Drugs and pharmaceutical compositions for treating APLAID

Abstract

The present invention relates to the field of biotechnology, and specifically to drugs and pharmaceutical compositions for treating APLAID. The drugs are inhibitors targeting the IFNβ signaling pathway, and the pharmaceutical compositions comprise inhibitors targeting the IFNβ signaling pathway, as well as pharmaceutically acceptable carriers and / or excipients. Molecular mechanism studies conducted using PLCγ2p.Leu845Ser mutant macrophages and mouse models indicate that upregulated IFNβ in macrophages is a key driver of APLAID. The p.Leu845Ser mutant PLCγ2 upregulates IFNβ expression by enhancing its binding to IRF5 and promoting IRF5 nuclear translocation. This invention reveals for the first time that activated IRF5 / IFNβ signaling in macrophages is a key driver of APLAID development and provides a new therapeutic target for APLAID patients.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a medicine and a pharmaceutical composition for treating APLAID. Background Art

[0002] APLAID is a rare autoinflammatory disease characterized by early onset, recurrent inflammatory bowel and eye disease, blistering skin lesions, joint pain, and pulmonary inflammatory lesions. Because the molecular mechanisms underlying APLAID remain unclear, there are no standard treatment guidelines for patients with APLAID. Existing treatments primarily focus on immunosuppressive therapies, which have limited efficacy and are associated with side effects. Therefore, there is an urgent need to identify new therapeutic strategies, particularly those targeting key molecular targets involved in the molecular mechanisms and pathological processes of the disease.

[0003] Studies have shown that PLCγ2 (phosphatidylinositol-specific phospholipase Cγ2) is a key immune signaling molecule, and its mutation is closely associated with the development of APLAID. Our previous studies have found that the p.Leu845Ser mutation in the PLCγ2 gene can enhance binding to IRF5 (interferon regulatory factor 5), promote IRF5 nuclear translocation, and ultimately lead to overexpression of IFNβ. IFNβ (interferon β) is an important cytokine in the immune system, widely involved in immune and inflammatory responses. Our study is the first to show that overexpression of IFNβ is a key factor in the development and progression 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 novel method and pharmaceutical composition for the treatment of APLAID. Specifically, the drug is an inhibitor targeting the IFNβ signaling pathway, effectively intervening in abnormal IFNβ activation and related signaling, reducing the inflammatory response in macrophages and other immune cells, thereby alleviating the clinical symptoms of APLAID. Using a PLCγ2p.Leu845Ser mutant mouse model and macrophage experiments, the present invention reveals for the first time 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 shortcomings and deficiencies in the prior art, the present invention aims to provide a drug and a pharmaceutical composition for treating APLAID.

[0006] The object of the present invention is achieved through the following technical solution: use 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 IFNβ antibodies, IFNAR antagonists, JAK inhibitors, STAT inhibitors and IRF inhibitors. IFNβ antibodies can directly neutralize IFNβ and prevent it from binding to IFNAR, such as anti-IFNβ monoclonal antibodies. IFNAR antagonists can block IFNAR1 or IFNAR2 and inhibit IFNβ signal transduction, such as Anifrolumab. JAK inhibitors can inhibit JAK1 and TYK2 kinases and block IFNβ downstream signal transduction. Such as Tofacitinib (JAK1 / 3 inhibitor), Baricitinib (JAK1 / 2 inhibitor), Deucravacitinib (TYK2 inhibitor). STAT inhibitors can inhibit the phosphorylation or dimerization of STAT1 or STAT2 and block the formation of ISGF3. IRF inhibitors can inhibit IRF family members and block the function of ISGF3.

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

[0009] Another object of the present invention is achieved through the following technical solution: a drug for treating APLAID, wherein 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 aniluomab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144.

[0012] Another object of the present invention is achieved through the following technical solution: a pharmaceutical composition for treating APLAID, comprising an inhibitor targeting the IFNβ signaling pathway and 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 aniluomab, 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 through the following technical solution: use of siRNA targeting PLCγ2 in the preparation of a drug for treating APLAID.

[0017] The beneficial effects of the present invention are as follows: Molecular mechanism studies conducted using PLCγ2p.Leu845Ser mutant macrophages and mouse models indicate that upregulated IFNβ in macrophages is an important driver of APLAID. The p.Leu845Ser mutant PLCγ2 upregulates IFNβ expression by enhancing its binding to IRF5 and promoting IRF5 nuclear translocation. Importantly, targeting IRF5 or IFNβ can significantly reverse the inflammatory phenotype of PLCγ2 mutant macrophages and mice. This invention reveals for the first time that activated IRF5 / IFNβ signaling in macrophages is a key driver of APLAID development and provides a new therapeutic target for APLAID patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the PLCγ2 mutation that causes obvious macrophage infiltration; among them, A is the routine blood examination result of APLAID patients with PLCγ2p.Leu845Ser mutation; B is the HE staining and IHC detection of CD68 and MPO expression in the patient's intestinal tissue; C is the laser confocal microscopy detection of PLCγ2 and CD68 expression and localization in intestinal tissue; D is the routine blood examination result of PLCγ2p.Leu845Ser mutant mice; E is the HE staining and IHC detection of CD68 and MPO expression in the inflammatory site of PLCγ2 mutant mice; F is the laser confocal microscopy detection of PLCγ2 and F4 / 80 expression and localization in the inflamed skin tissue of PLCγ2 mutant mice.

