Use of the inhibitor anti-CD90.2

Abstract

The present invention discloses the use of an inhibitor anti-CD90.2, which can reduce the expression of innate lymphoid cells and thus can be used as an anti-pneumonia preparation targeting ILC for post-infarct pneumonia. The expression of ILC2 cells in patients with acute cerebral infarction increases. After the clearance of ILC, differential expression of pneumonia-related indicators is observed in patients with acute cerebral infarction, indicating that ILC2 promotes pneumonia after cerebral infarction. The inhibitor anti-CD90.2 (250 μg / mouse) was injected into Rag- / - mice 48 hours before modeling. The expression of ILC2 was measured 24 hours after MCAO modeling and was found to decrease, indicating that the inhibitor anti-CD90.2 can play an anti-pneumonia role by reducing the expression of ILC2 in mice with acute cerebral infarction. The present invention provides a new target for the prevention and treatment of pneumonia, a complication after acute cerebral infarction surgery. At the same time, it is proposed that the inhibitor anti-CD90.2 reduces the expression of ILC2 and thus can be used as an anti-pneumonia preparation targeting ILC2, achieving an effective combination of theoretical research and practical application.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the use of an inhibitor anti-CD90.2, which can be used as an anti-pneumonia preparation targeting ILC2 by reducing the number of ILC2 cells in lung tissue after cerebral infarction. Background Art

[0002] Stroke is the second most common cause of death in the world and is a cerebrovascular disease with a relatively high recurrence rate and poor prognosis. The significant increase in stroke is related to the aging of the population, and acute ischemic stroke (AIS) accounts for 80% of the total cases. It is reported that more than 30% of stroke-infected patients die of secondary complications within 1 week after the onset of stroke, with bacterial pneumonia being the most common. In 2003, Hilker et al. proposed the concept of stroke-associated pneumonia (SAP). Many studies have shown that immunosuppression caused by severe stroke can lead to significant changes in the levels of inflammatory factors, increase the apoptosis of immune cells in multiple organs, and result in serious infection complications in patients. The prophylactic use of broad-spectrum antibiotics to reduce stroke-related bacterial infections, however, has not been successful. In addition, with the increasing problem of antibiotic resistance, future treatment methods to reverse post-stroke immune damage by enhancing host immunity may be a viable alternative.

[0003] Innate lymphoid cells may play an important role. ILCs are mainly tissue-resident lymphocytes, and ILCs are divided into five subsets: NK cells, ILC1, ILC2, ILC3, and LTi cells. ILC2 is the most abundant ILC subset present in the lung. ILC2 is necessary for expelling intestinal worms, driving allergic pneumonia, and maintaining tissue homeostasis. ILC2 can directly or indirectly interact with other immune cells, such as T cells and B cells, and act as a bridge between innate immunity and adaptive immunity. ILC2 is a key driver of type 2 immunity. Under the action of IL-33, IL-25, and TSLP released by lung epithelial cells, ILC2 can secrete a large amount of type 2 cytokines, promoting the recruitment and activation of eosinophils, fibroblasts, and macrophages. It also plays a key role in lung diseases by promoting Th2 cell differentiation, activating B cell class switching, survival, and the secretion of IgG1 and IgE. In different disease responses, ILC2 may play different roles, including pathogenic and protective effects on the body. However, whether ILC2 is involved in the occurrence and development of stroke-related pulmonary infections has not been reported. Therefore, in view of the fact that most infections occur in the first few days after stroke and the established role of ILC2 in early lung diseases, we attempted to determine whether ILC2 is affected by ischemic stroke and whether it leads to infection susceptibility or inhibits infection.

[0004] Reported anti-infective drugs include penicillins, cephalosporins, macrolides, quinolones, etc. Whether most anti-infective drugs are effective also depends on the patient's health, postoperative recovery, or other conditions that occur during surgery, thus limiting their potential clinical use. Therefore, finding anti-infective drugs with fewer side effects and stronger targeted clearance ability is the common goal pursued in the global research field of postoperative infection complications of cerebral infarction.

[0005] anti-CD90.2 is a broad-spectrum ILC inhibitor that acts on ILC1, ILC2, and ILC3 and is widely used in research related to ILC cell subsets. ILC2 is a cell related to inflammation and plays an important role in regulating the intestine and pneumonia. Moreover, ILC2 cells also act by secreting related cytokines. Studies have shown that the clearance of ILC2 inhibits the occurrence related to allergic pneumonia, and the clearance of the ILC2 subset also inhibits asthma impairment and dysregulation. Various studies have revealed a close relationship between ILC2 and pneumonia.

