Application of CSF1-CD47 in the preparation of early transplantation immune rejection inhibitors

Abstract

The present invention discloses the use of CSF1-CD47 in the preparation of an early transplantation immune rejection inhibitor. The inventors have found that CSF1 / CD47 NVs can specifically bind to the corresponding ligands CSF1R and SIRPα expressed by macrophages, and on the one hand, promote bone marrow-derived monocytes to Ly6G by enhancing the CSF1 / CSF1R signaling pathway. ‑ Ly6C ‑ On the other hand, it inhibits macrophage differentiation by inhibiting macrophage phagocytosis of nanovesicles by enhancing the CD47 / SIRPα signaling pathway, prolonging the action time of CSF1, and also inhibiting the phagocytosis and antigen presentation of mononuclear macrophages to transplant antigens. In the in vivo mouse heart transplant model, CSF1 / CD47 NVs also showed more significant effects than single-target NVs in alleviating immune rejection and prolonging the survival curve of transplanted hearts. The combined use of CSF1 / CD47 can effectively alleviate early immune rejection of transplantation and improve the success rate of organ transplantation.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to the application of CSF1 / CD47 in the preparation of an early transplantation immune rejection inhibitor. Background Art

[0002] Organ transplantation is an effective treatment for end-stage organ failure. The rejection of the recipient's immune system to the organ transplant is the main challenge after transplantation. Immunosuppressive therapy is the cornerstone of successful organ transplantation. To prevent rejection, the recipient must receive immunosuppressant therapy after organ transplantation.

[0003] Currently, the immunosuppressants commonly used in clinical practice mainly target specific immune cells, especially T cells, such as cyclosporine A and tacrolimus, which inhibit the T cell activation pathway "Calcineurin-NFAT". However, it is difficult for such drugs to reestablish immune tolerance, and long-term high-dose use can also cause serious liver and kidney toxicity. Therefore, it is of great significance to prepare specific, safe and effective inhibitors for innate immune cells in the early stage of immune rejection.

[0004] Colony-stimulating factor-1 (CSF1), also known as macrophage colony-stimulating factor (M-CSF), is a secreted cytokine that is regulated by most tissue macrophages and osteoclasts. The effects of CSF1 are mediated by the CSF1 receptor tyrosine kinase (CSF1R), through autophosphorylation of CSF1R and subsequent phosphorylation of downstream molecules. Triggering this phosphorylation cascade increases gene transcription and protein translation and induces cytoskeletal remodeling through several signaling pathways, leading to the survival, proliferation, and differentiation of target cells. CSF1-regulated tissue macrophages are important for innate immunity and tissue development and function. Since CSF1 regulates the survival, proliferation and chemotaxis of macrophages and supports their activation, this factor is associated with the pathogenesis of many diseases (Pixley FJ, Stanley ER. CSF1 regulation of the wandering macrophage: complexity inaction[J]. Trends in cell biology, 2004, 14(11): 628-638., Hume DA, MacDonald K P A. Therapeutic applications of macrophage colony-stimulating factor-1(CSF1) and antagonists of CSF1 receptor (CSF1R) signaling[J]. Blood, TheJournal of the American Society of Hematology, 2012, 119(8): 1810-1820., Hamilton JA. CSF-1 signal transduction[J]. Journal of leukocyte biology,1997, 62(2): 145-155., Sweet MJ, Hume DA. CSF1 as a regulator of macrophageactivation and immune responses[J]. ARCHIVUM IMMUNOLOGIAE ET THERAPIAEEXPERIMENTALIS-ENGLISH EDITION-, 2003, 51(3): 169-178.).

[0005] CD47 is a receptor for members of the platelet protein family, a ligand for the transmembrane signaling protein SIRPα, and a component of a supramolecular complex containing specific integrins, heterotrimeric G proteins, and cholesterol. Peptides containing the VVM motif in the C-terminal domain of platelets are agonists of CD47, initiating heterotrimeric Gi protein signaling and enhancing the function of integrins of the β1, β2, and β3 families, thereby regulating a range of cellular activities, including platelet activation, cell motility and adhesion, and leukocyte adhesion, migration, and phagocytosis. CD47 is widely considered to be a “don’t eat me” signal that helps maintain immune tolerance of non-malignant cells under physiological conditions, but this molecule can help cancer cells survive in different cancer types. In many cancer types, CD47 bound to signal regulatory protein α (SIRPα) initiates inhibitory signaling pathways that cause malignant cells to evade phagocytosis by macrophages. Therapies that inhibit CD47 signaling in cancer cells may promote phagocytosis of tumor cells by macrophages, thereby limiting tumor growth (Russ A, Hua AB, Montfort WR, et al. Blocking “don't eat me”signal of CD47-SIRPα in hematological malignancies, an in-depth review[J]. Blood reviews, 2018, 32(6): 480-489.、Brown EJ, Frazier W A. Integrin-associated protein (CD47) and its ligands[J]. Trends in cell biology, 2001,11(3): 130-135.、Eladl E, Tremblay-LeMay R, Rastgoo N, et al. Role of CD47 inhematological malignancies[J]. Journal of Hematology & Oncology, 2020, 13(1):1-14.、Chao MP, Weissman IL, Majeti R. The CD47–SIRPα pathway in cancerimmune evasion and potential therapeutic implications[J]. Current opinion inimmunology, 2012, 24(2): 225-232.).

