Use of ezh2 specific inhibitors in the treatment of asthma

Abstract

The present application relates to the treatment of asthma by an inhibitor specific for EZH2, preferably GSK343.

Description

TECHNICAL FIELD

[0001] The present invention relates to the treatment of asthma by an EZH2 specific inhibitor and pharmaceutical compositions comprising the same. In particular, the present invention relates to the treatment of asthma by GSK343 and pharmaceutical compositions comprising the same. BACKGROUND

[0002] Asthma is one of the most common chronic inflammatory lung diseases in children. The development of asthma involves many types of immune cells, such as T cells, B cells, eosinophils and neutrophils 1 . The production of T helper 2 (Th2) cytokines plays a major role in the pathogenesis of asthma. Th2 lymphocytes also increase the synthesis of IgE by stimulating the humoral immune response 2 . Invariant natural killer T (iNKT) cells are characterized by expressing an invariant T cell receptor (TCR) a chain composed of Vα14 / Jα18 in mice or Vα24 / Jα18 in humans, recognizing glycolipid antigens, and releasing large amounts of type 1 and type 2 cytokines. iNKT cells drive the development of mouse asthma-like lung pathology 3 , and the increase of iNKT cells after allergen challenge was found in asthma patients 4 , suggesting that iNKT cells play an important role in asthma. Although the role of iNKT cells in asthma has been studied for many years, how iNKT cells regulate the pathogenesis of this disease has not been clearly elucidated.

[0003] Progranulin (PGRN), also known as acrogranin-epigen, was first recognized as a growth factor for wound healing. Subsequently, PGRN was found to be expressed in other cell types, including macrophages, neurons and chondrocytes 5 . In rheumatoid arthritis (RA), PGRN exerts an anti-inflammatory effect by binding to tumor necrosis factor (TNF) receptors, thereby antagonizing TNF-α signaling 6,7 . In addition, serum PGRN levels are lower in asthma patients than in healthy controls 8 . Furthermore, macrophage-derived PGRN induces Th2 cytokine production in mouse NKT cells, which is crucial in the development of allergic airway inflammation 9 . However, how PGRN regulates iNKT cells and their Th2 cytokine production remains unknown.

[0004] EZH2 (Enhancer of zeste homolog 2) belongs to the polycomb repressive complex 2 (PRC2) family of proteins, which is capable of methylating lysine 27 of histone-H3 (H3K27me3) 10It can also modify non-histone proteins, such as VAV1 and TALIN, which are important for actin polymerization and cell migration. 11,12 Previous studies have shown that EZH2 inhibits iNKT cell-induced Th2 cytokine production, driving asthma-like lung lesions in mice, through methylation-dependent ubiquitination of proleukemic zinc fingers (PLZF) in iNKT cells. 13 .

[0005] In this invention, we investigated the molecular mechanism by which PGRN regulates Th2 responses in iNKT cells using PGRN knockout (KO) mice. In these mice, we found that although the proportion of iNKT cells increased, the number of iNKT cells and IL-4 production decreased. Interestingly, EZH2 expression increased, leading to downregulation of PLZF. Furthermore, expression of the EZH2 inhibitor GSK343 ​​rescued defects in NKT2 cell differentiation, IL-4 production, and PLZF expression. All these studies elucidate a novel pathway by which PGRN regulates NKT2 cells through upregulation of EZH2 expression. This study also identifies a potential new drug target for asthma treatment. Summary of the Invention

[0006] In a first aspect, the present invention provides the use of an EZH2-specific inhibitor, preferably GSK343, in the preparation of a medicament for treating asthma.

[0007] In a second aspect, the present invention provides a pharmaceutical composition for treating asthma, comprising an EZH2-specific inhibitor, preferably GSK343, and a pharmaceutically acceptable carrier. Attached Figure Description

[0008] Figure 1 PGRN deficiency impairs the development of iNKT cells.

[0009] (A and B) Flow cytometry data of iNKT cells from WT and PGRN KO mice. CD1d was not loaded as a negative control (n=11). (CE) Flow cytometry data of cells in phases 0-3 from WT and PGRN KO mice (n=9). (F) Flow cytometry data of iNKT cells from WT and PGRN KO chimeric mice. (G) Flow cytometry data of cells in phases 0-3 from WT and PGRN KO chimeric mice. Percentage of cell populations selected in (H) and (F) (n=3). Percentage of cell populations selected in (I) and (G) (n=3). (J) Expression of PGRN in iNKT cells in phases 0-3 from WT mice. The flowchart is representative of three independent experiments. Data are expressed as mean ± SEMs. *p<0.05; **p<0.01; **p<0.001

[0010] Figure 2 PGRN deficiency leads to impaired NKT2 cell proliferation.