[0019] Figure 2PLCγ2 mutation activates the IFNβ pathway in macrophages; 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; CG is qPCR detection of STAT1, STAT2, ISG15, CXCL10 and TNFSF10 expression in THP1mut and THP1nc cells in M0 (PMA) or M1 (PMA / LPS) state; H is Wb evaluation of the protein levels of PLCγ2, STAT1, STAT2, pSTAT1 and pSTAT2 in THP1nc and THP1mut cells; (IJ is Elisa detection of IFNβ and IFNα concentrations in THP1mut and THP1nc cells in M0 or M1 state; KP is qPCR detection of STAT1, STAT2, IRF7, IFNβ, C in PBMCs of APLAID patients (P) and healthy donors (H) treated with PMA or LPS. Expression of STAT1, STAT2, pSTAT1, and pSTAT2 in PBMCs of PMA-treated APLAID patients (P) and healthy donors (H) were evaluated by Wb; R was used to detect the expression of IFNβ, p-STAT1, and ISG15 in the intestinal tissues of APLAID patients by IHC; SX was used to detect the mRNA levels of STAT1, STAT2, IFNβ, ISG15, CXCL10, and TNFSF10 in the skin tissues of PLCγ2 mutant mice by qPCR; Y was used to detect the protein levels of STAT1 and pSTAT1 in the skin tissues of PLCγ2 mutant mice by Wb; Z was used to detect the expression of IFNβ, pSTAT1, and ISG15 in the skin tissues of PLCγ2 mutant mice by IHC; qPCR and ELISA data were shown as mean ± SD, and Student's t-test was used. The statistical significance was ***P < 0.001, **P < 0.01, and *P < 0.05, ns, not significant.

[0020] Figure 3It is the mutant PLCγ2 that promotes IFNβ expression; A is qPCR detection of PLCG2 expression in THP1mut cells transfected with siRNA or non-coding siRNA targeting PLCG2; B is Elisa evaluation of IFNβ concentration in THP1mut cells transfected with siRNA or non-coding siRNA targeting PLCG2; CF is qPCR analysis of the effect of PLCG2 knockout on the expression of STAT1, STAT2, CXCL10 and TNFSF10 in THP1mut cells; G is luciferase reporter gene assay evaluation of the effect of PLCG2 knockout on IFNβ promoter activity; H is Wb analysis of the expression of PLCG2, STAT1, S The protein levels of TAT2, P-STAT1 and P-STAT2; IL and MP were qPCR detection of the mRNA levels of STAT1, STAT2, CXCL10 and TNFSF10 in THP1mut cells treated with type I interferon receptor inhibitor (Anifro) or Upadacitinib (Upa), respectively; QR was Wb detection of the protein levels of STAT1, STAT2, P-STAT1 and P-STAT2 in THP1mut cells treated with Anifro or Upa; qPCR data were shown as mean ± standard deviation, and statistical significance was determined using Student's t-test, with ***P<0.001, **P<0.01, *P<0.05, ns, not significant.

[0021] Figure 4PLCγ2 mutant macrophages inhibit the proliferation of intestinal epithelial cells; A is a co-culture model diagram using the supernatant of THP1mut or THP1nc cells and NCM460 cells; BC is the KEGG pathway enrichment of differentially expressed genes in NCM460 cells treated with THP1mut supernatant or IFNβ; D is the EDU method for detecting the proliferation of NCM460 cells treated with THP1mut cells, THP1nc cells, and 1 μg / mL IFNβ supernatant; E is the EDU detection of the proliferation of NCM460 cells treated with the supernatant of THP1mut cells treated with PLCG2 siRNA; JK Figure 3 EDU assay for the proliferation of NCM460 cells treated with the supernatant of THP1-mut cells treated with Anifro or Upa; F, H, L, N statistically analyzed the proportion of EDU-positive cells in the EDU assay in groups D, E, J, and K; G, I, M, and O detected the cell viability of NCM460 cells treated with the supernatant of THP1mut cells treated with Anifro or Upa; CCK8 and EDU statistics are shown as mean ± SD, and statistical significance was determined using Student's t-test, with ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant.

[0022] Figure 5Mutant PLCγ2 upregulates IFNβ by enhancing IRF5 nuclear localization; A is immunoprecipitation of THP1mut protein lysate using Flag antibody, and Wb analysis of the content of PLCγ2 in the protein complex obtained from IP; B is mass spectrometry analysis of proteins in the IP complex, and pathway enrichment of identified proteins; C is a heat map showing the abundance of proteins related to type I interferon in the IP protein complex; D is immunoprecipitation of THP1mut protein lysate using Flag or IRF5 antibodies, and Wb analysis of the content of PLCγ2 and IRF5 in the protein complex obtained from IP; E is laser confocal microscopy detection of the distribution and colocalization of PLCγ2 and IRF5 in THP1-mut and THP1nc cells; F is co-transfection of FlagPLCγ2 in 293T cells (wt) / IRF5 or FlagPLCβ2(mut) / IRF5-overexpression plasmid, 293T protein lysates were immunoprecipitated using Flag or IRF5 antibodies, and the content of PLCγ2 and IRF5 in the protein complexes obtained from IP was analyzed by Western blotting; G, laser confocal microscopy was used to detect the colocalization of PLCγ2 and IRF5 in 293T cells, which were co-transfected with FlagPLCγ2(wt) / IRF5 or FlagPLCβ2(mut) / IRF5-overexpression plasmid; H, laser confocal microscopy was used to analyze the distribution of PLCγ2 and IRF5 in the cytoplasm and nucleus of co-transfected 293T cells; IJ, laser confocal microscopy was used to detect the effect of IRF5 inhibitor YE6144 on the colocalization of PLCγ2 and IRF5 in THP1mut cells and co-transfected 293T cells.

