Application of TIPE2 protein in preparation of preparation for inhibiting iNKT cell activation

Abstract

The invention belongs to the technical field of protein functions and application, and particularly relates to application of TIPE2 protein in preparation of a preparation for inhibiting iNKT cell activation, and a nucleotide sequence for coding the TIPE2 protein is shown as SEQ ID NO.1. The TIPE2 protein provided by the invention can prevent excessive activation of iNKT cells to avoid further autoimmune diseases. The key effect of the TIPE2 in liver related diseases is expanded, and the unknown value of the TIPE2 in the iNKT cell biological function of immune-mediated liver injury is disclosed. It is proved that the TIPE2 is a key regulatory factor of liver homeostasis, and a clue is provided for the TIPE2 to become a potential therapeutic target for regulating immune-mediated liver injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein function and application, and specifically relates to an application of TIPE2 protein in preparing a preparation for inhibiting iNKT cell activation. Background Art

[0002] iNKT cells, whose Chinese name is invariant natural killer T cells, originate from hematopoietic stem cells. After they are generated in the bone marrow, they migrate to the thymus for further development. iNKT cells have functions such as immune regulation, anti-tumor, anti-viral infection, and anti-bacterial infection. Among them, the regulatory effect of iNKT cells helps to maintain autoimmune tolerance. However, when iNKT cells are abnormally activated, it may cause immune imbalance and trigger autoimmune diseases. For example, in autoimmune diseases such as systemic lupus erythematosus, iNKT cells may be overactivated, produce excessive cytokines, and destroy autoimmune tolerance. These cytokines may stimulate autoreactive B cells to produce autoantibodies, attack their own tissues and organs, and lead to the occurrence and development of the disease.

[0003] Autoimmune hepatitis, or AIH, is an inflammatory disease of unknown etiology that is often associated with liver damage and severe mortality. Pathologically, AIH is characterized by immune-mediated destruction of hepatocytes associated with immune cell infiltration and activation leading to progressive necrotic inflammation in the liver. Concanavalin A, ConA, administration induces acute liver injury and systemic immune activation in mice, similar to the pathology of human immune-mediated hepatitis. Hepatic iNKT cells are highly enriched in the mouse liver and are required for ConA-induced hepatitis. Emerging evidence suggests that experimental conditions that induce aggravation or alleviation of liver injury are associated with an increase or decrease in iNKT cells or their function, respectively. Importantly, activation of iNKT cells is associated with the degree of apoptosis, which occurs rapidly after induction by ConA or the iNKT cell-specific ligand α-galactosylceramide, α-GalCer. iNKT cell-mediated injury is associated with the rapid release of multiple cytokines. The activation of iNKT cells is triggered by the ligation of the semi-invariant TCR along with a series of co-stimulatory and co-inhibitory receptors, which are subsequently regulated by intracellular proteins and immunomodulatory cytokines produced by other immune cells or by the iNKT cells themselves.

[0004] At present, drugs that prevent abnormal activation of iNKT cells include: cell surface receptor antagonists such as anti-T cell receptor antibodies and immunosuppressants such as glucocorticoids. Taking anti-T cell receptor antibody drugs will have a certain impact on normal T cell function, which requires precise targeting and control; long-term use of glucocorticoids can lead to a variety of adverse reactions, the most common of which is Cushing's trait, and can also aggravate osteoporosis and cause related bone diseases. At the same time, long-term use of glucocorticoids is related to the occurrence of type 2 diabetes, cataracts, hypertension, infection and mental illness. Immunosuppressants can also bring some adverse reactions, which can cause bone marrow suppression and a decrease in blood cells.

[0005] In summary, there is an urgent need to provide a preparation that prevents abnormal activation of iNKT cells. Summary of the invention

[0006] The purpose of the present invention is to provide an application of TIPE2 protein in the preparation of a preparation for inhibiting iNKT cell activation, so as to prevent abnormal activation of iNKT cells.

[0007] The technical solution adopted by the present invention is:

[0008] The present invention provides an application of a TIPE2 protein in preparing a preparation for inhibiting iNKT cell activation. The nucleotide sequence encoding the TIPE2 protein is shown in SEQ ID NO.1.

[0009] Preferably, the iNKT cells are iNKT + TNF-α + NKT cells + IFN-γ + cell.

[0010] Preferably, the preparation consists of TIPE2 and pharmaceutically acceptable excipients.