[0006] There are no reports on the application of CSF1 and CD47 in organ transplantation. Summary of the invention

[0007] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide an application of CSF1 / CD47 in the preparation of an early transplantation immune rejection inhibitor.

[0008] The technical solution adopted by the present invention is:

[0009] The invention discloses an application of a composition in preparing an early stage transplantation immune rejection inhibitor, wherein the composition contains CSF1 and CD47 and / or can express CSF1 and CD47 in vivo.

[0010] In some application examples, the composition contains components for expressing CSF1 and CD47 in vivo, specifically by translating the mRNA of CSF1 and CD47 into CSF1 and CD47 in vivo.

[0011] In some application examples, the component is cell membrane nanovesicles. As a natural biomaterial, cell membrane nanovesicles (NV) have the advantages of long circulation, good biocompatibility and low toxicity and side effects.

[0012] In some application examples, the cell membrane nanovesicles are derived from stem cells, which are infected with CSF1 and CD47 lentiviruses respectively, and then stimulated with IFN-γ and post-processed. Stem cells can produce more cell membrane nanovesicles.

[0013] In some application examples, the stem cells are iPSCs-MSCs. iPSCs-MSCs have a wide range of sources and are easier to obtain.

[0014] In some application examples, the stimulating concentration of IFN-γ is 10-30 ng / mL, which has good stimulating ability.

[0015] In some application examples, the IFN-γ stimulation time is 24 to 48 hours.

[0016] In some application examples, the post-processing includes mechanical grinding, gradient centrifugation and extrusion.

[0017] The beneficial effects of the present invention are:

[0018] The inventors found that CSF1 / CD47 NVs can specifically bind to the corresponding ligands CSF1R and SIRPα expressed by macrophages, and promote bone marrow-derived monocytes to Ly6G by enhancing the CSF1 / CSF1R signaling pathway. - Ly6C -On the other hand, it inhibits macrophage differentiation by inhibiting macrophage phagocytosis of nanovesicles by enhancing the CD47 / SIRPα signaling pathway, prolonging the action time of CSF1, and also inhibiting the phagocytosis and antigen presentation of mononuclear macrophages to transplant antigens. In the in vivo mouse heart transplant model, CSF1 / CD47 NVs also showed more significant effects than single-target NVs in alleviating immune rejection and prolonging the survival curve of transplanted hearts. The combined use of CSF1 / CD47 can effectively alleviate early immune rejection of transplantation and improve the success rate of organ transplantation.

[0019] In some examples of the present invention, high-quality iPSCs-MSC cell membrane-derived CSF1 / CD47 nanovesicles were obtained for the first time by utilizing IFN-γ-stimulated iPSC-MSC cell membrane-derived nanovesicles to target early innate immune cells in transplant rejection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Different transplant days Csf1 and CD47 mRNA expression levels.

[0021] Figure 2 The effect of different concentrations of IFN-γ stimulation on the immunosuppressive effect of iPSC-MSC.

[0022] Figure 3 The expression of CSF1 and CD47 in CSF1 / CD47 NVs was verified.

[0023] Figure 4 This is the result of the effect of CSF1 / CD47 NVs on immunosuppression.

[0024] Figure 5 The effect of different treatments on the survival days of mouse heart transplantation.

[0025] Figure 6 Different treatments have an impact on the transplanted heart Tnf-α , Il-6 Effects of inflammatory cytokine gene expression.

[0026] Figure 7 Is the effect of different treatments on inflammatory Ly6G - Ly6C + Macrophage polarization to Ly6G - Ly6C - Influence of suppressive macrophages. DETAILED DESCRIPTION

[0027] The invention discloses an application of a composition in preparing an early stage transplantation immune rejection inhibitor, wherein the composition contains CSF1 and CD47 and / or can express CSF1 and CD47 in vivo.

[0028] In some examples of applications, the composition comprises components that express CSF1 and CD47 in vivo.

