Medicine and medicine composition for treating APLAID

Abstract

The invention relates to the technical field of biology, in particular to a medicine and a medicine composition for treating APLAID, the medicine is an inhibitor of a targeted IFN beta signal channel, and the medicine composition comprises the inhibitor of the targeted IFN beta signal channel and further comprises a pharmaceutically acceptable carrier and / or auxiliary materials. Molecular mechanism research carried out by utilizing a PLCgamma2p. Leu845Ser mutant macrophage and a mouse model shows that IFN beta up-regulated in the macrophage is an important driving factor of APLAID, and expression of the IFN beta is up-regulated by enhancing combination of the PLCgamma2p. Leu845Ser mutant macrophage and IRF5 and promoting nuclear translocation of the IRF5. The invention reveals that IRF5 / IFN beta signal transduction activated in macrophages is a key driving factor of APLAID development for the first time, and provides a new therapeutic target for APLAID patients.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to drugs and pharmaceutical compositions for treating APLAID. Background Art

[0002] APLAID is a rare autoinflammatory disease. The main clinical features of APLAID include early onset, recurrent inflammatory bowel disease and eye diseases, blistering skin lesions, joint pain, and pulmonary inflammatory lesions. Since the molecular mechanism of APLAID is still unclear, there is no standard treatment guideline for APLAID patients. Existing treatment methods mainly focus on immunosuppressive therapy, with limited effects and certain side effects. Therefore, there is an urgent need to find new treatment strategies, especially for the key molecular targets in the molecular mechanism and pathological process of the disease.

[0003] Studies have shown that PLCγ2 (phosphatidylinositol-specific phospholipase Cγ2) is a key immune signaling molecule, and its mutation is closely related to the occurrence of APLAID. Our previous study found that the p.Leu845Ser mutation of the PLCγ2 gene can enhance the binding to IRF5 (interferon regulatory factor 5), promote the nuclear translocation of IRF5, and ultimately lead to the overexpression of IFNβ. IFNβ (interferon β) is an important cytokine in the immune system and is widely involved in immune responses and inflammatory reactions. We first found that the overexpression of IFNβ is a key factor in the occurrence and development of this disease. Therefore, targeting the IFNβ signaling pathway, especially by regulating the PLCγ2 / IRF5 / IFNβ signaling axis, may provide new ideas and strategies for the treatment of APLAID.

[0004] The present invention aims to provide a new method and pharmaceutical composition for treating APLAID. Specifically, the drug is an inhibitor targeting the IFNβ signaling pathway, which can effectively intervene in the abnormal activation of IFNβ and related signal transduction, reduce the inflammatory responses of macrophages and other immune cells, and thus relieve the clinical symptoms of APLAID. Through the PLCγ2 p.Leu845Ser mutant mouse model and macrophage experiments, the present invention first reveals the key role of IFNβ in APLAID and provides a new drug target for the treatment of APLAID. Summary of the Invention

[0005] In order to overcome the disadvantages and deficiencies existing in the prior art, the purpose of the present invention is to provide drugs and pharmaceutical compositions for treating APLAID.

[0006] The purpose of the present invention is achieved by the following technical solution: the application of an inhibitor targeting the IFNβ signaling pathway in the preparation of a drug for treating APLAID.

[0007] Preferably, the inhibitor targeting the IFNβ signaling pathway includes any one of an IFNβ antibody, an IFNAR antagonist, a JAK inhibitor, a STAT inhibitor, and an IRF inhibitor. The IFNβ antibody can directly neutralize IFNβ and prevent it from binding to IFNAR, such as an anti-IFNβ monoclonal antibody. The IFNAR antagonist can block IFNAR1 or IFNAR2 and inhibit IFNβ signal transduction, such as Anifrolumab. The JAK inhibitor can inhibit JAK1 and TYK2 kinases and block downstream IFNβ signal transduction. Such as: Tofacitinib (JAK1 / 3 inhibitor), Baricitinib (JAK1 / 2 inhibitor), Deucravacitinib (TYK2 inhibitor). The STAT inhibitor can inhibit the phosphorylation or dimerization of STAT1 or STAT2 and block the formation of ISGF3. The IRF inhibitor can inhibit IRF family members and block the function of ISGF3.

[0008] Preferably, the IFNAR antagonist is anifrolumab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144.

[0009] Another object of the present invention is achieved by the following technical solution: a drug for treating APLAID, and the drug is an inhibitor targeting the IFNβ signaling pathway.

[0010] Preferably, the inhibitor targeting the IFNβ signaling pathway includes any one of an IFNβ antibody, an IFNAR antagonist, a JAK inhibitor, a STAT inhibitor, and an IRF inhibitor.

[0011] Preferably, the IFNAR antagonist is anifrolumab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144.

[0012] Yet another object of the present invention is achieved by the following technical solution: a pharmaceutical composition for treating APLAID, the pharmaceutical composition includes an inhibitor targeting the IFNβ signaling pathway, and further includes a pharmaceutically acceptable carrier and / or excipient.

[0013] Preferably, the inhibitor targeting the IFNβ signaling pathway includes at least one of an IFNβ antibody, an IFNAR antagonist, a JAK inhibitor, a STAT inhibitor, and an IRF inhibitor.

[0014] Preferably, the IFNAR antagonist is anifrolumab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144.

[0015] Preferably, the pharmaceutical composition further comprises wogonin.

[0016] Another object of the present invention is achieved by the following technical solution: the use of siRNA targeting PLCγ2 in the preparation of a medicament for treating APLAID.

[0017] The beneficial effects of the present invention are as follows: Molecular mechanism studies using PLCγ2 p.Leu845Ser mutant macrophages and mouse models show that upregulated IFNβ in macrophages is an important driver of APLAID, and the p.Leu845Ser mutant PLCγ2 upregulates the expression of IFNβ by enhancing its binding to IRF5 and promoting the nuclear translocation of IRF5. Importantly, targeting IRF5 or IFNβ can significantly reverse the inflammatory phenotypes of PLCγ2 mutant macrophages and mice. The present invention for the first time reveals that the activated IRF5 / IFNβ signaling in macrophages is a key driver for the development of APLAID and provides a new therapeutic target for APLAID patients. Description of the Drawings

[0018] Figure 1 PLCγ2 mutation leads to obvious infiltration of macrophages; among them, A is the blood routine examination result of APLAID patients with PLCγ2 p.Leu845Ser mutation; B is the expression of CD68 and MPO detected by HE staining and IHC in the intestinal tissue of patients; C is the expression and localization of PLCγ2 and CD68 detected by laser confocal microscopy in the intestinal tissue; D is the blood routine examination result of PLCγ2 p.Leu845Ser mutant mice; E is the expression of CD68 and MPO detected by HE staining and IHC in the inflammatory sites of PLCγ2 mutant mice; F is the expression and localization of PLCγ2 and F4 / 80 detected by laser confocal microscopy in the inflammatory skin tissue of PLCγ2 mutant mice.