[0011] Preferably, the pharmaceutically acceptable excipients include one or more of a water-soluble filler, a pH adjuster, a stabilizer, water for injection and an osmotic pressure regulator.

[0012] Preferably, the preparation at least comprises a recombinant plasmid or a recombinant virus containing the nucleotide sequence shown in SEQ ID NO.1.

[0013] Preferably, the recombinant virus is composed of an original viral vector and a nucleotide sequence as shown in SEQ ID NO.1, and the original viral vector includes an adenoviral vector, a lentiviral vector or an adeno-associated viral vector.

[0014] Preferably, the adenoviral vector includes an Ad-CMV vector or an Ad-CAG vector; the lentiviral vector includes a pLVX-Puro vector, a pLenti-CMV-GFP-P2A-Puro vector or a pLVTHM vector; and the adeno-associated viral vector includes an AAV1 vector, an AAV2 vector, an AAV5 vector, an AAV8 vector or an AAV9 vector.

[0015] Preferably, the recombinant plasmid is composed of an original plasmid and a nucleotide sequence as shown in SEQ ID NO.1, and the original plasmid includes a pcDNA3 plasmid, a pCEP4 plasmid, a pcDNA5 / FRT / TO-TOPO plasmid or a pMT2 plasmid.

[0016] Preferably, the use further comprises using the preparation to prepare a drug for treating autoimmune liver disease caused by iNKT cell activation.

[0017] Preferably, the autoimmune liver disease is autoimmune hepatitis.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides an application of a TIPE2 protein in the preparation of a preparation for inhibiting the activation of iNKT cells, wherein the TIPE2 protein prevents the overactivation of iNKT cells and avoids further inducing autoimmune diseases, and the nucleotide sequence encoding the TIPE2 protein of the present invention is shown in SEQ ID NO.1. The present invention expands the key role of TIPE2 in liver-related diseases and reveals the unknown value of TIPE2 in the biological function of iNKT cells in immune-mediated liver damage. It is confirmed that TIPE2 is a key regulator of liver homeostasis, providing clues for it to become a potential therapeutic target for regulating immune-mediated liver damage.

[0019] In the present invention, Tipe2 is determined - / - Mice are more sensitive to ConA-induced hepatitis. It was determined that TIPE2 protects against immune-mediated hepatitis through an iNKT cell-dependent mechanism. It was further confirmed that the loss of TIPE2 enhances the activation of iNKT cells in a cell-intrinsic manner, indicating that TIPE2 is a cell-intrinsic regulator of iNKT cell activation. AAV8-mediated TIPE2 overexpression significantly protects against ConA-induced hepatitis in vivo, and TIPE2 plays an indispensable role in experimental hepatitis induced by D-GalN / LPS and APAP. In summary, the present invention provides clues for TIPE2 to become a potential therapeutic target for regulating immune-mediated liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Figure 2 shows the effect of TIPE2 deficiency on the composition of immune cells in the liver. A: gating strategy for flow cytometric analysis of liver mononuclear cells, LMNCs, and bone marrow cells; B-G: WT and Tipe2 - / - Myeloid cell subsets CD45 in mouse LMNCs + CD11c + 、CD45 + CD11c - CD11b + 6G + 、CD45 + CD11c - CD11b + 6G - Siglec + 、CD45 + CD11c - CD11b + 6G - CX3CR1 + Ly6C high 、CD45 + CD11c - CD11b + 6G - CX3CR1 - Ly6C low and CD11b + 6G - CX3CR1 + Ly6C - Percentage; H~M: WT and TIPE2 - / - Lymphocyte subsets CD45 in mouse LMNCs + CD4 + TCRβ + 、CD45 + CD8 + TCRβ + 、CD45 + CD19 + TCRβ - 、CD45 + NK1.1 + TCRβ - 、CD45 + CD1d + TCRβ + and CD45 + TCRγδ + TCRβ - Percentage of cells; N and O represent juvenile WT and TIPE2 - / -Hematoxylin-eosin (H&E) staining of mouse liver sections; P and Q are young WT and TIPE2, respectively. - / - Macroscopic observation of mouse liver; R: P and Q correspond to juvenile WT and TIPE2 - / - Serum alanine aminotransferase ALT levels in mice.

