Application of targeted cell membrane receptor SLAMF7 in preparation of macrophage activation syndrome inhibitor

Abstract

The invention relates to an application of a targeted cell membrane receptor SLAMF7 in preparation of a macrophage activation syndrome inhibitor. Experiments prove that interferon gamma (IFN-gamma) can remarkably increase the expression level of an SLAMF7 receptor on a neutrophile granulocyte membrane, and expression increase of SLAMF7 can further promote neutrophile granulocytes to release neutrophile granulocyte extracellular traps (NETs), so that inflammatory reaction is activated to participate in the pathogenesis process of AOSD-MAS. Based on the discovery, the SLAMF7 not only can be used as a novel biomarker of AOSD secondary MAS, but also can participate in IFN-gamma induced NETs release and inflammatory response in the development process of the disease, so that the preparation of the inhibitor targeting the IFN-gamma-SLAMF7-NETs axis has a good market application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of adult Still's disease, and particularly relates to the application of targeting cell membrane receptor SLAMF7 in the preparation of macrophage activation syndrome inhibitors. Background Art

[0002] Adult-onset Still's disease (AOSD) is a rare and complex systemic autoinflammatory disease, the core feature of which is the continuous activation of the innate immune pathway, usually triggered by viral infections. In AOSD patients, the number of neutrophils is usually significantly increased, and a large number of studies have confirmed its important role in the pathogenesis of AOSD. AOSD patients may develop various severe complications such as fulminant liver failure, diffuse alveolar hemorrhage, and macrophage activation syndrome (MAS). MAS is a severe and fatal inflammatory response syndrome that complicates AOSD patients. IFN-γ plays a key role in the development mechanism of MAS, which is generally considered to be characterized by monocyte / macrophage activation, while the role of neutrophils in the occurrence and development of MAS has not been fully elucidated. Our previous studies have revealed the interaction between neutrophils and macrophages, and the activation of NLRP3 inflammasome by neutrophils releasing NETs, thus exacerbating the inflammatory response. Inflammatory factors may self-regulate the inflammatory cascade reaction by regulating downstream molecules. In-depth understanding of the mechanism of inflammatory regulation of AOSD secondary to MAS is crucial for the diagnosis, evaluation, and discovery of potential therapeutic targets of MAS. Through transcriptome sequencing of neutrophils from AOSD patients, we found that the expression of signaling lymphocytic activation molecule family 7 (SLAMF7) was significantly increased. Experiments confirmed that SLAMF7 is a cell transmembrane receptor that can be significantly upregulated by interferon γ (IFN-γ). It is intended to study the important role of SLAMF7 on neutrophils in AOSD secondary to MAS in IFN-γ-induced NETs and inflammatory responses. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide the application of cell membrane receptor SLAMF7 in the preparation of macrophage activation syndrome inhibitors. Experiments have confirmed that IFN-γ can significantly increase the expression level of SLAMF7 receptor on the neutrophil membrane, and the increased expression of SLAMF7 will further promote neutrophils to release neutrophil extracellular traps (NETs), thereby activating the inflammatory response and participating in the pathogenesis of AOSD-MAS.

[0004] The present invention provides the application of targeting cell membrane receptor SLAMF7 in the preparation of macrophage activation syndrome inhibitors.

[0005] Furthermore, the expression level of the cell membrane receptor SLAMF7 is increased in neutrophils of patients with AOSD complicated by MAS and in MAS model mice, and is significantly correlated with MAS-related indicators; stimulation of neutrophils with SLAMF7 recombinant protein in vitro leads to an increase in NETs production; knockdown of SLAMF7 in the neutrophil-like NB4 cell line results in a decrease in NET production and a decline in inflammatory indicators.

[0006] Furthermore, the inhibitor also comprises a pharmaceutically acceptable carrier and / or excipient.

[0007] Furthermore, the pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0008] Beneficial effects

[0009] The present invention focuses on SLAMF7 on the neutrophil membrane as a novel biomarker for adult-onset Still's disease (AOSD) secondary to macrophage activation syndrome (MAS), and the innovative application of targeting the "IFN-γ—SLAMF7—NETs" axis in the preparation of drugs for inhibiting AOSD-MAS. The present invention experimentally confirms that interferon γ (IFN-γ) can significantly increase the expression level of the SLAMF7 receptor on the neutrophil membrane, and the increased expression of SLAMF7 will further promote the release of neutrophil extracellular traps (NETs) by neutrophils, thereby activating the inflammatory response and participating in the pathogenesis of AOSD-MAS. Based on this discovery, SLAMF7 can not only be used as a novel biomarker for AOSD secondary to MAS, but also participate in IFN-γ-induced NETs release and inflammatory response during the development of this disease. Therefore, the preparation of inhibitors targeting the "IFN-γ—SLAMF7—NETs" axis has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figures 1 - 3 Sequencing results of neutrophils and PBMCs of AOSD patients and HCs.

