Application of CD177 in diagnosis and treatment of inflammatory lung injury

Abstract

The invention discloses application of CD177 in diagnosis and treatment of inflammatory lung injury. The research determines that the neutrophil surface molecule CD177 is obviously and abnormally increased in a patient with acute respiratory distress syndrome (ARDS) and is a reliable immune target for diagnosing lung injury; animal experiments prove that the CD177 has a key therapeutic effect in acute lung injury: the deficiency of the CD177 can obviously inhibit excessive infiltration of neutrophil in lung tissues, and the acute lung injury of mice is relieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of CD177 in the diagnosis and treatment of inflammatory lung injury. Background Technology

[0002] Differentiation antigen cluster 177 (CD177, also known as human neutrophil alloantigen 2a and NB1) is a glycosylated phosphatidylinositol (GPI)-coupled N-glycosylated transmembrane protein, heterogeneously expressed on the surface of human neutrophils. In the state of infection, CD177 increases in response to inflammatory stimulation and is considered an adhesion molecule mediating the recruitment of human neutrophils to a chemokine gradient. CD177 has been shown to play an important role in mediating the release of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) in inflammatory diseases such as inflammatory bowel disease (IBD) and acute pancreatitis, but the specific mechanism of action of CD177 in ALI remains unclear.

[0003] Dendritic cells (DCs), as the most potent antigen-presenting cells, are highly activated in ALI, serving as a bridge between innate and adaptive immunity. Furthermore, the activation of neutrophils can further activate DCs, inducing IL-17-mediated immune responses. IL-17 not only possesses a remarkable ability to recruit neutrophils but also directly increases alveolar epithelial permeability, thus dominating the disease progression of ALI / ARDS. This suggests that dendritic cells and their downstream IL-17 inflammatory signaling pathway play a crucial role in ALI. Neutrophils are the most abundant immune cells in the human body, forming the first line of defense in innate immunity and participating in the regulation of many physiological processes, such as acute injury and repair, angiogenesis, coagulation, autoimmunity, and chronic inflammation. Neutrophils play a vital role in various inflammatory diseases, acting as a central force in initiating and maintaining these diseases by attacking tissues, creating inflammatory environments, and forming new epitopes.

[0004] Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a difficult-to-treat clinical respiratory critical illness. Despite treatments such as mechanical ventilation and extracorporeal membrane oxygenation (ECMO), the mortality rate remains high. The essence of ALI / ARDS is lung tissue damage caused by an uncontrolled inflammatory response, primarily driven by a complex cytokine network, including the infiltration of various immune cells, mainly neutrophils, and the release of large amounts of inflammatory mediators. This, in turn, regulates the continuous transduction of pro-inflammatory pathways, ultimately leading to irreversible end-organ damage. Therefore, identifying novel molecular targets of neutrophils in ALI / ARDS to explore more precise immunotherapies is particularly urgent. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by providing an application of CD177 in the diagnosis and treatment of inflammatory lung injury.

[0006] To achieve its objective, the present invention employs the following technical solution:

[0007] The first aspect of the present invention provides the use of a reagent for detecting CD177 in the preparation of diagnostic tools for inflammatory lung injury or neutrophil-associated inflammatory diseases.

[0008] A second aspect of the invention provides the use of CD177 as a target in screening drugs for the treatment of inflammatory lung injury or neutrophil-related inflammatory diseases.

[0009] CD177 serves as an immune target.

[0010] The CD177 mentioned above is the neutrophil CD177.

[0011] The CD177 mentioned above refers to CD177 in neutrophils from peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue.

[0012] The inflammatory lung injury or neutrophil-associated inflammatory disease mentioned above includes acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).

[0013] The drug includes at least one of the following functions:

[0014] (1) Reduce alveolar permeability and alleviate neutrophil infiltration in lung tissue;

[0015] (2) Reduces the transcriptional and protein levels of inflammatory cytokines IL-1β, IL-6, TNF-α, CXCL1 and CXCL2 in lung tissue;

[0016] (3) Reduce the level of pathological damage to lung tissue, delay weight loss and prolong survival.

[0017] A third aspect of the present invention provides a reagent for interfering with or inhibiting CD177 expression, said reagent being a protein, compound, or nucleic acid molecule.

[0018] Preferably, the reagent is a nucleic acid molecule shRNA, siRNA, or sgRNA that interferes with or inhibits CD177 expression.

[0019] A fourth aspect of the present invention provides a medicament for treating inflammatory lung injury or neutrophil-associated inflammatory diseases, said medicament comprising the above-mentioned reagents that interfere with or inhibit CD177 expression.

[0020] The beneficial effects of this invention are:

[0021] This invention identifies the crucial role of the neutrophil surface molecule CD177 in acute lung injury. CD177 deficiency significantly alleviates acute lung injury in mice and inhibits neutrophil infiltration in lung tissue. However, this is not because CD177 deficiency affects the autonomous transvascular endothelial inflammatory tissue recruitment capacity of neutrophils. CD177's functions in promoting neutrophil adhesion, stretching deformation and polarization, directional chemotaxis, and in vitro induced release of NETs all exhibited levels comparable to WT, but reduced the overall ROS level. CD177 deficiency significantly inhibited the IL-23 / IL-17 signaling axis. IL-17 has a remarkable ability to recruit neutrophils during lung tissue injury and is strictly transduced by the upstream factor IL-23; dendritic cells are considered the main source of IL-23. Our further investigation revealed that CD177 deficiency significantly inhibited dendritic cell maturation in an ALI mouse model. This may further lead to a reduction in activated dendritic cells in lung tissue and impair the messenger function of IL-23 / IL-17, thereby reducing inflammatory recruitment of neutrophils to the lungs, alleviating pathological damage to lung tissue, and prolonging survival. This inhibitory effect caused by CD177 deficiency can be blocked by exogenous BMDC infusion and recombinant IL-23 and IL-17 supplementation. This invention delves into the molecular mechanism by which CD177, as a novel neutrophil molecular target, mediates acute lung injury. Neutrophil CD177 promotes lung immunopathology by inducing dendritic cell maturation and driving downstream IL-23 / IL-17 signal transduction. This invention enriches the ALI / ARDS-neutrophil-dendritic cell-inflammation regulatory network and provides a novel immunotherapeutic target for ALI / ARDS. Attached Figure Description

[0022] Figure 1 The study showed that CD177 expression in neutrophils was significantly upregulated during lung inflammation: (A) Analysis of differentially expressed genes in peripheral blood neutrophils of ARDS using public databases showed that CD177 was the most significantly upregulated; (B) Flow cytometry was used to detect CD177 expression levels in peripheral blood neutrophils of healthy volunteers (n=13) and ARDS patients (n=29); (C) ELISA was used to detect the protein content of CD177 in plasma; (D) Analysis of mRNA expression levels in circulating neutrophils before and after endotoxin infusion using public databases; (E) Analysis of mRNA expression levels in airway neutrophils before and after endotoxin infusion using public databases.

