Application of METTL3 inhibitor in preparation of medicine for enhancing antibacterial immunity of lung

Abstract

The invention relates to the technical field of biological medicines, in particular to application of an METTL3 inhibitor in preparation of a medicine for enhancing antibacterial immunity of lungs. The m6A modification level of the AMs is regulated and controlled through an active immune enhancement scheme based on in-vivo pretreatment of an m6A inhibitor (STC-15), so that the ability of the AMs to swallow and remove bacteria is enhanced, and a brand-new host-oriented treatment thought is provided for resisting pulmonary infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of METTL3 inhibitors in the preparation of drugs that enhance pulmonary antibacterial immunity. Background Technology

[0002] Respiratory and pulmonary infections pose a significant threat to global health. The diverse and easily mutated pathogens they cause present serious challenges to existing pathogen-targeting treatment strategies. Alveolar macrophages (AMs), as a core component and first line of defense of the lung's innate immune system, play an irreplaceable and crucial role in maintaining pulmonary homeostasis and resisting respiratory infections. The functional state of AMs is the "critical trigger" and "central controller" determining the outcome of pulmonary infections. Well-functioning and appropriately responsive AMs can rapidly control and clear pathogens, achieving a perfect balance between immune silencing and tissue homeostasis. Conversely, functionally suppressed, depleted, or dysregulated AMs can lead to uncontrolled pathogen replication and excessive inflammation, potentially developing into severe pneumonia, sepsis, or even respiratory failure. Therefore, developing therapeutic strategies that enhance AMs' antimicrobial phagocytosis, optimize their inflammatory output, or prevent their immune depletion has become a novel and highly attractive therapeutic target for combating respiratory pathogens such as drug-resistant bacteria, influenza viruses, and the novel coronavirus. Empowering AMs through pharmacological or immunological means holds promise for paving the way for next-generation broad-spectrum anti-lung infection therapies.

[0003] In recent years, m6A RNA methylation modification has been confirmed as an important regulator of immune responses, affecting macrophage inflammatory activation. However, existing research is mostly limited to conventional macrophage models, and its specific regulatory mechanism on alveolar macrophages (AMs) in lung antibacterial infections remains a blank. Whether and how m6A RNA methylation modification specifically regulates the immune function of this special cell subset in lung antibacterial infections is unclear. Research on the role of m6A modification in tissue-specific macrophage function is limited. Current studies on m6A regulation of macrophages mainly focus on cultured bone marrow-derived macrophages (BMDM) or cell lines. These models cannot fully simulate the unique developmental origin, tissue microenvironment, and functional characteristics of in situ tissue-resident macrophages (such as alveolar macrophages). Whether m6A modification has a specific regulatory function in AMs has not been reported. Existing research focuses on revealing the basic regulatory mechanisms of m6A in immunity, but there is very little research on translating it into an active immune enhancement strategy, especially through pharmacological interventions, such as using m6A inhibitors to directly target the lungs to enhance host defense capabilities. The possibility of "empowering" pulmonary endothelial cells (AMs) by regulating m6A levels to enhance their phagocytic and bactericidal capabilities is a therapeutic concept worth exploring. Faced with the increasingly serious problem of antibiotic resistance, the medical community urgently needs a "host-directed therapy" that does not directly target pathogens but instead enhances the host's own immunity to combat infection. Currently, there is a lack of drugs or methods that can specifically and efficiently activate local innate immunity in the lungs without causing excessive systemic inflammatory responses.

[0004] In-depth research into the functional mechanisms of m6A modification in alveolar macrophages (AMs) is not only of significant scientific importance but also provides new directions for developing novel immune interventions for respiratory infections. Therefore, there is an urgent need to develop a novel broad-spectrum therapeutic strategy that enhances the host's innate immune response, particularly the function of alveolar resident macrophages (AMs). Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a scheme to target lung m6A modification to enhance the lung's innate antibacterial immunity.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the use of METTL3 inhibitors in the preparation of drugs that enhance pulmonary antibacterial immunity.

