Method for regulating and controlling expression level of planopodia protein on surface of macrophage

Abstract

The invention relates to a method for regulating and controlling the expression level of planopodia protein on the surface of macrophage, and belongs to the technical field of biological medicine. The regulation and control method provided by the invention comprises the step of promoting or inhibiting the expression level of PDPN on the surface of the macrophage, wherein the promotion is realized by improving the expression level of an ADAP gene, improving the expression quantity of the ADAP, improving the phosphorylation level of 571th tyrosine of the ADAP or promoting the expression of signal transduction and a transcription activator STAT3; the inhibition is realized by reducing the expression level of the ADAP gene, reducing the expression quantity of the ADAP protein, reducing the phosphorylation level of tyrosine at the 571st site of the ADAP protein or inhibiting signal transduction and expression of a transcription activator STAT3. By using the method provided by the invention, the expression level of the planofoot protein on the surface of the macrophage can be flexibly regulated and controlled, and a new development direction is provided for treatment of sepsis and preparation of sepsis treatment medicines.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for regulating the expression level of podoplanin on the surface of macrophages. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by the body's dysregulated response to infection. It is a serious complication of patients with critical illnesses such as infection, burns / trauma, and shock. At present, sepsis affects more than 19 million people worldwide each year, with a mortality rate of more than 30%. Immune response dysfunction is the core mechanism of the occurrence and development of sepsis. The host's immune response is usually highly inflammatory in the early stage of sepsis, accompanied by the release of a large number of inflammatory mediators, while causing damage to tissues and immune cells, and then enters the late stage of sepsis characterized by immunosuppression. Therefore, current research focuses on trying to regulate the immune status of sepsis patients, control cytokine storms, reduce the risk of secondary infections, and promote the repair of damaged tissues to improve the prognosis of sepsis patients. Although the early mortality rate of sepsis has been alleviated with the effective intervention of related antibiotics and the gradual improvement of auxiliary supportive treatment. However, the incidence of sepsis is still rising, and almost one in five sepsis patients will die, especially in developing countries, and it is still one of the main causes of death in critically ill patients. Due to the lack of understanding of the clear underlying mechanisms of sepsis, there is still a lack of effective sepsis treatment drugs. Therefore, it is crucial to deeply explore the pathogenesis of sepsis, and it is urgent to develop safe and effective methods and drugs to treat sepsis.

[0003] At present, the treatment of sepsis mainly focuses on the early stage of the disease, such as fluid resuscitation, removal of infection sources, blood pressure maintenance, glucocorticoid treatment, antibiotic treatment, etc. Since sepsis is mostly caused by bacterial infection, antibiotic treatment is an indispensable clinical treatment method. Studies have shown that the vast majority of surviving sepsis hosts show immunosuppression within a few days or even weeks after the onset of the disease. During this period, the host is very likely to develop secondary infections and increase mortality. However, most of the secondary infection pathogens are multi-drug resistant bacteria, which greatly increases the difficulty of infection control. The existence of multiple broad-spectrum antibiotic-resistant bacteria seriously threatens human life safety, taking the lives of hundreds of thousands of patients every year, and the trend is increasing year by year. Therefore, it is urgent to develop new drugs for the treatment of sepsis to cope with the severe challenges brought by antibiotic resistance. In addition, the long-term use of antibiotics will lead to an increase in drug-resistant bacteria and affect the balance of intestinal flora. Therefore, it is only suitable for short-term emergency sterilization, which is another limitation of existing sepsis treatment. At present, there is a lack of effective medical methods in the existing technology to alleviate the progression of sepsis. Dysregulation of macrophages and their mediated innate immune function is one of the important mechanisms for the development of sepsis. Patients with sepsis are usually accompanied by severe impairment of macrophage function. Macrophages play a variety of roles in various stages of sepsis. Studies have shown that the functional status of macrophages is closely related to the mortality rate of patients with sepsis. Therefore, exploring new treatments for macrophages may be an effective strategy for preventing and treating sepsis.

