Application of ANXA1 agonist or inhibitor to regulation of peripheral IgE level

Abstract

The invention relates to application of an inhibitor or agonist of ANXA1 to regulation of IgE level, in particular to the effect of promoting or recovering the IgE level. The ANXA1 plays a key role in immune cell functions, especially in a T cell differentiation process. Through a series of in-vitro experiments, the effect of the ANXA1 and the potential of the ANXA1 in regulating the IgE level are discussed, and the ANXA1 is found to have an important function in regulating immune response and IgE production. The research result of the invention provides a theoretical basis for regulating the IgE level by using the ANXA1 agonist or inhibitor. The ANXA1 agonist can be used for promoting synthesis of IgE, and the ANXA1 inhibitor can be used for reducing the level of IgE, so that the ANXA1 has a wide application prospect, and particularly provides a new treatment strategy and target spot in clinical treatment of allergic diseases, immune disorder and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to the use of an ANXA1 agonist or inhibitor in regulating peripheral IgE levels, and in particular to the use of an ANXA1 inhibitor or agonist in the preparation of a drug for treating autosomal recessive hereditary high IgE syndrome caused by DOCK8 mutation. Background Art

[0002] The normal function of the immune system depends on the effective migration, differentiation, function and survival of different immune cells. DOCK8 (Dedicator of Cytokinesis 8) is a key cell signaling molecule that plays a role in multiple important responses of the immune system. DOCK8 deficiency or mutation leads to abnormal function of immune cells, especially affecting innate and adaptive immune responses. Dock8-deficient mice show lymphopenia, CD4+T cell outflow disorder, and lack of normal thymocyte migration to the peripheral blood. This indicates the role of Dock8 in the MST kinase signaling pathway and emphasizes the importance of Dock8 for the migration of single positive thymocytes to the peripheral circulation. In addition, DOCK8-deficient CD4+T cells show a bias towards Th2-type differentiation, which may be related to the occurrence of allergic diseases. DOCK8 is also essential for the function of CD8+T cells and directly affects their ability to form long-term and effective memory responses. DOCK8 deficiency is closely related to an immunodeficiency disease called hyper IgE syndrome (HIES). Studies have found that Th2 cells play a central role in IgE-mediated allergic reactions. They regulate B cell differentiation and eosinophil activation by secreting cytokines, which contribute to the production and maintenance of IgE.

[0003] Although DOCK8 deficiency and mutation play an important role in the occurrence of immunodeficiency and HIES, its specific molecular mechanism is still not fully understood. In order to better understand the changes in the immune system in the absence of DOCK8, researchers have begun to explore the role of other key molecules, among which ANXA1 (Annexin A1) has received increasing attention as a possible important regulator. ANXA1 is a calcium-dependent membrane-bound protein that has been shown to play an important role in immune response, inflammation regulation and cell function. ANXA1 plays a significant role in the migration and differentiation of immune cells, inhibiting inflammatory response and maintaining immune homeostasis. In recent years, researchers have gradually discovered that ANXA1 may play an important role in regulating IgE levels and related immune responses. In IgE-mediated allergic reactions, ANXA1 may indirectly or directly participate in the production of IgE by affecting T cells and their differentiation. Studies have shown that ANXA1 can not only regulate the function of immune cells, but also promote or inhibit immune responses by regulating related cytokines (such as IL-4, IL-5, etc.) and the behavior of immune cells. Therefore, the development of ANXA1 inhibitors or agonists may provide a new therapeutic strategy by regulating IgE levels and restoring the balance and function of the immune system in the immunodeficiency state caused by DOCK8 deficiency. Summary of the invention

[0004] The present invention relates to the field of immunology, and in particular to a strategy for regulating the differentiation of DOCK8 mutant T cells, aiming to promote or inhibit the differentiation of Th2 cells by regulating the expression or activity of ANXA1, thereby providing a method for treating DOCK8 mutation-related immune diseases.

[0005] According to a first aspect of the present invention, there is provided use of an ANXA1 agonist or an ANXA1 inhibitor in regulating peripheral IgE levels.

[0006] Preferably, the ANXA1 agonist is used to promote IgE synthesis.

[0007] Preferably, the ANXA1 agonist is used to promote the increase of the proportion of Th2 cells in T cells.

[0008] Preferably, the ANXA1 inhibitor is used to reduce IgE levels.