[0011] (A) Flow cytometry data of NKT2, NKT17, and NKT1 cells from WT and PGRN KO mice. (B) Flow cytometry data of NKT2, NKT17, and NKT1 cells from WT and PGRN KO chimeric mice. (C) Percentage and absolute number of cell populations selected from (A) (n=10). (D) Percentage of cell populations selected from (B) (n=3). (E) Expression of PGRN in NKT1, NKT2, and NKT17 cells of WT mice. Data are presented as mean ± SEM. *p<0.05; ***p<0.001

[0012] Figure 3 PGRN is crucial for the effector function of NKT2 cells.

[0013] (A) Flow cytometry analysis of IL-4, IL-17, TNF-α, and IFN-γ expression in enriched thymic iNKT cells after 5 hours of in vitro stimulation with PMA and ionomycin. (B) Flow cytometry analysis of IL-4, IL-17, TNF-α, and IFN-γ cells in enriched iNKT cells from WT and PGRN KO chimeric mice after PMA and ionomycin stimulation. (C) Percentage of the selected cell population in (A), (n=8). (D) Percentage of the selected cell population in (B), (n=3). (E) Proportion of IL-4, IL-17, TNF-α, and IFN-γ cells in enriched thymic iNKT cells stimulated with α-Galcer and PMA+ionomycin. (F) IL-4 mRNA expression in thymic iNKT cells (n=3). (G) Serum titers of IL-4 after in vivo treatment with α-Galcer or methylcholine (n=3). (H)(E) Percentage of selected cell populations (n=3). (I) Airway resistance data in WT and PGRN KO mice after methacholine stimulation (n=3). Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001

[0014] Figure 4 Loss of PGRN increases apoptosis in stage 1 iNKT cells.

[0015] (A and B) Proportion of Ki67+ cells in iNKT, NKT1, NKT2, and NKT17 cells of WT and PGRN KO mice (n=6). (C and D) Proportion of Ki67+ cells in iNKT, NKT1, NKT2, and NKT17 cells of chimeric mice (n=3). (E and F) Flow cytometry analysis of adductin V+ cells in iNKT, phase 0, 1, 2, and 3 cells of WT and PGRN KO mice (n=6). (G and H) Flow cytometry analysis of adductin V+ cells in iNKT, phase 0, 1, 2, and 3 cells of chimeric mice (n=3). Data are expressed as mean ± SEM. *p<0.05; **p<0.01

[0016] Figure 5 PGRN regulates NKT2 differentiation by promoting PLZF expression and nuclear entry.

[0017] (A) Expression of Egr2 mRNA in iNKT cells of the thymus of WT and PGRN KO mice (n=3). (B) Expression of Irf4 mRNA in iNKT cells of the thymus of WT and PGRN KO mice (n=3). (C) Expression of Zbtb16 mRNA in iNKT cells of the thymus of WT and PGRN KO mice (n=3). Data are presented as mean ± SEM of (AC). (D) Flow cytometry analysis of the expression of PLZF, Tbet, RORγt, and GATA3 in iNKT cells of the thymus of WT and PGRN KO mice (n=3). (E) Flow cytometry data from NK1.1-PLZF+ iNKT cells of the thymus of WT and PGRN KO mice. (F) Percentage and number of cells in the gated population in (E), (n=3). (GI) Categorized iNKT cells from the thymus of WT and PGRN KO mice were cultured with 125 ng / ml α-Galcer or uncultured for 72 h and stained with PLZF. Cells were analyzed using confocal microscopy, and the MFI of PLZF was measured. Data are representative images from three independent experiments, with mean ± SDs of more than 50 cells plotted. Scale bar, 2.5 μm. (I) The ratio of nuclear PLZF MFI in iNKT cells before and after stimulation was plotted (n = 3). *P < 0.05; **P < 0.01. Data were analyzed by two-way ANOVA in (H).

[0018] Figure 6 PGRN coupling with EZH2 regulates PLZF expression and stability.

[0019] (A) Expression of EZH2 mRNA in iNKT cells of the thymus of WT and PGRN KO mice (n=3). (B and C) EZH2 staining of iNKT cells in the thymus and measurement of EZH2 MFI using confocal microscopy. Scale bar, 2.5 μm. **p<0.01 in (C) by Mann-Whitney U test. (D) Immunoblotting of CD4+ cell lysates from the thymus of WT and PGRN KO mice to detect EZH2; β-actin as a loading control. The image is representative of three independent experiments. (E and F) Flow cytometry analysis of IgE expression in B cells of the spleen of WT and PGRN KO mice (n=3). (G) Thymic cells of WT and PGRN KO mice labeled with CD4, then stimulated with PMA (50 ng / ml) and ionomycin (500 ng / ml) for specified time, and stained for phospho-IKKα / β and phospho-p65. MFI values ​​were analyzed using FlowJo software. (H) Phosphorylation of p65 and IKKα / β in iNKT cells of WT and PGRN KO mice. (I) iNKT cells were isolated from WT mice, stimulated with or without anti-PGRN antibody, and then stained with EZH2. EZH2 expression levels were analyzed by flow cytometry. (JO) PGRN KO mice were treated with GSK343 ​​intraperitoneally for 14 days. Flow cytometry data of iNKT (J) and NKT2 (L) cells in thymocytes of WT and PGRN KO mice are shown, along with the frequencies and cell numbers of K (selected from J) and M (selected from L), (n=4). (N and O) PGRN KO mice were treated with GSK343 ​​for 14 days. Flow cytometry data of iNKT cells expressing IL-4, IL-17, TNF-α, and IFN-γ after 5 hours of stimulation with PMA and iomycin (n=4). (P) PGRN KO mice were treated with GSK343 ​​for 14 consecutive days. PLZF expression in thymic iNKT cells was analyzed by flow cytometry (n=4). (Q) Lung resistance in WT, PGRN KO, WT+GSK343, and KO+GSK343 ​​mice after methacholine stimulation (n=3). (R) Serum IgE titers in WT, PGRN KO, and PGRN KO mice after 14 consecutive days of intraperitoneal injection of GSK343 ​​(n=4). Data were analyzed using a two-tailed unpaired Student's t-test (A, F) and one-way ANOVA (K, M, Q, R). Data are expressed as mean ± SEM. *P<0.05; **P<0.01.

