Use of targeting skin dendritic cells in preparation of drugs for improving physiological thymus structure and function abnormalities

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a targeted skin dendritic cell in preparation of a drug for improving physiological thymus structure and function abnormality. The application discloses an immune regulation method for improving age-related thymus physiological atrophy and function recession. The method improves volume atrophy, tissue structure disorder and T cell output reduction caused by thymus physiological aging by targeting exhaustion or inhibition of dendritic cells in the skin, thereby helping to maintain thymus related structure and function. Specifically, the method can alleviate thymus degenerative changes that occur with age, improve the ratio of thymus cortex and medulla, and improve the level of new output T cell related indexes. The application provides a new intervention strategy for thymus immune aging.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of targeting skin dendritic cells in the preparation of drugs that improve physiological thymus structure and function abnormalities. Background Technology

[0002] During human development, the thymus gradually matures in late fetal development and maintains relatively active growth from birth to adolescence. Upon entering adulthood, the thymus gradually shrinks in size and undergoes structural remodeling with age, exhibiting significant physiological degeneration. This process is considered a key marker of immunosenescence, characterized by decreased integrity of the thymic epithelial structure, an imbalance in the cortical-medullic ratio, and a weakened capacity for sustained production of new T cells. With changes in the thymic microenvironment and its reduced support for T cell development, the efficiency of naïve T cell production gradually declines, and the coverage of the T cell receptor (TCR) pool also shrinks. This change limits the body's ability to establish a sufficient immune response to new antigenic stimuli, and is one of the important foundations for the age-related decline in immune function. This makes it difficult for older individuals to form a sufficient and stable immune response to new infections or tumor-associated antigens, thereby increasing the likelihood of infection and reducing immune surveillance capabilities.

[0003] Existing research on interventions for immunosenescence largely focuses on improving immune function by enhancing or replacing T-cell production capacity, such as through hormone regulation, cytokine supplementation, or intervention in the hematopoietic process. However, these methods often rely on systemic administration or systemic regulation, which leads to significant non-targeting effects, cumulative side effects, and difficulty in maintaining long-term efficacy, thus limiting their application in immunosenescence interventions.

[0004] Recent studies have gradually revealed that the structure and function of the thymus are not entirely determined by its own microenvironment; a long-term and stable regulatory relationship may exist between peripheral tissues and the thymus, a central immune organ. As the largest barrier tissue in the body that is constantly in contact with the external environment, the skin plays a crucial role in systemic immune regulation. Various immune cells distributed in the skin participate in maintaining local homeostasis and can influence the systemic immune environment through cell migration or the release of signaling molecules. Among them, dendritic cells, as important antigen-presenting cells, play a key role in maintaining local skin immune homeostasis and participating in the regulation of immune responses. Changes in the local immune status of the skin can affect the systemic immune environment through immune cell migration and cytokine release. However, systematic research conclusions are still lacking regarding whether local skin immune cells, especially dendritic cells (DCs), participate in regulating the structural integrity of the thymus, maintaining the stability of thymic function, and influencing the physiological degeneration of the thymus with age. Furthermore, a feasible technical solution based on local skin immune regulation for improving or delaying age-related thymic functional decline has not yet been developed.

[0005] Based on the above problems, there is an urgent need to explore a new approach to immune regulation, which can improve the thymus-related structures and functional phenotypes through local intervention or non-invasive methods, while avoiding the side effects of systemic immune intervention, thereby delaying the physiological decline of thymus function with age and making up for the shortcomings of existing technologies. Summary of the Invention

[0006] Objective: This invention aims to address the shortcomings of existing interventions for age-related physiological thymic degeneration, which primarily rely on systemic immune regulation, lack targeted targeting, and struggle to maintain thymic function long-term. It provides an application of targeting skin dendritic cells in the preparation of drugs to improve physiological thymic structural and functional abnormalities. This invention utilizes an immunomodulatory method that regulates local skin dendritic cells (DCs) to improve the structural and functional state of the thymus, offering a new technical approach to delaying immunosenescence and maintaining the function of central immune organs. By regulating local skin DCs to target and deplete or inhibit their function, this invention improves age-related thymic structural and functional degeneration and contributes to improving the regulation of physiological thymic immunosenescence.

[0007] To address the aforementioned technical problems, this invention discloses the application of targeting dermal dendritic cells in the preparation of drugs to improve physiological thymus structure and function abnormalities. This invention is the first to propose the application of targeting depleted or inhibited local dermal dendritic cells (DCs) in the preparation of products for improving or delaying age-related physiological thymus degeneration. The specific technical solution is as follows: In a first aspect, the present invention provides the application of targeting skin dendritic cells in the preparation of drugs that improve physiological thymus structure and function abnormalities.