[0009] Preferably, the ANXA1 inhibitor is used to reduce the proportion of Th2 cells in DOCK8 mutant T cells.

[0010] Preferably, the ANXA1 inhibitor is used for preparing a drug for treating immune diseases caused by DOCK8 mutation.

[0011] Preferably, the ANXA1 inhibitor is used for preparing a drug for treating autosomal recessive hereditary hyper-IgE syndrome caused by DOCK8 mutation.

[0012] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0013] (1) In the present invention, flow cytometry analysis revealed that the expression of ANXA1 in Dock8 mutant T cells was significantly increased compared with the control group. Further experimental results showed that the active peptide fragment Ac2-26 of ANXA1 can promote the differentiation of Th2 cells. In the in vitro induced Th2 differentiation study, the proportion of Th2 cells (IL-4+) significantly increased after Ac2-26 treatment in both the control group and the Dock8 mutant group's CD4+T cells, indicating that ANXA1 can indeed promote T cell differentiation toward Th2.

[0014] (2) To further study the regulatory effect of ANXA1 on the differentiation of Dock8 mutant T cells, the cells were treated with the ANXA1 receptor inhibitor Boc. The results showed that in the control group, the proportion of Th2 cells induced by Boc treatment decreased significantly. More importantly, in the Dock8 mutant group, the proportion of Th2 cells after Boc treatment was significantly lower than that in the untreated group, indicating that the inhibitor of ANXA1 can restore the proportion of Th2 cells in DOCK8 mutant T cells.

[0015] (3) The research of the present invention provides a new direction for the clinical application of ANXA1 as a target. By using ANXA1 agonists or inhibitors, IgE levels can be restored or regulated in immunodeficiency, allergic diseases and related immunopathological states. This treatment strategy can not only improve the symptoms of immunodeficiency caused by DOCK8 deficiency, but also provide new drug development ideas for the treatment of other immune-related diseases. Through targeted treatment of ANXA1, it may be possible to effectively improve IgE-mediated allergic reactions, restore immune balance, reduce the immune burden of patients, and promote the precision treatment of allergic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Dock8 1281&1282TGThe deletion mutation leads to premature termination of DOCK8 protein translation and increased serum IgE levels, mimicking the patient phenotype. A: Construction strategy of Dock8 mutant mice, deleting the thymine-guanine (TG) bases at positions 1281 and 1282 in the Dock8 gene. B: Agarose gel electrophoresis of PCR products of Dock8 mutant mice and littermate control mice. The bands correspond to the expected DNA fragments, indicating successful amplification. M: DNA molecular marker; WT: wild-type control, 376 bp; Mut: mutant, 374 bp. C: Sanger sequencing results of wild-type and Dock8 mutant mice, and the amino acid sequence encoded by the DNA product. D: Dock8 mutant mice show dermatitis on the back. E: The level of IgE in serum was analyzed by enzyme-linked immunosorbent assay (ELISA) (n=5). F: The mRNA level of Dock8 in wild-type and Dock8 mutant mice was analyzed by qPCR (n=3).

[0017] Figure 2 : Effects of Dock8 mutation on T cell differentiation and homeostasis. A: Flow cytometric analysis of the expression of TCR-β+ total T cells in the thymus, spleen, peripheral lymph nodes (pLn), and mesenteric lymph nodes (mLn). Representative illustrations of flow cytometry. B: Flow cytometric analysis of the expression of CD4+ and CD8+ T cells in the thymus, spleen, peripheral lymph nodes (pLn), and mesenteric lymph nodes (mLn). Representative illustrations of flow cytometry. C: Flow cytometric analysis of CD4+ naive and activated T cells from the thymus, spleen, peripheral lymph nodes (pLn), and mesenteric lymph nodes (mLn) of WT and Dock8 mutant mice, representative illustrations of flow cytometry. D: Flow cytometric analysis of CD8+ naive and activated T cells from the thymus, spleen, peripheral lymph nodes (pLn), and mesenteric lymph nodes (mLn) of WT and Dock8 mutant mice, representative illustrations of flow cytometry. EG: Quantitative analysis of the percentage and number of TCR-β+ (E), CD4+ (F) and CD8+ (G) T cells (n=6). HJ: Quantitative analysis of the percentage and number of CD4+ naive T cells (H), CD4+ activated T cells (I) and CD4+ memory T cells (J) (n=6). KM: Quantitative analysis of the percentage and number of CD8+ naive T cells (K), CD8+ activated T cells (L) and CD8+ memory T cells (M) (n=6).