[0019] Figure 2It is the PLCγ2 mutation that activates the IFNβ pathway in macrophages; among them, A is a heat map showing the differential expression of cytokines in THP1mut and THP1nc cells; B is the KEGG pathway enrichment of differentially expressed genes in THP1mut cells; C-G are qPCR to detect the expression of STAT1, STAT2, ISG15, CXCL10, and TNFSF10 in THP1mut and THP1nc cells in the M0 (PMA) or M1 (PMA / LPS) state; H is Wb to evaluate the protein levels of PLCγ2, STAT1, STAT2, pSTAT1, and pSTAT2 in THP1nc and THP1mut cells; (I-J are Elisa to detect the concentrations of IFNβ and IFNα in THP1mut and THP1nc cells in the M0 or M1 state; K-P are qPCR to detect the expression of STAT1, STAT2, IRF7, IFNβ, CXCL10, and TNFSF10 in PBMCs of APLAID patients (P) and healthy donors (H) treated with PMA or LPS; Q is Wb to evaluate the protein levels of STAT1, STAT2, pSTAT1, and pSTAT2 in PBMCs of APLAID patients (P) and healthy donors (H) treated with PMA; R is IHC to detect the expression of IFNβ, P-STAT1, and ISG15 in the intestinal tissue of APLAID patients; S-X are qPCR to detect the mRNA levels of STAT1, STAT2, IFNβ, ISG15, CXCL10, and TNFSF10 in the skin tissue of PLCγ2 mutant mice; Y is Wb to detect the protein levels of STAT1 and pSTAT1 in the skin tissue of PLCγ2 mutant mice; Z is IHC to detect the expression of IFNβ, pSTAT1, and ISG15 in the skin tissue of PLCγ2 mutant mice; among them, qPCR and Elisa data are shown as mean ± standard deviation, using the student t-test, and the statistical significance is ***P<0.001, **P<0.01, *P<0.05, ns, not significant.

[0020] Figure 3It is the mutant PLCγ2 that promotes IFNβ expression; among them, A shows the expression of PLCG2 in THP1mut cells transfected with siRNA targeting PLCG2 or non-coding siRNA detected by qPCR; B evaluates the concentration of IFNβ in THP1mut cells transfected with PLCG2 siRNA or non-coding siRNA by Elisa; C-F analyze the effects of PLCG2 knockout on the expression of STAT1, STAT2, CXCL10, and TNFSF10 in THP1mut cells by qPCR; G evaluates the effect of PLCG2 knockout on the IFNβ promoter activity by luciferase reporter gene assay; H analyzes the protein levels of PLCG2, STAT1, STAT2, P-STAT1, and P-STAT2 in THP1mut cells transfected with PLCG2 siRNA by Wb; I-L and M-P respectively detect the mRNA levels of STAT1, STAT2, CXCL10, and TNFSF10 in THP1mut cells treated with type I interferon receptor inhibitor (Anifro) or Upadacitinib (Upa) by qPCR; Q-R detect the protein levels of STAT1, STAT2, P-STAT1, and P-STAT2 in THP1mut cells treated with Anifro or Upa by Wb; among them, the qPCR data are shown as mean ± standard deviation, and the student t-test is used. The statistical significance is determined as ***P<0.001, **P<0.01, *P<0.05, ns, not significant.

[0021] Figure 4PLCγ2 mutant macrophages inhibit the proliferation of intestinal epithelial cells. Among them, A is a co-culture model diagram of using the supernatant of THP1mut or THP1nc cells and NCM460 cells; B-C are the KEGG pathway enrichment of differentially expressed genes in NCM460 cells treated with THP1mut supernatant or IFNβ; D is the detection of the proliferation of NCM460 cells treated with THP1mut cells, THP1nc cells, and 1 μg / mL IFNβ supernatant by the EDU method; E is the detection of the proliferation of NCM460 cells treated with the supernatant of THP1mut cells treated with PLCG2 siRNA by EDU; J-K are the detection of the migration of NCM460 cells treated with the supernatant of THP1-mut cells treated with Anifro or Upa by EDU; F, H, L, N are the statistical analysis of the proportion of EDU-positive cells in the EDU assay in groups D, E, J, K; G, I, M, O are the detection of the cell viability of NCM460 cells treated with the supernatant of THP1mut cells treated with Anifro or Upa by CCK8 assay. Among them, the CCK8 and EDU statistical data are shown as mean ± standard deviation, and the student t-test is used. The statistical significance is determined as ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant.

[0022] Figure 5Mutant PLCγ2 upregulates IFNβ by enhancing the nuclear localization of IRF5; among them, A shows immunoprecipitation of THP1mut protein lysates using a Flag antibody, and the content of PLCγ2 in the protein complex obtained from IP was analyzed by Wb; B shows mass spectrometry analysis of the proteins in the IP complex and pathway enrichment of the identified proteins; C shows a heatmap showing the abundance of type I interferon-related proteins in the IP protein complex; D shows immunoprecipitation of THP1mut protein lysates using a Flag or IRF5 antibody, and the content of PLCγ2 and IRF5 in the protein complex obtained from IP was analyzed by Wb; E shows the distribution and co-localization of PLCγ2 and IRF5 in THP1-mut and THP1nc cells detected by laser confocal microscopy; F shows co-transfection of FlagPLCγ2(wt) / IRF5 or FlagPLCβ2(mut) / IRF5-overexpression plasmids in 293T cells, immunoprecipitation of 293T protein lysates using a Flag or IRF5 antibody, and the content of PLCγ2 and IRF5 in the protein complex obtained from IP was analyzed by Wb; G shows the co-localization of PLCγ2 and IRF5 in 293T cells detected by laser confocal microscopy, which were co-transfected with FlagPLCγ2(wt) / IRF5 or FlagPLCβ2(mut) / IRF5-overexpression plasmids; H shows the distribution of PLCγ2 and IRF5 in the cytoplasm and nucleus of co-transfected 293T cells analyzed by Wb; I-J shows the effect of the IRF5 inhibitor YE6144 on the co-localization of PLCγ2 and IRF5 in THP1mut cells and co-transfected 293T cells detected by laser confocal microscopy.

[0023] Figure 6It is an IRF5 inhibitor that inhibits IFNβ transcription and function; among them, A is the effect of YE6144 on the IFNβ concentration in THP1mut cells in M0 and M1 states analyzed by Elisa; B is the effect of YE6144 on the IFNβ promoter activity in MCF7 cells analyzed by luciferase reporter; C-G are the mRNA levels of IFNβ, STAT1, STAT2, CXCL10, and TNFSF10 in THP1mut cells treated with YE6144 detected by qPCR; H is the protein levels of STAT1, STAT2, pSTAT1, and pSTAT2 in THP1mut cells treated with YE6144 analyzed by Wb; I-J are the proliferation of NCM460 cells treated with the supernatant of M0 or M1 state THP1mut cells treated with YE6144 detected by EDU assay; K-L are the statistical analysis of the proportion of EDU-positive cells in the EDU assay in Figures I-J; M-N are the cell viability of NCM460 cells treated with the supernatant of M0 or M1 state THP1mut cells treated with YE6144 detected by CCK8 assay; among them, the data in Figures A-G, K-N are shown as mean ± standard deviation, and the student t-test is used. The statistical significance is determined as ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant.

[0024] Figure 7 Anifrolumab improved the inflammatory phenotype of APAID mice; A is the blood routine examination results of PLCγ2p.Leu845Ser mutant mice with or without Anifro treatment; B is the pathological state of the inflammatory tissues of PLCγ2p.Leu845Ser mutant mice analyzed by HE staining; C is the protein levels of STAT1 and pSTAT1 in the inflammatory skin tissues analyzed by Wb; D is the expression of STAT1 and F4 / 80 in the inflammatory tissues of PLCγ2p.Leu845Ser mutant mice detected by IHC; E is the expression of PLCγ2 and F4 / 80 in the inflammatory tissues of PLCγ2p.Leu845Ser mutant mice detected by laser confocal microscopy; F-I are the mRNA levels of STAT1, CXCL10, TNFSF10, and ISG15 in the inflammatory skin tissues of PLCγ2p.Leu845Ser mutant mice with or without Anifro treatment detected by qPCR; among them, the data in Figures F-I are shown as mean ± standard deviation, and the statistical significance is ***P < 0.001, **P < 0.01.