[0006] Currently, there are no reports on whether the anti-CD90.2 inhibitor can affect the expression of ILC2, whether ILC2 can regulate pneumonia after cerebral infarction, and the anti-pneumonia drug related to anti-CD90.2 targeting ILC2. Given the continuous increase in postoperative complications related to cerebral infarction and the few reports on drugs for anti-pneumonia after cerebral infarction, therefore, the development of anti-pneumonia drugs with low side effects and high specificity after cerebral infarction has a long way to go. The present invention provides a new target for preventing and treating inflammation-related diseases induced by cerebral infarction in lung cells, and also proposes that the inhibitor anti-CD90.2 clears the expression of ILC2, so it can be used as an anti-pneumonia preparation targeting ILC2, achieving an effective combination of theoretical research and practical application. Summary of the Invention

[0007] In order to solve the problem of preventing and treating pneumonia, a postoperative complication of cerebral infarction, the present invention provides a use of the inhibitor anti-CD90.2, that is, anti-CD90.2 can be used as an anti-pneumonia preparation after cerebral infarction by reducing the expression of ILC2.

[0008] The object of the present invention can be achieved by the following technical solutions:

[0009] The present invention first discloses the use of the inhibitor anti-CD90.2, which can be used as an anti-pneumonia preparation by reducing the expression of ILC2.

[0010] The inhibitor anti-CD90.2 has the effect of reducing the expression of ILC2. The realization mechanism is as follows: the expression of ILC2 increases 24 hours after cerebral infarction in C57 / BL6 mice. In cerebral infarction mice, 250 μg / mouse of the inhibitor anti-CD90.2 is injected in advance and after 48 hours of treatment, the expression of ILC2 in the lung tissue of cerebral infarction mice decreases.

[0011] The inhibitor anti-CD90.2 is used as an anti-pneumonia preparation. The realization mechanism is as follows: the expression of ILC2 increases in the lung tissue after cerebral infarction. After clearing ILC2, the inflammation of the lung tissue weakens, indicating that ILC2 has the effect of promoting lung tissue inflammation. Adding the inhibitor anti-CD90.2 can clear the expression of ILC2. At the same time, detecting pneumonia-related indicators shows that the inflammation of the lung tissue weakens after anti-CD90.2 treatment, that is, anti-CD90.2 has the anti-pneumonia effect targeting ILC2 and can be used as an anti-pneumonia preparation after cerebral infarction.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] 1. The inventors found through research that the inhibitor anti-CD90.2 plays an anti-pneumonia role by reducing the expression of ILC2 in the lung tissue after cerebral infarction. The expression of ILC2 increases in the lung tissue after cerebral infarction. After clearing ILC2, differential expression of inflammation-related indicators in the lung tissue is determined, indicating that ILC2 has the effect of promoting pneumonia after cerebral infarction. Treating cerebral infarction model mice with 250 μg / mouse of the inhibitor anti-CD90.2 48 hours in advance reduces the expression of ILC2, that is, the inhibitor anti-CD90.2 can play an anti-pneumonia role by reducing the expression of ILC2 in pneumonia after cerebral infarction. Therefore, it can be used as an anti-pneumonia preparation targeting ILC2, realizing the effective combination of theoretical research and practical application.

[0014] 2. The inhibitor anti-CD90.2 used in the present invention reduces the expression of ILC2, targets ILC2, and achieves the effect of anti-pneumonia after cerebral infarction. It has higher specificity compared with some natural product anti-pneumonia drugs and has no obvious effect on other organs, showing the potential for practical application. Brief Description of the Drawings