[0018] Figure 3 : Dock8 1281&1282TG The deletion mutation did not alter the proliferation and apoptosis of thymic and peripheral T cells.AB: Quantitative analysis of the mean fluorescence intensity (MFI) of KI-67 (A) and Annexin V (B) in CD4+ and CD8+ T cells (n=6).

[0019] Figure 4 :Dock8 mutations disrupt immune homeostasis by inhibiting Treg cell differentiation. AB: Flow cytometric analysis of CD4+CD25+Treg cells (A) and CD4+Foxp3+Treg cells (B) in the thymus, spleen, peripheral lymph nodes (pLn), and mesenteric lymph nodes (mLn), representative illustrations of flow cytometry. CD: Quantitative analysis of the percentage and number of CD4+CD25+Treg cells (C) and CD4+Foxp3+Treg cells (D). EF: Flow cytometric analysis of the expression of pre-Treg cells (CD4+CD25+Foxp3-) in the thymus, spleen, peripheral lymph nodes (pLn), and mesenteric lymph nodes (mLn) from CD4+CD45.1+ (E) and CD4+CD45.2+ (F) mice, representative illustrations of flow cytometry. GI: Quantitative analysis of the percentages of pre-Treg cells (CD4+CD25+Foxp3-) (G), CD4+CD25+Treg cells (H), and CD4+Foxp3+Treg cells (I) from CD45.2+ cells (n=6).

[0020] Figure 5 : Dock8 mutation disrupts immune homeostasis by inhibiting Treg cell differentiation. AC: Quantitative analysis of the percentage of pre-Treg cells (CD4+CD25+Foxp3-) (A), CD4+CD25+Treg cells (B), and CD4+Foxp3+Treg cells (C) from CD45.1+ cells (n=6). D: Quantitative analysis of the mean fluorescence intensity (MFI) of Foxp3 in CD4+Foxp3+Treg cells (n=6). EF: Quantitative analysis of the mean fluorescence intensity (MFI) of CTLA4 (E) and ICOS (F) in CD4+CD25+Treg cells (n=6). GH: Quantitative analysis of the mean fluorescence intensity (MFI) of CD103 (G) and NRP-1 (H) in CD4+Foxp3+Treg cells (n=6). I: Gating strategy for pre-Treg cells (CD4+CD25+Foxp3-), CD4+CD25+Treg cells, and CD4+Foxp3+Treg cells.

[0021] Figure 6:Dock8 mutation leads to imbalance of Th1 and Th2 differentiation. AE: Flow cytometric analysis of cytokine expression of CD4+ and CD8+ T cells in thymus, spleen, peripheral lymph nodes (pLn) and mesenteric lymph nodes (mLn). Representative illustrations of flow cytometry (AC is TCR-β+CD4+, DE is TCR-β+CD8+). FI: Quantitative analysis of the percentage of IL-2 (F), IFN-γ (G), IL-4 (H) and IL-17A (I) in CD4+ T cells (n=5). JL: Quantitative analysis of the percentage of IL-2 (J), IFN-γ (K) and IL-17A (L) in CD8+ T cells (n=5).

[0022] Figure 7 :Dock8 mutation promotes IgE secretion by regulating Th2 and Tfh cells. AD: Sorted CD4+T cells were stimulated with anti-CD3 / 28, IL-4, anti-IFN-γ and anti-IL-2, and five days later were stimulated with PMA, Ionomycin and GolgiStop for five hours, followed by flow staining. IL-4+ represents Th2 cells (A), IFN-γ+ (B) and IL-2+ (C) represent Th1 cells, and IL-17A+ represents Th17 cells (D). Representative illustrations of flow cytometry. E: Quantitative analysis of the percentages of Th1 cells, Th2 cells, and Th17 cells (n=5). FG: Flow cytometric analysis of CXCR5+ICOS+Tfh cells (F) and CXCR5+PD-1+Tfh cells (G) in the spleen. Representative illustrations of flow cytometry. H: Quantitative analysis of the percentages of CXCR5+ICOS+ and CXCR5+PD-1+Tfh cells (n=5). I: Quantitative analysis of the mean fluorescence intensity (MFI) of ICOS, CXCR5 and PD-1 in Tfh cells (n=5).