[0023] Figure 6IRF5 inhibitors inhibit IFNβ transcription and function; A is an Elisa analysis of the effect of YE6144 on IFNβ concentration in THP1mut cells in M0 and M1 states; B is a luciferase reporter analysis of the effect of YE6144 on IFNβ promoter activity in MCF7 cells; CG is a qPCR detection of the mRNA levels of IFNβ, STAT1, STAT2, CXCL10, and TNFSF10 in THP1mut cells treated with YE6144; H is a Wb analysis of STAT1, STAT2, pSTAT1, and pSTAT2 in THP1mut cells treated with YE6144. Protein levels; IJ is EDU assay detecting the proliferation of NCM460 cells treated with supernatant of M0 or M1stateTHP1mut cells treated with YE6144; KL is statistical analysis of the proportion of EDU-positive cells in EDU assay in Figure IJ; MN is CCK8 assay detecting the cell viability of NCM460 cells treated with supernatant of M0 or M1stateTHP1mut cells treated with YE6144; Among them, the data in Figures AG, KN are shown as mean ± SD, and the statistical significance was determined by Student's t-test, and ***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 treated with or without Anifro; B is the HE staining analysis of the pathological state of the inflammatory tissue of PLCγ2p.Leu845Ser mutant mice; C is the Wb analysis of the protein levels of STAT1 and pSTAT1 in the inflamed skin tissue; D is the IHC detection of STAT1 and F4 / 80 in the inflammatory tissue of PLCγ2p.Leu845Ser mutant mice Expression; E is the laser confocal microscopy detection of the expression of PLCγ2 and F4 / 80 in the inflammatory tissues of PLCγ2p.Leu845Ser mutant mice; FI is the qPCR detection of the mRNA levels of STAT1, CXCL10, TNFSF10 and ISG15 in the inflammatory skin tissues of PLCγ2p.Leu845Ser mutant mice with or without Anifro treatment; Among them, the data in Figure FI are shown as the mean ± standard deviation, and the statistical significance is ***P<0.001, **P<0.01.

[0025] Figure 8Figure 2 is the expression of PLCγ2 and IFNβ in damaged intestinal tissues of patients with inflammatory bowel disease; Figure A is the IHC detection of PLCγ2 expression in damaged intestinal tissues of patients with inflammatory bowel disease; Figure B is the IHC detection of IFNβ expression in damaged intestinal tissues of patients with inflammatory bowel disease.

[0026] Figure 9 Infliximab and wogonin improve inflammation in APAID mice; AD are qPCR detections of the expression of STAT1, STAT2, CXCL10, and TNFSF10 in THP1mut cells treated with infliximab (Rem) and wogonin; E is Wb detection of the protein levels of STAT1, STAT2, pSTAT1, and pSTAT2 in THP1mut cells treated with infliximab (Rem) and wogonin. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of those skilled in the art, the following embodiments and accompanying drawings are provided. Figure 1-9 The present invention is further described, and the contents mentioned in the embodiment are not intended to limit the present invention.

[0028] The present invention relates to the use of an inhibitor targeting the IFNβ signaling pathway in the preparation of a medicament for treating APLAID. In some specific embodiments, 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. In some more specific embodiments, the IFNAR antagonist is aniluomab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144.

[0029] The present invention provides a drug and pharmaceutical composition for treating APLAID. The drug is an inhibitor targeting the IFNβ signaling pathway. The pharmaceutical composition includes an inhibitor targeting the IFNβ signaling pathway and a pharmaceutically acceptable carrier and / or excipient. In some specific embodiments, 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. In some more specific embodiments, the IFNAR antagonist is anirutumab, the JAK inhibitor is upadacitinib, and the IRF inhibitor is the IRF5 inhibitor YE6144. In some specific embodiments, the pharmaceutical composition also includes wogonin.

[0030] The present invention provides an application of a siRNA targeting PLCγ2 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 sequence of the control group is:

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

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

[0036] Example 1

[0037] 1. PLCγ2p.Leu845Ser mutation leads to significant macrophage infiltration

[0038] Previously, we found a patient with APLAID caused by the PLCγ2p.Leu845Ser mutation who presented with severe IBD. We collected damaged intestinal tissue and peripheral blood from the patient and performed pathological and immune cell analyses, respectively. Routine blood tests showed a significant increase in monocytes and a slight increase in neutrophils ( Figure 1 A). At the same time, pathological examination of the patient's inflammatory bowel tissue showed the presence of a large number of macrophages (CD68+) and a small number of neutrophils (MPO+) ( Figure 1 B). Consistent with this finding, the number and proportion of peripheral blood mononuclear cells in PLCγ2p.Leu845Ser mutant mice were significantly increased ( Figure 1 D). Subsequent histopathological examination (HE) experiments confirmed that PLCγ2p.Leu845Ser mutant mice showed obvious inflammation in the ears ( Figure 1 E), accompanied by extensive infiltration of macrophages (F4 / 80+) in the inflammatory area ( Figure 1 E). Immunofluorescence analysis further showed that PLCγ2 was mainly expressed in macrophages infiltrating the inflammatory tissues of APAID patients and mice ( Figure 1 C and Figure 1 F).