[0008] The targeted skin dendritic cells refer to the depletion of skin dendritic cells and / or the inhibition of skin dendritic cell proliferation or function.

[0009] The targeted skin dendritic cells refer to the administration of substances that deplete skin dendritic cells and / or inhibit their proliferation or function. In some embodiments of the present invention, the substances that deplete skin dendritic cells and / or inhibit their proliferation or function include anti-CD11c antibodies. These substances, which target or inhibit local skin dendritic cells, act as key factors in improving or delaying age-related physiological degeneration of the thymus.

[0010] Secondly, this invention provides the application of anti-CD11c antibodies in the preparation of drugs that improve physiological thymus structure and function abnormalities.

[0011] The improvement of physiological thymus structure and function abnormalities includes any one or more combinations of the following (1) to (3): (1) Improves thymus atrophy; (2) Improves the ratio of thymic cortex to medulla; (3) Enhance the output capacity of new T cells in the thymus.

[0012] The aforementioned physiological thymus structural and functional abnormalities refer to physiological thymus structural and functional abnormalities caused by aging.

[0013] Specifically, in a naturally aging / age-related mouse model, this invention, through selective depletion of local dendritic cells (DCs) in the skin, found that this local intervention significantly improved the structural and functional phenotypes related to the thymus. Specifically, the size and volume of the atrophied thymus were partially restored; the structural ratio of the thymic cortex to medulla was improved; and the levels of indicators related to newly generated exporting T cells in the peripheral circulation increased. Experimental results indicate that targeted regulation of local skin dendritic cells can improve the age-related structural and functional changes of the thymus without inducing systemic immunosuppression, helping to maintain T cell homeostasis and delay the immune aging process.

[0014] The drug contains a pharmaceutically acceptable carrier or excipient.

[0015] Wherein, the pharmaceutically acceptable carrier or excipient is selected from any one or more combinations of diluents, disintegrants, precipitation inhibitors, flow aids, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, ion exchangers, flavoring agents, or antioxidants.

[0016] The drug can be administered via any one of the following routes: intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or oral. Preferably, the drug is administered via different routes to target and intervene in local dendritic cells (DCs) on the skin.

[0017] Beneficial effects: Existing interventions for age-related physiological decline of the thymus mostly rely on systemic immune regulation or hormone-related interventions, which suffer from insufficient targeting, significant systemic side effects, and difficulty in maintaining thymic function long-term. This invention provides a novel strategy for maintaining thymic function based on local skin immune regulation. By targeting local skin dendritic cells, it effectively improves the physiological changes in the structure and function of the thymus that occur with age. The method described in this invention features local action, strong targeting, and minimal systemic side effects, helping to maintain the structural integrity and functional stability of the thymus, promoting the maintenance of indicators related to the output of new T cells, thereby delaying the decline in immune function with age. This method provides a new technical approach for the intervention of immunosenescence and the maintenance of thymic function, and has promising application prospects. Attached Figure Description

[0018] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0019] Figure 1 This study investigated the changes in thymus morphology and weight in Itgax DTR / + mice after two months of targeted depletion of local dendritic cells in the skin.

[0020] Figure 2 This study investigated the hematoxylin-eosin (H&E) staining results and changes in the cortex / medulla ratio of the thymus tissue of Itgax DTR / + mice after two months of targeted depletion of local dendritic cells in the skin.

[0021] Figure 3 To continuously deplete the local dendritic cells in the skin of Itgax DTR / + mice for 2 months, and to reduce the initial CD4 count in the peripheral circulation of mice. + Changes in the proportion of T cell subsets and the relative content of T cell receptor excision loop DNA (sjTRECs) in peripheral circulation, where A represents initial CD4+. + Changes in the proportion of T cell subsets, B represents the change in the relative content of sjTRECs.

[0022] Figure 4To investigate the changes in thymus morphology and weight in C57BL / 6 mice after pharmacological intervention with CD11c neutralizing antibody on local dendritic cells of the skin for two consecutive months.

[0023] Figure 5 To investigate the changes in the relative content of T cell receptor excision loop DNA (sjTRECs) in the peripheral circulation of C57BL / 6 mice after pharmacological intervention with CD11c neutralizing antibody on local dendritic cells of the skin for two consecutive months. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments, but it should not be construed that the scope of the present invention is limited to the following embodiments. Various substitutions and modifications made based on common technical knowledge and methods in the art without departing from the above-described technical concept of the present invention should be included within the scope of protection of the present invention.

[0025] The experimental drugs and reagents used in the following examples—physiological saline, diphtheria toxin, anti-mouse CD11c monoclonal antibody, isotype control monoclonal antibody, 4% paraformaldehyde, OCT embedding agent, and DNA extraction kit—are all commercially available.