[0023] Figure 8: ANXA1 promotes the induction of Dock8 mutant Th2 cells. A: Expression of ANXA1 in T cells (TCR-β+, CD4+, CD8+, Treg cells). Representative illustrations of flow cytometry. B: Mean fluorescence intensity (MFI) of ANXA1 in T cells (TCR-β+, CD4+, CD8+, Treg cells) (n=5). CE: Sorted CD4+ T cells were stimulated with anti-CD3 / 28, IL-4, anti-IFN-γ and anti-IL-2 (C), or treated with anti-CD3 / 28, IL-4, anti-IFN-γ and anti-IL-2 and Ac2-26 (active peptide fragment of ANXA1) (D), or treated with anti-CD3 / 28, IL-4, anti-IFN-γ and anti-IL-2 and Boc (ANXA1 receptor inhibitor) (E). After five days, cells were stimulated with PMA, Ionomycin and GolgiStop for five hours before flow staining, IL-4, IFN-γ, IL-2, IL-17A staining and quantification of cell percentage (n=5). FI: Ratio of Th1, Th2 and Th17 cells in the three groups (basic: anti-CD3 / 28, IL-4, anti-IFN-γ and anti-IL-2; Ac2-26: anti-CD3 / 28, IL-4, anti-IFN-γ, anti-IL-2 and Ac2-26; Boc: anti-CD3 / 28, IL-4, anti-IFN-γ, anti-IL-2 and Boc) (n=5).

[0024] Fig. 9 : Summary diagram of the pattern in which ANXA1 affects Th2 differentiation and thus IgE production in the Dock8 mutant high IgE mouse model. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0026] The agonist or inhibitor of ANXA1 in the present invention can regulate the level of Th2 cells in HIES disease.

[0027] Furthermore, HIES is AR-HIES caused by DOCK8 gene mutation.

[0028] Example 1 Construction of a mouse model simulating DOCK8 mutation patients

[0029] Clinically, patients with DOCK8 gene mutations usually present with large deletions of the coding region, although smaller point mutations or missense mutations can also be observed. Different mouse models have been constructed to study the consequences of DOCK8 mutations. In this study, a targeted mutation mouse model was constructed in exon 11 of the Dock8 gene using sgRNA and CRISPR / Cas9 technology, which resulted in the appearance of a premature stop codon. The mutation strategy was to introduce a deletion of thymine-guanine (TG) bases at positions 1281 and 1282, resulting in a frameshift mutation and the appearance of an early stop codon (TAA) after the mutation site ( Figure 1 To confirm that the mutation was successfully generated, a fragment of exon 11 containing the mutation site was amplified. PCR analysis showed no significant difference in product size ( Figure 1 B in the figure). Subsequently, the amplified PCR products were Sanger sequenced and compared with the sequences in the NCBI database using the BLAST tool, confirming the deletion of the TG bases at positions 1281 and 1282. Translating the corresponding nucleotide sequences into amino acid sequences showed that in wild-type (WT) mice, the codon at position P427 was CCT (encoding proline), while the codon at position G428 was GGG (encoding glycine). After the deletion of TG at positions 1281 and 1282, the codon at position P427 changed to CCG (still encoding proline), while the codon at position P428 changed to GGT (encoding glycine). Downstream, the original codon at position P429 changed from GTT (encoding valine) to TGG (encoding tryptophan), while the codon at position P430 changed from GTT (encoding proline) to TAA, which is a stop codon. This change resulted in a single peak, confirming that the mutation was a pure mutation ( Figure 1 C). These results confirm the frameshift mutation and its subsequent premature termination.

[0030] To determine whether the mutant mice exhibited the IgE elevation phenotype that is unique to DOCK8 mutation patients, serum IgE levels were measured using ELISA. The results showed that the serum IgE levels of mutant mice were significantly higher than those of the control group ( Figure 1 In addition, during the feeding of these mice, signs of skin inflammation were observed ( Figure 1 D in the figure), which is consistent with the clinical features associated with Dock8 mutation. Finally, spleen cells were extracted from Dock8 mutant mice and quantitative PCR (qPCR) analysis was performed, and the results showed that the expression of Dock8 mRNA in mutant mice was significantly lower than that in the control group ( Figure 1 F). Taken together, these findings confirm that Dock8 1281&1282TGThe deletion mouse model successfully mimicked the HIES phenotype observed in clinical cases of DOCK8 mutations and was accompanied by a decrease in Dock8 expression.