[0029] In some application examples, the component is a cell membrane nanovesicle.

[0030] In some application examples, the cell membrane nanovesicles are derived from stem cells, which are obtained by infecting the stem cells with CSF1 and CD47 lentiviruses respectively, followed by stimulation with IFN-γ and post-processing.

[0031] In some application examples, the stem cells are iPSCs-MSCs.

[0032] In some application examples, the stimulating concentration of IFN-γ is 10-30 ng / mL.

[0033] In some application examples, the IFN-γ stimulation time is 24 to 48 hours.

[0034] The technical solution of the present invention is further explained below in conjunction with experiments.

[0035] Construction of mouse heart transplant model:

[0036] The heart of the donor BALB / c mouse (male) was transplanted into the neck of the recipient 8-week-old C57BL / 6 (male) mouse. The survival period of transplant rejection was determined by monitoring the beating of the transplanted heart in the neck every day. Using the cannula method, heterotopic heart transplantation in the neck of mice was established. Recipient preparation: Isolate and free the external jugular vein and common carotid artery of the recipient mouse, ligate the distal end of the vein, clamp the proximal end with a vascular clamp, pass the external jugular vein through the venous cannula, turn the vascular intima outside, and fix it with 8-0 sutures; the same operation was performed on the common carotid artery, and the recipient preparation was completed. Donor preparation: Open the abdominal cavity of the donor mouse, inject 1 mL of heparinized saline into the mouse through the inferior vena cava, heparinize the mouse systemically, open the mouse chest cavity, free the common artery and pulmonary artery, and cut them at the distal end. Ligate other vascular branches of the heart with 8-0 sutures, and cut all tissues between the transplanted heart and the donor mouse, and remove the transplanted heart for use. Connection: Wrap the pulmonary artery of the transplanted heart around the external jugular vein of the recipient mouse that has just been prepared, and fix it with 8-0 sutures. Wrap the common artery of the transplanted heart around the common carotid artery of the recipient mouse that has just been prepared, and fix it with 8-0 sutures. Open the venous clamp first, then the arterial clamp, and allow the transplanted heart to be filled with blood and resume beating.

[0037] The inventors extracted transplanted hearts from mice on different days and used qPCR to detect Csf1 and CD47 The results showed that the overall Csf1The expression level of α-glucose significantly decreased on the fifth day after transplantation compared with the third day after transplantation ( Figure 1 a), CD47 The mRNA expression level of Figure 1 b).

[0038] Induction of CSF1 / CD47 nanovesicles

[0039] Since the cell membrane can be modified to express complex structural membrane proteins, and can simultaneously express multiple targets with natural active proteins, its operation is relatively simple and safe, which provides the possibility of expressing multiple innate immune targets, enhancing negative immune regulation, and promoting immune tolerance reconstruction. In addition, the inherent properties of cell membranes of different cell origins can also be used to enhance the targeting and immunotherapeutic properties of nanovesicles, providing more flexible selectivity for the construction of multifunctional cell membrane nanovesicles. The inventors used cell membrane nanovesicle technology to achieve more simultaneous expression of CSF1 and CD47 targets for early transplant rejection.

[0040] At present, exosomes released by mesenchymal stem cells are believed to carry active substances in MSCs for intercellular communication, have obvious inhibitory effects on different effector cells involved in innate and adaptive immune responses, and have made significant progress in the treatment of autoimmune diseases. Now more and more evidence shows that in addition to the important role of paracrine in MSCs, direct cell-to-cell communication mediated by surface molecules may help to achieve maximum immunosuppression. Similar to MSCs, iPSCs-MSCs can also induce the formation of a variety of cell types, and can carry multipotent stemness for unlimited proliferation, becoming a favorable tool for research. IPSCs-MSCs are not only similar to MSCs in phenotype and function, but also have higher proliferation potential, longer survival time and lower heterogeneity, and it has been proven that the exosomes they release also have an effect on inflammation.

[0041] For the first time, the inventors used CSF1 / CD47 nanovesicles derived from the cell membrane of iPSCs-MSCs stimulated by IFN-γ to target innate immune cells in the early stages of transplant rejection.

[0042] qPCR results showed that when IFN-γ 20 ng / mL was added to iPSC-MSC cells, the immunosuppressive molecules Ido as well as Pd-l1 The expression of IFN-γ has reached the maximum value compared with the control, proving that IFN-γ gives iPSC-MSC more immunosuppressive effect ( Figure 2 ).