[0011] Figure 4 The mRNA level of SLAMF7 in neutrophils of AOSD patients is higher than that of HCs.

[0012] Figure 5 The level of SLAMF7 receptor on neutrophils of AOSD patients is higher than that of HCs.

[0013] Figures 6 - 8 The expression level of SLAMF7 receptor on neutrophils of AOSD patients is higher than that of patients in the stable phase and HCs, and is significantly correlated with disease activity and clinical laboratory indicators.

[0014] Figures 9 - 11 The expression level of SLAMF7 receptor on neutrophils in patients with AOSD complicated with MAS is higher than that in non-MAS patients, and it is significantly correlated with the MAS score HScore and MAS-related indicators.

[0015] Figures 12 - 15 The plasma soluble SLAMF7 level in AOSD patients is higher than that in other rheumatic diseases such as SLE, RA, and HLH caused by other diseases.

[0016] Figures 16 - 18 It is the test result of MAS mice.

[0017] Figures 19 - 21 After in vitro IFN-γ stimulation upregulates SLAMF7 on HC neutrophils and then recombinant SLAMF7 protein stimulation, it can significantly increase the formation of neutrophil NETs and the level of inflammatory factors.

[0018] Figures 22 - 24 It is the relevant experimental result after shRNA knockdown of SLAMF7 expression in NB4 cell line.

[0019] Figure 25 It is the experimental result after pretreatment of healthy human neutrophils with PAD4 inhibitor / NE inhibitor / DPI in vitro.

[0020] Figure 26 It is a schematic diagram of the role mode of IFN-γ-stimulated neutrophils highly expressing SLAMF7 in the inflammatory regulation of AOSD-MAS by promoting NETs formation. Specific implementation manners

[0021] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0022] Example 1

[0023] In this example, neutrophils and PBMC cells from AOSD patients and healthy controls (HC) were collected for transcriptome sequencing. Through methods such as PCA analysis, GO enrichment analysis, and LASSO regression analysis, it was found that the expression of SLAMF7 was significantly increased on neutrophils from AOSD patients. Next, the expression of SLAMF7 in neutrophils from AOSD patients was detected by RT-qPCR, Western Blot, or flow cytometry. The correlation between the expression of SLAMF7 and the clinical characteristics and related laboratory indicators of MAS patients was analyzed by Spearman correlation test. The expression of SLAMF7 on neutrophils was detected in a MAS mouse model stimulated by TLR9 agonist. In vitro, after upregulating SLAMF7 in HC neutrophils by IFN-γ stimulation and then stimulating with recombinant SLAMF7 protein, the neutrophil NETs level was detected by PicoGreen method and immunofluorescence. Finally, an SLAMF7-knockdown neutrophil-like NB4 cell line was constructed using shRNA lentivirus to explore the effects of SLAMF7 knockdown on NET and inflammation. Thus, the specific mechanism by which IFN-γ induces the expression of SLAMF7 on neutrophils and then promotes NET formation and amplifies inflammation in AOSD patients with secondary MAS was revealed.

[0024] The results are as follows:

[0025] Figure 1 It showed significant transcriptomic heterogeneity in neutrophils and PBMC in the transcriptome sequencing of neutrophils and PBMC from AOSD patients.

[0026] Figure 2 It showed the most significantly expressed genes among the DEGs upregulated in neutrophils from AOSD patients compared to HC.

[0027] Figure 3 It showed that the expression level of SLAMF7 in neutrophils from AOSD patients was significantly higher than that in HC in the neutrophil transcriptome sequencing.

[0028] Figure 4 It showed that the mRNA level of SLAMF7 in neutrophils from AOSD patients was significantly higher than that in HC.

[0029] Figure 5 It showed that the protein expression level of SLAMF7 on the cell membrane of neutrophils from AOSD patients was increased compared to HC.

[0030] Figure 6 It showed that the expression level of SLAMF7 receptor on the cell membrane of neutrophils from active AOSD patients was increased compared to stable-phase patients and HC.

[0031] Figure 7 It showed that the mRNA expression level of SLAMF7 in neutrophils from AOSD patients decreased after treatment.

[0032] Figure 8 It is shown that the expression of SLAMF7 receptor in neutrophils of AOSD patients is positively correlated with disease activity and clinical inflammatory indicators.

[0033] Figure 9 It is shown that the expression of SLAMF7 receptor in neutrophils of AOSD-MAS patients is higher than that in non-MAS patients.

[0034] Figure 10 It is shown that the expression of SLAMF7 receptor in neutrophils of AOSD-MAS patients is positively correlated with MAS score HScore.