[0026] The experimental animals used in the following examples were: Itgax DTR / + mice and C57BL / 6 mice from the same littermate, aged 0–12 weeks and weighing approximately 5–30 g, which were commercially available. The Itgax DTR / + mice expressed diphtheria toxin receptors under the influence of the Itgax promoter. Selective depletion of dendritic cells could be achieved by administering diphtheria toxin (DT) at specific time points or time periods.

[0027] The animals were housed under the following conditions: room temperature 18–20 °C, humidity 50–60%, alternating light and dark (12 h), moderate light intensity, and good ventilation and cleanliness. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee of the Institute of Dermatology, Chinese Academy of Medical Sciences (CASD).

[0028] Example 1 Selective depletion of local dendritic cells in the skin and experimental grouping: Diphtheria toxin was administered intradermally to Itgax DTR / + mice to achieve selective depletion of local dendritic cells in the skin. The concentration of diphtheria toxin used was 0.0015 mg / mL, with an injection volume of 200 μL each time, administered every two days until the experimental endpoint. According to the experimental design, Itgax DTR / + mice were randomly divided into the following three groups, with 6 mice in each group: Young control group: At the start of the experiment, there were newborn 0-week-old Itgax DTR / + mice that were not treated with diphtheria toxin or saline and were simply fed until the end of the experiment. Age control group: At the start of the experiment, 6-week-old Itgax DTR / + mice were injected intradermally with an equal volume of physiological saline; Dendritic cell depletion group: The experiment started with 6-week-old Itgax DTR / + mice, which were injected intradermally with diphtheria toxin.

[0029] In this study, the dendritic cell depletion group and the age control group received intradermal injections every two days starting from the beginning of the experiment (day 0), with the treatment period lasting for 2 months (60 days). At the end of the experiment, the young control group mice were approximately 8 weeks old, and the age control group and the dendritic cell depletion group mice were approximately 14 weeks old.

[0030] On day 60 of the experiment, mice were euthanized by cervical dislocation after anesthesia. Thymus and spleen tissues were collected from each group of mice at the same time point using sterile surgical instruments. The thymus tissue was weighed, and the changes in the ratio of thymic cortex to medulla were observed by hematoxylin and eosin (H&E) staining. At the same time, spleen tissue was examined to analyze the relative content of signal joint T-cell receptor excision circles (sjTRECs) and changes in the proportion of naïve T cells in peripheral immune organs.

[0031] The results of thymus tissue weighing are as follows Figure 1 As shown, after targeted depletion treatment of local dendritic cells in the skin for approximately two months, the size and morphology of the thymus in mice underwent significant changes. Compared with the young control group, the thymus weight of the age-controlled mice decreased by approximately 51% (thymus weight of the young control group was 54.15±1.99 mg, and the thymus weight of the age-controlled group was 26.4 g±2.45 mg), exhibiting physiological atrophy characteristics with age. In contrast, in the local dendritic cell depletion treatment group, the thymus volume and weight of the mice recovered compared with the age-controlled group, increasing by approximately 59% (thymus weight of the depletion group was 41.97±1.18 mg), suggesting that targeted regulation of local dendritic cells in the skin can, to some extent, improve the atrophic phenotype of the thymus with age.

[0032] The results of hematoxylin-eosin (H&E) staining of mouse thymus tissue are as follows: Figure 2As shown, compared with the young control group, the thymic cortex of the mice in the age control group was significantly thinner, the medullary region was relatively expanded, and the ratio of cortex to medulla was changed; while in the skin dendritic cell depletion treatment group, the above structural changes were improved to some extent, indicating that targeted regulation of skin dendritic cells helps maintain the relative stability of thymic tissue structure.

[0033] The changes in the relative content of T cell receptor cleavage loops and the proportion of naïve T cells in peripheral immune organs are as follows: Figure 3 As shown in B and A, in the skin dendritic cell targeted depletion experiment, compared with the young control group, the relative content of T cell receptor cleavage loops (sjTRECs) in the peripheral immune organs of age-controlled mice was decreased, suggesting a decline in the output capacity of thymic neoplastic T cells with age; while in the depletion group, the relative content of peripheral sjTRECs was increased, suggesting a certain degree of improvement in the output of thymic neoplastic T cells. Meanwhile, the peripheral immune cell subset analysis results showed that, compared with the young control group, the peripheral initial CD4+ of age-controlled mice was higher. + T cells (CD62L) hi CD44 lo and CD62L hi CD44 lo Qa2 lo The proportion decreased; while in the depleted group, the initial CD4 content decreased. + The proportion of T cells was higher than that of the age-matched control group, suggesting that targeted regulation of local dendritic cells in the skin helps improve the homeostasis of the peripheral T cell population.