[0025] Figure 8Expression of PLCγ2 and IFNβ in damaged intestinal tissues of patients with inflammatory bowel disease; A shows the expression of PLCγ2 in damaged intestinal tissues of patients with inflammatory bowel disease detected by IHC; B shows the expression of IFNβ in damaged intestinal tissues of patients with inflammatory bowel disease detected by IHC.

[0026] Figure 9 Improvement of inflammation in APAID mice by infliximab and wogonin; A - H show the expression of STAT1, STAT2, CXCL10, and TNFSF10 in THP1mut cells treated with infliximab (Rem) and wogonin detected by qPCR; I - J show the protein levels of STAT1, STAT2, pSTAT1, and pSTAT2 in THP1mut cells treated with infliximab (Rem) and wogonin detected by Wb. Detailed implementation manners

[0027] For the convenience of those skilled in the art, the following combines examples and attached Figures 1-9 to further illustrate the present invention. The content mentioned in the implementation manner does not limit the present invention.

[0028] Use of the inhibitor targeting the IFNβ signaling pathway of the present invention in the preparation of a drug for treating APLAID. As some specific implementation manners, the inhibitor targeting the IFNβ signaling pathway includes any one of an IFNβ antibody, an IFNAR antagonist, a JAK inhibitor, a STAT inhibitor, and an IRF inhibitor. As some more specific implementation manners, the IFNAR antagonist is anifrolumab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144.

[0029] Drug and pharmaceutical composition for treating APLAID of the present invention. The drug is an inhibitor targeting the IFNβ signaling pathway, and the pharmaceutical composition includes an inhibitor targeting the IFNβ signaling pathway, and also includes a pharmaceutically acceptable carrier and / or excipient. As some specific implementation manners, the inhibitor targeting the IFNβ signaling pathway includes any one of an IFNβ antibody, an IFNAR antagonist, a JAK inhibitor, a STAT inhibitor, and an IRF inhibitor. As some more specific implementation manners, the IFNAR antagonist is anifrolumab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144. As some specific implementation manners, the pharmaceutical composition further includes wogonin.

[0030] Use of the siRNA targeting PLCγ2 of the present invention in the preparation of a drug for treating APLAID. The sequence of the siRNA targeting PLCγ2 is:

[0031] si-PLCG2-F: 5'-GCAUGACUUCCAGAGGUUUTT-3';

[0032] si-PLCG2-R: 5'-AAACCUCUGGAAGUCAUGCTT-3'.

[0033] The control group sequence is:

[0034] si-NC-F: 5'-UUCUCCGAACGUGUCACGUTT-3';

[0035] si-NC-R: 5'-ACGUGACACGUUCGGAGAATT-3'.

[0036] Example 1

[0037] 1. PLCγ2 p.Leu845Ser mutation leads to significant macrophage infiltration

[0038] Previously, we identified a patient with APLAID caused by PLCγ2 p.Leu845Ser mutation who presented with severe IBD. We collected the patient's damaged intestinal tissue and peripheral blood and performed pathological and immune cell analyses respectively. The results of blood routine examinations showed a significant increase in monocytes and a slight increase in neutrophils ( Figure 1 A). Meanwhile, pathological examination of the patient's inflamed intestinal tissue showed a large presence of macrophages (CD68+) and a small number of neutrophils (MPO+) ( Figure 1 B). Consistent with this finding, the number and proportion of peripheral blood monocytes in PLCγ2 p.Leu845Ser mutant mice were significantly increased ( Figure 1 D). Subsequently, histopathological examination (HE) experiments confirmed that PLCγ2 p.Leu845Ser mutant mice showed obvious inflammatory manifestations in the ears ( Figure 1 E), accompanied by extensive infiltration of macrophages (F4 / 80+) at the inflammatory sites ( Figure 1 E). Immunofluorescence analysis further showed that PLCγ2 was mainly expressed in macrophages infiltrating the inflamed tissues of APAID patients and mice ( Figure 1 C and Figure 1 F).

[0039] These findings indicate that macrophages with high expression of PLCγ2 may play a crucial role in the development of IBD associated with APLAID. In addition, we detected the expression of PLCγ2 in intestinal tissue samples from 16 IBD patients clinically diagnosed with ulcerative colitis or Crohn's disease. Notably, we found that the expression of PLCγ2 was significantly positive in the intestinal tissues of 9 patients ( Figure 8A), further indicating a correlation between high expression of PLCγ2 and the occurrence of IBD.

[0040] 2. The PLCγ2 p.Leu845Ser mutation activates the IFNβ pathway in macrophages

[0041] To investigate the role of macrophages in APLAID, we established THP1 cells stably expressing PLCγ2 p.Leu845Ser (named THP1mut) and control cells (named THP1nc). First, we performed transcriptome sequencing to analyze differentially expressed genes between THP1mut and THP1nc cells. The transcriptome sequencing results showed that various inflammatory factors (including CXCL10, CCL2, TNFSF10, and CSF1) were significantly upregulated in THP1mut cells ( Figure 2 A). Pathway enrichment analysis of differentially expressed genes indicated that the type I interferon pathway was significantly enriched ( Figure 2 B). In addition, qPCR experiments confirmed the significant upregulation of type I interferon-related genes such as ISG15, STAT1, STAT2, CXCL10, and TNFSF10 in THP1mut cells ( Figure 2 C-2G).

[0042] Extensive studies have shown that type I interferon enhances the expression of inflammatory factors by activating the JAK / STAT signaling pathway. To evaluate the activation status of the JAK / STAT pathway in THP1mut cells, we performed Western blotting (Wb) and found that the expression and phosphorylation levels of STAT1 and STAT2 in THP1mut cells were significantly stronger than those in THP1nc cells ( Figure 2 H). Given that type I interferon is mainly composed of IFNα and IFNβ, to determine which one is responsible for activating the type I interferon pathway, we performed ELISA assays and observed a ten-fold increase in the secretion of IFNβ in THP1mut cells, while the secretion of IFNα did not change significantly ( Figure 2 I-2J).

[0043] We further investigated the activation status of the IFNβ pathway in peripheral blood mononuclear cells of APLAID patients carrying the PLCγ2 p.Leu845Ser mutation. qPCR results showed that the expression of type I interferon-related genes such as IFNβ, ISG15, STAT1, STAT2, CXCL10, and TNFSF10 increased in peripheral blood mononuclear cells of APLAID patients compared with those of healthy controls ( Figure 2 K-2P). Wb results indicated that the expression and phosphorylation levels of STAT1 and STAT2 increased in peripheral blood mononuclear cells of APLAID patients ( Figure 2Q). IHC experiments showed that the expressions of IFNβ, P-STAT1, and ISG15 in the diseased intestinal tissues of APLAID patients were significantly positive ( Figure 2 R). In addition, we detected the expression of IFNβ in the intestinal tissues of another 16 patients clinically diagnosed with IBD. We found that among 9 IBD patients with positive PLCγ2, 8 were simultaneously positive for IFNβ ( Figure 8 B), which further verified the regulatory relationship between PLCγ2 and IFNβ.