[0004] Podoplanin (PDPN), also known as PA2.26 antigen, gp38, M2A, T1 and D2-40, is a highly conserved small molecule type I transmembrane mucin-like glycoprotein in vertebrates. PDPN is the only known endogenous receptor for platelet C-type lectin-like receptor 2 (CLEC-2), which can interact with CLEC-2 on the surface of platelets and has received extensive attention in regulating the pathogenesis of sepsis. Studies on mouse models of sepsis induced by LPS or cecal ligation and puncture have shown that the interaction between macrophage PDPN and platelet CLEC-2 can promote the recruitment of macrophages to the site of infection and accelerate the clearance of bacteria. After specific knockout of hematopoietic cell PDPN, the number and proportion of macrophages recruited to the peritoneal infection site were reduced, but the number of neutrophils did not change significantly. Platelets participate in regulating related acute inflammatory responses through CLEC-2. CLEC-2 interacts with PDPN on the surface of alveolar F4 / 80-positive macrophages, which attenuates pneumonia tissue damage in mice with acute respiratory distress syndrome induced by LPS. A recent report showed that both platelet CLEC-2 and recombinant CLEC-2-Fc protein can increase the expression of macrophage PDPN and CD44, promote interaction with intracellular ERM (Ezrin-Radixin-Moesin) proteins, and accelerate the migration of inflammatory macrophages from the peritoneal site to the mesenteric lymph nodes to activate T cell responses, thereby reducing peritoneal inflammation. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for regulating the expression level of podoplanin on the surface of macrophages. The regulation method provided by the present invention includes promoting or inhibiting the expression level of podoplanin PDPN on the surface of macrophages, wherein the promotion is achieved by increasing the expression level of the ADAP gene, increasing the expression amount of the ADAP protein, increasing the phosphorylation level of the 571st tyrosine of the ADAP protein, or promoting the expression of the signal transduction and transcription activator STAT3; the inhibition is achieved by reducing the expression level of the ADAP gene, reducing the expression amount of the ADAP protein, reducing the phosphorylation level of the 571st tyrosine of the ADAP protein, or inhibiting the expression of the signal transduction and transcription activator STAT3.

[0006] The first object of the present invention is to provide a method for regulating the expression level of podoplanin on the surface of macrophages, wherein the regulation includes promoting or inhibiting the expression of podoplanin, and the promotion is achieved by any of the following methods:

[0007] (1) increasing the expression level of the ADAP gene or the expression amount of the ADAP protein;

[0008] (2) increasing the phosphorylation level of tyrosine 571 of the ADAP protein whose amino acid sequence is shown in SEQ ID NO.1;

[0009] (3) Promote the expression of signal transducer and transcription activator STAT3;

[0010] The inhibition is achieved by any of the following methods:

[0011] (1) reduce the expression level of ADAP gene or the expression of ADAP protein;

[0012] (2) inhibiting the phosphorylation level of tyrosine 571 of the ADAP protein whose amino acid sequence is shown in SEQ ID NO.1;

[0013] (3) Inhibit the expression of signal transducer and transcription activator STAT3.

[0014] The second object of the present invention is to provide use of a STAT3 agonist in the preparation of a drug for treating sepsis.

[0015] The third object of the present invention is to provide use of a STAT3 inhibitor in reducing the expression level of podoplanin on the surface of macrophages.

[0016] The fourth object of the present invention is to provide a preparation for increasing the expression level of ADAP gene or ADAP protein for use in increasing the expression level of podoplanin on the surface of macrophages or in preparing a drug for treating sepsis.

[0017] The fifth object of the present invention is to provide a preparation for increasing the tyrosine phosphorylation level at position 571 of the ADAP protein with an amino acid sequence such as that shown in SEQ ID NO.1, and its use in increasing the expression level of podoplanin on the surface of macrophages or in preparing a drug for treating sepsis.

[0018] The sixth object of the present invention is to provide the use of a preparation for reducing the expression level of ADAP gene or ADAP protein in reducing the expression level of podoplanin on the surface of macrophages.

[0019] The seventh object of the present invention is to provide a method for reducing the tyrosine phosphorylation level of position 571 of the ADAP protein with an amino acid sequence as shown in SEQ ID NO.1 and its use in reducing the expression level of podoplanin on the surface of macrophages.

[0020] The eighth object of the present invention is to provide the use of a kinase in increasing the expression level of podoplanin on the surface of macrophages or in preparing a drug for treating sepsis, wherein the kinase is Btk kinase or PI3K / mTOR kinase.

[0021] Furthermore, the Btk kinase increases the tyrosine phosphorylation level at position 571 of the ADAP protein having an amino acid sequence as shown in SEQ ID NO.1.

[0022] Furthermore, the PI3K / mTOR kinase increases the phosphorylation level of STAT3, further increasing the expression of podoplanin.

[0023] Beneficial effects of the present invention:

[0024] Podoplanin PDPN is an important LPS response protein in macrophages. Macrophages that highly express PDPN on the cell surface are PDPN-positive macrophages, which play an important role in the progression of sepsis. They have higher phagocytic activity than other subtypes of macrophages and have potential for treating sepsis. The method provided by the present invention can flexibly regulate the expression level of PDPN protein on the surface of macrophages, providing a new development direction for the treatment of sepsis and the preparation of sepsis treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0026] Figure 1 The effect of ADAP on LPS-induced PDPN protein expression in mouse peritoneal macrophages in Example 1 of the present invention, wherein A is the RT-qPCR result showing that PDPN mRNA expression is significantly downregulated after ADAP deficiency, B is the Western Blot result showing that PDPN protein expression is significantly downregulated after ADAP deficiency, and C is the expression of PDPN protein on the surface of macrophages after ADAP protein is detected by flow cytometry;