[0031] Example 2 DOCK8 mutation does not affect thymic T cell development, but disrupts peripheral T cell homeostasis and is accompanied by autoimmune disease characteristics

[0032] Clinically, patients with DOCK8 mutations usually show a decrease in the total number of peripheral blood T cells. To further study this phenomenon, this article performed flow cytometric analysis of the total number of T cells in the thymus, spleen, peripheral lymph nodes (pLN), and mesenteric lymph nodes (mLN) of Dock8 mutant mice. The results showed that the proportion of TCR-β+T cells in the spleen of mutant mice was significantly decreased, while the proportion of T cells in the thymus increased. In contrast, the proportion of T cells in the peripheral lymph nodes and mesenteric lymph nodes was almost the same as that in the control group. Interestingly, there was no significant change in the number of cells in the thymus, but the number of cells in the spleen decreased, while the number of cells in the peripheral lymph nodes and mesenteric lymph nodes increased ( Figure 2 A and E in Figure 1). Further analysis of CD4+ and CD8+ T cells showed that the ratio of CD4+ and CD8+ T cells in the spleen did not differ significantly between the two groups, but the number of cells in the spleen decreased, while the number of cells in the mesenteric lymph nodes increased. However, the ratio of CD8+ T cells in the thymus increased, the ratio decreased in the peripheral lymph nodes, and there was no difference in the spleen and mesenteric lymph nodes ( Figure 2 To further clarify the reasons for the changes in mutant T cells in different immune tissues, we examined the proliferation and apoptosis of T cells in the thymus, spleen, peripheral lymph nodes, and mesenteric lymph nodes. The results showed that the proliferation and apoptosis of T cells in mutant mice were comparable to those in control mice ( Figure 3 A and B in Figure 1). This phenomenon suggests that DOCK8 mutations do not affect the development of thymic T cells, but rather affect the migration of T cells. The results showed that DOCK8 mutations disrupted the migration of CD8+ T cells from the thymus to peripheral lymph nodes, while the development of T cells remained intact.

[0033] CD44 and CD62L are key adhesion molecules on T cells and are involved in important processes such as T cell activation, migration, homing and memory formation. To further evaluate the activation level of T cells, we examined the expression of CD44 and CD62L on the surface of T cells. The results showed that the DOCK8 mutation did not affect the activation state of CD4+T cells. The proportions of initial (CD44 low CD62L high), active (CD44 high CD62L low) and memory (CD44 high CD62L high) CD4+T cells in mutant mice were comparable to those in the control group ( Figure 2C, HJ in ). However, CD8+ T cells showed a higher activation state. Further analysis showed that the proportions of initial, active, and memory CD8+ T cells in the thymus and spleen of DOCK8 mutant mice were similar to those of the control group. However, in the peripheral lymph nodes and mesenteric lymph nodes, the proportion of CD8+ initial T cells was significantly reduced, while the proportion of activated and memory T cells was significantly increased ( Figure 2 D, KM in Figure 3). These results suggest that DOCK8 mutations lead to a decrease in the initial ability of the immune response, while enhancing the persistence and intensity of the immune response. Taken together, these results suggest that DOCK8 mutations lead to a complex state of immunosuppression and autoimmune response, suggesting that they may be associated with the development of autoimmune diseases.

[0034] Example 3 DOCK8 mutation inhibits Treg cell differentiation but does not affect Treg precursor cells

[0035] Treg cells play an important role in autoimmune diseases by maintaining immune homeostasis and preventing excessive immune responses, thereby avoiding tissue damage and inflammation. These specialized T cells are characterized by the expression of the transcription factor Foxp3, which is essential for the development and function of Treg cells. To further investigate the effects of Dock8 mutations on Treg cells, we investigated changes in Treg subsets in the thymus, spleen, peripheral lymph nodes (pLN), and mesenteric lymph nodes (mLN) of Dock8 mutant mice. The results showed that CD25+Treg cells ( Figure 4 A, C) and Foxp3+Treg cells ( Figure 4 The Dock8 mutation reduced the Treg cell population, as the proportion of wild-type bone marrow (CD45.1+) cells and Dock8 mutant bone marrow (CD45.2+) cells was significantly decreased in the co-transplanted CD45.1+ cells. To further verify this result, we generated mixed bone marrow chimeras and compared the development of wild-type bone marrow (CD45.1+) cells with Dock8 mutant bone marrow (CD45.2+) cells. Compared with the co-transplanted CD45.1+ cells, the bone marrow of Dock8 mutant mice developed fewer Treg cells ( Figure 4 GI in Figure 5 These results suggest that Dock8 mutations have a cell-self-sufficiency effect on Treg cells, supporting a role for Dock8 in promoting cell-self-sufficiency in Treg cell populations.