[0043] CSF1 and CD47 lentivirus were used to infect iPSC-MSCs, respectively, and stable cell lines were selected. Then, 20 ng / mL IFN-γ was added to stimulate for 48 hours. Nano-sized cell membrane vesicles were further obtained by mechanical grinding, gradient centrifugation, and extrusion. The vesicle samples and cell samples were further verified by Western blotting. The results showed that compared with the control sample, the constructed CSF1 / CD47 NVs did express CD47-mcherry ( Figure 3 a) and EGFP-CSF1 ( Figure 3 b).

[0044] To further verify whether CSF1 and CD47 on the nanovesicles can bind to the corresponding ligands CSF1R and SIRPα. The inventors first used lentiviral infection to construct HEK293T cells that stably overexpress CSF1R and SIRPα. Then the inventors co-incubated iPSCs-MSC cell-derived CSF1 NVs (50 μg / mL) with HEK293T cells expressing CSF1R-mcherry, and CD47NVs (50 μg / mL) with HEK293T cells expressing SIRPα. Confocal photographs showed that the red fluorescent CD47 NVs could co-localize with the green fluorescent Sirpα on the HEK293T cell membrane, and the green fluorescent CSF1 NVs could co-localize with the red fluorescent CSF1R on the HEK293T cell membrane ( Figure 3 c).

[0045] The inventors also co-incubated the nanovesicles with the target cells RAW264.7 for verification. The inventors co-incubated CD47-mcherryNVs (50 μg / mL) with the green cell membrane of RAW264.7 stained with WGA488, and EGFP-CSF1 NVs (50 μg / mL) with the red cell membrane of RAW264.7 stained with WGA594 for 30 min. The samples were prepared for confocal microscopy. The results showed that compared with the control mcherry NVs, CD47-mcherry NVs and EGFP-CSF1 NVs were able to effectively bind to the surface of RAW264.7 ( Figure 3 d).

[0046] The inventors co-incubated different nanovesicle treatment groups stained with cy5.5 with RAW264.7 cells, and flow cytometry showed that the macrophage phagocytosis of CD47 NVs and CSF1 / CD47 NVs groups was significantly less than that of NC and mcherry NVs, which proved that CD47 expressed by the vesicles could interact with SIRPα expressed by RAW264.7, reducing the phagocytosis of nanovesicles by macrophages ( Figure 4 a, Figure 4 b).

[0047] The inventors expressed Ly6C in the bone marrow + Monocytes were co-incubated with CSF1 / CD47 NVs for 72 h. The addition of CSF1 / CD47 NVs significantly promoted the expression of Ly6G - Ly6C + Monocytes express Ly6G - Ly6C - Polarization of immunosuppressive cells of monocytic origin ( Figure 4 c).

[0048] After the inventors established a mouse model of heterotopic heart transplantation in the mouse neck, they injected the following into the recipient mice through the tail vein at a total of 80 μg: PBS, Free NVs, CSF1 NVs, CD47 NVs, CSF1+CD47 NVs, and CSF1 / CD47 NVs. Tail vein injections were performed every day for the first three days, and then once every other day until the heart stopped beating. Importantly, the dual-target CSF1 / CD47 NVs extended the number of days of mouse heart transplantation more significantly than CSF1 NVs, CD47 NVs, and CSF1+CD47 NVs ( Figure 5 ).

[0049] Using qPCR to detect transplanted heart samples, it was found that CSF1 / CD47 NVs also significantly inhibited the expression of CSF1 / CD47 in transplanted hearts. Tnf-α , Il-6 Inflammatory cytokine gene expression ( Figure 6 ).

[0050] The inventors performed flow cytometry on the hearts on the fifth day after transplantation. The representative flow cytometry graphs show that CSF1 / CD47NVs promotes inflammatory Ly6G - Ly6C + Macrophage polarization to Ly6G - Ly6C - Suppressive macrophages have the best effect ( Figure 7 ).

[0051] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. Use of a composition in the preparation of an early immune rejection inhibitor for organ transplantation, the composition comprising components that express CSF1 and CD47 in vivo, the components being cell membrane nanovesicles, the cell membrane nanovesicles being derived from stem cells, the stem cells being infected with CSF1 and CD47 lentiviruses respectively, followed by IFN-γ stimulation, and post-processing to obtain the cell membrane nanovesicles, the stem cells being iPSCs-MSCs.

2. The use according to claim 1, characterized in that: The organ transplant is a heart transplant.

3. The use according to claim 1, characterized in that: The stimulation concentration of IFN-γ is 10-30 ng / mL.

4. The use according to claim 1 or 3, characterized in that: The IFN-γ stimulation time is 24 to 48 hours.

5. The use according to claim 1, characterized in that: The post-processing includes mechanical grinding, gradient centrifugation and extrusion.

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