[0021] Figure 2 Results of the effect of TIPE2 on immune-mediated liver injury. A: WT and TIPE2 24 hours after ConA injection - / - Macroscopic observation of mouse liver; B: Liver from WT and TIPE2 24 hours after ConA injection - / - Representative injury areas of mouse liver sections stained with H&E; C: Statistical results corresponding to Figure A; D: WT and TIPE2 4 hours and 24 hours after ConA injection - / - Serum ALT levels in mice; E and F: WT and TIPE2 8 hours after ConA injection, respectively - / - IFN-γ and TNF-α levels in mouse serum; G: RNA-seq analysis of WT and TIPE2 8 hours after ConA injection - / - Heat map of genes in mouse liver tissue; H: CD45 measured by flow cytometry - and CD45 + TIPE2-GFP expression in two cell populations; I: serum ALT levels in chimeric mice 24 hours after ConA injection.

[0022] Figure 3 For transcriptome data analysis, WT and TIPE2 - / - Liver tissue was collected from mice. A: GO terms based on differentially expressed genes; B: KEGG pathway analysis.

[0023] Figure 4 For WT and TIPE2 - / - The results of mice injected with ConA and α-GalCer, A is the flow cytometry analysis of WT and TIPE2 4 hours after ConA injection - / - iNKT in mouse LMNCs, B: iNKT cell frequency; C: iNKT absolute cell number, D: Annexin V + iNKT cells; E and F: WT and TIPE2 cells 24 hours after α-GalCer injection - / - Representative injury areas and statistical results of H&E staining of liver sections of mice; G: WT and TIPE2 24 hours after injection of α-GalCer - / -Serum ALT levels of mice; H: WT and TIPE2 12 and 24 hours after α-GalCer injection - / - Serum IFN-γ levels in mice; I and J: analysis and statistical results of serum ATL activity 24 hours after injection of α-GalCer and anti-CD 1d or PBS; K and L: analysis and statistical results of serum ATL activity 48 hours after NK1.1 mAb administration; - / - Mouse NK1.1 + Changes in cell number and serum ATL levels.

[0024] Figure 5 Results showing that TIPE2 deficiency affects ConA-induced lymphocyte influx into the liver. A: WT and TIPE2 4 hours after ConA injection. - / - Flow cytometric analysis of mouse LMNCs, B and C: CD4 + Lymphocyte frequency and absolute cell count; D and E: CD8 corresponding to Figure A + Lymphocyte frequency and absolute cell number; F: WT and Tipe2 4 hours after ConA injection - / - Flow cytometric analysis of CD4+ and CD8+ lymphocyte activation in mouse LMNCs, G and H: CD4 corresponding to F. + C69 + Cell frequency and absolute cell number; I: WT and TIPE2 4 hours after ConA injection - / - CD4+ cells producing TNF-α and IFN-γ in mouse LMNCs + Flow cytometric analysis of lymphocytes, J and K: CD4 + C69 + Cell frequencies and absolute cell numbers.

[0025] Figure 6 For the WT and Tipe2 gfp / + TIPE2 expresses GFP on iNKT cells in the thymus, spleen, and liver of mice + Results analysis, where A: comparison of the results from WT and Tipe2 gfp / + TIPE2 expresses GFP on iNKT cells in the thymus, spleen, and liver of mice + Representative flow cytometry analysis of WT and Tipe2 - / - Flow cytometric analysis of the developmental stages of mouse thymic iNKT cells; C: The percentage of iNKT cells in stages 1, 2, and 3 in the thymus; D-F: The percentage of iNKT cells in the thymus, spleen, and liver, respectively; G: WT and Tipe2 - / -Surface protein expression of Fas, FasL and CD40L on mouse liver iNKT cells; H and I: before and after PMA / ionomycin stimulation for the indicated time in vitro, isolated from Tipe 2 gfp / + TIPE2 expresses GFP on iNKT cells in mouse liver + Representative histograms and summary graphs of flow cytometry analysis.

[0026] Figure 7 The results of TIPE2 regulating cytokine production, where A and B are respectively WT and Tipe2 2 hours after injection of ConA - / - Flow cytometric analysis results and statistical graphs of IL-4 expression in mouse liver iNKT cells: C-E: PMA and ionomycin were used to stimulate WT and Tipe2 cells in vitro. - / - Flow cytometric analysis results and statistical graphs of IFN-γ and TNF-α expression in iNKT cells 5 hours after LMNC in mice; F: irradiated WT recipient CD45.1 + / CD45.2 + 3 hours later, the cells were treated with - / - Schematic diagram of the experimental design of 1:1 mixed bone marrow cell transfer in mice and treatment of mice 8 weeks later; G: Representative flow cytometry images corresponding to Figure F show spleen CD11b + 6G + Neutrophils and liver CD1d + TCRβ + CD45.1 and CD45.2 staining in iNKT cells; H: CD1d from liver + TCRβ + Representative flow cytometry images of CD45.1 and CD45.2 staining of iNKT cells and representative flow cytometry images of IFN-γ in iNKT cells in the liver after ConA injection; I: Summary image of IFN-γ depicted in G.