[0039] These findings suggest that macrophages with high PLCγ2 expression may play a crucial role in the development of IBD associated with APLAID. In addition, we examined 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 intestinal tissues of 9 patients were significantly positive for PLCγ2 expression ( Figure 8A), further indicating that there is a correlation between high expression of PLCγ2 and the occurrence of IBD.

[0040] 2. PLCγ2p.Leu845Ser mutation activates the IFNβ pathway in macrophages

[0041] To investigate the role of macrophages in APLAID, we established THP1 cells stably expressing PLCγ2p.Leu845Ser (named THP1mut) and control cells (named THP1nc). First, we performed transcriptome sequencing to analyze the differentially expressed genes between THP1mut and THP1nc cells. 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 showed 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 interferons are mainly composed of IFNα and IFNβ, to determine which one is responsible for activating the type I interferon pathway, we performed an ELISA assay and observed a tenfold increase in IFNβ secretion in THP1mut cells, while IFNα secretion 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 PLCγ2p.Leu845Ser mutation. qPCR results showed that compared with healthy controls, the expression of type I interferon-related genes such as IFNβ, ISG15, STAT1, STAT2, CXCL10, and TNFSF10 was increased in peripheral blood mononuclear cells of APLAID patients ( Figure 2 K-2P). Wb results showed that the expression and phosphorylation levels of STAT1 and STAT2 were increased in peripheral blood mononuclear cells of APLAID patients ( Figure 2Q). IHC experiments showed that the expression of IFNβ, P-STAT1 and ISG15 was significantly positive in the intestinal tissues of APLAID patients ( Figure 2 R). In addition, we examined the expression of IFNβ in the intestinal tissues of 16 other patients clinically diagnosed with IBD. We found that among the 9 IBD patients who were PLCγ2 positive, 8 were also IFNβ positive ( Figure 8 B), which further validates the regulatory relationship between PLCγ2 and IFNβ.

[0044] Furthermore, we confirmed the activation status of the IFNβ signaling pathway in PLCγ2p.Leu845ser mutant mice. Similarly, we observed a significant increase in the expression of IFNβ and IFNβ pathway activation-related genes STAT1, STAT2, ISG15, CXCL10, and TNFSF10 in PBMCs of PLCγ2 mutant mice ( Figure 2 S-2X). Wb results showed that the expression and phosphorylation of STAT1 in 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 inflammatory tissues of PLCγ2 mutant mice ( Figure 2 Taken together, these findings suggest that activation of the IFNβ pathway in macrophages driven by the PLCγ2p.Leu845Ser mutation may play an important role in the development of PLCγ2 mutation-associated APLAID.

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

[0046] To further confirm that the PLCγ2p.Leu845Ser mutation leads to upregulation of IFNβ and activation of the type I interferon pathway in macrophages, we initially used two PLCγ2 siRNAs to knock down PLCγ2 expression in THP1mut cells. qPCR and ELISA assays showed that PLCγ2 knockdown led to 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 gene assay showed that reducing the expression of PLCγ2 could significantly inhibit the luciferase activity of the IFNβ promoter ( Figure 3 G). Western blot analysis showed that PLCγ2 knockdown significantly reduced the expression and phosphorylation of STAT1 and STAT2 in THP1mut cells ( Figure 3 H).

[0047] We further investigated the effects of inhibiting the type I interferon pathway on PLCγ2 mutant macrophages using the type I interferon inhibitor Anifrolumab and the JAK / STAT pathway inhibitor Upadacitinib. qPCR results showed that both Anifrolumab and Upadacitinib significantly reversed the upregulation of STAT1, STAT2, CXCL10, and TNFSF10 in PLCγ2 mutant macrophages ( Figure 3 Western blot 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 effects of wogonin on PLCγ2p.Leu845Ser mutant macrophages. Our results showed that wogonin significantly inhibited 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, antiproliferative and immunomodulatory properties. To explore the effects of elevated IFNβ in macrophages carrying the PLCγ2p.Leu845Ser mutation on intestinal tissue, human normal intestinal epithelial cells NCM460 were exposed to 30% supernatant of THP1nc cells or THP1mut cells, and complete culture medium containing 10ng / mL recombinant human IFNβ was used as a positive control. We initially performed transcriptome sequencing on NCM460 cells treated with the above conditions. Pathway enrichment analysis of differentially expressed genes highlighted 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 with NCM460 cells treated with THP1nc culture supernatant ( Figure 4 D and Figure 4 F-4G). Our further studies 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] A large number of literatures have shown that not only can type I interferon inhibit cell proliferation, but also various inflammatory factors produced by the activation of 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 NCM460 cell proliferation ( Figure 4 These findings suggest that PLCγ2p.Leu845Ser mutation-induced activation of the type I interferon pathway plays a key role in the pathogenesis of PLCγ2 mutation-associated IBD, primarily by inhibiting intestinal epithelial cell proliferation.