[0007] Alveolar macrophages (AMs), as the core of pulmonary immune defense, play a crucial role in controlling infection through multiple mechanisms, including pathogen recognition, phagocytic clearance, inflammation regulation, and maintenance of tissue homeostasis. This invention demonstrates through in vivo experiments that pretreatment of the lungs with a specific inhibitor of m6A methyltransferase METTL3 (STC-15) via intratracheal infusion can serve as an active immune intervention strategy, pre-training and "empowering" alveolar macrophages (AMs), thereby significantly enhancing the host's defense capabilities against bacterial infection. This represents a novel concept and therapeutic approach of "immune enhancement." This invention, through multi-dimensional in vitro and in vivo experiments, confirms the remarkable effects of STC-15 pretreatment: significantly reducing bacterial load, effectively reducing the recruitment level of pro-inflammatory effector cells in the lungs, and alleviating infection-induced tissue damage. This indicates that enhancing early immune clearance can effectively prevent pathological damage to lung tissue caused by excessive inflammation in the later stages of infection, thus facilitating prognosis and recovery.

[0008] This invention provides a host-guided therapy that does not rely on direct bacterial killing. Current anti-infective treatments mainly rely on antibiotics to directly kill pathogens, which easily leads to drug resistance. The strategy of this invention does not directly target pathogens, but rather indirectly eliminates infection by modulating the function of the host's own immune cells. By inhibiting METTL3 and reducing m6A modification levels, the post-transcriptional regulatory network of immune-related genes in AMs is altered, thereby enhancing their phagocytic and bactericidal abilities. This host-guided therapy offers a promising alternative to address the increasingly serious problem of antibiotic resistance, and is particularly suitable for treating infections caused by drug-resistant bacteria such as multidrug-resistant Pseudomonas aeruginosa.

[0009] This invention not only has significant scientific value for a deeper understanding of the post-transcriptional regulatory mechanisms of pulmonary immune defense, but also provides a solid theoretical basis and new drug targets for developing novel broad-spectrum treatment strategies against pulmonary bacterial infections.

[0010] Preferably, the METTL3 inhibitor comprises STC-15 or a pharmaceutically acceptable salt thereof.

[0011] Preferably, the drug enhances the phagocytic and antibacterial immune capabilities of alveolar macrophages by regulating the m6A modification level of alveolar macrophages.

[0012] Secondly, the present invention provides the use of METTL3 inhibitors in the preparation of medicaments for the prevention and / or treatment of bacterial pulmonary infections.

[0013] Preferably, the METTL3 inhibitor comprises STC-15 or a pharmaceutically acceptable salt thereof.

[0014] Preferably, the bacterial infection includes Pseudomonas aeruginosa infection.

[0015] Thirdly, the present invention provides a pharmaceutical composition for enhancing pulmonary antibacterial immunity, wherein the active ingredient of the pharmaceutical composition comprises STC-15 or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0016] Preferably, the drug is administered locally via tracheal instillation or nebulized inhalation.

[0017] Preferably, the pharmaceutical composition is formulated into a dosage form suitable for local administration to the lungs, including a nebulized inhaler, a dry powder inhaler, or a tracheal instillation solution.

[0018] Fourthly, the present invention provides a method for enhancing the host's lung antibacterial immunity, comprising applying an effective amount of METTL3 inhibitor locally to the host's lungs before or during bacterial infection to reduce the m6A modification level of alveolar macrophages and enhance the immune function of alveolar macrophages.

[0019] Preferably, the METTL3 inhibitor comprises STC-15 or a pharmaceutically acceptable salt thereof.

[0020] Preferably, the local application is performed by intratracheal instillation, nebulized inhalation, or intrabronchial perfusion.