[0027] Figure 8 Results of the effects of TIPE2 overexpression on mouse hepatitis, where A: Schematic diagram of the experimental model; B and C: Representative images and statistical results of liver histology of control AAV and TIPE2 OE mice 24 hours after ConA injection, C: Serum ALT level; E and F: Flow cytometric analysis of IFN-γ and TNF-α in liver iNKT cells from control AAV and TIPE2 OE mice 2 hours after ConA injection.

[0028] Fig. 9Figure 2 shows the effect of TIPE2 overexpression on mouse LMNCs. A shows the activation of iNKT cells in LMNCs isolated from WT mice infected with control adenovirus and adenovirus overexpressing TIPE2 after stimulation with PMA+Ionomycin for 2 h in vitro. B shows the CD1d + CD69 + Cell statistics.

[0029] Fig.10 Figure 2 is the level of cytokine secretion by iNKT cells in LMNCs isolated from WT mice after infection with control adenovirus and adenovirus overexpressing TIPE2 and stimulation with PMA+Ionomycin for 4 h in vitro. A is the flow cytometry analysis result of cytokines secreted by iNKT cells, and B and C are the corresponding iNKT + IFN-γ + and iNKT + TNF-α + Cell number statistics. DETAILED DESCRIPTION

[0030] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0031] The inventive concept of the present invention is as follows:

[0032] iNKT cells play an important role in immune regulation, anti-tumor, anti-viral infection, and anti-bacterial infection. In the immune regulation function, iNKT cells can secrete a variety of cytokines, such as IFN-γ and IL-4 to balance the Th1 / Th2 cell response. iNKT cells can also regulate the functions of other immune cells. For example, the IFN-γ it secretes can activate macrophages and enhance their phagocytic and bactericidal abilities. At the same time, iNKT cells also have a regulatory effect on dendritic cells, which can promote the maturation and antigen presentation ability of dendritic cells, thereby better initiating adaptive immune responses. However, when iNKT cells are abnormally activated, they will produce too many cytokines, destroy autoimmune tolerance, lead to immune imbalance, and trigger autoimmune diseases.

[0033] Based on this, the present invention proposes an application of TIPE2 protein in the preparation of a preparation for inhibiting iNKT cell activation. The nucleotide sequence encoding the TIPE2 protein is shown in SEQ ID NO.1.

[0034] SEQ ID NO.1:

[0035]

[0036] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0037] The abbreviations used in the present invention are shown in Table 1.

[0038] Table 1 Abbreviations

[0039]

[0040] Example 1

[0041] Application of TIPE2 protein in the preparation of preparations for inhibiting iNKT cell activation

[0042] (1) The experimental method is as follows:

[0043] 1.1ConA was administered to establish an acute hepatitis model.

[0044] ConA was dissolved in PBS and injected intravenously into mice at a dose of 10 mg / kg. α-GalCer purchased from Enzo LifeSciences was dissolved in 0.5% Tween-20 and diluted in sterile PBS. 1 μg / 50 μL of α-GalCer was injected intravenously into mice. D-galactosamine / lipopolysaccharide D-GalN / LPS-induced hepatitis was injected intraperitoneally with 5 μg / kg LPS and 200 mg / kg D-GalN dissolved in PBS. For mouse survival studies, mice were injected intraperitoneally with 10 μg / kg LPS and 200 mg / kg D-GalN. Acetaminophen-induced hepatitis was performed by intraperitoneal injection of 300 mg / kg APAP after an overnight fast. To neutralize CD1d, 200 μg of anti-CD1d antibody was given intraperitoneally to mice 24 hours before α-GalCer injection. To deplete NK and iNKT cells, mice were intraperitoneally injected twice with 200 μg of anti-NK1.1 antibody 48 and 24 h before α-GalCer administration. The depletion rate was determined by flow cytometric analysis 40 h after treatment. To assess liver injury, alanine aminotransferase activity in serum was determined using a commercial kit based on the method recommended by the International Federation of Clinical Chemistry.