[0020] Figure 7

[0021] (A and B) Flow cytometry data of splenic iNKT cells from WT and PGRN KO mice; CD1d unloaded as a negative control (n=4). (C) Flow cytometry analysis of NKT2, NKT17, and NKT1 cells from the spleen of WT and PGRN KO mice. (D) Percentages of IL-4, IL-17, TNF-α, and IFN-γ cells enriched in splenic iNKT cells after 5 hours of stimulation with PMA and iomycin. (E) Percentage and absolute number of gated populations in (C) (n=3). (F) Percentage of cell populations selected from (D) (n=4). (G) Classified iNKT cells from the thymus of WT mice stained with anti-PGRN antibody to show cell localization. Data are presented as mean ± SEMs. *p<0.05 Example

[0022] This application will be explained in detail below.

[0023] It is well known that T helper 2 (Th2) cytokines produced by invariant natural killer T (iNKT) cells are involved in the progression of asthma, but the regulation of Th2 cytokines in iNKT cells remains unknown. Although gamma globulin (PGRN) is known to induce Th2 cytokine production in iNKT cells in vivo, the underlying mechanism is unclear. Therefore, one of the objectives of this invention is to investigate the role of PGRN in iNKT cells.

[0024] In this invention, the effect of PGRN on iNKT cell differentiation was detected by flow cytometry. Further studies on iNKT cell stimulation and airway resistance were performed to assess the function of PGRN on iNKT cells. In addition, the mechanism by which PGRN regulates iNKT cells was investigated using RT-PCR, Western blotting, and confocal microscopy.

[0025] In our study, we found that in PGRN KO mice, despite an increased percentage of iNKT cells, the absolute number of iNKT cells was reduced. Furthermore, analysis of iNKT cell subsets revealed a decrease in NKT2 cells and their IL-4 production. We further discovered that the reduction in NKT2 cells in PGRN KO mice was caused by increased expression of the zeste homolog enhancer 2 (EZH2), which in turn led to increased PLZF degradation and altered nuclear localization. Interestingly, we found that the PGRN signaling pathway reduced EZH2 expression, and we treated PGRN KO mice with EZH2-specific inhibitors (such as GSK343). We suggest that EZH2-specific inhibitors like GSK343 ​​can repair defects in NKT2 differentiation, IL-4 production, and PLZF expression.

[0026] The EZH2 gene encodes a member of the PcG family. PcG family members form multimeric protein complexes involved in maintaining transcriptional repression of genes across successive cell generations. This protein is associated with embryonic ectodermal developmental proteins, VAV1 oncoprotein, and X-linked nucleoprotein. This protein may play a role in hematopoiesis and the central nervous system. Several alternative splicing variants of this gene have been identified.

[0027] Zest homolog 2 enhancer (EZH2), also known as histone lysine n-methyltransferase EZH2, is a histone methyltransferase with an added methyl group (-CH) located at the 27th lysine residue of histone 3 (H3K27).

[0028] Diseases associated with EZH2 include Weaver syndrome and lymphoma. The relevant pathways for EZH2 involve pkmt methylation of histone lysine residues and activated PKN1 stimulation of transcription of AR (androgen receptor) regulatory genes KLK2 and KLK3. Gene ontology (GO) annotations associated with this gene include sequence-specific DNA binding and chromatin binding. An important homolog of this gene is EZH1. It is currently unclear whether diseases associated with EZH2 include asthma.

[0029] Surprisingly, in current applications, we have found that EZH2-specific inhibitors such as GSK343 ​​can be used to treat asthma. GSK343 ​​is the preferred EZH2-specific inhibitor.

[0030] In our current application, we have discovered a novel pathway (PGRN-EZH2-PLZF) that regulates the Th2 response of iNKT cells, providing a potential new target for asthma treatment.