[0044] In addition, we confirmed the activation status of the IFNβ signaling pathway in PLCγ2p.Leu845ser mutant mice. Similarly, we observed a significant increase in the expressions of IFNβ and genes related to the activation of the IFNβ pathway, including STAT1, STAT2, ISG15, CXCL10, and TNFSF10, in the PBMCs of PLCγ2 mutant mice ( Figure 2 S-2X). The results of Wb showed that the expression and phosphorylation of STAT1 in the PBMCs of PLCγ2 mutant mice were significantly enhanced ( Figure 2 Y). IHC experiments further confirmed that IFNβ, P-STAT1, and ISG15 were significantly positively expressed in the inflamed tissues of PLCγ2 mutant mice ( Figure 2 Z). In summary, these findings indicate that the activation of the IFNβ pathway in macrophages driven by the PLCγ2p.Leu845Ser mutation may play an important role in the development of PLCγ2 mutation-related APLAID.

[0045] 3. Mutant PLCγ2 promotes the expression of IFNβ in macrophages

[0046] To further confirm that the PLCγ2p.Leu845Ser mutation can lead to the upregulation of IFNβ and the activation of the type I interferon pathway in macrophages, we initially used two PLCγ2 siRNAs to knockdown the expression of PLCγ2 in THP1mut cells. qPCR and ELISA assays showed that the knockdown of PLCγ2 led to a significant downregulation of IFNβ and genes related to the type I interferon pathway, including STAT1, STAT2, CXCL10, and TNFSF10 ( Figure 3 A-3F). Dual-luciferase reporter assays showed that reducing the expression of PLCγ2 could significantly inhibit the luciferase activity of the IFNβ promoter ( Figure 3 G). Wb analysis showed that the knockdown of PLCγ2 significantly reduced the expression and phosphorylation of STAT1 and STAT2 in THP1mut cells ( Figure 3 H).

[0047] We further investigated the effect of inhibiting the type I interferon pathway on PLCγ2 mutant macrophages by the type I interferon inhibitor Anifrolumab and the JAK / STAT pathway inhibitor Upadacitinib. The results of qPCR experiments showed that both Anifrolumab and Upadacitinib significantly reversed the upregulation of STAT1, STAT2, CXCL10, and TNFSF10 in PLCγ2 mutant macrophages ( Figure 3 I-3P). Wb analysis showed that Anifrolumab and Upadacitinib significantly inhibited the expression and phosphorylation levels of STAT1 and STAT2 in THP1mut cells ( Figure 3 Q-3R).

[0048] Wogonin has been reported to block the PLCG2 / PKC signaling pathway. Here, we further tested the effect of wogonin on PLCγ2p.Leu845Ser mutant macrophages. Our research results showed that wogonin could significantly inhibit the mRNA expression of STAT1, STAT2, CXCL10, and TNFSF10; in addition, the phosphorylation of STAT1 and STAT2 was also significantly inhibited ( Figure 9 ).

[0049] 4. PLCγ2 mutant macrophages inhibit intestinal epithelial cell proliferation by upregulating IFNβ

[0050] Numerous studies have shown that type I interferon is a multifunctional cytokine with antiviral, anti-proliferative, and immunomodulatory properties. To investigate the effect of elevated IFNβ in macrophages carrying the PLCγ2p.Leu845Ser mutation on intestinal tissues, human normal intestinal epithelial cells NCM460 were exposed to the supernatants of 30% THP1nc cells or THP1mut cells, and complete medium containing 10 ng / mL recombinant human IFNβ was used as a positive control. We initially performed transcriptome sequencing on NCM460 cells treated under the above conditions. Pathway enrichment analysis of differentially expressed genes highlighted the significant enrichment of pathways related to cell proliferation and type I interferon signaling ( Figure 4 A-4C). CCK8 and EDU cell proliferation assays showed that the proliferation activity of NCM460 cells treated with THP1mut culture supernatant or 10 ng / mL recombinant human IFNβ was significantly reduced compared to NCM460 cells treated with THP1nc culture supernatant ( Figure 4 D and Figure 4 F-4G). Our further research showed that knocking down the expression of PLCγ2 in THP1mut cells using siRNA significantly reversed the inhibitory effect of THP1muts culture supernatant on NCM460 cells ( Figure 4E and Figure 4 H-4I).

[0051] Numerous studies have shown that not only can type I interferons inhibit cell proliferation, but also various inflammatory factors activated by the type I interferon pathway, such as CXCL10 and TNFSF10, can significantly inhibit cell proliferation. To further verify the inhibitory effect of inflammatory factors involved in the activation of the type I interferon signaling pathway on intestinal epithelial cell proliferation, we pretreated THP1mut cells with the type I interferon inhibitor Anifrolumab and the JAK / STAT pathway inhibitor Upadacitinib, and then collected the culture supernatant to treat NCM460 cells. CCK8 and EdU cell proliferation assays showed that both Anifrolumab and Upadacitinib could significantly reverse the inhibitory effect of THP1mut cells on the proliferation of NCM460 cells ( Figure 4 J-4O). These findings indicate that the activation of the type I interferon pathway induced by the PLCγ2p.Leu845Ser mutation plays a key role in the pathogenesis of PLCγ2 mutation-related IBD, mainly by inhibiting the proliferation of intestinal epithelial cells.

[0052] 5. Mutant PLCγ2 upregulates IFNβ by increasing the transcriptional activity of IRF5

[0053] To further clarify the molecular mechanism by which the PLCγ2p.Leu845Ser mutation induces IFNβ upregulation, we performed co-immunoprecipitation (Co-IP) experiments on the protein lysates of THP1mut cells expressing flag PLCγ2 (p.Leu845Ser) using a flag antibody. Mass spectrometry and pathway enrichment analysis were performed on the protein complexes obtained from the Co-IP experiments. The results showed that the PLCγ2 (p.Leu845Ser) protein complex contained many proteins involved in immune regulation, among which the abundance of the type I interferon regulatory gene IRF5 was very high ( Figure 5 A-5C). This gene has been shown to be involved in the transcriptional regulation of type I interferons. Wb experiments also confirmed that IRF5 is a component of the PLCγ2 (p.Leu845Ser) protein complex ( Figure 5 D). Similarly, the protein complex obtained using the IRF5 antibody for Co-IP could specifically detect PLCγ2 ( Figure 5 D). In addition, laser confocal imaging clearly showed the co-localization of PLCγ2 and IRF5, and macrophages expressing p.Leu845Ser mutant PLCγ2 showed enhanced nuclear localization of the PLCγ2 / IRF5 complex ( Figure 5 E).

[0054] In addition, we overexpressed IRF5 and wild-type or p.Leu845Ser mutant PLCγ2 simultaneously in 293T cells. Through Co-IP experiments and laser confocal imaging, we further confirmed that the p.Leu845Ser mutant of PLCγ2 had enhanced binding ability to IRF5 and increased nuclear localization of the PLCγ2 / IRF5 complex ( Figure 5 F-5G). In addition, we used cytoplasmic and nuclear fractionation techniques to evaluate the distribution of PLCγ2 and IRF5 between the cytoplasmic and nuclear compartments. The Wb results showed that the levels of PLCγ2 and IRF5 in the nuclei of 293T cells overexpressing the p.Leu845Ser mutant of PLCγ2 were significantly higher than those overexpressing wild-type PLCγ2 ( Figure 5 H). Finally, our study treated THP1mut cells and 293T cells overexpressing PLCγ2 (p.Leu845Ser) / IRF5 with the IRF5 inhibitor YE6144. The results of laser confocal imaging showed that YE6144 significantly inhibited the nuclear localization of the PLCγ2 / IRF5 complex ( Figure 5 I-5J). These data indicate that the increased nuclear translocation of IRF5 caused by the PLCγ2 p.Leu845Ser mutation may play an important role in driving the upregulation of IFNβ transcription.