[0021] The beneficial effects of this invention are as follows: 1. This invention is the first to target lung m6A modification to enhance innate immunity. Existing research largely focuses on elucidating the immunomodulatory mechanisms of m6A at the in vitro cellular level. This invention, however, organically combines "local administration of m6A inhibitors to the lungs," "alveolar macrophage functional remodeling," and "in vivo antibacterial immune enhancement." In vivo experiments demonstrate that pretreatment of the lungs with a specific inhibitor of m6A methyltransferase METTL3 (STC-15) via intratracheal instillation can serve as an active immune intervention strategy, pre-training and "empowering" alveolar macrophages (AMs), thereby significantly enhancing the host's defense capabilities against bacterial infections. This represents a novel concept and therapeutic approach to "immune enhancement."

[0022] 2. This invention provides a host-guided therapy that does not rely on direct bactericidal action. Current anti-infective treatments mainly rely on antibiotics to directly kill pathogens, which easily leads to drug resistance. The strategy of this invention does not directly target pathogens, but rather indirectly eliminates infection by modulating the function of the host's own immune cells. By inhibiting METTL3 and reducing m6A modification levels, the post-transcriptional regulatory network of immune-related genes in AMs is altered, thereby enhancing their phagocytic and bactericidal capabilities. This host-guided therapy offers a promising alternative to address the increasingly serious problem of antibiotic resistance, and is particularly suitable for treating infections caused by drug-resistant bacteria such as multidrug-resistant Pseudomonas aeruginosa.

[0023] 3. This invention has demonstrated the superior effects of STC-15 pretreatment through multidimensional in vitro and in vivo experiments: significantly reducing bacterial load, effectively reducing the recruitment level of pro-inflammatory effector cells in the lungs, and alleviating infection-induced tissue damage. This indicates that enhancing early immune clearance can effectively prevent pathological damage to lung tissue caused by excessive inflammation in the later stages of infection, thus facilitating prognosis and recovery.

[0024] 4. Advantages of Local Administration: This invention employs a local administration method via tracheal infusion, directly delivering the drug to the lung target organ, enabling STC-15 to act efficiently on alveolar macrophages and other cells within the lung. This local administration method significantly reduces the risk of systemic exposure, avoiding systemic immune dysregulation or other potential side effects that may result from systemic administration of m6A inhibitors, thus improving the safety and controllability of the treatment strategy. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the experimental procedure for infecting PA with STC-15 pretreated mice.

[0026] Figure 2 After pretreatment with STC-15 or adjuvant for 2 days, P. aeruginosa (PA) Stimulation curve of mouse body weight change after 16 days.

[0027] Figure 3 H&E sections and histochemical scores of lungs of mice that were pretreated with STC-15 or adjuvant for 2 days and then intratracheally instilled with PA for 12 hours.

[0028] Figure 4 Lung lavage fluid of mice that were pretreated with STC-15 or adjuvant for 2 days and then instilled with PA 12 h via intratracheal instillation.

[0029] Figure 5 The results of neutrophil content assay in the lungs of mice after tracheal instillation of PA 12 h following 2 days of pretreatment with STC-15 or adjuvant (neutrophils were specifically labeled with APC-Ly-6G and PE-CD11b).

[0030] Figure 6 Plates were prepared from diluted BALF solution in mice that had been pretreated with STC-15 or adjuvant for 2 days and then infused with PA 12 h via intratracheal instillation.

[0031] Figure 7 Results of BALF absorbance testing in mice after tracheal instillation of PA 12 h following 2 days of pretreatment with STC-15 or adjuvant.

[0032] Figure 8 Results of BALF protein content detection in mice that were pretreated with STC-15 or adjuvant for 2 days and then infused with PA 12 hours via tracheal infusion. Detailed Implementation

[0033] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. It should be noted that these descriptions of embodiments are for understanding the invention but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the invention described below can be combined with each other as long as they do not conflict with each other.

[0034] Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the materials and reagents used are commercially available products that can be obtained through commercial channels.