[0023] Figure 2The following diagram shows the expression of CD177 in bone marrow and peripheral blood neutrophils in mice under homeostatic conditions: (A) Abundance of CD177 expression in mouse tissues and cells; (B) Abundance of CD177 expression in human immune cells; (C) Flow cytometry analysis of CD177 expression levels in mouse bone marrow and peripheral blood neutrophils, lymphocytes and monocytes.

[0024] Figure 3 The results showed that CD177 expression in neutrophils was significantly upregulated in ALI mice: (A, B) flow cytometry detection of CD177 expression and mean fluorescence intensity in peripheral blood of ALI mice; (C, D) flow cytometry detection of CD177 expression and mean fluorescence intensity in BALF of ALI mice; (E, F) flow cytometry detection of CD177 expression and mean fluorescence intensity in lung tissue of ALI mice; (GH) immunofluorescence detection of in situ abundance of CD177 in mouse lung tissue sections; all experiments were repeated three times, and representative images are shown, n=6.

[0025] Figure 4 The study demonstrated that CD177 knockout alleviated acute lung injury in mice: (A) flow cytometry analysis of CD177 knockout efficiency in neutrophils; (B) q-PCR analysis of CD177 knockout efficiency at the gene expression level; (C) survival experiments of CD177 knockout and littermate controls in an ALI model; (D) changes in body weight of CD177 knockout and littermate controls in an ALI model; (E) HE staining of lung tissue sections (400X); (F) pathological damage scores of lung tissue. Representative images were shown for all experiments, n≥6.

[0026] Figure 5 The results show that CD177 deficiency inhibits inflammatory cell infiltration in mouse lung tissue: (A) Quantitative analysis of total protein concentration in bronchoalveolar lavage fluid (BALF) using BCA; (B) Flow cytometry analysis of neutrophils in BALF and (C) Absolute count of macrophages; (D) Flow cytometry analysis of neutrophils and quantification in lung tissue, (E) Macrophages (F) and number (G); Representative images of all experiments are shown.

[0027] Figure 6 The study showed that CD177 deficiency reduces the release of inflammatory mediators in the lungs: ELISA was used to detect the protein levels of IL-1β (A), IL-6 (B), TNF-α (C), MPO (D), CXCL1 (E), and CXCL2 (F) in bronchoalveolar lavage fluid (BALF); q-PCR was used to detect the transcriptional levels of IL-1β (G), IL-6 (H), TNF-α (I), MPO (J), CXCL1 (K), and CXCL2 (L) in lung tissue; all experiments are presented with representative images, n≥5. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0029] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0030] Example 1

[0031] This embodiment of the experiment validated CD177 (GenBank: AB237911.1) as a candidate immune target for ARDS using data from public databases, ARDS patients, and mouse ARDS models: 1) First, analysis of the ARDS peripheral blood database showed that CD177 was the most differentially upregulated gene, consistent with the significantly increased plasma protein levels detected; 2) Similarly, the absolute specificity of CD177 expression on neutrophils under mouse homeostasis laid the foundation for subsequent CD177 gene editing; 3) CD177 expression was significantly upregulated in peripheral and lung tissue neutrophils of ARDS mice. Furthermore, by constructing knockout mice, we further clarified the promoting effect of CD177 on ARDS: the massive infiltration of neutrophils and the high level of inflammatory pathology in ARDS were largely salvaged by CD177 knockout. The results of this embodiment suggest that neutrophil CD177, as a specific, effective, and accurate potential target for ARDS, provides a basis for predicting the occurrence of ARDS and neutrophil-related inflammatory diseases. Specific research is as follows:

[0032] 1. Materials and Methods

[0033] 1.1 Experimental Materials

[0034] 1.1.1 Laboratory animals

[0035] Wild-type C57BL / 6J mice (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. CD177 knockout (KO) - CD177-KO mice were successfully constructed by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (formerly Nanjing Model Animal Center). In all experiments, the mice were matched for weight and age, and littermates of the same age and sex were used as controls for all CD177-KO mice. All mice were housed separately (N≤5) under specific pathogen-free (SPF) conditions, with constant temperature (23±3℃), constant relative humidity (65±5%), and a strict 12-hour day-night cycle. Mice had free access to food and water.

[0036] All operations were conducted in accordance with the Chongqing Science and Technology Commission's guidelines for animal research and were approved by the Animal Protection Committee of Chongqing Medical University. We made every effort to minimize the number of animals used and to reduce their suffering.

[0037] 1.1.2 Collection of Clinical Samples

[0038] This study was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (No. 2020-117). Informed consent was obtained from each participant upon enrollment. Bronchoalveolar lavage fluid and peripheral blood samples were collected from ALI / ARDS patients (acute exacerbation phase) at the Department of Respiratory Medicine, Jiangnan Campus of the Second Affiliated Hospital of Chongqing Medical University. Peripheral venous blood was collected within 24 hours of admission and placed in anticoagulant tubes, incubated at room temperature for 20 min, and centrifuged at 3000 rpm for 10 min. The supernatant plasma was collected and stored at -80℃.