[0015] Figure 1 It is a diagram of the pneumonia susceptibility results in the acute cerebral infarction model in Example 1 of the present invention. Among them Figure 1 A shows the expression rates of three ILC subsets analyzed by flow cytometry in healthy individuals and ACI patients, confirming that the ILC subsets change significantly during acute cerebral infarction (p < 0.01, statistically significant); Figure 1B shows the TTC staining of C57BL / 6 wild-type mice after MCAO surgery; white is the infarct area. The infarct area of the brain tissue of acute cerebral infarction mice was detected by TTC, and it was found that the white infarct area was significantly increased in the model group through infarct area analysis (p < 0.001, statistically significant). Figure 1 C shows that at 0, 6, 12, and 24 h after reperfusion, lung specimens of sham-operated group (SS) and MCAO-operated group (MCAO) C57BL / 6 wild-type mice were taken, and neutrophil infiltration was measured by MPO activity, indicating that neutrophil infiltration in the lung tissue of mice after cerebral infarction was significantly increased (p < 0.001, statistically significant). Figure 1 D shows the bacteriological analysis of bacterial loads in the blood and lungs of sham mice and the operated group at 6, 12, 24, and 36 hours after reperfusion. The numbers represent the number of colony-forming units (CFU) per milliliter of blood and per milligram of tissue, demonstrating that bacteria are produced in the blood and lungs 24 hours after cerebral infarction (*P < 0.05, ***P < 0.001, ns indicates no statistically significant difference).

[0016] Figure 2 This is the flow cytometry gating strategy diagram of ILC2 subsets in Example 2 of the present invention. Among them, FSC and SSC gate out the lymphocyte population, FSC-H and FSC-A gate out the mononuclear cells, SSC and Live gate out the live cells, CD45 and Lin gate out the total ILC subsets, RORγt and Thy1.2 gate out the T cell-related ILCs, and NK1.1 and ST2 gate out the ILC2s.

[0017] Figure 3 This shows that the proportion of ILC2 in the lung after stroke is up-regulated in Example 2 of the present invention. Figure 3 A shows the expression of ILC2 in the lungs of wild-type C57BL / 6 mice, showing the expression rates of ILC2 cells in the lung tissues of sham-operated group and model group mice at different time points. Figure 3 B shows the expression of IL-33 and TSLP at the mRNA level in the lung tissues of mice at different time points after modeling. The results show that ILC2 may promote the occurrence of bacterial infection, and ILC2 may respond to the stimulation of TSLP factor.

[0018] Figure 4 shows that depletion of ILC2 with inhibitor anti-CD90.2 can reduce stroke-related pulmonary infection in Example 3 of the present invention. Among them, Figure 4A This is the flow chart of clearing ILC2 with inhibitor anti-CD90.2. After treating with 250 μg / mouse anti-CD90.2 for 48 h, an MCAO model was established, and lung tissues were taken for experiments 24 h later. Figure 4BTo measure the expression rate of ILC cells in the lung tissue after cerebral infarction by flow cytometry, anti-CD90.2 effectively depleted ILC cells with CD90.2 on the surface. Flow cytometry was used to further characterize the expression of ILC2 after treatment with anti-CD90.2, and the expression of ILC2 was significantly decreased. Figure 4C The figure shows the results of colony counts and neutrophil infiltration in the lung tissue and blood after pneumonia. The results of pneumonia-related colony group counts showed that the number of colonies in the lung tissue was significantly decreased after treatment with anti-CD90.2. The MPO results of the lung tissue showed that neutrophil infiltration in the lung tissue was significantly decreased after treatment with anti-CD90.2, that is, pulmonary inflammation was reduced.

[0019] Figure 5 In Example 3 of the present invention, the inhibitor anti-CD90.2 depleted ILC2 can protect mice from stroke-related pulmonary infections. Figure 5 A shows the HE staining of the lung tissue without injection and injection of the inhibitor anti-CD90.2. It can be seen that there will be exudation in the lung tissue after modeling. The HE staining of the sham group showed no abnormality, and the exudation of the lung tissue injected with the inhibitor anti-CD90.2 was reduced. Figure 5 B is the lung tissue injury score. The lung tissue injury score was reduced after injection of the inhibitor anti-CD90.2, and there was a significant difference compared with the lung tissue without injection of the inhibitor anti-CD90.2 (p < 0.05, statistically significant). Figure 5 C shows the observation of neutrophil infiltration by immunofluorescence. Neutrophils in the lung tissue were stained with Ly6g. After modeling, an increase in the number of neutrophils was observed, and neutrophils were significantly reduced after injection of the inhibitor anti-CD90.2. Figure 5 D is the statistical chart of the positive proportion of neutrophils. There was a significant reduction in neutrophils after injection of the inhibitor anti-CD90.2 compared with that without injection of the inhibitor anti-CD90.2 (p < 0.001, statistically significant). Figure 5 E is the immunohistochemical staining of neutrophils in the lung tissue. There was a significant reduction in neutrophils after injection of the inhibitor anti-CD90.2 compared with that without injection of the inhibitor anti-CD90.2 (p < 0.001, statistically significant). Detailed implementation mode

[0020] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with embodiments and drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and do not limit the present invention.