[0035] Example 1: Establishment of a mouse lung model infected with Pseudomonas aeruginosa Establishing a mouse model of bacterial lung infection pretreated with STC-15 ( Figure 1 ): Preparation of STC-15 solution: Centrifuge 1 mg of STC-15 (Selleck; E1728) powder at 13000 g for 3 min to allow the powder to accumulate at the bottom of the tube. Add 8 μL of DMSO to the tube and place it in a 56°C water bath to dissolve completely. After the powdered solid has completely dissolved, add 32 μL of PEG 300, 4 μL of Tween 80, and 36 μL of DEPC H2O sequentially, and shake thoroughly to mix. If a small amount of precipitate still exists, break it up using ultrasound. After the solution has completely dissolved, place it in 56°C warm water for later use. The control group adjuvant was prepared according to the above proportions, containing no STC-15, and after thorough shaking, placed in warm water for later use as well.

[0036] Healthy mice of appropriate weight and age (6-8 weeks) were randomly divided into groups and acclimatized for one week. After examination and confirmation of no obvious abnormalities, experiments were conducted. The weight of the mice was measured using a small animal weighing balance and recorded. The required dosage of anesthetic was calculated, and the mice were anesthetized via intraperitoneal injection. In a clean bench, a mouse fixation board was erected vertically. The anesthetized mice were suspended vertically by tying their upper incisors with surgical sutures. The mouth was opened with forceps, and the tongue was removed to expose the trachea. A vascular catheter was inserted deep into the trachea. Freshly prepared STC-15 solution and adjuvant were slowly added to the catheter. After both groups of mice had completely inhaled the STC-15 solution and adjuvant, they were kept suspended for 1-2 minutes. They were then slowly turned and shaken to ensure that the solution fully and evenly covered the alveoli and tracheal surface.

[0037] Two days later, the mice were anesthetized and fixed in the same manner, and then subjected to Pseudomonas aeruginosa (Pseudomonas aeruginosa) treatment. P. aeruginosa ) suspension (5×107 Mice were perfused with CFU (25 g). As the mice breathed, the perfused fluid was gradually absorbed into their lungs. After the mice were fully recovered, they were returned to their cages. Their weight was monitored for 16 consecutive days, and a weight curve was plotted. Results are as follows... Figure 2 As shown.

[0038] Simultaneously, the model mice were perfused with a suspension of Pseudomonas aeruginosa (5 × 10⁻⁶). 7 CFU (25 g) was completely absorbed. Mice were euthanized after 12 hours of overdose anesthesia. The mice were disinfected, dissected, and their thoracic cavities were exposed. The right second lung was carefully removed and immediately placed in 4% paraformaldehyde tissue fixative at 4°C overnight. Embedding, sectioning, dewaxing, staining, dehydration, and mounting were then performed. After mounting, the slides were ready for microscopic observation. The results are as follows: Figure 3 As shown.

[0039] The results showed that the pathological damage and lesion severity of the lungs of mice were significantly reduced after STC-15 pretreatment. Histopathological scoring of the lung HE sections of the two groups of mice also showed that the histopathological scores of the lungs of mice after STC-15 pretreatment were lower, which indicates that the degree of lung damage and inflammation level were significantly reduced after STC-15 pretreatment.