[0045] 1.2 RNA sequencing for gene enrichment and pathway analysis.

[0046] Liver tissues were isolated from ConA-stimulated WT and Tipe2- / - mice, and total RNA was extracted using the mirVana miRNA isolation kit. RNA integrity was assessed using an Agilent 2100 Bioanalyzer. Samples with an RNA integrity number RIN ≥ 7 were sequenced by Solexa High-Throughput Sequencing Services. Differentially expressed genes were identified using the DESeq 2012 functions estimateSizeFactors and nbinomTest. The log-fold changes of mRNA up- or down-regulation between the two groups were selected, with a significance threshold of P < 0.05. Only genes with a greater than two-fold change and a P value less than 0.05 were considered for further analysis. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed based on the differentially expressed gene DEGs to investigate groups of genes involved in common biological responses or with related functions.

[0047] 1.3 Flow cytometry detection of WT and TIPE2 - / - The composition of mouse liver immune cells, TIPE2-GFP expression of immune and non-immune cell populations, cytokine secretion levels in acute liver injury, acute lymphocyte activation levels, and WT and TIPE2 after acute liver injury - / - Annexin V in mouse LMNCs + The proportion of iNKT cells.

[0048] To isolate liver mononuclear cells from the liver, perfuse the liver with 20 mL of PBS via the portal vein and then mince and pass through a stainless steel mesh. The filtrate was centrifuged at 50 g for 5 min, and the supernatant was washed once with PBS. The supernatant containing LMNCs was resuspended in 40% Percoll in RPMI 1640 medium. The cell suspension was gently overlaid on 70% Percoll and centrifuged at 750 g for 20 min at room temperature. Wash twice in PBS and resuspend in flow cytometry buffer or RPMI 1640 medium containing 2% FBS.

[0049] After blocking with anti-mouse CD 16 / CD 32 antibodies, cells were stained with the indicated fluorescent conjugated antibodies. Dead cells were stained with the Live / Dead fixable aqua dead cell stain kit and gated in the analysis. For analysis of surface markers, cells were stained with appropriate surface antibodies in PBS containing 2% FBS. For analysis of cytokines, including IL-4, TNF-α, and IFN-γ, Cytofix / Cytoperm and Perm / Wash solutions were used according to the manufacturer's instructions. Annexin V staining was performed according to the manufacturer's instructions. Flow cytometry data were acquired on a FACS Canto or AttuneNxT instrument and analyzed with FlowJo software.

[0050] 1.4H&E staining was used to detect liver tissue damage.

[0051] For liver histology, liver tissue samples were fixed in 4% paraformaldehyde solution overnight and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E) to assess the severity of inflammation and immune cell infiltration.

[0052] 1.5ALT detects serum transaminase levels.

[0053] To assess liver injury, alanine aminotransferase activity in serum was measured using a commercial kit based on the method recommended by the International Federation of Clinical Chemistry.

[0054] 1.6ELISA was used to detect the levels of cytokines in serum.

[0055] The levels of TNF-α and IFN-γ in serum were determined by ELISA kits according to the manufacturer's instructions.

[0056] 1.7 AAV 8-mediated TIPE2 overexpression significantly protects against ConA-induced hepatitis in vivo.

[0057] To overexpress TIPE2 in vivo, an adeno-associated virus serotype 8, AAV8, delivery system was established according to standard molecular biology methods. The complete open reading frame encoding TIPE2, without an intervening stop codon, was cloned into the AAV8-CMV-EGFP vector to generate AAV-TIPE2. WT mice at 8 weeks of age were injected intravenously with control, AAV8-CMV-EGFP; Genechem, or TIPE2 overexpressing virus AAV8-CMV-TIPE2-IRES-EGFP; Genechem dissolved in PBS, 2 × 10 11 vg was overexpressed, and the hepatitis model was induced 3 weeks after AAV injection.

[0058] (2) Experimental results are as follows:

[0059] 2.1TIPE2 - / - Mice are more susceptible to ConA-induced hepatitis.