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

[0053] To further elucidate the molecular mechanism by which the PLCγ2p.Leu845Ser mutation induces IFNβ upregulation, we used a flag antibody to perform co-immunoprecipitation (Co-IP) experiments on protein lysates from THP1mut cells expressing flagPLCγ2(p.Leu845Ser). The protein complexes obtained from the Co-IP experiments were subjected to mass spectrometry and pathway enrichment analysis. 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 interferon. 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 can specifically detect PLCγ2 ( Figure 5 D). In addition, laser confocal imaging clearly demonstrated the colocalization of PLCγ2 and IRF5, and macrophages expressing the p.Leu845Ser mutant PLCγ2 exhibited enhanced nuclear localization of the PLCγ2 / IRF5 complex ( Figure 5 E).

[0054] In addition, we simultaneously overexpressed IRF5 and wild-type or p.Leu845Ser mutant PLCγ2 in 293T cells. Through Co-IP experiments and laser confocal imaging, we further confirmed that the p.Leu845Ser mutant 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. Western blot results showed that compared with 293T cells overexpressing wild-type PLCγ2, the levels of PLCγ2 and IRF5 in the nucleus of cells overexpressing the p.Leu845Ser mutation were significantly increased ( Figure 5 H). Finally, our study used the IRF5 inhibitor YE6144 to treat THP1mut cells and 293T cells overexpressing PLCγ2(p.Leu845Ser) / IRF5. Laser confocal imaging results showed that YE6144 exhibited a significant inhibitory effect on the nuclear localization of the PLCγ2 / IRF5 complex ( Figure 5 These data suggest that the increased nuclear translocation of IRF5 caused by the PLCγ2p.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 function of IFNβ.

[0056] Based on the hypothesis that the p.Leu845Ser mutation in PLCγ2 enhances IFNβ transcription by promoting IRF5 nuclear translocation, we initially used the IRF5 inhibitor YE6144 to inhibit IRF5 function and performed qPCR and dual-luciferase reporter assays to assess the effect of IRF5 inhibition on IFNβ translation. Our results showed that after IRF5 function inhibition, both IFNβ expression and promoter activity were significantly reduced ( Figure 6 A-6C). In addition, the effect of YE6144 on the activation of the JAK / STATs pathway was also studied. qPCR assays showed that YE6144 significantly inhibited the expression of multiple inflammatory factors such as STAT1, STAT2, CXCL10, and TNFSF10 in THP1mut macrophages ( Figure 6 D-6G). Consistent with this, Wb experiments 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 THP1mut macrophage supernatant treated with YE6144 on NCM460 cell proliferation was significantly reduced ( Figure 6Together, these data suggest that the IRF5 / IFNβ regulatory axis could serve as a therapeutic target for individuals with APLAID caused by the PLCγ2p.Leu845Ser mutation.

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

[0058] To elucidate whether the type I interferon pathway could serve as a potential therapeutic target for APLAID individuals carrying the PLCγ2p.Leu845Ser mutation, we evaluated the efficacy of the IFNAR antagonist anifrolumab in APLAID mice. Complete blood cell counts showed that the number and ratio of monocytes in mutant mice treated with anifrolumab were significantly reduced compared with untreated mice ( 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). Western blot analysis showed that Anifrolumab significantly reduced the expression and phosphorylation of STAT1 protein in the inflammatory 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 inflammatory tissues of APLAID mice treated with Anifrolumab was significantly reduced, and the expression of PLCγ2 was significantly decreased ( Figure 7 H). qPCR assays showed that Anifrolumab significantly inhibited the expression of IFNβ pathway-related genes such as STAT1, TNFSF10, CXCL10, and ISG15 in the inflammatory tissues of APLAID mice ( Figure 7 F-7I). Taken together, these findings indicate that anifrolumab can effectively alleviate the inflammatory response and improve the pathological conditions of APLAID mice caused by the PLCγ2p.Leu845Ser mutation by inhibiting the type I interferon pathway.

[0059] Based on the pathological features of APLAID patients caused by the PLCγ2 (p.Leu845Ser) mutation, this study established a PLCγ2 mutant cell model and a mouse model to comprehensively investigate the molecular mechanisms of the pathogenesis of APLAID caused by the PLCγ2 p.Leu845Ser mutation. Our main findings are as follows:

[0060] 1. We revealed for the first time 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 IFNβ expression by enhancing the binding of IFNβ to IRF5 and promoting IRF5 nuclear translocation. Functional inhibition of IRF5 can significantly reverse the inflammatory phenotype of PLCγ2 mutant macrophages.

[0062] 3. Our experiments showed for the first time that anifrolumab, a type 1 interferon receptor antibody drug 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 the PLCγ2 (p.Leu845Ser) mutation.

[0063] Although there have been many reports of APLAID cases caused by PLCγ2 gene mutations worldwide, most APLAID patients will develop 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 used IHC to analyze the expression of PLCγ2 in the diseased intestinal tissues of 16 IBD patients for the first time and found that approximately 60% of the IBD patients' diseased intestinal tissues were PLCγ2 positive ( Figure 8 A). Therefore, our study preliminarily revealed the correlation between PLCγ2 and the development of IBD.