[0040] Example 2: Analysis of neutrophil surface markers in mouse bronchoalveolar lavage fluid CD11c is a transmembrane protein belonging to the immunoglobulin superfamily. It is primarily expressed on the surface of activated monocytes, macrophages, and some dendritic cells. In the immune system, CD11c is closely related to phagocytosis, inflammatory responses, and antigen presentation. As an adhesion molecule, it participates in the activation, migration, and phagocytosis of immune cells through interactions with other molecules. Ly6G (Lymphocyte antigen 6 complex, locus G) is a highly glycosylated glycosylphosphatidylinositol (GPI) anchoring protein. As a GPI anchoring protein, Ly6G may be involved in regulating neutrophil adhesion, migration, and recruitment at inflammatory sites. It may work synergistically with other transmembrane proteins to influence cell-vascular endothelial interactions, and may form complexes with other receptors on the cell membrane (such as integrins, like CD11b / CD18) to participate in intracellular signal transduction, thereby regulating neutrophil activation, phagocytosis, or oxidative burst functions. Studies have shown that anti-Ly6G antibodies can interfere with neutrophil-mediated host defense, suggesting that they may play a role directly or indirectly in antimicrobial immunity. It is expressed almost exclusively on mature neutrophils; in mice, other immune cells (such as monocytes and lymphocytes) do not express Ly6G. Therefore, Ly6G is often used in combination with another myeloid cell marker, CD11b, to distinguish neutrophils from other myeloid cells.

[0041] This embodiment describes the flow cytometry detection of cells in mouse lung lavage fluid.

[0042] method: (1) Take 1000 g of pulmonary lavage fluid, centrifuge at 4℃ for 10 min, and carefully discard the supernatant; (2) Resuspend in 200 μL of erythrocyte lysis buffer and let stand for 3 min to break down the red blood cells; (3) Centrifuge at 2000 g, 4℃ for 5 min, and resuspend in 200 μL of filtered 1×PBS; (4) Take 150 μL of cell resuspension and add it to the flow cytometry antibody dilution solution for neutrophils, and incubate in the dark for 30 min; (5) Centrifuge at 2000 g, 4℃ for 5 min, carefully discard the supernatant, and resuspend in 300 μL filtered 1×PBS to prevent non-specific over-binding of the antibody. (6) Add the resuspended solution to the flow cytometer and it is ready for testing.

[0043] Depend on Figure 5The results of flow cytometry showed that the number of neutrophils in the STC-15 pretreatment group was significantly lower than that in the adjuvant pretreatment control group, indicating that the recruitment of neutrophils in the lungs of the STC-15 pretreatment group was reduced when stimulated by bacteria, thereby further reducing the degree of lung inflammation and pathological changes.

[0044] Example 3: Determination of bacterial load and protein concentration in mouse lung lavage fluid Alveolar resident macrophages (AMs) are core regulators in maintaining lung structural and functional homeostasis. Through their sophisticated immunomodulatory functions, AMs play an indispensable role in suppressing excessive inflammation, promoting tissue repair, and maintaining the balance of the alveolar microenvironment. For example, after respiratory viral infections or allergen stimulation, AMs can effectively inhibit excessive neutrophil activation and abnormal lymphocyte responses, thereby reducing immunopathological damage and preventing acute lung injury or chronic fibrosis. Simultaneously, AMs maintain immune tolerance by clearing apoptotic cells and cellular debris, preventing secondary immune responses caused by self-antigen exposure, and ensuring the rapid recovery of lung tissue's structural and functional balance after responding to challenges. Furthermore, AMs can regulate the barrier function and repair process of alveolar epithelial cells, promoting the proliferation and differentiation of type II alveolar epithelial cells by expressing various growth factors and remodeling mediators, enhancing alveolar integrity, and preventing irreversible tissue damage. Therefore, AMs are not only effector cells of innate immunity but also guardians of the lung microenvironment; their normal function directly affects whether lung tissue can effectively respond to endogenous and exogenous stimuli and maintain physiological balance.

[0045] To further determine whether the antibacterial ability of mouse lungs was significantly improved after STC-15 pretreatment, this example measured the OD value, plate smear, and protein content of the lung lavage fluid from model mice.