[0031] The EZH2-specific inhibitors that can be used in this invention include, but are not limited to, GSK343, GSK503, GSK126, PF-06821497, CPI-169, CPI-360, CPI-1205, and other EZH2-specific inhibitor groups known in the art. Preferably, the EZH2-specific inhibitor used in this invention is GSK343.

[0032] GSK343 ​​has the following structure:

[0033]

[0034] Materials and methods

[0035] mice

[0036] Six- to eight-week-old PGRN- / - mice (PGRN KO) (Jackson Laboratory) and age-matched C57BL / 6 wild-type (WT) mice (controls) were used in the experiments. All mice were housed in individually ventilated cages at Chongqing Medical University Children's Hospital. All animal work was reviewed and approved by the Institutional Animal Care and Use Committee of Chongqing Medical University Children's Hospital, and all experiments were conducted in accordance with medical ethics principles and the Declaration of Helsinki. Detailed methodologies for bone marrow chimeric mice, quantitative RT-PCR, and Western blotting experiments are available as supplementary materials.

[0037] Flow cytometry

[0038] Spleen cells and thymocytes were isolated according to a previously published protocol. 14 Monoclonal antibodies against CD44 (clone: ​​IM7), anti-annexin V, anti-CD4 (clone: ​​RM4-5), anti-NK1.1 (clone: ​​PK136), anti-Tcr-β (clone: ​​H57-597), anti-CD24 (clone: ​​M1 / 69), anti-CD8 (clone: ​​53-6.7), anti-CD45.2 (clone: ​​104), and anti-CD45.1 (clone: ​​A20) were incubated in PBS + 2% FBS at room temperature for 30 min. Spleen cells were incubated with FcγR blocking antibody, labeled with anti-b220 (clone: ​​RA3-6B2), fixed and permeabilized with Foxp3 staining buffer (eBioscience), and stained with anti-IgE (clone: ​​RME-1) to detect IgE expression in spleen cells. All antibodies were purchased from BioLegend.

[0039] For intracellular staining, cells were fixed and infiltrated using Foxp3 staining buffer (eBioscience) according to the manufacturer's instructions, followed by staining with APC-anti-RORγt (clone: ​​AFKJ5-9, eBioscience), anti-T-bet (clone: ​​AFKJ5-9, eBioscience), anti-PK136 (BioLegend), anti-gata3 (clone: ​​10E10A23, BioLegend), anti-ki-67 (clone: ​​SolA15, eBioscience), anti-ezh2 (clone: ​​D2C9, Cell Signaling Technology), anti-pgrn (clone: ​​ERP18539-59, abcam), anti-plzf(D-9)(mouse)(Santa Cruz), and FITC-labeled rat anti-mouse IgG1 secondary antibody (clone: ​​M1-14D12, eBioscience).

[0040] To detect phosphorylated proteins, we labeled iNKT cells and pulsed them at 37°C with phorbol 12-myristate-13-acetate (50 ng / ml) and iomycin (500 ng / ml) for different time periods. After activation, the cells were fixed and infiltrated with Phosflow Perm Buffer III (BD Biosciences), and then stained with rabbit monoclonal antibodies against phosphorylated nf-κB p65 (Ser536) (clone: ​​93H1) and phosphorylated ikkα / β (Ser176 / 180) (clone: ​​16A6). All primary antibodies were derived from Cell Signaling Technologies. The secondary antibody used was AF488 goat anti-rabbit antibody (Life Technologies).

[0041] Bone marrow chimeric mice

[0042] For the chimeric study, bone marrow cells (1 × 10⁷) were derived from WT and PGRN- / - mice in a 1:1 ratio and then intravenously injected into CD45.1 mice irradiated with a sublethal dose (6 Gy). Eight weeks later, the recipient mice were euthanized for experimental use.

[0043] Stimulation of iNKT cells

[0044] Thymocytes were isolated under in vitro stimulation with α-galactosylceramide (α-galcer) and seeded in 24-well plates. They were incubated with RPMI + 10% fetal bovine serum for 72 hours with or without 125 ng / ml α-galcer, followed by 50 ng / ml PMA and 500 ng / ml iomycin for the last 5 hours. Intracellular markers were then stained using loaded CD1D, anti-7aad (BBI Life Sciences), and anti-tcr-β (clone: ​​MP6-XT22, BioLegend), anti-il-4 (clone: ​​11B11, BioLegend), anti-ifn-γ (clone: ​​XMG1.2, BioLegend), and anti-il-17a (clone: ​​TC11-18H10.1, BioLegend). For anti-pgrn stimulation, iNKT cells were isolated from WT mice, stimulated with or without anti-pgrn antibody, and then EZH2 cells were stained. Flow cytometry was used to detect the expression level of EZH2.

[0045] Immunoblotting

[0046] Detection of EZH2 expression in thymic CD4+ + cell count: 3 × 10-1 6 Thymus cells were sorted from WT and PGRN knockout mice. After lysis and protein denaturation, immunoblotting analysis was performed using SDS-PAGE and anti-ezh2 antibody. β-Actin was used as a loading control.