[0055] 6. Functional inhibition of IRF5 inhibits the transcription and biological functions of IFNβ.

[0056] Regarding the hypothesis that the p.Leu845Ser mutant of PLCγ2 enhances IFNβ transcription by promoting IRF5 nuclear translocation, we initially used the IRF5 inhibitor YE6144 to inhibit the function of IRF5 and performed qPCR and dual-luciferase reporter assays to evaluate the effect of IRF5 inhibition on IFNβ translation. Our research results showed that after the functional inhibition of IRF5, both the expression and promoter activity of IFNβ were significantly decreased ( Figure 6 A-6C). In addition, the effect of YE6144 on the activation of the JAK / STATs pathway was also investigated. qPCR detection showed that YE6144 significantly inhibited the expression of various inflammatory factors such as STAT1, STAT2, CXCL10, and TNFSF10 in THP1mut macrophages ( Figure 6 D-6G). Consistent with this, the Wb experiment further demonstrated that YE6144 significantly inhibited the expression and phosphorylation of STAT1 and STAT2 ( Figure 6 H). In addition, both EDU staining and CCK8 cell proliferation assays showed that the inhibitory effect of the supernatant of THP1mut macrophages treated with YE6144 on the proliferation of NCM460 cells was significantly reduced ( Figure 6I-6N). In summary, these data indicate that the IRF5 / IFNβ regulatory axis can serve as a therapeutic target for individuals with APLAID caused by the PLCγ2 p.Leu845Ser mutation.

[0057] 7. Targeting the IFNβ signaling pathway can significantly improve the inflammatory phenotype of PLCγ2 mutant mice.

[0058] To clarify whether the type I interferon pathway can be a potential therapeutic target for individuals with APLAID carrying the PLCγ2 p.Leu845Ser mutation, we evaluated the therapeutic effect of the IFNAR antagonist Anifrolumab on APLAID mice. Complete blood count results showed that compared with untreated mice, the number and ratio of monocytes in mutant mice treated with Anifrolumab were significantly reduced ( Figure 7 A). Histopathological examination (HE) results showed that Anifrolumab significantly improved the inflammatory cell infiltration in the ear lesion area of APLAID mice ( Figure 7 B). Wb analysis showed that Anifrolumab significantly reduced the expression and phosphorylation of STAT1 protein in the inflamed tissues of APLAID mice ( Figure 7 C). Consistent with this, IHC experiments also showed that the level of phosphorylated STAT1 in the inflamed tissues of mice treated with Anifrolumab was significantly reduced ( Figure 7 D). In addition, laser confocal imaging showed that the number of F4 / 80-positive macrophages in the inflamed tissues of APLAID mice treated with Anifrolumab was significantly reduced, and the expression of PLCγ2 was significantly decreased ( Figure 7 H). qPCR detection showed that Anifrolumab significantly inhibited the expression of IFNβ pathway-related genes such as STAT1, TNFSF10, CXCL10, and ISG15 in the inflamed tissues of APLAID mice ( Figure 7 F-7I). In summary, these findings indicate that Anifrolumab can effectively reduce the inflammatory response and improve the pathological condition of APLAID mice caused by the PLCγ2 p.Leu845Ser mutation by inhibiting the type I interferon pathway.

[0059] Based on the pathological characteristics of APLAID patients caused by the PLCγ2 (p.Leu845Ser) mutation, the present invention comprehensively studied the molecular mechanism of the pathogenesis of APLAID with the PLCγ2 p.Leu845Ser mutation by establishing a PLCγ2 mutant cell model and a mouse model. Our main findings are as follows:

[0060] 1. We first revealed that the occurrence of APLAID caused by PLCγ2 (p.Leu845Ser) mutation is closely related to the activation of the IFNβ pathway in macrophages.

[0061] 2. The PLCγ2 (p.Leu845Ser) mutation promotes the expression of IFNβ by enhancing the binding of IFNβ to IRF5 and promoting the nuclear translocation of IRF5. The functional inhibition of IRF5 can significantly reverse the inflammatory phenotype of PLCγ2 mutant macrophages.

[0062] 3. Our experiments first showed that the type 1 interferon receptor antibody drug Anifrolumab approved by the US Food and Drug Administration can significantly reverse the inflammatory phenotype of APLAID mice, providing an effective treatment strategy for individuals with APLAID caused by PLCγ2 (p.Leu845Ser) mutation.

[0063] Although multiple cases of APLAID caused by PLCγ2 gene mutation have been reported globally, most APLAID patients present with typical multi-organ inflammatory lesions such as skin inflammation and inflammatory bowel disease (IBD). However, the relationship between PLCγ2 and the occurrence of IBD has not been reported. In this study, we first used IHC to analyze the expression of PLCγ2 in diseased intestinal tissues of 16 IBD patients and found that the diseased intestinal tissues of approximately 60% of IBD patients were positive for PLCγ2 ( Figure 8 A). Therefore, our study preliminarily revealed the correlation between PLCγ2 and the occurrence of IBD.

[0064] Case reports of APLAID caused by PLCγ2 mutations showed that the proportions of neutrophils and monocytes in the peripheral blood of APLAID patients were significantly increased. Seth L. Masters et al. used a mouse model to study the molecular mechanism of APLAID caused by the PLCγ2 p.Ser707Tyr mutation. Their pathological results showed that neutrophils were the main inflammatory cells present in the PAWS, ears, and spleens of PLCγ2 p.Ser707Tyr mutant mice, while macrophages were only confined to the spleen. Unfortunately, this observation was not confirmed in the inflamed tissues of APLAID patients carrying the PLCγ2 p.Ser707Tyr mutation. Their molecular mechanism study showed that the elevation of G-CSF in the peripheral blood was the main cause of the autoinflammatory disease in PLCγ2 p.Ser707Tyr mutant individuals. In contrast, we observed a significant increase in the absolute number and proportion of monocytes in the peripheral blood of PLCγ2 p.Leu845Ser mutant patients and mice, while the neutrophil level remained unchanged. In addition, pathological analysis of the inflamed tissues of APLAID patients and mice caused by the PLCγ2 p.Leu845Ser mutation showed that macrophages were the main inflammatory cells, while the localization of neutrophils in the inflamed tissues was weak. In addition, our mechanism study showed that the abnormal activation of the IFNβ signaling pathway in macrophages played a crucial role in the pathogenesis of APLAID caused by the PLCγ2 p.Leu845Ser mutation. Overall, our study provided new insights into the role of macrophages in APLAID caused by the PLCγ2 p.Leu845Ser mutation and identified upregulated IFNβ as the key driver.

[0065] Numerous studies have shown that the transcriptional regulation of IFNβ is a complex process involving multiple transcription factors and signaling pathways, including type I interferon regulatory factors IRF3, IRF7, IRF5, TLR, and NF-κB. We used immunoprecipitation and mass spectrometry techniques to analyze the proteins specifically bound to PLCγ2 with the p.Leu845Ser mutation. Consistent with previous studies, we found that PLCγ2 with the p.Leu845Ser mutation could bind multiple type I interferon-related transcription factors, including IRF5, IRF3, and NF-κB, with IRF5 being the most abundant ( Figure 5A-5C). Functional experiments showed that the IRF5 inhibitor significantly reversed the upregulation of IFNβ transcription and the activation of downstream inflammatory pathways caused by the PLCγ2 p.Leu845Ser mutation. To our knowledge, our study for the first time revealed that mutant PLCγ2 with p.Leu845Ser promoted the upregulation of IFNβ by enhancing its binding to IRF5 and promoting the nuclear translocation of IRF5, and for the first time demonstrated that the IRF5 / IFNβ regulatory axis could be a potential therapeutic target for PLCγ2 p.Leu845Ser mutant APLAID.