[0039] 1.2 Main Experimental Reagents

[0040]

[0041] 1.3 Experimental Methods

[0042] 1.3.1 Establishment of a mouse model of lipopolysaccharide-induced acute lung injury

[0043] Lipopolysaccharide (LPS)-induced acute lung injury is a classic model of pulmonary inflammatory disease. Eight- to twelve-week-old, body-matched, same-sex wild-type or CD177 knockout mice (Jicui Pharmaceutical, China) were selected and stabilized in an SPF-grade environment for 5-7 days to avoid stress. 2% sodium pentobarbital was dissolved in D-PBS and vortexed until dissolved. Mice were anesthetized by intraperitoneal injection at 50 mg / kg. After deep anesthesia was confirmed by pupillary reflexes and respiration, the mice were placed in a prone position, and their airways were opened by suspending their teeth on a wire fixed to a surgical board. An endotracheal tube was inserted, and LPS dissolved in physiological saline was administered via the tube at 5 mg / kg body weight. Control mice received the same volume of sterile physiological saline. The mice were then held upright for 1 minute and placed in a warm environment to await awakening, thus completing the modeling process. Mice were anesthetized and euthanized 24 hours after modeling for subsequent testing.

[0044] 1.3.2 Extraction of bronchoalveolar lavage fluid from mice

[0045] Twenty-four hours after LPS modeling, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital based on their body weight. The mice were then fixed in a supine position on a board, with their teeth lifted and secured to the surgical board to expose as much of the neck as possible (head down). After disinfecting the neck hair with 75% alcohol, the neck tissue was dissected to expose the trachea, and a cotton suture was threaded under the tracheal tissue using hemostatic forceps. Next, a V-shaped incision (point down) was made in the trachea, and a 22G plastic endotracheal tube (approximately 5 cm) was inserted into the tracheal lumen approximately 0.5 cm, secured with sutures. Then, 0.5 ml of pre-chilled sterile PBS solution (containing 100 μM EDTA) was loaded into a 1 ml syringe. The loaded syringe was connected to the catheter, and the PBS solution was gently injected into the lungs. After all the saline solution had been injected, the solution was gently withdrawn while massaging the chest. The injection and retrieval of PBS should be gentle to maximize BALF fluid recovery (approximately 70%-80%), and the solution was kept on ice until needed. After centrifugation in BALF (800 g, 5 min), the supernatant was stored at -80℃ (for ELISA detection of inflammatory factors; the cell pellet was resuspended in sterile D-PBS and placed on ice for use in subsequent experiments such as cell counting or flow cytometry).

[0046] 1.3.3 Preparation of single-cell suspension from mouse lung tissue

[0047] The mouse was immobilized in a supine position with the chest cavity fully open. An incision was made in the right atrial appendage, and the heart was held in place with forceps. Then, 10 mL of pre-cooled PBS was injected into the left ventricle via a syringe and pushed in for perfusion. Successful perfusion was indicated by the lungs turning white. Because the lung tissue has a rich vascular system, this procedure aims to flush away intravascular immune cells not adhering to the lung tissue. The entire lung tissue was then removed and placed in an ice-cold petri dish containing D-PBS. The lung tissue was weighed and photographed. (Primary cell culture in a clean bench begins from this step.) Take lung tissue and wash three times with D-PBS solution containing 1% penicillin and antibiotics. Perform whole-lung tissue mincing and digestion (1 mg / mL type I collagenase: collagenase dissolved in 1640 empty culture medium containing 3% inactivated serum). Collect the minced lung tissue in the digestion solution and incubate at 37°C for 45 minutes to 1 hour, manually inverting and mixing the digestion solution every 10 minutes. After digestion, most of the lung tissue should settle to the bottom of the liquid, indicating complete digestion. Filter all digestion solution through a 70 μm filter to remove tissue. Centrifuge horizontally at 350 g for 5 min, wash twice with D-PBS, and centrifuge at 350 g for 5 min. Resuspend the cells in ammonium chloride-erythrocyte lysate buffer, lyse erythrocytes at room temperature for 3 min, terminate erythrocyte lysate 3 times the volume of D-PBS (Staining Buffer) containing 3% FBS, centrifuge at 350 g for 5 min, wash twice with D-PBS, and centrifuge at 350 g for 5 min. Resuspend in Staining Buffer. Place the buffer on ice until needed.

[0048] 1.3.4 Public Database Analysis

[0049] By searching published literature on ARDS and cross-referencing it with neutrophil-related function and inflammation as keywords, the GSE76293 database was ultimately selected. Differentially expressed genes were screened using the online tool GEO2R. The data were corrected for Benjamini & Hochberg (False Discovery Rate), and the cut-off criteria were set as follows: a corrected p-value less than 0.05 (adj. p < 0.05); and a fold change in gene expression greater than 2 (|logFC| > 2). Volcano plots of differentially expressed genes were generated using the ggplot2 package.

[0050] 1.3.5 Enzyme-Linked Immunosorbent Assay (ELISA)

[0051] Serum CD177 levels were measured using a commercially available ELISA kit (ThermoFisher, EH80RB) according to the manufacturer's instructions.

[0052] 1.3.6 Experiment on isolation and extraction of mouse bone marrow neutrophils

[0053] 1) Kill mice aged approximately 8-12 weeks by cervical dislocation and soak them in 75% alcohol for about 1 minute.

[0054] 2) Deglove the skin of the mouse's leg along the ankle joint, cut the ligaments, remove the muscles, separate the mouse's humerus, femur and tibia, and wash twice in sterile phosphate buffered saline (PBS).

[0055] 3) Mix Percoll stock solution with 10×D-Hanks solution at a volume ratio of 9:1, and label the mixture as 100% Percoll solution. Dilute with D-Hanks solution to obtain 45%, 60%, and 81% Percoll solutions.

[0056] 4) After cutting open both ends of the joint, use a sterile syringe to draw 1 mL of Hanks-1 solution to flush the bone marrow cavity. Collect all the solution into a 15 mL centrifuge tube, centrifuge at 500 g for 5 min, and discard the supernatant. Add 5 mL of erythrocyte lysis buffer, pipette the bone marrow into a single-cell suspension, lyse at room temperature for 5 min, add 10 mL of Hanks-1 solution to stop lysis, centrifuge at 500 g for 5 min at room temperature, remove the supernatant, wash the cells once more with 10 mL of Hanks-1 solution, and resuspend the cells in 45% Percoll solution.