[0034] Example 2 Pharmacological intervention and experimental grouping of local dendritic cells in the skin: C57BL / 6 mice were administered CD11c neutralizing antibodies via intradermal injection to achieve pharmacological intervention of local dendritic cells in the skin. The neutralizing antibody concentration was 0.35 mg / mL, with an injection volume of 200 μL per injection, administered twice weekly until the experimental endpoint. According to the experimental design, C57BL / 6 mice were randomly divided into three groups of six each: Control group: Newborn C57BL / 6 mice at the start of the experiment were raised until the end of the experiment without antibody treatment; IgG-Ab group: The experiment started with 6-week-old C57BL / 6 mice, which were injected intradermally with an equal dose of isotype control antibody; CD11c-Ab group: The experiment started with 6-week-old C57BL / 6 mice, which were injected intradermally with CD11c neutralizing antibody.

[0035] The IgG-Ab and CD11c-Ab groups were treated with intradermal injections twice a week from the start of the experiment for a period of 2 months (60 days). At the end of the experiment, the mice in the control group were approximately 8 weeks old, and the mice in the IgG-Ab and CD11c-Ab groups were approximately 14 weeks old.

[0036] On day 60 of the experiment, mice were euthanized by cervical dislocation after anesthesia. Thymus and spleen tissues were collected from each group of mice at the same time point using sterile surgical instruments. The thymus tissue was weighed, and the changes in the ratio of thymic cortex to medulla were observed by hematoxylin and eosin (H&E) staining. Simultaneously, the spleen tissue was examined to analyze the relative content of T cell receptor cleavage loops in peripheral immune organs and the changes in the proportion of naïve T cells.

[0037] The results of thymus tissue weighing are as follows Figure 4 As shown, after two months of pharmacological intervention on local dendritic cells of the skin using CD11c neutralizing antibodies, the thymus volume and weight of mice increased by approximately 17.4% compared to the IgG-Ab group (thymus weight in the CD11c antibody group was 52.58±2.34 mg, and thymus weight in the IgG-Ab group was 44.78±1.36 mg), and recovered approximately 30.1% of the thymus weight loss. This suggests that pharmacological intervention on local dendritic cells of the skin can also improve the physiological thymus atrophy-related phenotype to some extent.

[0038] The relative content of T-cell receptor cleavage loops in peripheral immune organs is as follows: Figure 5 As shown, further, in the CD11c neutralizing antibody pharmacological intervention experiment, compared with the control group, the relative content of sjTRECs in the peripheral circulation of mice in the IgG-Ab group decreased by about 25.7%, while the relative content of sjTRECs in the peripheral circulation of mice in the CD11c-Ab group was significantly higher than that in the IgG-Ab group, suggesting that after local application of CD11c neutralizing antibody to intervene in local dendritic cells of the skin, the output capacity of thymic neoplastic T cells was also restored to a certain extent.

[0039] The results in summary indicate that targeted regulation or pharmacological intervention of local dendritic cells in the skin can, to some extent, improve the structural and functional changes of the thymus with age without relying on systemic immune intervention, and have a positive impact on T cell generation and peripheral T cell population homeostasis.

[0040] This invention provides a concept and method for targeting dermal dendritic cells in the preparation of drugs to improve physiological thymus structure and function abnormalities. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The application of targeting skin dendritic cells in the preparation of drugs to improve physiological thymus structure and function abnormalities, wherein the targeting skin dendritic cells are depleted skin dendritic cells and / or inhibit the proliferation or function of skin dendritic cells.

2. The application according to claim 1, characterized in that, The targeted skin dendritic cells refer to the administration of substances to the body that deplete skin dendritic cells and / or inhibit the proliferation or function of skin dendritic cells.

3. The application according to claim 2, characterized in that, The substances that deplete skin dendritic cells and / or inhibit the proliferation or function of skin dendritic cells include anti-CD11c antibodies.

4. Application of anti-CD11c antibody in the preparation of drugs to improve physiological thymus structure and function abnormalities.

5. The application according to claim 1 or 4, characterized in that, The improvement of physiological thymus structure and function abnormalities includes any one or more combinations of the following (1) to (3): (1) Improves thymus atrophy; (2) Improves the ratio of thymic cortex to medulla; (3) Enhance the output capacity of new T cells in the thymus.

6. The application according to claim 1 or 4, characterized in that, The aforementioned physiological abnormalities in thymus structure and function refer to abnormalities in thymus structure and function caused by aging.

7. The application according to claim 1 or 4, characterized in that, The drug contains a pharmaceutically acceptable carrier or excipient.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable carrier or excipient is selected from any one or more combinations of diluents, disintegrants, precipitation inhibitors, flow aids, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, ion exchangers, flavoring agents, or antioxidants.

9. The application according to claim 1 or 4, characterized in that, The drug can be administered via any one of the following methods: intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intravenous administration, or oral administration.

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