[0066] Since the molecular mechanisms of the pathogenesis are still unclear, individuals with APLAID caused by PLCγ2 mutations lack precise therapeutic drugs. Clinical therapeutic drugs for APLAID mainly include immunosuppressants such as glucocorticoids, cyclosporine, tacrolimus, and a few biological agents such as infliximab. These drugs are difficult to maintain long-term remission of the disease. Based on our research results, we tested the therapeutic effect of the type I interferon receptor inhibitor Anifrolumab on PLCγ2 p.Leu845Ser mutant mice, and we found that Anifralumab significantly inhibited the inflammatory symptoms of APLAID mice. Here, our study for the first time showed that Anifrolumab, a drug approved by the US Food and Drug Administration for the treatment of systemic lupus erythematosus (SLE), is a potential therapeutic agent for individuals with APLAID caused by PLCγ2 p.Leu845Ser mutations. In addition, considering that gain-of-function mutations in PLCγ2 can lead to excessive release of DAG, resulting in abnormal activation of inflammatory pathways, studies have shown that the Chinese herbal medicine extract wogonin can inhibit the production of DAG. On this basis, we tested the effect of wogonin in PLCγ2 p.Leu845Ser mutant cells. Our results showed that wogonin could significantly inhibit the expression of inflammatory factors and the activation of the STAT1 pathway in PLCγ2 p.Leu845Ser mutant macrophages ( Figure 9 ). This indicates that wogonin-based drugs may also become potential therapeutic agents for PLCγ2 p.Leu845Ser mutant APLAID.

[0067] Experimental Materials and Methods

[0068] The detailed information of the reagents used in the present invention is shown in Table 1, and the primer sequences used in the present invention are shown in Table 2.

[0069] Table 1 Detailed Information of the Reagents Used in the Present Invention

[0070]

[0071]

[0072] Table 2 Primer sequences used in the present invention

[0073]

[0074]

[0075] Cell culture: THP1, NCM460, 293T, and MCF7 cell lines were purchased from Hysigen Biosciences in Suzhou, China. THP1, NCM460, and MCF7 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, while 293T cells were cultured in DMEM medium supplemented with 10% FBS. All cells were cultured in an environment of 5% CO 2 2, 37 °C. The reagents used in the experiments are listed in Table 1.

[0076] Lentivirus and stable cell line generation: The THP1mut cell line and THP1nc cell line expressing the p.Leu845Ser mutant PLCγ2 were constructed by Xunxun Biopharmaceutical Technology Co., Ltd. in Guangzhou, China. Lentiviruses expressing the p.Leu845Ser mutant PLCγ2 or wild-type PLCγ2 were purchased from ICE Biotechnology Co., Ltd. in Guangzhou. ICE Biotechnology Co., Ltd. in Guangzhou constructed plasmids including pcDNA3.1-PLCγ2, pcDNA3.1-BLCγ2 (p.Leu845Ser), pcDNA3.1-IRF5, pcDNA 3.1 vector, and pGL3basic IFNβ promoter.

[0077] Macrophage polarization: THP1-mut and THP1-nc cells were cultured in 6-well plates at a density of 1x10 6 cells per well. After induction with 100 ng / mL PMA for 24 hours, M0 macrophages were obtained, and then fresh 361 medium containing 100 ng / mL LPS was added for another 24 hours to obtain M1 macrophages.

[0078] Drug treatment: M0 macrophages were treated with 10 μM or 50 μM wogonin, 500 nM Upadacitinib, and 18 μg anti-IFNAR1 for 24 hours. 1 μM YE6144 was used to treat M0 and M1 macrophages as well as 293T cells.

[0079] Isolation of human peripheral blood mononuclear cells: Peripheral blood (10 mL) was collected from healthy individuals and patients, and PBMCs were obtained using a human monocyte isolation kit according to the operation instructions. The detailed information of the reagents is shown in Table 1.

[0080] siRNA interference: PLCγ2 siRNA was designed and synthesized by Shanghai GenePharma. Twenty-four hours before transfection, THP1 mut M0 macrophages were cultured in 6-well plates at a density of 8x10 5 cells per well. siRNA was transfected using RNAiMAX and the cells were cultured for another 48 hours, then the supernatant was collected and RNA and proteins were extracted. The detailed information of the transfection reagent is shown in Table 1. The siRNA sequences are listed in Table 2.

[0081] Co-culture experiment: Twenty-four hours before co-culture, NCM460 cells were cultured in 6-well plates at a density of 5x10 5 cells per well. The next day, the cell supernatant of THP1mut or THP1nc macrophages was added to NCM460 cells at a ratio of 30%, with 1 μg / mL IFNβ as the positive control. The cells were cultured for another 48 hours, then harvested for cell proliferation detection and transcriptome sequencing.

[0082] CCK8 assay: The CCK-8 kit was used to analyze the cell proliferation activity. Briefly, the CCK8 solution was added to the cell culture medium at a volume ratio of 1:10, incubated at 37 °C for 1 hour, and the absorbance was read at 450 nm using a microplate reader. Each cell type was tested three times. The detailed information of the CCK8 kit is shown in Table 1.

[0083] Edu assay: According to the BeyoClick TM EdU-488 operation instructions, the cells were incubated with EdU for 2 hours. Subsequently, fixation and permeabilization were performed. Finally, the cell nuclei were stained with DAPI and fluorescence microscope images were captured. Three replicate wells were set for each group of cells and the proportion of EdU-positive cells was calculated. The detailed information of the BeyoClick TM EdU-488 kit 392 is shown in Table 1.

[0084] RNA extraction and qPCR detection: The RNA purification kit was used to extract total RNA from cell and tissue samples, and the RNA concentration was measured using NanoDrop. 1 μg of RNA was reverse transcribed into cDNA using the Color reverse transcription kit. qPCR reagents were used to detect the expression levels of target genes, and the 2-ΔΔCt method was used for evaluation compared with β-actin as the control gene. The primer sequences of the target genes are listed in Table 2.

[0085] Co-IP assay: Using Pierce TMTotal cellular proteins were extracted with IP lysis buffer. Flag and IRF5 IP-specific antibodies were mixed with protein lysis buffer at a ratio of 1:50 and incubated overnight at 4°C. The corresponding protein complexes were obtained using G protein magnetic beads and then used for proteomic detection and Western blot detection. The detailed information of the Co-IP reagents is shown in Table 1.

[0086] Dual-luciferase reporter assay: Guangzhou ICE Biotechnology Company designed and constructed the IFNβ promoter expression vector pGL3basic IFNβ and the pcDNA3.1-IRF5 overexpression vector. MCF7 cells were cultured in 6-well plates and treated with PLCγ2 siRNA for 48 hours. 1 μg of pGL3-IFNβ and 0.1 μg of pRL-TK were transfected into siRNA-treated and untreated MCF7 cells using lipo3000. Untreated cells were treated for 48 hours without 1 μM IRF5 inhibitor YE6144. Then, the dual-luciferase reporter assay was performed according to the instructions of the dual-luciferase reporter kit. The fluorescence values of firefly and Renilla were read using a Victor Nivo multi-functional microplate reader.