[0047] confocal microscope

[0048] Sorted iNKT cells were placed on polylysine-coated slides. After cell fixation and permeabilization, they were stained for 1 h with anti-plzf (Santa Cruz Biotechnology), anti-ezh2 (clone: ​​D2C9, Cell Signaling Technology), and af488-anti-actin (Invitrogen) antibodies, respectively, using af546-goat anti-mouse antibody (1:400) as a secondary antibody. Nuclear staining was performed using 1.5 μg / ml DAPI (Beyotime). iNKT cells were incubated with α-Galcer (125 ng / ml) in 96-well plates for 72 h and then stained using the same method. Images were acquired using a confocal fluorescence microscope (Nikon A1R).

[0049] Airway hyperresponsiveness

[0050] In short, 2 μg of α-Galcer was added to 50 μl of PBS and administered intranasally. Mice were anesthetized 24 h later. A tracheostomy cannula was inserted and connected to a computer-controlled ventilator. Acetylcholine (10 mg / ml, 25 mg / ml, 50 mg / ml, and 100 mg / ml) was added to 0.9% sodium chloride solution via nebulizer, and airway resistance was subsequently measured. Airway resistance was then measured and plotted (lung resistance cmH2O / mL / s).

[0051] Quantitative RT-PCR

[0052] Thymocytes were stained with PE-CD1d and anti-tcr-β, and then iNK T cells were sorted using a BD FACS Aria III sorter. Total RNA was isolated from the sorted iNK T cells using TRIzol reagent (BioTeke) and reverse transcribed into cDNA using a PrimeScript RT kit (US, Takara). Gene expression was then measured using the following primer pairs on a CFX96 Real-Time PCR system (Bio-Rad):

[0053] Irf4:F 5'-GGAAGACAAGATTACGATGTGC-3';R 5"-AATCCTGTACACCTTGTATGGG-3"

[0054] Egr2:F 5'-GATCCTTCAGCATTCTTATCGC-3'; R 5"-GATCATAGGAATGAGACCTGGG-3"

[0055] Ezh2:F 5'-ATGAAGCAGACAGAAGAGGAAA-3';R 5"-GGATAGCCCTCTTAGCAAAGAT-3"

[0056] Zbtb16:F 5'-CGCCACCTTCGCTCACATACAG-3';R 5"-TGGTGCTTGAGGCTGAACTTCTTG-3"

[0057] confocal microscope

[0058] The confocal microscopy examination method is as described above. 15 Airway hyperresponsiveness

[0059] We assessed airway hyperresponsiveness as described previously. 16

[0060] ELISA method

[0061] Serum IgE and IL-4 levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience). Statistical analysis was performed.

[0062] Statistical significance was assessed using a two-tailed unpaired t-test in Prism 7 software (*p<0.05; *p<0.01; **p<0.001), unless otherwise stated.

[0063] result

[0064] PGRN deficiency can impair the development of iNKT cells.

[0065] To investigate the role of PGRN in iNKT cell development, we compared the percentage and total number of thymic iNKT cells in PGRN KO mice and WT mice under the same background. Using CD1d and TCR-β staining, PGRN KO mice showed a higher percentage of iNKT cells but a lower absolute number of cells compared to WT controls. Figure 1 A, B). iNKT cells originate from double-positive thymocytes and are divided into four populations (phases 0-3). 17 The iNKT cell phases were determined using CD1d, CD24, CD44, and NK1.1 as markers. 17 We found no significant difference in the percentage and absolute number of phase 0 (CD1d+CD24+) and phase 1 (CD44-NK1.1-) and phase 3 (CD44+NK1.1+) cells between WT and KO mice. In PGRN KO cells, the expression level of CD44 was significantly reduced. Figure 1To determine whether PGRN KO iNKT cell development is intrinsically linked, we transferred CD45.2WT or 1:1 PGRN KO:CD45.1WT bone marrow cells into WT CD45.1 recipient mice, thus generating bone marrow chimeric mice. After 8 weeks, we found that the percentage of CD45.2 PGRN KO iNKT cells was higher than that of CD45.2 WT iNKT cells (CE). Figure 1 F,H). In chimeric mice, the proportion of CD45.2 PGRN KO iNKT cells in phase 0 and 1 was also reduced, but there was no difference in the proportion of iNKT cells in phase 2 and 3. Figure 1 G,I). These data indicate that the effect of PGRN on iNKT development is cell-autonomous. We also performed quantitative analysis of splenic iNKT cells in WT and PGRNKO mice, finding a significant increase in the percentage of iNKT cells in KO mice, but no significant difference in absolute cell number. Figure 7 AB). Furthermore, we found that PGRN expression was higher in phases 0 and 1 than in phases 2 and 3 (AB). Figure 1 These findings suggest that PGRN is important for the development and differentiation of iNKT cells in phase 0 and 1.

[0066] PGRN deficiency leads to impaired NKT2 cell expansion.