[0036] We hypothesized that Dock8 mutations affect Treg development by acting on the Treg precursor cell population. Previous studies have shown that Foxp3+Treg cells are differentiated from precursor cells (CD4+CD25+Foxp3-). However, in our bone marrow chimera analysis, we found that there was no significant difference in the proportion of CD4+CD25+Foxp3- precursor cell populations between Dock8 mutant mice and control mice ( Figure 4 EG in, Figure 5 A and I in Figure 2). To further explore the mechanism of Treg cell reduction, we evaluated the expression level of Foxp3 in CD4+Foxp3+Treg cells. The results showed that there was no significant difference in the mean fluorescence intensity (MFI) of Foxp3 expression between the mutant group and the control group ( Figure 5 D in Figure 1). Next, we examined the expression of key molecules CTLA4 and ICOS in Treg cells. CTLA4 mainly acts as an inhibitory molecule that can downregulate T cell activation and promote immune tolerance; while ICOS plays a dual role by enhancing T cell activation and promoting Treg cell function. Our results showed that in the thymus of mutant mice, the expression of CTLA4 on the surface of Treg cells was significantly higher than that in the control group ( Figure 5 E), while the expression of ICOS was significantly lower than that of the control group ( Figure 5 F). This difference suggests that the differential expression of CTLA4 and ICOS in mutant mice jointly impairs the function of Treg cells. Notably, this phenomenon was not observed in the spleen, peripheral lymph nodes, and mesenteric lymph nodes.

[0037] Although the proportion of Treg cells in mLN was reduced, the expression of migration-related adhesion molecules CD103 and NRP1 in the mutant group was higher than that in the control group ( Figure 5 G and H in Figure ). This finding suggests that Dock8 mutations may enhance Treg cell migration to inflamed tissues and their retention therein, thereby exerting their immunosuppressive function. Taken together, these results indicate that Dock8 mutations lead to an imbalance in immune homeostasis by regulating Treg cell function, further supporting its important role in regulating immune responses.

[0038] Example 4 DOCK8 mutation inhibits Treg cell differentiation but does not affect Treg precursor cells

[0039] Studies have shown that Treg cells can inhibit the differentiation and function of Th2 cells, thereby preventing excessive Th2-mediated immune responses and abnormal B cell activation. However, cytokines secreted by T cell subsets also play a vital role in immune responses. In this study, we measured the levels of IL-2 and IFN-γ secreted by Th1 cells, IL-17A secreted by Th17 cells, and IL-4 secreted by Th2 cells in mutant mice. Flow cytometry results showed that IL-4 levels were significantly increased in CD4+T cells ( Figure 6 C and H). However, the proportions of IFN-γ+ and IL-2+ T cells did not show significant differences ( Figure 6 In CD8+ T cells, IFN-γ levels were significantly increased ( Figure 6 D and E, GK), IL-2 levels showed no significant difference ( Figure 6 J in Figure 3). There was no difference in IL-17A levels between CD4+ and CD8+ T cells ( Figure 6 These findings suggest that Dock8 mutations affect T cell differentiation and immune homeostasis, leading to widespread alterations in T cell differentiation, function, and activation, and support the conclusion that Treg cells inhibit Th2 differentiation and function.