[0087] Cytoplasmic and nuclear fractionation assay: 5 μg of the pcDNA3.1-PLCγ2 or pcDNA3.1-BLCγ2 (p.Leu845Ser) overexpression vector was co-transfected with 5 μg of the pcDNA3.1-IRF5 overexpression vector into 293T cells. After 48 hours, cytoplasmic and nuclear proteins were extracted according to the instructions of the nuclear and cytoplasmic protein extraction kit. The detailed information of the nuclear and cytoplasmic protein extraction kit is shown in Table 1.

[0088] Western blot: Proteins were extracted from cells and mouse tissues using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were detected using an enhanced BCA protein assay kit. Protein samples were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% non-fat milk in TBST, primary antibodies including STAT1, STAT2, pSTAT1, pSTAT2, PLCγ2, IRF5, and GAPDH were incubated overnight at 4°C at a ratio of 1:1000. Subsequently, the membranes were incubated with goat anti-mouse IgG and goat anti-rabbit IgG at a ratio of 1:2500 for 1 hour at room temperature, and finally images were obtained. The detailed information of the antibodies is shown in Table 1.

[0089] HE staining: Tissues from patients and mice were fixed with 4% paraformaldehyde for three days, and paraffin sections were prepared. All tissue sections were dewaxed and stained with hematoxylin and eosin according to the method of the hematoxylin and eosin staining kit, and then dehydrated with gradient alcohol, cleared with xylene and sealed with neutral gum before photography. The detailed information of the hematoxylin and eosin staining kit is shown in Table 1.

[0090] Immunohistochemistry: According to the instructions of the SABC immunohistochemistry kit, following the protocol of the SABC immunohistochemistry kit (mouse / rabbit), tissue sections underwent a sequential process of dewaxing, EDTA antigen retrieval, H 2 O 2 blocking of endogenous peroxidase and blocking with 5% BSA. Antibodies including IFNβ, pSTAT1, PLCγ2, IRF5, F4 / 80, CD68, and MPO were diluted at a ratio of 1:200 (diluent: 5% BSA) and incubated overnight at 4°C. Subsequently, biotinylated goat anti-mouse / rabbit IgG was incubated at 37°C for 30 minutes, and then SABC was incubated at 37°C for 30 minutes. After DAB staining, hematoxylin counterstaining, dehydration, clearing, mounting, and finally photography were performed. The detailed information of the antibodies and the SABC kit is shown in Table 1.

[0091] Laser confocal imaging: For THP1 and 293T cell samples, initially, the test cells were seeded on glass slides. Subsequently, they were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.3% Triton X-100 for 10 minutes, blocked with 5% BSA, incubated overnight with the primary antibody solution at 4°C, and incubated with the secondary antibody solution at room temperature for 30 minutes. Finally, they were sealed with a mounting reagent containing DAPI. For tissue samples, after antigen retrieval, blocking of endogenous peroxidase and blocking with 5% BSA, they were incubated with the primary and secondary antibody solutions prepared above, and then sealed with a mounting reagent containing DAPI. The sealed cells and tissue sections were imaged using a Leica Stellaris5 laser confocal microscope.

[0092] The PLCγ2 and IRF5 (sc-56714) antibodies were mixed with a 3% BSA solution at a volume ratio of 1:200 and used as the primary antibody reaction solution. Goat anti-mouse IgG-488 and goat anti-rabbit IgG-594 were mixed with a 3% BSA solution at a volume ratio of 1:500 and used as the secondary antibody reaction solution. The detailed information of the antibodies is listed in Table 1.

[0093] Enzyme-linked immunosorbent assay: The contents of interferon α and interferon β in the collected supernatant were detected using human interferon α ELISA kit and human interferon β ELISA kit. Each sample was tested three times, and the concentrations of IFNα and IFNβ were calculated using the standard curve. The detailed information of the ELISA kit is listed in Table 1.

[0094] Animal experiments: F0 generation PLCγ2p.Leu845Ser mutant mice were constructed by Shanghai Model Organisms Center, and SPF-grade C57BL / 6 mice were purchased from Guangdong Medical Experimental Animal Center. All mice were housed in an SPF-grade environment. 7-week-old male F0 generation gene mutant mice were mated with female C57BL / 6 mice of the same age to obtain F1 generation gene mutant mice. At 3 weeks of age, the F1 generation gene mutant mice were treated by intraperitoneal injection of 30 μg anti-IFNAR1 for 5 consecutive days. Other F1 generation gene mutant mice and C57BL / 6 mice of the same age were injected with the same volume of PBS as a control. After the mice reached 5 weeks of age, blood was collected from the eyeballs for routine blood analysis. Immunohistochemistry, confocal laser scanning detection, Western blotting and qPCR analysis were performed on the tissue samples of the mice. All animal experiments were approved by the Ethics Review Committee of Life Sciences, Guangdong Medical University.

[0095] Statistical analysis: Statistical analysis was performed using GraphPad Prism 8.0, and Student's t-test was performed on all experiments. Statistical significance was defined as P < 0.05. The significance levels were as follows: * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. ns was used to indicate no statistically significant difference.

[0096] Method for constructing THP1 cells stably expressing PLCγ2p.Leu845Ser: The PLCG2 point mutation gene vector was synthesized and handed over to Guangzhou Aiji Biotechnology Co., Ltd. to construct lentivirus. On this basis, THP1 cells were infected, and the THP1-Mut(p.Leu845Ser) cell line was obtained by screening with puromycin.

[0097] I. The gene of PLCG2(2534T>C) was synthesized by gene synthesis method. The synthesized PCG2 gene and pCDH-CMV lentiviral vector were digested with restriction endonucleases respectively, and ligated with T4 DNA ligase to obtain pCDH-CMV-PLCG2-845-3xFLAG-EF1-puro plasmid. The plasmid was detected by nucleic acid gel electrophoresis;

[0098] II. The constructed plasmid was sequenced and packaged into lentivirus

[0099] 1. Cell preparation

[0100] 1) On the first day, thaw one vial of 293T cells;

[0101] 2) On the second day, change the medium of the thawed cells;

[0102] 3) On the third day, passage the 293T cells at a ratio of 2.0x10 6 cells / T75;

[0103] 4) On the fifth day, seed the 293T cells into a 10-cm diameter culture dish at a cell density of 4.5x10 6 ;

[0104] 2. Lentivirus packaging

[0105] 1) On the day of transduction, observe whether the cells can package the virus. The main observation indicators are: whether the cells are evenly seeded and whether the cell density reaches 80% - 90%. If the cell state is appropriate, remove the culture medium from the 293T cells and add 10 ml of culture medium for virus packaging;

[0106] Note: To avoid cell detachment, add the fresh culture medium along the wall of the flask.

[0107] 2) Prepare the DNA-transfection reagent complex:

[0108] A. Add serum-free Opti-MEM medium, helper plasmid mixture (PSPAX2 and PMD2G), and lentiviral expression plasmid to a 15-mL centrifuge tube and mix well by gentle shaking;

[0109] B. Prepare another 15-mL centrifuge tube, add serum-free Opti-MEM medium and plasmid transfection reagent, and mix gently by shaking.

[0110] Incubate at room temperature for 10 minutes;

[0111] C. After 10 minutes, add the serum-free Opti-MEM medium containing plasmid DNA to the serum-free Opti-MEM medium containing plasmid transfection reagent, mix gently by shaking, and incubate at room temperature for 10 - 15 minutes;

[0112] 3) Add the DNA complex to the 293T cells and gently rock the culture dish back and forth to mix the complex. Incubate in a 37°C, 5% CO 2 incubator with saturated humidity;

[0113] 4) After 4 - 6 hours of transduction, change to fresh medium and incubate in a 37°C, 5% CO 2 incubator with saturated humidity.