[0057] 5) Slowly add 3 mL of 81% Percoll solution and 4.5 mL of 60% Percoll solution sequentially to a 15 mL centrifuge tube. Then, slowly add a 45% Percoll suspension containing cells to the top layer. A clear boundary line should be visible between the three different concentrations. Centrifuge at 1500 g for 30 min with zero acceleration / deceleration. Neutrophils should be located between the 60% and 81% Percoll solution layers.

[0058] 6) Aspirate the neutrophil layer and resuspend the cells in 15 mL of Hanks-1 solution. Centrifuge the cell suspension at 1250 g for 5 min. Remove the supernatant and wash the cells once more with 15 mL of D-Hanks solution. Centrifuge at 750 g for 5 min and remove the supernatant to obtain mouse bone marrow neutrophils. Count the cells using a cell counter for later use.

[0059] 1.3.7 Isolation and extraction of mouse neutrophils

[0060] Mouse bone marrow single-cell suspensions were obtained using the method described above. Percoll stock solution was mixed with 10×D-Hanks solution at a volume ratio of 9:1, labeled as 100% Percoll solution. D-Hanks solution was added for dilution to obtain 45%, 60%, and 81% Percoll solutions. Bone marrow single-cell suspensions from two mice in the same experimental group were mixed and then centrifuged at 500 g for 5 min at room temperature. After centrifugation, the supernatant was discarded, and the cells were washed once with 10 mL of Hanks-1 solution, followed by centrifugation at 500 g for 5 min at room temperature. The cells were resuspended in 45% Percoll solution after centrifugation. 81% and 60% Percoll solutions were slowly added sequentially to 15 mL centrifuge tubes, and a 45% Percoll suspension containing bone marrow cells was slowly added to the top layer. A clear boundary line was visible between the three different concentrations. Centrifugation was performed at 1500 g for 30 min with zero acceleration / deceleration; neutrophils were located between the 60% and 81% Percoll solution layers. Aspirate the neutrophil layer and resuspend the cells in 15 mL of Hanks-1 solution. Centrifuge the cell suspension at 1250 g for 5 min. Remove the supernatant and wash the cells once more with 15 mL of D-Hanks solution. Centrifuge at 750 g for 5 min and remove the supernatant to obtain bone marrow neutrophils.

[0061] 1.3.8 Flow cytometry detection of neutrophil surface proteins

[0062] The cells to be tested were diluted to 1-2 × 10⁻⁶. 6Cells were incubated in 100 μL of purified anti-mouse CD16 / CD32 antibody (Mouse BD Fc Block) in staining buffer (1×PBS containing 2% FBS). Fluorescein-conjugated antibodies were used to label cell surface antigens, including FITC-anti-mouse CD45 (0.5 mg / mL), PE-anti-mouse Ly6G (0.2 mg / mL), APC-anti-mouse CD11b (0.2 mg / mL), and Alexa Fluor 647-anti-mouse CD177 (0.2 mg / mL). Diluted antibodies were added to EP tubes containing cells and incubated at 4°C in the dark for 30 min, mixing every 10 min during incubation. After incubation, 1 mL of analysis buffer was added to wash away excess fluorescent dye. Cells were centrifuged at 500 g at 4°C for 5 min to remove the supernatant and collect the cells. Cells were resuspended in 300 μL of analysis buffer and stored on ice in the dark for analysis. Stained cells were analyzed using a Meet NL-CLC flow cytometer (Cytek Biosciences), a Guava easyCyte system (Luminex Corporation), or a CytoFLEX system (Beckman Coulter). Data were analyzed using FlowJo software (Tree Star).

[0063] 1.3.9 Real-time quantitative PCR

[0064] 1) Total RNA extraction from cells: After washing the collected bone marrow neutrophils twice with D-PBS to remove serum or other impurities, each 2×10⁶ cells were extracted. 6 Add 1 mL of TRIzol to each cell and repeatedly pipette to dissolve it. Store at -80°C.

[0065] 2) Total RNA extraction from mouse lung tissue: Weigh 10-20 mg of lung tissue using a precision balance. After washing away excess blood with D-PBS, blot dry on sterile filter paper. Add 300 μL of TRIzol to disperse the tissue, then homogenize the lung tissue using an IKA electric tissue homogenizer. The entire process is performed on ice. After homogenization, add another 700 μL of TRIzol and mix well. Incubate at room temperature for 5 min to allow for complete tissue lysis. Centrifuge at 13400 g for 15 min, collect the supernatant on ice for subsequent operations.

[0066] 3) Melt the TRIzol solution containing cells or tissues on ice, add 200 μL of chloroform, vortex, centrifuge briefly using a microcentrifuge, and let stand at room temperature for 10 min.

[0067] 4) After standing, the layers will be visible. Centrifuge at 12,000 rpm and 4℃ for 15 min. Transfer the upper aqueous phase to a 1.5 mL enzyme-free EP tube, add an equal volume of isopropanol, invert and mix well, and then place in a -20℃ refrigerator for 30 min.

[0068] 5) Centrifuge at 12000 rpm, 4℃ for 15 min. A white precipitate will be visible at the bottom of the tube. Aspirate the supernatant, add 75% ethanol prepared with DEPC (diethylpyrocarbonate, DEPC) and water, gently tap the precipitate, centrifuge at 7500 g, 4℃ for 10 min, remove the supernatant, add DEPC and water again to prepare 75% anhydrous ethanol, gently tap the precipitate, centrifuge at 7500 rpm, 4℃ for 10 min, remove the supernatant to obtain the RNA precipitate.

[0069] 6) Turn on the ultra-clean table fan and open the EP tube in the table to air dry the sediment until it becomes transparent. Add 25μL of enzyme-free water and blow to fully dissolve the sediment.

[0070] 7) Determine RNA concentration using Nanodrop2000: Prepare a 2% agarose gel, replace the electrophoresis buffer with freshly prepared 1×TAE, mix 1 μL of RNA solution with 1 μL of gel loading, electrophoresis at 130V for 8 minutes, and observe the RNA bands using a UV gel imaging system. If the result shows three bands without dragging, then this RNA can be used for subsequent experiments.

[0071] 8) Add 3 μL of 5 gDNA digester buffer and 12 μL of RNA solution to a 200 μL PCR tube, mix well, and incubate at 42℃ for 2 min to remove gDNA.