[0064] Case reports of APLAID caused by PLCγ2 mutations have shown that the proportions of neutrophils and monocytes in the peripheral blood of APLAID patients are significantly increased. Seth L. Masters et al. used a mouse model to investigate the molecular mechanisms of APLAID caused by the PLCγ2p.Ser707Tyr mutation. Their pathological findings demonstrated that neutrophils were the predominant inflammatory cells present in the PAWS, ears, and spleen of PLCγ2p.Ser707Tyr mutant mice, while macrophages were confined to the spleen. Unfortunately, this observation was not confirmed in the inflammatory tissues of APLAID patients carrying the PLCγ2p.Ser707Tyr mutation. Their molecular mechanistic studies demonstrated that elevated G-CSF in the peripheral blood was the primary cause of the autoinflammatory disease in individuals with the PLCγ2p.Ser707Tyr mutation. In contrast, we observed a significant increase in both the absolute number and proportion of monocytes in the peripheral blood of patients and mice carrying the PLCγ2p.Leu845Ser mutation, while neutrophil levels remained unchanged. Furthermore, pathological analysis of inflammatory tissues from patients and mice with APLAID caused by the PLCγ2p.Leu845Ser mutation revealed that macrophages were the predominant inflammatory cells, while neutrophils were less well localized in the inflamed tissues. Furthermore, our mechanistic studies revealed that aberrant activation of the IFNβ signaling pathway in macrophages plays a crucial role in the pathogenesis of APLAID caused by the PLCγ2p.Leu845Ser mutation. Collectively, our studies provide new insights into the role of macrophages in APLAID caused by the PLCγ2p.Leu845Ser mutation and identify upregulated IFNβ as a 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, TLRs, and NF-κB. We used immunoprecipitation and mass spectrometry to analyze proteins that specifically bind to PLCγ2 with the p.Leu845Ser mutation. Consistent with previous studies, we found that PLCγ2 with the p.Leu845Ser mutation can bind to multiple type I interferon-related transcription factors, including IRF5, IRF3, and NF-κB, with IRF5 being the most abundant ( Figure 5Functional experiments demonstrated that an IRF5 inhibitor significantly reversed the upregulation of IFNβ transcription and activation of downstream inflammatory pathways caused by the PLCγ2 p.Leu845Ser mutation. To our knowledge, our study is the first to reveal that the p.Leu845Ser mutant PLCγ2 promotes IFNβ upregulation by enhancing its binding to IRF5 and promoting IRF5 nuclear translocation. It also suggests that the IRF5 / IFNβ regulatory axis could serve as a potential therapeutic target for APLAIDs of the PLCγ2 p.Leu845Ser mutant.

[0066] Because the molecular mechanisms of pathogenesis remain unclear, individuals with APLAID caused by PLCγ2 mutations lack precise therapeutics. Clinical treatments for APLAID primarily include immunosuppressants such as glucocorticoids, cyclosporine, and tacrolimus, and a few biologics such as infliximab, but these drugs are ineffective in maintaining long-term remission. Based on our findings, we tested the efficacy of the type 1 interferon receptor inhibitor anifrolumab in mice harboring the PLCγ2p.Leu845Ser mutation and found that anifrolumab significantly suppressed inflammatory symptoms in APLAID mice. Here, our study demonstrates for the first time that anifrolumab, a US Food and Drug Administration-approved drug for the treatment of systemic lupus erythematosus (SLE), is a potential therapeutic agent for individuals with APLAID caused by the PLCγ2p.Leu845Ser mutation. Furthermore, given that gain-of-function PLCγ2 mutations lead to excessive release of DAG, which in turn leads to abnormal activation of inflammatory pathways, studies have shown that the herbal extract wogonin can inhibit DAG production. On this basis, we tested the effect of wogonin in PLCγ2p.Leu845Ser mutant cells. Our results showed that wogonin could significantly inhibit the expression of inflammatory factors and the activation of STAT1 pathway in PLCγ2p.Leu845Ser mutant macrophages ( Figure 9 This suggests that wogonin-based drugs may also be potential therapeutic agents for the PLCγ2p.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 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, 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 atmosphere of 5% CO2 at 37°C. Reagents used in the experiments are listed in Table 1.

[0076] Lentivirus and Stable Cell Line Generation: THP1mut and THP1nc cell lines expressing the p.Leu845Ser mutant PLCγ2 were constructed by Guangzhou Xunxun Biopharmaceutical Technology Co., Ltd., China. Lentivirus expressing the p.Leu845Ser mutant PLCγ2 or wild-type PLCγ2 was purchased from Guangzhou ICE Biotechnology Co., Ltd. Plasmids including pcDNA3.1-PLCγ2, pcDNA3.1-BLCγ2 (p.Leu845Ser), pcDNA3.1-IRF5, pcDNA 3.1 vector, and pGL3basic IFNβ promoter were constructed by Guangzhou ICE Biotechnology Co., Ltd.

[0077] Macrophage polarization: THP1-mut and THP1-nc cells were cultured at 1x10 cells per well. 6 The cells were cultured in 6-well plates at a density of 100 cells / mL. M0 macrophages were obtained after induction with 100 ng / mL PMA for 24 hours, and 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 and 293T cells.

[0079] Human Peripheral Blood Mononuclear Cell Isolation: Peripheral blood (10 mL) was collected from healthy individuals and patients, and PBMCs were isolated using a human monocyte isolation kit according to the manufacturer's instructions. Detailed information about the reagents is provided in Table 1.

[0080] siRNA interference: PLCγ2 siRNA was designed and synthesized by Shanghai Gene Pharmaceuticals. 24 hours before transfection, THP1 mut M0 macrophages were plated in 6-well plates at 8×10 5 Cells were cultured at a density of 100 cells / mL. siRNA was transfected using RNAiMAX and the cells were cultured for an additional 48 hours. The supernatant was then collected and RNA and protein were extracted. Details of the transfection reagent are provided in Table 1. siRNA sequences are listed in Table 2.