[0067] Based on the differential expression of transcription factors that produce IFN-γ, IL-4, and IL-17, iNKT cells can be divided into NKT1, NKT2, and NKT17 cells. To investigate the role of PGRN in the differentiation of NKT1, NKT2, and NKT17 cells, we stained thymic iNKT cells for intracellular expression of PLZF, Tbet, GATA3, and RORγt. We found that the percentage and absolute number of NKT2 cells were decreased in PGRN-treated KO mice, but there was no significant difference in NKT17 and NKT1 cells between WTs and KOs. Figure 2 A, C). We also used chimeric mice to investigate whether the changes in NKT2 in PGRN KOs were intrinsic to the cells. The results consistently showed that the percentage of NKT2 cells was also reduced in CD45.2 PGRN KO mice, but there was no difference in the percentages of NKT1 and NKT17 cells between WT and PGRN KO mice. Figure 2 B, D). These results indicate that the changes in NKT2 cells in KO mice are intrinsic to the cell. Furthermore, we found a decrease in the number of splenic NKT2 cells in PGRN KO mice, but no difference in the number of NKT1 and NKT17 cells between WTs and KOs. Figure 7We also examined PGRN expression in different iNKT cell subsets of wild-type mice and found that PGRN expression was highest in NKT2 cells. Figure 2 All these results indicate that PGRN is important for NKT2 cell differentiation.

[0068] PGRN is crucial for the effector function of NKT2 cells.

[0069] NKT2 cells produce IL-4 and play an important role in certain diseases, such as allergic asthma-like lung lesions observed in mice. 14 We stimulated thymic and splenic iNKT cells from WT and PGRN KO mice in vitro for 5 hours with PMA and iomycin, and examined cytokine production by flow cytometry. Compared with the corresponding WT cells, IL-4 production was reduced in both PGRN KO thymic and splenic iNKT cells, TNF-α expression was increased in PGRN KO thymic iNKT cells, while there was no significant difference in IL-17 and IFN-γ. Figure 3 A, C, Figure 7 D, F). By using chimeric mice, we found that the reduced IL-4 production in the thymus of PGRN KO mice was intrinsic to the cell. Figure 3 (B, D). To further confirm the effects of PGRN on iNKT cell cytokine production under physiological conditions, thymic iNKT cells were stimulated with α-Galcer in vivo and in vitro. Flow cytometry analysis showed that in PGRN KO mice, IL-4 production was reduced, but the production of IL-17, TNF-α, and IFN-γ remained unchanged. Figure 3 E, H), indicating that PGRN is crucial for α-Galcer-induced IL-4 production. Furthermore, we examined IL-4 mRNA expression in thymic iNKT cells and found that it was also decreased in KO mice (E, H). Figure 3 F). Since IL-4 produced by iNKT cells has previously been shown to induce allergic asthma-like lung lesions in mice. 14 We measured serum IL-4 levels in mice after stimulation with α-Galcer and methacholine, and found that the levels in PGRN KO mice were lower than those in WT mice. Figure 3 G). We then tested airway resistance after stimulation with aerosol methacholine. We found that baseline high reactivity was comparable in WT and PGRN KO mice, and airway resistance increased in both genotypes after stimulation with aerosolized methacholine. However, airway resistance decreased in KO mice compared to the WT control group. Figure 3 I). These results indicate that the lack of PGRN leads to impaired function of NKT2 cells.

[0070] The loss of PGRN upregulated apoptosis in stage 1 iNKT cells.

[0071] We investigated whether the reduction in thymic iNKT and NKT2 cells in PGRN KO mice was caused by changes in proliferation or apoptosis. First, we examined iNKT cell proliferation by flow cytometry and found no difference in the proportions of Ki67+NKT1, NKT2, NKT17, and total iNKT cells between WT and PGRN KO mice. Figure 4 A, B). We also found that the proportion of Ki67+ cells in CD45.2 PGRNKO iNKT, NKT1, NKT2, and NKT17 cells did not change ( Figure 4 C, D). We then examined the expression of adjuvant V in total cells and iNKT cells in stages 0–3, finding an increased proportion of adjuvant V+ cells in stage 1 iNKT cells of PGRN KO mice, most of which were NKT2 cells. No difference was observed in total iNKT cells and iNKT cells in stages 0–3 with adjuvant V+. Figure 4 E, F). Furthermore, we found that the percentage of adnexin V+ cells in CD45.2 PGRN KO mice was higher than in the WT control group. This suggests that the increase in adnexin V in stage 1 iNKT cells is endogenous. Figure 4 G, H) may be the reason for the reduced number of NKT2 cells. Therefore, we conclude that the loss of PGRN increases apoptosis in stage 1 iNKT cells.

[0072] PGRN regulates NKT2 cell differentiation by promoting PLZF expression.