[0072] 9) Add 5 μL of 4×SuperRT-Mix (containing gDNA digester inhibitor) to the one-step RNA product, mix well by pipetting, and then centrifuge by jogging.

[0073] 10) Perform reverse transcription according to the following procedure: 25℃, 5 min; 55℃, 15 min; 85℃, 2 min. After reverse transcription is completed, add 40 μL of enzyme-free water to dilute the cDNA. Store the PCR product at -20℃ for later use.

[0074] 11) Perform real-time quantitative PCR amplification according to the following system and procedure:

[0075] PCR system: 2×Sybr 5 μL, primer F (10 μM) / primer R (10 μM) 0.2 μL each, cDNA 4.6 μL.

[0076] PCR program: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, 40 cycles; 95℃ for 15s, 60℃ for 60s, 95℃ for 15s, and store at 12℃.

[0077] 12) Data statistics are performed according to Formula 2. –ΔΔCT Perform the calculation.

[0078] 1.3.10 Preparation of mouse spleen single-cell suspension and peripheral leukocyte suspension

[0079] 1) Isolation of single cells from mouse spleen: After cervical dislocation of mice, spleen tissue was harvested while maintaining the integrity of the spleen's outer membrane. The spleen was then placed on ice, and ice-cold D-PBS was added. The membrane was torn open, and the spleen was cut into tissue pieces with scissors. Cells were released by gently grinding the tissue with the end of a syringe. The membrane was filtered through a 70μm filter to obtain a single-cell suspension. After centrifugation, the cell pellet was obtained. Red blood cell lysis was performed in the same manner as for lung tissue. The cells were washed twice with D-PBS and resuspended on ice for later use.

[0080] 2) Preparation of mouse peripheral blood leukocyte suspension: After anesthetizing mice, blood was collected from the tail vein into an anticoagulant blood collection tube containing EDTA (EDTA content accounted for 10% of the blood volume). After inverting and mixing, 10 times the volume of lysin was added, and the mixture was mixed by pipetting. The mixture was then lysed at room temperature for 5 min, centrifuged at 400 g for 5 min, washed twice with D-PBS, and resuspended in D-PBS on ice for later use.

[0081] 1.3.11 Detection of mouse bone marrow neutrophil viability

[0082] Myeloid granulocytes were cultured for 3 h in the presence of 100 ng / mL lipopolysaccharide (LPS). After 3 h, the cells were washed. Apoptosis levels were detected using a fluorescein-labeled membrane protein V antibody (Annexin V) and a 7-AAD double staining method.

[0083] 1.3.12 Statistical Methods

[0084] All data are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 9.2.0 software. Comparisons between groups were performed using the two-tailed Students't test, one-way ANOVA with Tukey's post-hoc test, or two-way repeated measures ANOVA with Bonferroni's post-hoc test. Statistical significance was indicated by *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, respectively.

[0085] 2 Results

[0086] 2.1 CD177 expression was significantly upregulated in ARDS patients.

[0087] LPS- or bacterial-induced lung inflammation is a classic inflammatory model for studying the role of neutrophils in innate immune regulation. Therefore, to explore the potential role of neutrophil CD177 in inflammatory states, we chose LPS-induced ARDS as the primary disease model for our study.

[0088] First, through analysis of publicly available blood neutrophil microarray data (GSE76293), we found that CD177 was the differentially expressed gene with the highest upregulation rate in ARDS patients compared to healthy volunteers. Figure 1 A). Subsequently, we collected peripheral blood from ARDS patients and healthy volunteers, and flow cytometry analysis revealed that the expression level of CD177 was significantly upregulated in neutrophils in the peripheral blood of ARDS patients. Figure 1 B), in addition, plasma CD177 protein levels are also significantly upregulated in ARDS patients ( Figure 1 C).

[0089] To further confirm the upregulation level of CD177 in the inflammatory state of ARDS, we analyzed a published microarray dataset (GSE2322). Neutrophils were extracted from peripheral blood and bronchoalveolar lavage fluid (BALF) before and after LPS stimulation, and the changes in CD177 expression before and after stimulation were analyzed. The results showed that compared with unstimulated neutrophils, CD177 expression in peripheral blood and BALF neutrophils was significantly upregulated after LPS stimulation. Figure 1 (D, E) This indicates that neutrophil CD177 plays an important role in responding to lung infection.

[0090] 2.2 CD177-specific expression in mouse neutrophils

[0091] CD177 encodes a GPI-anchored neutrophil surface glycoprotein that plays a role in neutrophil activation. Generally, approximately 20%–80% of circulating neutrophils are CD177-positive, while 3%–5% of the population lacks CD177-positive neutrophils. However, individuals lacking CD177 expression still exhibit normal neutrophil counts and normal immune defense functions. Based on the above research on CD177 in human neutrophils, the function of CD177 in neutrophil biology remains largely unknown.

[0092] Therefore, we first clarified the expression specificity of CD177 in mouse immune cells. Through BioGPS and Immgen screening comparisons, we found that CD177 is also specifically expressed in bone marrow and peripheral granulocytes in both humans and mice. Figure 2 A, B). Flow cytometry analysis of CD177 expression in mouse bone marrow and peripheral blood revealed that CD177 was specifically highly expressed on neutrophils, but not on lymphocytes and monocytes. Furthermore, our results showed that CD177 expression was higher on mouse peripheral neutrophils than on bone marrow (…). Figure 2 (C) This result is consistent with the reported identification of CD177 as a marker of neutrophil activation.

[0093] 2.3 CD177 expression is upregulated in ARDS mice

[0094] To further investigate the specific mechanism of action of neutrophil CD177 in lung inflammation, we first established a mouse ARDS model using intratracheal instillation of LPS, while the control group received an equal volume of sterile saline. After 24 h, flow cytometry results showed that CD177 was specifically and highly expressed in BALF, lung, and peripheral blood neutrophils; and compared with PBS, the protein level of CD177 on the surface of peripheral blood neutrophils in the LPS group was significantly upregulated. Figure 3 A, B), and simultaneously, a large number of neutrophils recruited inflammatoryally from bronchoalveolar lavage fluid (BALF) and lung tissue were strongly positive for CD177. Figure 3 Immunofluorescence results of mouse lung tissue sections showed that the protein level of CD177 was significantly increased in the lung tissue of ARDS mice. Figure 3 G,H).