[0081] Co-culture experiments: 24 hours before co-culture, NCM460 cells were plated at 5×10 cells per well in a 6-well plate. 5 The cells were cultured at a density of 100 cells / mL. The next day, cell supernatants from THP1mut or THP1nc macrophages were added to NCM460 cells at a 30% ratio, with 1 μg / mL IFNβ serving as a positive control. The cells were cultured for an additional 48 hours and then harvested for cell proliferation assays and transcriptome sequencing.

[0082] CCK8 Assay: Cell proliferation activity was analyzed using a CCK-8 kit. Briefly, CCK8 solution was added to cell culture medium at a volume ratio of 1:10, incubated at 37°C for 1 hour, and absorbance was read at 450 nm using a microplate reader. Each cell type was assayed in triplicate. Details of the CCK8 kit are provided in Table 1.

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

[0084] RNA extraction and qPCR: Total RNA was extracted from cell and tissue samples using an RNA purification kit, and RNA concentration was measured using a NanoDrop. One microgram of RNA was reverse-transcribed into cDNA using a Color reverse transcription kit. qPCR was used to measure target gene expression levels, assessed using the 2-ΔΔCt method compared to β-actin as a control gene. Primer sequences for the target genes are listed in Table 2.

[0085] Co-IP experiment: using Pierce TMTotal cellular protein was extracted using 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. Protein G magnetic beads were used to obtain the corresponding protein complexes, which were then analyzed by protein profiling and Western blotting. Details of the Co-IP reagents are provided in Table 1.

[0086] Dual-luciferase reporter assay: Guangzhou ICE Biotechnology Co., Ltd. designed and constructed the IFNβ promoter-expressing vector pGL3basic IFNβ and the pcDNA3.1-IRF5 overexpression vector. MCF7 cells were cultured in six-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 in the absence of 1 μM IRF5 inhibitor YE6144. Dual-luciferase reporter assays were then performed according to the kit's instructions. Luminescence values ​​for firefly and nephrocytes were read using a Victor Nivo multi-function microplate reader.

[0087] For cytoplasmic and nuclear fractionation experiments, 293T cells were transfected with 5 μg of the pcDNA3.1-PLCγ2 or pcDNA3.1-BLCγ2 (p.Leu845Ser) overexpression vectors, along with 5 μg of the pcDNA3.1-IRF5 overexpression vector. Forty-eight hours later, cytoplasmic and nuclear proteins were extracted according to the instructions of the nuclear and cytoplasmic protein extraction kits. Details of the nuclear and cytoplasmic protein extraction kits are provided 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 determined using an enhanced BCA protein assay kit. Protein samples were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk in TBST, the membranes were incubated with primary antibodies, including STAT1, STAT2, pSTAT1, pSTAT2, PLCγ2, IRF5, and GAPDH, at a 1:1000 dilution overnight at 4°C. Subsequently, the membranes were incubated with goat anti-mouse IgG and goat anti-rabbit IgG at a 1:2500 dilution for 1 hour at room temperature before imaging. Details of the antibodies are provided 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 deparaffinized and stained with hematoxylin and eosin according to the hematoxylin and eosin staining kit protocol. They were then dehydrated with graded alcohols, cleared with xylene, and sealed with neutral glue before photography. Details of the hematoxylin and eosin staining kit are shown in Table 1.

[0090] Immunohistochemistry: Tissue sections underwent deparaffinization, EDTA antigen retrieval, H₂O₂ elimination of endogenous peroxidase, and 5% BSA blocking according to the SABC immunohistochemistry kit (mouse / rabbit) protocol. Antibodies, including IFNβ, pSTAT1, PLCγ2, IRF5, F4 / 80, CD68, and MPO, were diluted 1:200 in 5% BSA and incubated overnight at 4°C. Subsequently, biotinylated goat anti-mouse / rabbit IgG was incubated at 37°C for 30 minutes, followed by SABC incubation at 37°C for 30 minutes. Following DAB staining, sections were counterstained with hematoxylin, dehydrated, cleared, mounted, and photographed. Details of the antibodies and SABC kit are provided in Table 1.

[0091] Laser Confocal Imaging: For THP1 and 293T cell samples, the test cells were initially plated 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 with the primary antibody solution overnight at 4°C, and incubated with the secondary antibody solution for 30 minutes at room temperature. Finally, the slides were sealed with a DAPI-containing sealing reagent. For tissue samples, after antigen retrieval, elimination of endogenous peroxidases, and blocking with 5% BSA, they were incubated with the primary and secondary antibody solutions prepared above and then sealed with a DAPI-containing sealing reagent. The sealed cells and tissue sections were imaged using a Leica Stellaris 5 laser confocal microscope.

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

[0093] Enzyme-linked immunosorbent assay: Human interferon α ELISA kits and human interferon β ELISA kits were used to measure interferon α and interferon β in the collected supernatants. Each sample was tested in triplicate, and the concentrations of IFNα and IFNβ were calculated using a standard curve. Details of the ELISA kits are listed in Table 1.