[0021] Example 1:

[0022] Analyze the susceptibility of pneumonia in cerebral infarction. In acute cerebral infarction patients and MCAO model rats, flow cytometry was used to detect the changes in ILC subsets in acute cerebral infarction patients, and plate counting and MPO detection were used to detect neutrophil infiltration.

[0023] Figure 1 This is a result graph of the susceptibility of pneumonia in the acute cerebral infarction model, and its implementation method is as follows:

[0024] The clinical experiment was divided into an acute cerebral infarction group and a healthy control group. Compared with the control group, the expression rate of ILC1 increased, the expression rate of ILC2 decreased, and there was no significant change in ILC3 in the acute cerebral infarction group. The animal experiment was divided into a modeling group and a sham operation group. In the modeling group, TTC staining of brain tissue was used to determine the successful establishment of an acute cerebral infarction model, and the white part was the infarcted area. Then, the lung tissues of the modeling group and the sham operation group were used to detect the MPO level with an MPO kit, and the peripheral blood and lung tissues at different time points in the modeling group were plated on a plate to count the formed colonies.

[0025] Figure 1 A shows the expression rates of three ILC subsets in healthy individuals and ACI patients analyzed by flow cytometry, confirming that the ILC subsets change significantly during the occurrence of acute cerebral infarction (p<0.01, with statistical significance).

[0026] Figure 1 B is the TTC staining of C57BL / 6 wild-type mice after MCAO surgery; the white area is the infarcted area. The infarcted area of the brain tissue of acute cerebral infarction mice was detected by TTC, and it was found that the white infarcted area increased significantly in the model group through infarct area analysis (p<0.001, with statistical significance).

[0027] Figure 1 C shows that at 0, 6, 12, and 24 hours after reperfusion, lung specimens of sham operation group (SS) and MCAO operation group (MCAO) C57BL / 6 wild-type mice were taken, and neutrophil infiltration was measured by MPO activity, indicating that neutrophil infiltration in the lung tissue of mice after cerebral infarction increased significantly (p<0.001, with statistical significance).

[0028] Figure 1 D shows the bacteriological analysis of the bacterial load in the blood and lungs of sham mice and the operation group at 6, 12, 24, and 36 hours after reperfusion. The numbers represent the number of colony-forming units (CFU) per milliliter of blood and per milligram of tissue, proving that bacteria will be produced in the blood and lungs 24 hours after cerebral infarction (P<0.05, P<0.001, ns indicates no statistical significance of the difference).

[0029] This example shows an increased possibility of pneumonia infection in acute cerebral infarction.

[0030] Example 2:

[0031] Analysis shows that ILC2 levels increase in the lungs after stroke. In pneumonia following cerebral infarction, ILC2 first decreases and then increases in expression at 24 hours. The cytokines that stimulate ILC2 are IL-33 and TSLP. However, the changes in IL-33 at the time points of 0 h, 6 h, 12 h, and 24 h are not obvious, and the changes in TSLP at the time points of 0 h, 6 h, and 12 h are not obvious, but it increases significantly at 24 h, exerting a stimulatory effect on ILC2, in order to analyze the role of ILC2 in pneumonia after cerebral infarction.

[0032] Figure 2 It is a flow cytometry gating strategy diagram for ILC2 subsets. CD45 and Lin gate out the total ILC subset, RORγt and Thy1.2 gate out T cell-related ILC, and NK1.1 and ST2 gate out ILC2.

[0033] Figure 3 ILC2 levels increase in the lungs after stroke, and the implementation method is as follows:

[0034] The experiment was divided into a sham operation group and a model group. The expression rates of ILC2 cells in the lungs of the sham operation group and the model group at different time points were measured. In the sham operation group, there were no obvious changes in ILC2 at 0, 6, 12, and 24 hours. In the model group, the expression rate of ILC2 decreased at 0, 6, and 12 hours and increased at 24 hours. Total RNA was extracted from lung tissues, and reverse transcription technology and qPCR were used to detect the expression of cytokines that stimulate ILC2 and perform statistics.