[0095] 2.4 CD177 deficiency alleviates pathological damage in ARDS mice

[0096] The CD177 gene contains two transcripts. Exons 1-9 (ENSMUST00000063956.6) contain the transcription start codon ATG, which is the region edited and knocked out using CRISPR / Cas9 tools. To further clarify the impact of CD177 on the progression of ARDS, we successfully constructed CD177 knockout mice using CRISPR / Cas9 gene editing technology.

[0097] First, we performed flow cytometry and q-PCR on bone marrow cells from CD177 knockout mice and control mice. The results showed that CD177 was completely absent in neutrophils. Figure 4 A, B). Further survival experiments showed that CD177 knockout significantly improved the survival rate of ARDS mice compared to wild-type mice. Figure 4 C), CD177 knockout mice experienced less weight loss within 2 days after LPS modeling ( Figure 4 D); and the results of HE staining of lung tissue and lung injury scoring showed that CD177 knockout alleviated the pathological damage of lung tissue in ARDS mice ( Figure 4 E,F).

[0098] 2.5 CD177 deficiency improves lung immune cell infiltration in ARDS.

[0099] The essence of ARDS lung injury is a continuously amplified and uncontrolled inflammatory response, characterized by diffuse alveolar damage and predominantly neutrophil-mediated immune cell infiltration. Therefore, we further analyzed the effect of CD177 deficiency on neutrophil and macrophage infiltration in mouse ARDS lung tissue. Lung tissue and BALF were collected 24 hours after LPS modeling for flow cytometry analysis. Results showed that compared with the control group, CD177 deficiency reduced protein concentration in BALF, suggesting that CD177 deficiency alleviates the excessive increase in alveolar epithelial permeability induced by LPS. Figure 5 A); and the level of neutrophils infiltrating BALF was suppressed by CD177 deficiency ( Figure 5 B, C). Furthermore, the number of neutrophils and macrophages in the lung tissue of ARDS patients with CD177 deficiency was also significantly reduced. Figure 5 (DG), suggesting that CD177 deficiency significantly inhibits the infiltration of neutrophils and macrophages in the lungs of ARDS.

[0100] 2.6 CD177 deficiency inhibits the release of inflammatory mediators in the lungs of ARDS patients.

[0101] Pulmonary and systemic inflammation is a prominent feature of ARDS and a major cause of damage to lung epithelial and endothelial cells. Currently, clinical treatment often involves neutralizing inflammatory cytokines such as IL-6 and IL-1β with antibodies, but the efficacy is not ideal. Therefore, there is a need to identify more precise cytokine targets to achieve more accurate immunotherapy.

[0102] To investigate the impact of CD177 deficiency on inflammatory mediators in ARDS, we used ELISA to detect the protein levels of inflammatory mediators in BALF: IL-1β, IL-6, TNF-α, CXCL1, CXCL2 (neutrophil chemokines), and MPO (neutrophil myeloperoxidase). The results showed that CD177 deficiency significantly reduced the protein levels of these multiple inflammatory mediators and chemokines in BALF. Figure 6 Consistent with this, q-PCR analysis also showed significantly reduced transcriptional levels of IL-1β, IL-6, TNF-α, CXCL1, and CXCL2 in lung tissue, while MPO showed no significant difference. Figure 6 (GL). This suggests that CD177 deficiency reduces the overall level of inflammation in lung tissue.

[0103] 3. Analysis and Summary

[0104] CD177 is a polymorphic gene known to be associated with several important clinical diseases, including polycythemia vera, Wegener's granulomatosis, and neonatal neutropenia. The CD177 / NB1 glycoprotein was initially identified in several cases of neonatal neutropenia, where maternal antibodies specifically responded to the neonatal neutrophil-specific epitope HNA-2a derived from the NB1 glycoprotein. In several cases of transfusion-related acute lung injury, CD177-specific antibodies from donor blood were identified as the primary cause. Furthermore, several clinical studies have identified CD177 as a diagnostic marker for various myeloproliferative disorders, including thrombocytosis and idiopathic myelofibrosis. CD177 is expressed on neutrophil subsets as a 58-64 kDa glycophosphatidylinositol (GPI)-linked N-glycosylated cell membrane surface protein. Generally, 20%-80% of circulating neutrophils are CD177 positive, while 3%-5% of the population lacks CD177+ neutrophils. Despite such high individual heterogeneity and polymorphism, circulating CD177... + The percentage of neutrophils is constant in the same individual, while CD177 mRNA and protein expression increase rapidly in response to inflammatory stimuli.

[0105] ARDS (Acute Respiratory Disorders) is a clinical syndrome characterized by acute respiratory failure, resulting from lung tissue damage caused by various intrapulmonary and extrapulmonary factors. Although considerable experience has been accumulated in the past fifty years regarding its pathogenesis and life support techniques, the mortality rate remains as high as approximately 40%. This is inextricably linked to the difficulty in correcting refractory hypoxemia caused by damage to vascular endothelial cells and alveolar epithelial cells. The inflammatory response triggered by excessive neutrophil activation, ultimately leading to indiscriminate attack on various cells, is the central link in this process. However, previous interventions at various stages after neutrophil activation have not yielded satisfactory clinical benefits. Therefore, identifying key neutrophil molecules as intervention targets for ARDS is an urgent task.