[0114] 3. Collect and concentrate the virus

[0115] 1) At 48 hours after transduction, collect the culture supernatant containing the virus into a 50 mL centrifuge tube.

[0116] 2) Centrifuge at 4°C and 1500 x g for 4 minutes to remove cell debris and recover the supernatant.

[0117] 3) Filter the centrifuged virus supernatant through a 0.45 μM filter into a 50 mL centrifuge tube.

[0118] 4) Add the corresponding amount of PEG concentrate according to the volume of the filtrate. Mix the solution thoroughly and let it stand at 4°C for overnight precipitation.

[0119] 5) The next day, centrifuge at 4°C and 1500 x g for 30 minutes and then pour off the supernatant.

[0120] 6) Use a pipette to aspirate the excess supernatant, then resuspend the virus pellet with 1 mL of HBSS, and gently and thoroughly pipette the pellet with a pipette tip to form a single virus suspension. Aliquot the virus solution into cryotubes according to the regulations and store at -80°C.

[0121] III. Virus Titer Detection

[0122] 1. Virus Infection

[0123] 1) On the first day, seed 12-well plates with 1.5 x 10 5 cells per well.

[0124] 2) On the second day, observe the cell status and density, then change the medium and add Polybrene (final concentration 5 μg / mL).

[0125] 3) Add the corresponding amount of the project virus to the cells in the corresponding wells.

[0126] 4) After mixing, place it in an incubator at 37°C and 5% CO 2 with saturated humidity for culturing. Observe the cells after 4 - 6 h and change the medium.

[0127] 5) Take pictures of the cells 48 h after infection and collect the cells for DNA extraction.

[0128] 2. DNA Extraction

[0129] 1) Aspirate the cell culture medium, wash the cells with 1x PBS 2 - 3 times to remove residual serum, add an appropriate amount of Trypsi-EDTA, let it stand at room temperature for 5 minutes, then pipette the cells repeatedly and transfer the cells into a 1.5 mL EP tube.

[0130] 2) Centrifuge to remove the supernatant.

[0131] 3) Resuspend the cells with 350 μL of ElutionBuffer, and then add an equal volume of AVL solution to lyse the cells;

[0132] 4) Add 20 μL of proteinase K, invert and mix well thoroughly, incubate at 70 °C for 10 min, and briefly centrifuge to remove the water droplets on the inner wall of the tube;

[0133] 5) Add 350 μL of absolute ethanol, shake and mix well thoroughly for 15 s;

[0134] 6) Pipette the mixture and add it to the centrifugal column, centrifuge at 8000 rpm for 1 min, and discard the filtrate in the collection tube;

[0135] 7) Add 600 μL of Wash1 to the centrifugal column, centrifuge at 8000 rpm for 1 min, and discard the filtrate in the collection tube;

[0136] 8) Add 600 μL of Wash2 to the centrifugal column, centrifuge at 8000 rpm for 1 min, and discard the filtrate in the collection tube;

[0137] 9) Centrifuge at 12000 rpm without sample for 3 min to completely dry the adsorption membrane;

[0138] 10) Place the centrifugal column on a new 1.5 mL EP tube, add 50 μL of ElutionBuffer water to the center of the centrifugal column, cover the lid, and incubate at room temperature for 3 min;

[0139] 11) Centrifuge at 12000 rpm for 2 min, and store the obtained DNA at -20 °C;

[0140] 3. qPCR

[0141] 1) Reaction system: 2×Ipure SYBR Green Qper Master Mix: 10 μL, 10 μM forward primer: 1.0 μL, 10 μM reverse primer: 1.0 μL, sample solution 2.0 μL, ddH 2 O 6.0 μL, total volume 20.0 μL.

[0142] 2) Reaction conditions: A. Pre-denaturation at 95 °C for 10 min; B. Use the three-step method for amplification during the cycling process: denaturation at 95 °C for 15 s, annealing at 57 °C for 20 s, extension at 65 °C for 30 s, 40 cycles; C. Dissolution curve: 95 °C, 15 s; 60 °C, 60 s; 95 °C, 1 s.

[0143] IV. Construction of THP1-PLCG2(845) stable cell line

[0144] Before the experiment began, THP1 cells were cultured in a 96-well cell culture plate at a density of 10,000 cells per well. The next day, cells were taken and the virus was added to each well according to the formula: virus volume (μL) per well = MOI x number of cells / virus titer (TU / mL) x 1000. pCDH-CMV-3xFLAG-EF1-puro and pCDH-CMV-PLCG2-845-3xFLAG-EF1-puro were mixed with THP1 cells respectively. After incubation at 37°C for 30 min, the mixture was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The cells were then cultured in a 96-well plate. After 24 h, puromycin was added for screening at a final concentration of 1 μg / mL per well. The cells were further cultured for 96 h, then centrifuged at 1000 rpm for 10 min, and puromycin screening was performed again. After 10 generations of screening, Western-blot was used for identification.

[0145] Construction of the PLCG2 mutant mouse model: Knockin mice were established using the CRISPR / Cas9 technology. This technology involves designing guide RNAs targeting the genomic region of the target gene to guide the Cas9 nuclease to modify the genome at the insertion site, thereby increasing the homologous recombination efficiency in the gene modification region and homologous recombining the target fragment into the target site. The procedure for generating knockin mice using this technology is as follows:

[0146] 1. Design guide RNA target sequences for the genomic region of the target site and perform in vitro transcription according to the sequences to obtain guide RNAs for the gene.

[0147] 2. Construct a donor DNA recombinant plasmid for the recombination of the target fragment.

[0148] 3. Inject the in vitro transcribed guide RNAs and the donor DNA recombinant plasmid into fertilized eggs to obtain F0 generation mice.

[0149] 4. Genotype the obtained F0 generation mice by PCR and sequencing.

[0150] 5. Breed F0 generation mice with WT mice to obtain positive F1 generation heterozygous mice.

[0151] 6. Deliver 6-8-week-old positive F1 generation mice.

[0152] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious substitution without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. Application of inhibitors targeting IFNβ signaling pathway in the preparation of drugs for the treatment of APLAID.

2. The use according to claim 1, characterized in that: The inhibitor targeting the IFNβ signaling pathway includes any one of an IFNβ antibody, an IFNAR antagonist, a JAK inhibitor, a STAT inhibitor, and an IRF inhibitor.

3. The use according to claim 2, characterized in that: The IFNAR antagonist is aniluomab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144.

4. A drug for treating APLAID, characterized in that: The drug is an inhibitor targeting the IFNβ signaling pathway.

5. The drug according to claim 4, characterized in that: The inhibitor targeting the IFNβ signaling pathway includes any one of an IFNβ antibody, an IFNAR antagonist, a JAK inhibitor, a STAT inhibitor, and an IRF inhibitor.

6. The drug according to claim 5, characterized in that: The IFNAR antagonist is aniluomab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144.

7. A pharmaceutical composition for treating APLAID, characterized in that: The pharmaceutical composition comprises an inhibitor targeting the IFNβ signaling pathway and also comprises a pharmaceutically acceptable carrier and / or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that: The inhibitor targeting the IFNβ signaling pathway includes at least one of an IFNβ antibody, an IFNAR antagonist, a JAK inhibitor, a STAT inhibitor, and an IRF inhibitor.

9. The pharmaceutical composition according to claim 8, characterized in that: The IFNAR antagonist is aniluomab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144.

10. Use of siRNA targeting PLCγ2 in the preparation of drugs for treating APLAID.

Images