[0073] Next, we investigated how PGRN deficiency leads to a reduction in NKT2 cells. Several transcription factors affecting NKT2 cell development have been identified. Notably, GATA3 is a major transcription factor in NKT2 cells. 15 Strong TCR signaling induces high expression of PLZF, which is essential for NKT2 differentiation. 16 Egr2 directly binds to the Zbtb16 promoter and activates PLZF expression. 17 IRF4 promotes IL-4 production by activating the IL-4 promoter. 18 We first examined the expression of Egr2, Irf4, and Zbtb16 mRNA using RT-PCR and found no difference between PGRN WT and KO mice. Figure 5 AC). Next, flow cytometry analysis showed that the expression of GATA3, PLZF, and Tbet was comparable between WT and PGRN KO mice, but PLZF expression was significantly decreased in KO mice. Figure 5D). Furthermore, we found that the number of PLZF+NK1.1 cells in PGRN KO mice was significantly lower than that in the WT control group ( Figure 5 E, F). These results indicate that reduced PLZF expression in KO mice leads to the loss of NKT2 cells. Furthermore, the nuclear localization of PLZF is important for the development and function of iNKT cells. 19 Therefore, we tested whether PGRN affected the nuclear localization of PLZF using CFm. The results showed that PLZF was localized in both the nucleus and cytoplasm of iNKT cells, and the mean fluorescence intensity (MFI) of nuclear PLZF in KO mice was significantly lower than that in the WT control. After 3 days of stimulation with α-Galcer, we found that the nuclear localization of PLZF was increased in both genotypes of iNKT cells, but the nuclear PLZF MFI ratio in KO mice was lower than that in the WT control group. Figure 5 All these results indicate that PGRN regulates NKT2 differentiation and function by promoting PLZF expression and nuclear entry.

[0074] PGRN is coupled with EZH2 to regulate PLZF expression and stability.

[0075] The discrepancy between mRNA and protein expression levels leads us to speculate whether the reduced PLZF expression is caused by abnormal post-transcriptional modifications. EZH2 is a histone methyltransferase that methylates H3K27 and non-histone proteins (such as transcription factors), playing a crucial role in the development and stabilization of the immune system. 13 Previous studies have found that EZH2 deficiency in T cells leads to the expansion of NKT cells with high PLZF levels, which is attributed to PLZF methylation and degradation. 20 Here, we investigated whether the reduction in PLZF in PGRN KO mice was related to EZH2 expression. First, we examined EZH2 mRNA expression in iNKT cells and found that the EZH2 mRNA level in PGRN KO mice was significantly higher than that in WT mice. Figure 6 A). Next, we used confocal microscopy to analyze the expression of EZH2 protein by immunofluorescence, and found that EZH2 protein expression was increased in KO mice ( Figure 6 B, C). Furthermore, it was found that the EZH2 level in the CD4+ T cell lysate of PGRN KO mice was higher than that of WT mice (B, C). Figure 6 D). These results suggest that the reduced number of NKT2 cells and PLZF levels in PGRN KO mice may be caused by enhanced EZH2 expression.

[0076] EZH2 is believed to limit IgE production. 21Therefore, we examined IgE+ cells in WT and PGRN KO mice using flow cytometry and found that the frequency and number of IgE+ cells in the latter were decreased. Figure 6 E, F). Previous studies have shown that TNF-α depletion inhibits EZH2 expression (an autocrine TNF-α tumor), which is associated with the NF-κB pathway. 22 We asked whether PGRN binds to TNFR to promote EZH2 expression. Thymocytes were labeled with anti-CD4 antibody and stimulated in vitro with PMA and iomycin; we found that compared with WT mice, PGRN KO mice showed significantly increased phosphorylation of IKKB and p65, and this phosphorylation occurred downstream of the NF-α-TNFR signaling pathway. Figure 6 G). We also examined the phosphorylation of p65 and IKKB in thymic iNKT cells of WT and PGRN KO mice and found that both phosphorylation was increased in the latter mice. Figure 6 This result indicates that PGRN regulates EZH2 expression through the NF-κB pathway. Furthermore, using anti-PGRN immunofluorescence, we found that PGRN is simultaneously localized on the cell membrane and cytoplasm of iNKT cells (H). Figure 7 G). Then we activated WT iNKT cells with anti-PGRN antibody and found that EZH2 expression was downregulated (G). Figure 6 I) indicates that PGRN activation reduces EZH2 expression. To further confirm that PGRN couples with EZH2 to regulate PLZF expression and stability, PGRN KO mice were treated in vivo for 14 consecutive days with the EZH2 inhibitor GSK343. We found that iNKT, NKT2, and IL-4+ cells were rescued to the same levels as in WT mice through this treatment ( Figure 6 JO). Furthermore, we found that treatment with GSK343 ​​improved PLZF expression, hyperresponsiveness, and serum IgE titers to WT levels ( ). Figure 6 All these results indicate that PGRN, coupled with EZH2, regulates PLZF expression and stability, thereby further modulating NKT2 cell differentiation.