[0060] To elucidate the potential role of TIPE2 in liver homeostasis, we first investigated whether TIPE2 deficiency affects the immune cell composition in the liver. Figure 1 The results of flow cytometry analysis of liver immune cells showed that there was a significant difference between WT and TIPE2 - / - The percentage of myeloid cells including total dendritic cells CD45 + CD11c +, neutrophil CD45 + CD11c - CD11b + 6G + , hepatic macrophage CD45 + CD11c - CD11b + 6G - CX3CR1 - Ly6C low , bone marrow-derived mononuclear cells CD45 + CD11c - CD11b + 6G - CX3CR1 + Ly6C high , bone marrow-derived macrophages CD45 + CD11c - CD11b + Ly6G-CX3CR1 + Ly6C - and eosinophil CD45 + CD11c - CD11b + 6G - Siglec + Similar results were found in lymphocytes, including CD4 + T cell CD45 + CD4 + TCRβ + 、CD8 + T cell CD45 + CD8 + TCRβ + , B cell CD45 + CD19 + TCRβ - 、NK cell CD45 + NK1.1 + TCRβ - 、iNKT cell CD45 + CD1d + TCRβ + and γδ T cells CD45 + TCRγδ + TCRβ - As previously reported, young TIPE2 in the B6 background - / - The mice were relatively healthy, with normal hepatocyte distribution and normal structure under normal conditions. Liver damage measured by ALT activity was not affected by TIPE2 deficiency, indicating that TIPE2- / - The mice were not susceptible to spontaneous hepatitis. Altogether, these results suggest that loss of TIPE2 does not significantly alter liver homeostasis.

[0061] To investigate the functional contribution of TIPE2 to the development of immune-mediated liver injury, we performed a double-transcription analysis of WT and TIPE2 - / - Mice were treated with ConA, and hepatitis was evaluated by anatomical and histological examination of the liver and serum transaminase measurements, as Figure 2 After ConA injection, TIPE2 - / - Mice developed severe hepatitis characterized by diffuse hemorrhage, extensive lesions, inflammatory infiltration, and hepatocyte death. - / - Serum collected from mice also showed a significant increase in ALT activity levels, a marker of liver damage. Similarly, ELISA assays showed that TIPE2 - / - To investigate the possible mechanism by which TIPE2 may function during hepatitis, RNA-seq analysis was performed to compare ConA-treated WT and TIPE2 - / - Mouse liver transcriptome. Gene ontology term analysis showed that the most significantly altered biological processes were related to inflammatory responses, such as Figure 3 KEGG analysis revealed a predominance of immune-related pathways, among which the “cytokine-cytokine receptor interaction” pathway was the most significantly enriched. Consistent with these findings, heat map analysis showed that in TIPE2 - / - The expression of many genes related to inflammation in liver tissue, such as Lif, Cxcl2, Ccl3, Nos2 and Ccr1, was significantly increased.

[0062] Next, by using TIPE2 gfp / + Mice in which normal TIPE2 expression is indicated by eGFP expression were constructed to examine TIPE2 expression in different cell populations in the liver. In isolated LMNCs, TIPE2 was expressed only in CD45 + Expressed in immune cells but not in other cell types, such as CD45 - The contribution of TIPE2 to hematopoiesis was further demonstrated in chimeric mice by transfecting WT or TIPE2 - / - Interestingly, TIPE2 - / - Bone marrow cells of WT mice, i.e. TIPE2 - / -→WT, were more susceptible to ConA-induced liver injury than WT mice receiving WT bone marrow cells, i.e., WT→WT, as indicated by increased plasma ALT levels. These data suggest that WT and TIPE2 - / - The distinguishable susceptibility of mice to ConA-induced hepatitis is hematopoietic cell-dependent.

[0063] 2.2TIPE2 protects against immune-mediated hepatitis through an iNKT cell-dependent mechanism.

[0064] To determine TIPE2 - / - The cellular mechanisms underlying the increased susceptibility of mice were analyzed in the liver after ConA challenge. It is well documented that iNKT cells play a key role in the induction of ConA hepatitis and that iNKT cells act as the primary effector cells in this model, being rapidly and selectively depleted from the liver within hours. Compared with WT mice, TIPE2 - / - The level of iNKT cells in the liver of mice was low 4 hours after ConA injection, and the frequency and absolute number of iNK cells decreased at this time point, as shown in Figure 2. Figure 4 The present findings are consistent with previous studies, suggesting that activation-induced cell death, i.e., AICD, may be responsible for the rapid exhaustion of iNKT cells after ConA treatment. In fact, compared with iNKT cells isolated from WT mice after ConA injection, TIPE2 cells isolated from 4 h after ConA injection showed a significant decrease in AICD after ConA injection. - / - Liver iNKT cells isolated from mice showed significantly increased annexin V binding. Next, we investigated whether TIPE2 deficiency affects ConA-induced lymphocyte influx into the liver. Figure 5 As shown, after ConA injection, TIPE2 deficiency had an impact on the expression of CD4 + T cell accumulation was significantly affected, but CD8 + T cell accumulation was not affected. Next, WT and TIPE2 - / - Expression of activation marker CD69 and cytokine signaling between mice. - / - CD4 + T cells and CD8 + The expression of CD69 in activated T cells was not different from that in WT group, but the expression of CD69 in TIPE2 cells treated with ConA was - / - CD4 in mouse liver samples + The expression of TNF-α and IFN-γ in T cells was significantly increased.