[0027] Figure 2 The effect of overexpressing ADAP gene on PDPN protein expression in Example 1 of the present invention;

[0028] Figure 3 The effect of overexpressing ADAP or mutating the 571st tyrosine of the ADAP protein on the expression of the PDPN protein in Example 1 of the present invention;

[0029] Figure 4 The effect of STAT3 and STAT1 inhibitors on LPS-induced PDPN protein expression in macrophages in Example 2 of the present invention;

[0030] Figure 5 The effect of the STAT3 agonist colivelin on peritoneal PDPN-positive macrophages in septic mice in Example 2 of the present invention;

[0031] Figure 6The effect of the STAT3 agonist colivelin on the survival rate and bacterial load of septic mice in Example 2 of the present invention, wherein A is the change in the survival time of septic mice, and B is the difference in bacterial load in the peritoneal cavity and blood of septic mice;

[0032] Figure 7 This is a schematic diagram of the protein kinase inhibitor library screening process in Example 3 of the present invention;

[0033] Figure 8 The protein kinase inhibitor library screening results in Example 3 of the present invention;

[0034] Fig. 9 The results of the protein kinase inhibitor library screening in Example 3 of the present invention, in which 2 Btk inhibitors and 7 PI3K / mTOR inhibitors all showed inhibitory effects on PDPN expression;

[0035] Fig.10 The effect of the BTK inhibitor on PDPN expression in Example 3 of the present invention;

[0036] Fig.11 The Western Blot experiment in Example 3 of the present invention confirms the inhibitory effect of various mTOR inhibitors on PDPN expression. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0038] Example 1

[0039] Adhesion and degranulation-promoting adapter protein (ADAP) is a hematopoietic cell-specific immune adaptor protein that was first discovered and identified in T cells and plays an important role in T cell receptor signal transduction and integrin-mediated T cell adhesion. Subsequent studies have confirmed that ADAP is widely expressed in innate immune cells such as macrophages and plays a key role in regulating macrophage polarization and phagocytic activity.

[0040] To confirm the effect of ADAP on PDPN expression in macrophages, CRISPR technology was used to knock out the ADAP gene (NCBI gene ID: 23880) in mice to construct the Adap - / - In septic mice, WT or Adap - / -Mouse peritoneal macrophages were stimulated with LPS (100 ng / mL) for a time gradient, and the expression of PDPN in macrophages was analyzed by RT-qPCR and Western Blot. The results showed that the mRNA and protein levels of PDPN in WT macrophages increased with LPS stimulation, while Adap - / - The expression of PDPN in macrophages was significantly inhibited ( Figure 1 A and B). In addition, since PDPN is a type I transmembrane glycoprotein, the expression of PDP on the cell surface was detected by flow cytometry, e.g. Figure 1 As shown in C, the absence of ADAP also significantly inhibited the expression of PDPN on the surface of macrophages.

[0041] The cDNA encoding the full-length mouse ADAP (NCBI gene ID: 23880) was packaged with the psPAX2 plasmid (Addgene, 12260) and the pMD2.G plasmid (Addgene, 12259) in HEK293T cells, and the target cells were infected by lentiviral transduction in the presence of polybrene (8 μg / mL). The infected cells were selected with puromycin for at least 3 weeks before being used in the experiment. The expression of the ADAP gene was complemented in the ADAP-interfered RAW264.7 cells by lentiviral means. The results showed that the complementation of ADAP could rescue the LPS-induced PDPN expression in the ADAP-interfered RAW264.7 cells ( Figure 2 In addition, overexpression of wild-type ADAP was able to rescue ADAP-mediated inhibition of LPS-induced PDPN expression in macrophages, but overexpression of the ADAP Y571F mutant had no effect on PDPN expression ( Figure 3 ). Therefore, phosphorylation of ADAP at Y571 is essential for maintaining LPS-induced PDPN expression in macrophages.

[0042] Example 2

[0043] Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor that plays an important role in cell proliferation, differentiation, survival and immune response by regulating gene expression. Its abnormal activation is closely related to a variety of cancers and inflammatory diseases, and thus becomes an important therapeutic target. The development of inhibitors or agonists for STAT3 has important clinical significance. In addition, the tyrosine at position 571 of the ADAP protein is important for the activation of STAT3. Therefore, based on Example 1, it is explored whether STAT3 is involved in the expression and regulation of PDPN in LPS-stimulated macrophages.

[0044] The results showed that the STAT3 inhibitor Ruxolitinib significantly inhibited LPS-induced PDPN expression in macrophages, while the STAT1 inhibitor Fludarabine had no effect ( Figure 4 ). Therefore, these data indicate that STAT3 specifically regulates LPS-induced PDPN expression in macrophages.