[0046] 1. Bronchoalveolar lavage in mice and absorbance measurement of lavage fluid: (1) Select C57BL / 6 strain mice aged 6 to 8 weeks, acclimatize them for 7 days, and conduct experiments after no obvious abnormalities are found during the examination. (2) Weigh the mouse using a balance specifically for small animal weighing and record the weight, and calculate the amount of anesthetic required for overdose anesthesia; (3) The mice were euthanized by an overdose of anesthesia. After the mice died, they were disinfected by immersing them in 75% alcohol for 1 minute. After disinfection, the mice were fixed in a supine position on the dissection board. (4) Preheat the pulmonary lavage fluid to 37°C; (5) Wipe the mouse's abdomen with an alcohol swab, smooth the fur with tweezers, and then cut a small incision in the skin of the lower abdomen with scissors. Cut the skin straight from the incision to the mouse's lower jaw, and then separate the mouse's neck dilatation muscles to expose the trachea. (6) Insert an indwelling needle into the trachea and inject 2 mL of pulmonary lavage fluid into the lungs for lavage, 1 mL each time, for a total of ten times; (7) Collect about 10 mL of the flushed liquid, put the collected liquid into a centrifuge tube and place it on ice.

[0047] (8) Adjust the spectrophotometer to 600 nm, use the original lung lavage fluid for zeroing, add 500 μL of lavage fluid to a cuvette, and measure the OD value.

[0048] 2. Method for bacterial plating of mouse lung lavage fluid: (1) 5 mL of lavage fluid was lavaged from the lungs of the successfully modeled mice and mixed thoroughly. (2) Take 10 μL of lung lavage fluid and add it to 20 μL of filtered 1×PBS for resuspending; (3) Use a bacterial coating stick in a clean bench to coat antibiotic-free LB solid culture dishes; (4) Incubate overnight in a 37 ℃ bacterial culture incubator; (5) Once the colonies have grown to a suitable size, they can be taken out for observation and counting.

[0049] 3. Determination of protein concentration in mouse lung lavage fluid (1) 1 mL of rinsing fluid was irrigated from the lungs of mice that had successfully modeled the disease with 1×PBS and placed in a 1.5 mL centrifuge tube and centrifuged at 12000 g for 15 min. (2) Take 5 μL of the supernatant for BCA protein quantification; (3) Calculate and statistically analyze the protein concentration of the irrigation fluid.

[0050] Comparison of mouse lavage fluid and OD value measurement results are as follows: Figure 4 and Figure 7 As shown. The results indicate that BALF in mice treated with adjuvant was significantly better than that in mice pretreated with STC-15 after infection. P. aeruginosa The mice's BALF was redder and more turbid, and the OD value was also higher.

[0051] The results of bacterial smear and protein concentration assays in mouse BALF are as follows: Figure 6 and Figure 8 As shown, the bacterial load in the lungs of mice pretreated with STC-15 was significantly lower than that in the adjuvant-treated group.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of METTL3 inhibitors in the preparation of drugs that enhance pulmonary antibacterial immunity.

2. The application as described in claim 1, characterized in that, The METTL3 inhibitors include STC-15 or a pharmaceutically acceptable salt thereof.

3. The application as described in claim 1, characterized in that, The drug enhances the antibacterial phagocytic immune capacity of alveolar macrophages by regulating the m6A modification level of alveolar macrophages.

4. Application of METTL3 inhibitors in the preparation of drugs for the prevention and / or treatment of pulmonary bacterial infections.

5. The application as described in claim 4, characterized in that, The METTL3 inhibitors include STC-15 or a pharmaceutically acceptable salt thereof.

6. The application as described in claim 4, characterized in that, The bacterial infections include Pseudomonas aeruginosa infections.

7. A pharmaceutical composition for enhancing pulmonary antibacterial immunity, characterized in that, The active ingredient of the drug includes STC-15 or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, The drug is administered locally via tracheal instillation or nebulized inhalation.

9. A method for enhancing the host's lung antibacterial immunity, characterized in that, This includes administering an effective amount of METTL3 inhibitors locally to the host's lungs before or during bacterial infection, in order to reduce the m6A modification level of alveolar macrophages and enhance the immune function of alveolar macrophages.

10. The method as described in claim 9, characterized in that, The METTL3 inhibitors include STC-15 or a pharmaceutically acceptable salt thereof.

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