[0065] To evaluate the direct effect of TIPE2 on iNKT cells, another liver injury model induced by α-GalCer was established. α-GalCer administration resulted in punctate necrosis in the liver of WT mice and a mild increase in ALT and IFN-γ levels 24 hours after α-GalCer injection. - / - The α-GalCer-induced necrosis area in mice was larger, and the serum ALT and IFN-γ levels were significantly higher than those in WT mice. The present invention then studied whether the signal downstream of α-GalCer that TIPE2 regulates antigen dependence is presented on CD1d rather than from other cell surface activating receptors. Therefore, anti-CD1d monoclonal antibody was injected 24 hours before α-GalCer injection to block CD1d-mediated signal transduction, and ALT levels in serum were measured. Although the anti-CD1d monoclonal antibody did not eliminate the corresponding iNKT cell population, the anti-CD1dmAb-administered TIPE2 - / - The mice showed reduced liver damage and decreased ALT levels. To further confirm these results, the present invention used anti-NK1.1 mAb to deplete NK and iNKT cells. - / - In mice, treatment with anti-NK1.1 mAb completely depleted NK and iNKT cells and abolished the α-GalCer-induced increase in serum ALT levels. Taken together, these results suggest that TIPE2 protects against immune-mediated hepatitis through an iNKT cell-dependent mechanism.

[0066] 2.3TIPE2 is a cell-intrinsic regulator of iNKT cell activation.

[0067] To link TIPE2 to iNKT cell biology, we first examined the role of TIPE2 in iNKT cell proliferation and differentiation. gfp / + Expression on iNKT cells isolated from the thymus, spleen, and liver of reporter mice and WT control mice. The cell population of iNKT cells was gated according to previous reports, and CD45 was examined in primary and secondary lymphoid organs. + CD1d + TCRβ + GFP of TIPE2 on iNKT cells + Expression, such as Figure 6 As shown. The vast majority of iNKT cells were TIPE2 positive in the initial state. Next, we determined the role of TIPE2 in the development of iNKT cells and examined the frequency of iNKT cells. In the thymus, TIPE2 was expressed in WT mice compared with WT mice. - / - The developmental stages of mouse iNKT cells are defined as four stages: stage 0 - CD24 + CD44 - NK1.1- , Phase 1 - CD24 - CD44 - NK1.1 - , Phase 2-CD24 - CD44 + NK1.1 - and phase 3-CD24 - CD44 + NK1.1 + In addition, compared with WT mice, TIPE2 - / - There were no significant differences in the proportion of iNKT cells in the thymus, spleen, and liver of the mice, indicating that TIPE2 is not essential for the development and distribution of iNKT cells. - / - No significant differences in Fas, FasL, or CD40L expression were observed between iNKT cells of different phenotypes, indicating that under normal steady-state conditions, TIPE2 - / - There is no spontaneous activation in iNKT cells. Finally, changes in TIPE2 expression in iNKT cells were detected, and after in vitro stimulation with PMA / ionomycin, TIPE2 expression was significantly reduced in the early stages of the immune response. Since iNKT cells undergo apoptosis after activation, resulting in their rapid disappearance, changes in TIPE2 expression were only detected as early as 1 hour.