[0040] Example 5: Dock8 mutation promotes IgE secretion by regulating Th2 and Tfh cells

[0041] Th2 cells are key regulators of humoral immunity, mainly guiding B cells to produce immunoglobulins by secreting IL-4. In contrast, Tfh cells promote B cell affinity maturation by secreting IL-21. Th2 and Tfh cells are particularly important in promoting B cell antibody production, especially the production of IgE. In this study, in order to explore the specific mechanism by which Dock8 mutations cause hyper-IgE syndrome, the researchers purified CD4+ naive T cells from mice and induced Th2 differentiation in vitro using anti-CD3 / 28, IL-4, anti-IFN-γ, and anti-IL-2. After five days of culture, the cells were collected for further analysis. The proportions of IL-4+, IL-2+, IFN-γ+, and IL-17A+ cells were evaluated by flow cytometry. The results showed that the proportion of induced Th2 (IL-4+) cells was significantly higher than that of the control group ( Figure 7 In contrast, the proportions of IFN-γ+ and IL-2+ (Th1) cells, as well as IL-17A+ (Th17) cells, decreased or showed no significant difference ( Figure 7 BE in Dock8 mutant mice), indicating that Th2 cell differentiation was successfully induced in vitro. In addition, the proportion of Th2 cells in Dock8 mutant mice was also increased compared with the control group, suggesting that increased Th2 cells may help B cells secrete more IgE.

[0042] In addition, Tfh cells in CD4+T cells were identified using CXCR5, ICOS, and PD-1 markers. The results showed that the proportion of CXCR5+ICOS+ or CXCR5+PD-1+Tfh cells in T cells from Dock8 mutant mice was significantly higher than that in the control group ( Figure 7 At the same time, the expression of CXCR5 and ICOS in Tfh cells also increased ( Figure 7I in ). Tfh cells promote the differentiation of B cells into IgE-producing cells by providing co-stimulatory signals and cytokines, such as IL-4 and IL-21. IL-4 is essential for class switching of IgE, while IL-21 enhances the survival and proliferation of B cells. Therefore, Dock8 mutations may affect IgE secretion by regulating the number and function of Tfh and Th2 cells.

[0043] Example 6 ANXA1 promotes the differentiation of CD4+ T cells into Th2 cells in Dock8 mutant mice Previous studies have shown that ANXA1 plays a vital role in the differentiation of T cells. In this study, flow cytometry analysis showed that the expression level of ANXA1 in T cells of Dock8 mutant mice was higher than that in the control group ( Figure 8 A and B in the figure). Ac2-26 is an active peptide fragment of ANXA1, which is known to regulate Th2 cell differentiation. To further verify this, this experiment induced Th2 cell differentiation in vitro, and applied Ac2-26 treatment to CD4+T cells in the control and mutant groups. The results showed that in the Ac2-26-treated group, the proportion of induced Th2 (IL-4+) cells was significantly higher than that in the untreated group ( Figure 8 This result indicates that ANXA1 can indeed promote the differentiation of Th2 cells.

[0044] In addition, the study also used the ANXA1 receptor inhibitor Boc to treat the cells. In the control group, after Boc treatment, the proportion of induced Th2 cells decreased ( Figure 8 More importantly, the proportion of Th2 cells in the Dock8 mutant group treated with Boc was significantly reduced compared with the untreated Dock8 mutant group, indicating that the proportion of Th2 cells in Dock8 mutant mice can be restored by inhibiting ANXA1 ( Figure 8 These findings suggest that ANXA1 promotes the differentiation of CD4+ T cells into Th2 cells in Dock8 mutant mice and that this Th2 differentiation process can be reversed by inhibiting ANXA1 ( Fig. 9 The results of this study provide a theoretical basis for regulating immune balance in patients with DOCK8 mutations, and are expected to provide a new perspective for the development of future immune regulation treatment strategies.

[0045] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of ANXA1 agonists or ANXA1 inhibitors in regulating peripheral IgE levels.

2. The use according to claim 1, characterized in that The ANXA1 agonist is used to promote IgE synthesis.

3. The use according to claim 1, characterized in that The ANXA1 agonist is used to promote the increase of the proportion of Th2 cells in T cells.

4. The use according to claim 1, characterized in that The ANXA1 inhibitor is used to reduce IgE levels.

5. The use according to claim 4, characterized in that The ANXA1 inhibitor is used to reduce the proportion of Th2 cells in DOCK8 mutant T cells.

6. The use according to claim 5, characterized in that The ANXA1 inhibitor is used for preparing a drug for treating immune diseases caused by DOCK8 mutation.

7. The use according to claim 6, characterized in that The ANXA1 inhibitor is used for preparing a drug for treating autosomal recessive hereditary hyper-IgE syndrome caused by DOCK8 mutation.

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