[0077] discuss

[0078] PGRN can induce Th2 cytokine production in iNKT cells, but the underlying molecular mechanisms remain unclear. In our study, we investigated the role of PGRN in iNKT cells using PGRN-knockout (KO) mice. Following PGRN KO, the percentage of iNKT cells increased, but the total number of iNKT cells decreased. Interestingly, we found a significant reduction in stage 1 iNKT and NKT2 cells in KO mice, while the number of NKT1 and NKT17 cells remained unchanged. Using chimeric mice, we demonstrated that the changes in iNKT and NKT2 cells were endogenous. Furthermore, we found that NKT2 cell function was impaired in KO mice. Mechanistically, the reduction in NKT2 cell number was associated with increased EZH2 expression, which in turn led to increased PLZF methylation, regulated by overactivation of the TNF-α-TNFR2-mediated pathway. To our knowledge, we have identified a novel pathway by which PGRN regulates iNKT cell differentiation and function.

[0079] Although PGRN has been extensively studied in autoimmune diseases, cancer, and neurodegenerative diseases 23-26 Few studies have focused on immune cells. 27 A study showed that PGRN promotes the differentiation of CD4+ T cells into Treg cells and enhances the function of the latter. 6 PGRN is also highly expressed in neutrophil subsets, promoting antibody diversity in B cells. 28 Furthermore, PGRN has been shown to induce the expression of Th2 cytokines IL-4 and IL-5. 29 Since IL-4 is produced in both iNKT cells and activated CD4+ T cells, we first aimed to elucidate the role of PGRN in iNKT cells and the PGRN-dependent regulatory mechanisms on iNKT cell development and function. IL-4 is regulated upstream by several transcription factors, including GATA3, IRF4, and c-Maf. 30-32 Our study established another upstream regulatory axis (PGRN-EZH2-PLZF) to epigenetically control IL-4 expression, which may be utilized by other cell types besides iNKT cells. Therefore, we first established a negative correlation between PGRN signaling and EZH2 expression.

[0080] Previous studies have shown that NKT0 cells at stage 0 will develop into NKT2 cells, followed by NKT1 and NKT17 cells. 33In our study, we found a reduced number of phase 0 iNKT cells in PGRN-knockout (KO) mice, which may have impaired NKT2 cell differentiation. However, how PGRN affects the generation of phase 0 iNKT cells remains unclear. We also found increased NF-κB signaling in KO mice, suggesting that NF-κB signaling may influence early iNKT cell differentiation. PLZF is a key factor regulating iNKT cell development and function; deletion of PLZF in mice prevented the transition of iNKT cells from phase 1 to phase 2. Furthermore, the absence of PLZF in iNKT cells prevented the secretion of high levels of IL-4 and IFN-γ. 34,35 In our study, we found a significant decrease in iNKT cells in stages 0 and 1 upon PGRN KO, but no change in iNKT cell numbers in stages 2 and 3. Furthermore, we observed an increase in adjuvant V levels in stage 1 cells, indicating that the changes in NKT2 cells were due to increased apoptosis and decreased PLZF expression. A previous study showed that PLZF has anti-apoptotic effects in many cell types, such as Jurkat cells. 36 In our study, we found that downregulation of PLZF led to increased apoptosis in NKT2 cells, indicating that PLZF has similar anti-apoptotic activity in iNKT cells. Furthermore, we found no significant difference in IFN-γ production by activated iNKT cells between WT and PGRN KO mice, which may be due to residual PLZF expression in PGRN KO iNKT cells. However, changes in the expression of other proteins due to PGRN deficiency may also be a contributing factor, and other IFN-γ-producing cells may be able to compensate.

[0081] High expression of EZH2 can be observed in a variety of cancers, including B-cell and T-cell lymphomas. 37 Myc upregulates EZH2 by inhibiting miRNA-26a and miRNA-26b. 38 Downregulation of miRNA-138 leads to increased targeting of the Ezh2 gene. 39 NF-κB transcription factors Rel and IL-6 upregulate EZH2 expression in T-ALL and multiple myeloma cells. 40PI3K / AKT depletion reduces EZH2 expression. In our study, we found no difference in S6 ribosomal protein and Akt phosphorylation after iNKT cell stimulation between PGRN KO and WT mice (data not shown). However, in PGRN KO iNKT cells, NF-κB signaling was upregulated upon stimulation. Therefore, PGRN signaling is highly likely to inhibit NF-κB activation, leading to EZH2 downregulation. Reduced EZH2 expression upregulates PLZF, which in turn increases NKT2 and Th2 cell responses. Therefore, we constructed a regulatory network centered on EZH2 that modulates NKT2 cell differentiation and Th2 responses. The high expression of EZH2 in PGRN KO mice may be a result of increased TNF-α-TNFR signaling.

[0082] In summary, PGRN regulates NKT2 cell development and function by downregulating EZH2 expression and upregulating PLZF expression. This is a novel mechanism by which PGRN is linked to airway hyperactivity in mice, and may be a new target for asthma treatment.

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Claims

1. The use of the EZH2-specific inhibitor GSK343 ​​in the preparation of drugs for treating asthma.

Images