[0068] After activation, iNKT cells rapidly secrete large amounts of IL-4, TNF-α, and IFN-γ, all of which play an important role in ConA-induced liver injury. Next, we wanted to know whether TIPE2 regulates the production of these cytokines, and then used flow cytometry to analyze the intracellular cytokines secreted by liver iNKT cells, such as Figure 7 After injection of ConA, TIPE2 - / - iNKT cells in the WT group produced much more IL-4 than those in the TIPE2 group. - / - TNF-α and IFN-γ levels in iNKT cells were elevated after PMA / ionomycin stimulation in vitro. These observations suggest that TIPE2 on iNKT cells is critical for suppressing iNKT cell activation in vitro and in vivo. To further investigate whether TIPE2 is required for maintaining iNKT cells to function in a cell-intrinsic manner, we isolated 144 iNKT cells from 42 samples of the iNKT cell line at a 1:1 ratio of WT:CD45.1 + and TIPE2 - / - :CD45.2 + Bone marrow reconstitution of lethally irradiated WT recipient mice with CD45.1 + CD45.2 + , mixed bone marrow chimeric mice were generated. Two months after reconstitution, the spleens of recipient mice contained approximately a 1:1 ratio of CD45.1+ and CD45.2 + Neutrophil CD11b + 6G + , indicating equal reconstitution of these two types of hematopoietic stem cells. Likewise, the livers of recipient mice also contained approximately a 1:1 ratio of CD45.1 + and CD45.2 + iNKT cell CD1d + TCRβ + After injection of ConA into mixed chimeric mice, CD45.1 + Compared with WT iNKT cells, CD45.2 + TIPE2 - / - The secretion of IFN-γ in iNKT cells was significantly increased. These results demonstrate that the loss of TIPE2 enhances the activation of iNKT cells in a cell-intrinsic manner.

[0069] 2.4 Effect of TIPE2 overexpression on mouse hepatitis.

[0070] Based on the protective role of TIPE2 in immune-mediated hepatitis, we next explored the therapeutic feasibility and efficacy of experimentally overexpressing targeted TIPE2 in the liver. Figure 8 As shown. Therefore, control or AAV8-overexpressing TIPE2 was injected intravenously into WT mice, and ConA was administered three weeks later to induce hepatitis. Interestingly, TIPE2 overexpression was able to effectively reduce liver tissue necrosis and serum ALT levels compared with mice injected with control AAV. Notably, it was observed that iNKT cells producing TNF-α and IFN-γ were also reduced after TIPE2 overexpression. In summary, these data indicate that AAV8-mediated TIPE2 gene therapy significantly protects against ConA-induced hepatitis in vivo.

[0071] 2.5 Effect of TIPE2 overexpression on mouse LMNCs.

[0072] On the basis of the above 2.4, the present invention infected LMNCs isolated from WT mice with control adenovirus and adenovirus overexpressing TIPE2, and counted the activation of iNKT cells and the levels of cytokines secreted by iNKT cells after stimulation with PMA+ionomycin. The results are shown in Fig. 9 and Fig.10 In TIPE2-overexpressing LMNCs, iNKT cells producing TNF-α and IFN-γ were also reduced.

[0073] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0074] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. Application of TIPE2 protein in the preparation of a preparation for inhibiting iNKT cell activation, characterized in that: The nucleotide sequence encoding the TIPE2 protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The iNKT cells are iNKT + TNF-α + NKT cells + IFN-γ + cell.

3. The use according to claim 1, characterized in that: The preparation consists of TIPE2 and pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable excipients include one or more of a water-soluble filler, a pH regulator, a stabilizer, water for injection, and an osmotic pressure regulator.

5. The use according to claim 1, characterized in that: The preparation at least includes a recombinant plasmid or a recombinant virus containing a nucleotide sequence as shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that: The recombinant virus is composed of an original viral vector and a nucleotide sequence as shown in SEQ ID NO.1, and the original viral vector includes an adenoviral vector, a lentiviral vector or an adeno-associated viral vector.

7. The use according to claim 6, characterized in that: The adenovirus vector includes an Ad-CMV vector or an Ad-CAG vector; The lentiviral vector includes a pLVX-Puro vector, a pLenti-CMV-GFP-P2A-Puro vector or a pLVTHM vector; The adeno-associated virus vector includes an AAV1 vector, an AAV2 vector, an AAV5 vector, an AAV8 vector or an AAV9 vector.

8. The use according to claim 5, characterized in that: The recombinant plasmid is composed of an original plasmid and a nucleotide sequence as shown in SEQ ID NO.1, wherein the original plasmid includes a pcDNA3 plasmid, a pCEP4 plasmid, a pcDNA5 / FRT / TO-TOPO plasmid or a pMT2 plasmid.

9. The use according to claim 1, characterized in that: The application also includes using the preparation to prepare a drug for treating autoimmune liver disease caused by iNKT cell activation.

10. The use according to claim 9, characterized in that: The autoimmune liver disease is autoimmune hepatitis.

Images