[0035] Figure 11 It is shown that the expression of SLAMF7 receptor in neutrophils of AOSD-MAS patients is correlated with MAS-related indicators.

[0036] Figure 12 It is shown that the plasma SLAMF7 level in AOSD patients is significantly higher than that in other rheumatic diseases such as SLE, RA, and HC.

[0037] Figure 13 It is shown that the plasma SLAMF7 level in AOSD patients with concomitant MAS in active stage is higher than that in non-MAS patients, and higher than that in stable stage patients and HC.

[0038] Figure 14 It is shown that the plasma SLAMF7 level in AOSD patients significantly decreases after treatment.

[0039] Figure 15 It is shown that AOSD-MAS patients are higher than those with HLH caused by other diseases.

[0040] Figure 16 It is shown that the expression of SLAMF7 mRNA in the liver of MAS mice is significantly up-regulated compared with the control group.

[0041] Figure 17 It is shown that the proportion of SLAMF7+ cells in neutrophils in the liver of MAS mice is higher than that in the control group.

[0042] Figure 18 It is shown that the specific staining of Ly6G and SLAMF7 in neutrophils in the liver of MAS mice by immunohistochemistry is increased compared with the control group.

[0043] Figure 19 It is shown that IFN-γ stimulation of HC neutrophils in vitro can significantly up-regulate the expression of SLAMF7 and inflammatory factors.

[0044] Figure 20It is shown that after IFN-γ stimulates HC neutrophils to upregulate SLAMF7 in vitro and then recombinant SLAMF7 protein is added, the formation of neutrophil NETs can be significantly increased.

[0045] Figure 21 It is shown that after IFN-γ stimulates HC neutrophils to upregulate SLAMF7 and then recombinant SLAMF7 protein is added, the release of neutrophil NETs can be significantly increased.

[0046] Figure 22 It is shown that after knocking down SLAMF7 in the NB4 cell line using shRNA, the mRNA and protein levels of SLAMF7 in neutrophils are significantly decreased.

[0047] Figure 23 It is shown that after knocking down SLAMF7 in the NB4 cell line using shRNA, the protein level of SLAMF7 on the NB4 cell membrane and the mRNA levels of various inflammatory factors are significantly decreased.

[0048] Figure 24 It is shown that after knocking down SLAMF7 in the NB4 cell line using shRNA, the formation of NETs is reduced.

[0049] Figure 25 It is shown that after pre-treating healthy human neutrophils in vitro with PAD4 inhibitor / NE inhibitor / DPI and then adding recombinant SLAMF7 protein, the generation of NETs is significantly reduced.

[0050] Figure 26 It is a schematic diagram of the role mode of IFN-γ-stimulated neutrophils highly expressing SLAMF7 in the inflammatory regulation of AOSD-MAS by promoting the formation of NETs.

[0051] From the above results, it can be seen that in the neutrophils of AOSD patients, especially those with AOSD secondary to MAS, the mRNA and protein expressions of SLAMF7 are significantly upregulated, and there is a general positive correlation with disease activity, clinical inflammatory indicators, and cytokine levels. It is also confirmed in the MAS mouse model that the expression of SLAMF7 on neutrophils is significantly increased. In vitro experiments found that IFN-γ can significantly upregulate the expression of SLAMF7 on neutrophils, further promoting the increase in the formation of NETs and thus exacerbating inflammation. Knocking down SLAMF7 can relieve the excessive inflammatory response.

[0052] From the above results, it can be seen that SLAMF7 is involved in IFN-γ-induced neutrophil NET release and then activates the inflammatory response to participate in the pathogenesis of AOSD-MAS. Therefore, SLAMF7 can not only be used as a novel biomarker for secondary MAS in AOSD, but also participate in IFN-γ-induced NETs release and inflammatory response during the development of this disease. Therefore, the preparation of inhibitors targeting the "IFN-γ—SLAMF7—NETs" axis has good market application prospects.

Claims

1. Use of the cell membrane receptor SLAMF7 in the preparation of a macrophage activation syndrome inhibitor.

2. The application according to claim 1, characterized in that: The expression level of the cell membrane receptor SLAMF7 is increased in neutrophils of patients with AOSD complicated with MAS and MAS model mice, and is significantly correlated with MAS-related indicators; stimulation of neutrophils with SLAMF7 recombinant protein in vitro leads to an increase in NETs generation; after knocking down SLAMF7 in the neutrophil-like NB4 cell line, NET generation decreases and inflammatory indicators decline.

3. The application according to claim 1, wherein: The inhibitor further comprises a pharmaceutically acceptable carrier and / or excipient.

4. The application according to claim 3, characterized in that: The pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

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