[0045] The effect of STAT3 agonists on the formation of PDPN-positive peritoneal macrophages was tested in a mouse model of sepsis. Colivelin is a STAT3 agonist. One hour after the modeling of sepsis mice, the STAT3 agonist Colivelin (1 mg / kg) was injected intraperitoneally. The results showed that Colivelin treatment significantly increased the proportion of PDPN-positive peritoneal macrophage subpopulations induced in sepsis mice ( Figure 5 In addition, the STAT3 agonist Colivelin (1 mg / kg) significantly increased the survival time of septic mice and reduced the bacterial load in the peritoneal cavity and peripheral blood of septic mice ( Figure 6 ). Therefore, STAT3 agonists can effectively increase the number of PDPN-positive peritoneal macrophages in the peritoneal cavity of septic mice, thereby alleviating the progression of sepsis.

[0046] Embodiment 3:

[0047] To identify the key protein kinases involved in the ADAP-dependent induction of PDPN expression in macrophages, a library of 103 FDA-approved inhibitors targeting multiple protein kinases was screened ( Figure 7 and Figure 8 ). The results showed that 32 kinase inhibitors were able to inhibit LPS-induced PDPN expression in macrophages, including 2 Btk inhibitors and 7 PI3K / mTOR inhibitors, all of which showed inhibitory effects on PDPN expression ( Fig. 9 ). In vitro, mouse peritoneal macrophages stimulated by LPS were treated with the Btk kinase inhibitor Ibrutinib. The results showed that the Btk inhibitor significantly inhibited the LPS-induced PDPN expression in macrophages ( Fig.10 ). The reason is that Btk kinase enhances the phosphorylation level of tyrosine 571 of ADAP protein, thereby maintaining the expression of PDPN protein.

[0048] In vitro, mouse peritoneal macrophages stimulated by LPS were treated with multiple mTOR inhibitors. Western Blot results confirmed that multiple mTOR inhibitors could effectively inhibit LPS-induced PDPN expression in macrophages ( Fig.11), confirming the regulatory effect of the TLR4-PI3K-mTOR signaling axis on PDPN expression in macrophages. PI3K / mTOR kinase is related to STAT3 phosphorylation levels, so PI3K / mTOR inhibitors indirectly affect PDPN protein expression levels by acting on STAT3. Therefore, these data indicate that Btk kinase and PI3K / mTOR kinase can regulate the formation of PDPN-positive macrophage subpopulations.

[0049] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for regulating the expression level of podoplanin on the surface of macrophages, characterized in that: The regulation includes promoting or inhibiting the expression of podoplanin, and the promotion is achieved by any of the following methods: (1) increasing the expression level of the ADAP gene or the expression amount of the ADAP protein; (2) increasing the phosphorylation level of tyrosine 571 of the ADAP protein whose amino acid sequence is shown in SEQ ID NO.1; (3) Promote the expression of signal transducer and transcription activator STAT3; The inhibition is achieved by any of the following methods: (1) Reduce the expression level of ADAP gene or the expression of ADAP protein; (2) inhibiting the phosphorylation level of tyrosine 571 of the ADAP protein whose amino acid sequence is shown in SEQ ID NO.1; (3) Inhibit the expression of signal transducer and transcription activator STAT3.

2. Application of STAT3 agonists in the preparation of drugs for the treatment of sepsis.

3. Application of STAT3 inhibitors in reducing the expression level of podoplanin on the surface of macrophages.

4. Use of a preparation that increases the expression level of the ADAP gene or increases the expression amount of the ADAP protein in increasing the expression level of podoplanin on the surface of macrophages or in preparing a drug for treating sepsis.

5. Use of a preparation for increasing the tyrosine phosphorylation level at position 571 of the ADAP protein having an amino acid sequence as shown in SEQ ID NO.1 in increasing the expression level of podoplanin on the surface of macrophages or in preparing a drug for treating sepsis.

6. Use of a preparation that reduces the expression level of the ADAP gene or reduces the expression amount of the ADAP protein in reducing the expression level of podoplanin on the surface of macrophages.

7. Use of a preparation for reducing the tyrosine phosphorylation level at position 571 of the ADAP protein having an amino acid sequence as shown in SEQ ID NO.1 in reducing the expression level of podoplanin on the surface of macrophages.

8. Use of a kinase in increasing the expression level of podoplanin on the surface of macrophages or in preparing a drug for treating sepsis, wherein the kinase is Btk kinase or PI3K / mTOR kinase.

9. The use according to claim 8, characterized in that: The Btk kinase increases the tyrosine phosphorylation level at position 571 of the ADAP protein whose amino acid sequence is shown in SEQ ID NO.

1.

10. The use according to claim 8, wherein the PI3K / mTOR kinase increases the phosphorylation level of STAT3 and further increases the expression of podoplanin.

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