[0106] In this study, we clarified the promoting role of the neutrophil molecule CD177 in ARDS and investigated its effects on inflammatory cells and mediators in the lungs by constructing a CD177 knockout mouse ARDS model. Under physiological conditions, CD177 is still expressed only on neutrophils, not on monocytes or lymphocytes, and almost all neutrophils express CD177, unlike the heterogeneity of CD177 expression on human neutrophils. First, we identified CD177 as the most significantly upregulated gene during lung inflammation through analysis of public databases. Furthermore, the expression of CD177 protein in peripheral blood and on neutrophils in ARDS patients was significantly higher than in healthy volunteers. Similarly, in the mouse ARDS model, after 24 hours of LPS infusion via the trachea, CD177 expression in neutrophils in BALF, lung tissue, and peripheral blood showed significant upregulation. This indicates that CD177 expression on the neutrophil membrane surface increases in response to inflammatory stimulation. Although previous studies have reported that granulocyte colony-stimulating factor (G-CSF) strongly induces CD177 expression, CD177 expression is upregulated during pregnancy and severe bacterial infections. Our results indicate that CD177 upregulation is most significant under inflammatory stimulation, suggesting that CD177 responds most rapidly to inflammation. This may be related to its primary localization on the surface of the neutrophil cell membrane and granule inner membrane, allowing for rapid and direct eversion upon stimulation without de novo transcriptional synthesis. This supports the possibility that CD177 can rapidly participate in the regulation of acute inflammatory diseases.

[0107] To further explore the potential molecular mechanisms by which CD177 regulates ARDS, we constructed a CD177 gene-deficient mouse model. Notably, approximately 10% of peripheral blood neutrophils in normal individuals do not express CD177, suggesting that CD177 is not essential for development. Consistently, CD177 gene knockout in mice does not affect the normal development of various organs and immune cells. We found that compared to wild-type mice, CD177 knockout mice, after LPS-induced ARDS modeling via intratracheal injection, had longer survival times, less weight loss, and lower degrees of lung tissue pathological damage. CD177 knockout mice also exhibited decreased lung epithelial permeability, reduced neutrophil recruitment in the lungs, and decreased levels of IL-1β, IL-6, TNF-α, MPO (neutrophil myeloperoxidase), and CXCL1 and CXCL2 (neutrophil chemokine) secretion in BALF. Previous studies have shown that CD177 mediates neutrophil migration within endothelial cells through its interaction with proteinase 3. Furthermore, CD177 acts as an adhesion molecule, binding to Mac-1 and recognizing the platelet endothelial adhesion molecule PECAM-1 (CD31). These in vitro studies have demonstrated the role of CD177 in neutrophil migration. However, a recent study showed that CD177 and CD177-positive neutrophils accumulate similarly in the peritoneal cavity of human peritonitis patients, suggesting that CD177 expression in neutrophils does not provide an advantage for migration to this site. In summary, although CD177 / NB1 was identified in 1971, research on the biological functions of CD177 in neutrophil recruitment remains largely lacking. Therefore, considering our observation that CD177 deficiency leads to reduced lung injury in mice accompanied by a significant decrease in neutrophil infiltration, we further systematically explored the impact of CD177 on various aspects of neutrophil recruitment using in vivo mouse models and in vitro functional experiments with primary neutrophils. Experiments on neutrophil adhesion, stretching deformation, polarization, and chemotaxis, combined with adoptive migration experiments in neutrophil peritonitis and lung injury, revealed that CD177 does not participate in the regulation of autonomous inflammatory recruitment of neutrophils. Furthermore, CD177 has no significant effect on neutrophil phagocytosis or the formation of in vitro neutrophil traps (NETs), except for the release of reactive oxygen species (ROS). This indicates that other inflammatory mediators are involved in the regulation of neutrophil recruitment within the CD177-mediated mouse lung injury microenvironment, providing clues for further in-depth investigation.

[0108] Subsequently, we conducted further research, including: We used single-cell sequencing to analyze the immune landscape of neutrophil CD177-mediated ALI / ARDS, aiming to explore the impact of CD177 as a therapeutic target on immune cell-mediated microenvironmental changes and downstream immune molecules, thereby intervening in the pathological progression of ALI / ARDS. The main results of this subsequent study are as follows:

[0109] I. Effects of CD177 on inflammatory recruitment function of neutrophils

[0110] 1. Flow chamber experiments showed that CD177 deficiency does not affect the ability of neutrophils to adhere firmly to the surface of inflammatory-activated endothelial cells.

[0111] 2. Experimental results on stretching deformation showed that CD177 deficiency did not affect neutrophil FN-mediated stretching deformation, but it impaired the proportion of PK-dependent cell attachment and showed a smaller stretching area under f-MLF stimulation.

[0112] 3. CD177 deficiency does not affect neutrophil polarization levels, even in the presence of f-MLF.

[0113] 4. CD177 deficiency does not affect the in vitro chemotaxis of neutrophils. In the presence of the f-MLF chemokine gradient, there were no significant differences in the speed, distance, and angle of directional chemotaxis between WT and CD177-KO neutrophils in vitro.

[0114] 5. CD177 deficiency had no effect on the proportion and number of neutrophils adoptively migrating to the lungs in an acute lung injury model.

[0115] 6. CD177 deficiency impairs the release level of ROS in neutrophils.

[0116] 7. CD177 deficiency did not significantly affect the phagocytic capacity of neutrophils against bacteria in vitro.

[0117] 8. CD177 deficiency has no effect on PMA-induced neutrophil NETs formation.

[0118] II. CD177 regulates the IL-23 / IL-17 signaling axis in ALI, affecting neutrophil recruitment in ARDS.

[0119] 1. Analysis of public databases showed that cytokine-mediated signaling pathways, especially the IL-17 signaling pathway, were significantly enriched in transcripts from patients with high CD177 expression in the blood; meanwhile, flow cytometry results showed that CD177 deficiency significantly reduced IL-17A levels in mouse ALI lung tissue.

[0120] 2. The main source of IL-17A in the lung tissue of ALI mice is lymphocytes, especially pILC3 cells; while CD177 deficiency significantly inhibits the proportion and number of IL-17A+ lymphocytes in the lung tissue, and further analysis shows that the proportion of pILC3 is also significantly reduced.

[0121] 3. CD177 deletion significantly reduced the expression level of IL-23 in lung tissue; and significantly downregulated the transcriptional and protein expression levels of key pathway molecules STAT3, RORγt, and IL-23R, the functional receptor of IL-23, in lung tissue; at the same time, the results showed that the transcriptional levels of IL-17A and IL-22 were also inhibited.

[0122] 4. Supplementation with exogenous recombinant IL-23 and recombinant IL-17 can promote the lung tissue infiltration capacity of neutrophils, IL-17A+ lymphocytes and pILC3 in a CD177-deficient mouse ALI model, and enhance alveolar permeability, accompanied by more severe weight loss.

