondrias circulantes de la cláusula 5 CARACTERIZADO porque sirve para preparar un medicamento útil para contrarrestar la hiperactivación de linfocitos T CD4+.
6.- Use of the composition enriched in circulating mitochondria of clause 5 CHARACTERIZED because it serves to prepare a useful medicine to counteract the hyperactivation of CD4+ T lymphocytes.
7.- Uso de acuerdo con la cláusula 6 CARACTERIZADO porque sirve para preparar un medicamento útil para contrarrestar la hiperactivación de linfocitos T CD4+ en procesos inflamatorios agudos o en enfermedades autoinmunes. 7.- Use in accordance with clause 6 CHARACTERIZED because it serves to prepare a medicine useful to counteract the hyperactivation of CD4+ T lymphocytes in acute inflammatory processes or in autoimmune diseases.
8.- Uso de acuerdo con la cláusula 7 CARACTERIZADO porque las enfermedades autoinmunes se escogen entre: Lupus Eritematoso Sistémico (LES); Artritis reumatoide; Psoriasis; Síndrome antifosfolipídico; Esclerosis múltiple; Aterosclerosis; Osteoartritis; Diabetes tipo I; Síndrome de Sjogren; Miastenia gravis; Enfermedad de Crohn ; Colitis ulcerosa; Vasculitis; Vasculitis asociadas a anticuerpos anticitoplasma de neutrófilos; Enfermedad injerto contra huésped; Preeclamsia y/o Enfermedad celíaca. 8.- Use in accordance with clause 7 CHARACTERIZED because autoimmune diseases are chosen from: Systemic Lupus Erythematosus (SLE); Rheumatoid arthritis; Psoriasis; Antiphospholipid syndrome; Multiple sclerosis; Atherosclerosis; Osteoarthritis; Type I diabetes; Sjogren's syndrome; Myasthenia gravis; Crohn's disease ; Ulcerative colitis; Vasculitis; Vasculitis associated with antineutrophil cytoplasmic antibodies; Graft versus host disease; Preeclampsia and/or Celiac Disease.
9.- Uso de acuerdo con las cláusulas 6 a 8 CARACTERIZADO porque sirve para preparar un medicamento para administración parenteral.
9.- Use in accordance with clauses 6 to 8 CHARACTERIZED because it serves to prepare a medication for parenteral administration.
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