[0035] By comparing the differences between the sham operation group and the model group, it was found that the changes in IL-33 at the time points of 0 h, 6 h, 12 h, and 24 h were not obvious, and the changes in TSLP at the time points of 0 h, 6 h, and 12 h were not obvious, but it increased significantly at 24 h (p < 0.05, with statistical significance).

[0036] This example shows that ILC2 has a promoting effect on pneumonia after cerebral infarction.

[0037] Example 3:

[0038] This example discloses the application of an anti-CD90.2 inhibitor in reducing the expression of ILC2, and thus it can be used as an anti-pneumonia agent targeting ILC2.

[0039] Figure 4A It is a flow chart of the clearance of ILC2 by the inhibitor anti-CD90.2. After treating with 250 μg / mouse anti-CD90.2 for 48 h, an MCAO model was established, and lung tissues were taken for experiments 24 h later.

[0040] Figure 4BTo measure the expression rate of ILC cells in the lung tissue after cerebral infarction by flow cytometry, anti-CD90.2 effectively depleted ILC cells with CD90.2 on the surface. Flow cytometry was used to further characterize the expression of ILC2 after treatment with anti-CD90.2, and the expression of ILC2 was significantly decreased.

[0041] Figure 4C The figures show the results of colony counts and neutrophil infiltration in the lung tissue and blood after pneumonia. The results of pneumonia-related colony group counts indicate that the number of colonies in the lung tissue significantly decreased after treatment with anti-CD90.2. The results of MPO in the lung tissue show that neutrophil infiltration in the lung tissue significantly decreased after treatment with anti-CD90.2, that is, lung inflammation decreased.

[0042] Figure 5 A shows the HE staining of the lung tissue without injection and with injection of the inhibitor anti-CD90.2. It can be seen that there is exudation in the lung tissue after modeling, and no abnormality is seen in the HE staining of the sham group. The exudation in the lung tissue with injection of the inhibitor anti-CD90.2 decreased.

[0043] Figure 5 B is the lung tissue injury score. The lung tissue injury score decreased with injection of the inhibitor anti-CD90.2, showing a significant difference compared with the lung tissue without injection of the inhibitor anti-CD90.2 (p < 0.05, statistically significant).

[0044] Figure 5 C shows the observation of neutrophil infiltration by immunofluorescence. Neutrophils in the lung tissue were stained with Ly6g. After modeling, an increase in the number of neutrophils was observed, and the number of neutrophils significantly decreased after injection of the inhibitor anti-CD90.2.

[0045] Figure 5 D is the statistical chart of the positive proportion of neutrophils. The number of neutrophils significantly decreased with injection of the inhibitor anti-CD90.2 compared with without injection of the inhibitor anti-CD90.2 (p < 0.001, statistically significant).

[0046] Figure 5 E is the immunohistochemical staining of neutrophils in the lung tissue. The number of neutrophils significantly decreased with injection of the inhibitor anti-CD90.2 compared with without injection of the inhibitor anti-CD90.2 (p < 0.001, statistically significant).

[0047] This example shows that the anti-CD90.2 inhibitor can reduce the expression of ILC2 in the lung tissue after cerebral infarction, and the anti-CD90.2 inhibitor targets ILC2, thus playing an anti-pneumonia effect.

[0048] The above are the embodiments of the present invention. The above embodiments and the specific parameters in the embodiments are only for clearly expressing the invention verification process, and are not used to limit the patent protection scope of the present invention. The patent protection scope of the present invention still takes its claims as the criterion. All equivalent structural changes made by using the content of the specification and drawings of the present invention should similarly be included in the protection scope of the present invention.

Claims

1. Use of the inhibitor anti-CD90.2, characterized in that, The inhibitor anti-CD90.2 is used for preparing a preparation for preventing pneumonia, a complication after cerebral infarction surgery.

2. Use of the inhibitor anti-CD90.2 according to claim 1, characterized in that, The inhibitor anti-CD90.2 is used for preparing a preparation for preventing pneumonia, a complication after cerebral infarction surgery, which targets ILC2 cells.

3. Use of the inhibitor anti-CD90.2 according to claim 1 or 2, characterized in that, The inhibitor anti-CD90.2 plays a role in preventing pneumonia, a complication after cerebral infarction surgery, by reducing the number of ILC2 cells in lung tissue.

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