[0123] 5. Supplementation with exogenous recombinant IL-23 and recombinant IL-17 can upregulate the protein levels of IL-23 and IL-17A in the lung tissue of CD177-deficient mouse ALI model; as well as the protein levels of CXCL1, CXCL2, TNF-α and IL-6 in BALF; and aggravate the pathological damage of lung tissue.

[0124] III. CD177 regulates neutrophil recruitment mediated by the IL-23 / IL-17 signaling axis by modulating dendritic cell maturation.

[0125] 1. CD177 deficiency can specifically reduce the transluminal activity of CD11c+MHCII+ in mouse ALI lung tissue.

[0126] The proportion and absolute number of CD24+ dendritic cells; but it has no effect on the phenotypic differentiation of dendritic cell cDC1 and cDC2.

[0127] 2. CD177 deficiency has no effect on dendritic cells in the spleen, but significantly reduces the proportion of dendritic cell infiltration in MedLN.

[0128] 3. CD177 deficiency has no effect on the transvascular recruitment ability of exogenous BMDCs to lung tissue in the ALI model.

[0129] 4. CD177 deletion significantly reduced the expression levels of maturation markers CCR7, CD40, CD80 and MHCII on the surface of dendritic cells in lung tissue in a mouse ALI model.

[0130] 5. CD177 deficiency significantly reduced the expression levels of CD40 and CD80, maturation markers on the surface of BMDCs, after co-culturing BMDCs with WT and CD177-KO neutrophils under in vitro inflammatory conditions; it also inhibited the level of IL-23 secreted by BMDCs and the phosphorylation level of AKT / NF-κB.

[0131] 6. Intrapulmonary infusion of in vitro activated mature BMDCs promoted the infiltration of neutrophils, IL-17A+ leukocytes and type 3 innate lymphocytes pILC3 in the lung tissue of CD177-deficient ALI mice, enhanced alveolar permeability, and resulted in a greater reduction in body weight.

[0132] 7. Intrapulmonary infusion of in vitro activated mature BMDCs promoted the development of CD177-deficient ALI mice, upregulated the protein levels of IL-23-IL-17A in the lung tissue of CD177 knockout mice, and the protein levels of CXCL1, CXCL2, TNF-α and IL-6 in BALF; and aggravated the pathological damage of lung tissue.

[0133] The research results of this invention show that:

[0134] In addition to autonomous functional responses, neutrophil recruitment during inflammation requires interactions with endothelial cells to mediate a series of "exudative" behaviors, key of which include the expression of cell surface adhesion molecules and the release of chemokines in the microenvironment. IL-17, as a chemokine mediating neutrophil recruitment, can further amplify the chemotactic effect by promoting the production of CXCL1, CXCL2, and other similar molecules. This is consistent with our findings. The reduction in CXCL1 / CXCL2 due to CD177 deficiency is due to decreased IL-17A secretion levels in mouse lung tissue. This is dependent on the reduced number of ILC3 cells, which are less responsive to IL-23 signaling, leading to a decrease in the IL-23 / IL-17 signaling cascade and impaired neutrophil recruitment. Furthermore, we found that dendritic cells, the main regulatory cells of IL-23 / IL-17 signaling, have reduced infiltration in lung tissue. At the same time, the expression of their surface activation signals, CD40 / CD80 / MHCII, is also weakened, accompanied by decreased lymph node homing. This ultimately leads to the inhibition of a series of dendritic cell responses and activations under the molecular pattern of pathogen damage, thereby blocking the IL-23 / IL-17 signaling effect on neutrophil recruitment.

[0135] In summary, CD177 serves as a redundant marker for neutrophil development under homeostatic conditions, but it is crucial for neutrophil and mediated innate immune regulation during inflammation. In this study, CD177, in a mouse ARDS model, cascaded its regulation of dendritic cell maturation and homing, mediating the downstream IL-23 / IL-17 pathway, and ultimately reducing neutrophil recruitment to inflammatory tissues. This suggests that CD177's regulation of innate immunity is not limited to ARDS but may include various inflammatory diseases involving neutrophils. Furthermore, we further refined our understanding of the impact of CD177 on neutrophil biological function through various experimental methods. Therefore, our research on the mechanism of CD177-mediated ARDS enriches the neutrophil-involved inflammatory regulatory network and suggests that CD177 could be an ideal target for ARDS treatment, reducing leukocyte recruitment under uncontrolled inflammation without compromising host defense mechanisms, thus promoting the prognosis of inflammatory diseases.

Claims

1. Application of reagents for detecting CD177 in the preparation of diagnostic tools for inflammatory lung injury or neutrophil-related inflammatory diseases.

2. Application of CD177 as a target in screening drugs for the treatment of inflammatory lung injury or neutrophil-related inflammatory diseases.

3. The application according to claim 2, characterized in that: CD177 serves as an immune target.

4. The application according to claim 1 or 2, characterized in that: The CD177 mentioned is the neutrophil CD177.

5. The application according to claim 4, characterized in that: The CD177 mentioned refers to CD177 in neutrophils found in peripheral blood, bronchoalveolar lavage fluid, and lung tissue.

6. The application according to claim 1 or 2, characterized in that: The inflammatory lung injury or neutrophil-associated inflammatory disease includes acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).

7. The application according to claim 2, characterized in that: The drug includes at least one of the following functions: (1) Reduce alveolar permeability and alleviate neutrophil infiltration in lung tissue; (2) Reduces the transcriptional and protein levels of inflammatory cytokines IL-1β, IL-6, TNF-α, CXCL1 and CXCL2 in lung tissue; (3) Reduce the level of pathological damage to lung tissue, delay weight loss and prolong survival.

8. A reagent for interfering with or inhibiting CD177 expression, characterized in that: The reagent is a protein, compound, or nucleic acid molecule.

9. The reagent according to claim 8, characterized in that: The reagent is a nucleic acid molecule shRNA, siRNA, or sgRNA that interferes with or inhibits CD177 expression.

10. A medicament for treating inflammatory lung injury or neutrophil-associated inflammatory diseases, characterized in that: The drug comprises the reagent as described in claim 8 or 9.

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