[0094] Animal experimentation: F0-generation PLCγ2p.Leu845Ser mutant mice were established by the Shanghai Model Organisms Center, and SPF-grade C57BL / 6 mice were purchased from the Guangdong Medical Laboratory Animal Center. All mice were housed in an SPF-grade environment. Seven-week-old male F0-generation mutant mice were mated with female C57BL / 6 mice of the same age to generate F1-generation mutant mice. At 3 weeks of age, F1-generation mutant mice were treated with 30 μg of anti-IFNAR1 via intraperitoneal injection for 5 consecutive days. Other F1-generation mutant mice and C57BL / 6 mice of the same age were injected with the same volume of PBS as controls. After 5 weeks of age, blood was collected from the eyeballs of the mice for routine hematological analysis. Tissue samples were analyzed by immunohistochemistry, confocal laser scanning, Western blotting, and quantitative PCR. All animal experiments were approved by the Life Science Ethics Review Committee of Guangdong Medical University.

[0095] Statistical analysis: Statistical analysis was performed using GraphPad Prism 8.0, with Student's t-test performed for all experiments. Statistical significance was defined as P < 0.05. The significance levels are as follows: * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and *** indicates P < 0.0001. ns is 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 Aiki Biotechnology Co., Ltd. to construct a lentivirus, which was then used to infect THP1 cells. The THP1-Mut (p.Leu845Ser) cell line was obtained by adding puromycin for selection.

[0097] First, the PLCG2 (2534T>C) gene was synthesized by gene synthesis. The synthesized PLCG2 gene and the pCDH-CMV lentiviral vector were digested with restriction endonucleases and ligated with T4 DNA enzyme to obtain the pCDH-CMV-PLCG2-845-3xFLAG-EF1-puro plasmid. The plasmid was tested by nucleic acid gel electrophoresis.

[0098] 2. Sequence the constructed plasmid and package it with lentivirus

[0099] 1. Cell Preparation

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

[0101] 2) The next day, the recovered cells were cultured and the medium was changed;

[0102] 3) On the third day, 293T cells were cultured at 2.0x106 cell / T75 for passage;

[0103] 4) On the fifth day, 293T cells were cultured at 4.5x10 6 The cells were seeded into a 10 cm diameter culture dish at a cell density of

[0104] 2. Lentiviral packaging

[0105] 1) On the day of transduction, observe the cells for virus packaging. Key observations include: uniform cell seeding and a cell density of 80% to 90%. If the cells are in the correct condition, remove the culture medium from the 293T cells and add 10 ml of virus packaging medium.

[0106] Note: To prevent cells from detaching from the cell wall, fresh culture medium should be added along the cell wall.

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

[0108] A. Prepare a 15 mL centrifuge tube and add serum-free Opti-MEM medium, helper plasmid mixture (PSPAX2 and PMD2G), and lentiviral expression plasmid. Vortex thoroughly to mix.

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

[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, gently shake to mix, and incubate at room temperature for 10-15 minutes;

[0112] 3) Add the DNA complex to the 293T cells and gently shake the culture dish back and forth to mix the complex. Incubate in a 37°C, 5% CO2 saturated humidity incubator.

[0113] 4) After 4-6 hours of transduction, fresh culture medium was replaced and the cells were cultured in a 37°C, 5% CO2 saturated humidity incubator.

[0114] 3. Collect and concentrate viruses

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

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

[0117] 3) Filter the viral supernatant after centrifugation through a 0.45 μM filter into a 50 mL centrifuge tube;

[0118] 4) Add the appropriate amount of PEG concentrate according to the volume of the filtrate. Mix thoroughly and place at 4°C to precipitate overnight.

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

[0120] 6) Remove excess supernatant with a pipette, then resuspend the viral pellet in 1 mL of HBSS. Slowly and thoroughly pipette the pellet to a single viral suspension. Aliquot the viral suspension into cryovials according to the specified volume and store at -80°C.

[0121] 3. Virus Titer Detection

[0122] 1. Viral infection

[0123] 1) On the first day, press 1.5x10 5 Cells were seeded into each well of a 12-well plate;

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

[0125] 3) Add a certain amount of virus to the cells in the corresponding wells;

[0126] 4) After mixing, place the cells in a 37°C, 5% CO2 saturated humidity incubator. After 4-6 hours, observe the cells and change the medium.

[0127] 5) 48 hours after infection, the cells were photographed and collected for DNA extraction;

[0128] 2. DNA extraction

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

[0130] 2) Centrifuge and remove the supernatant;

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

[0132] 4) Add 20 μL of proteinase K and mix thoroughly by inversion. Incubate at 70°C for 10 minutes and centrifuge briefly to remove water droplets on the tube wall.

[0133] 5) Add 350 μL of anhydrous ethanol and shake thoroughly for 15 seconds;

[0134] 6) Pipette the mixed solution onto a centrifuge column, centrifuge at 8000 rpm for 1 min, and discard the filtrate in the collection tube;

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

[0137] 9) Centrifuge at 12000 rpm for 3 min to completely dry the adsorption film;

[0138] 10) Place the spin column in a new 1.5 mL EP tube, add 50 μL of ElutionBufcr water to the center of the spin column, cover it, and let it stand at room temperature for 3 minutes;

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

[0140] 3. qPCR

[0141] 1) Reaction system: 2× Ipure SYBR Green Qper Master Mix: 10 μL, 10 μM upstream primer: 1.0 μL, 10 μM downstream primer: 1.0 μL, sample solution 2.0 μL, ddH2O 6.0 μL, total volume 20.0 μL.

[0142] 2) Reaction conditions: A. Pre-denaturation at 95°C for 10 min; B. Three-step amplification: 40 cycles of denaturation at 95°C for 15 s, annealing at 57°C for 20 s, and extension at 65°C for 30 s; C. Melting curve: 95°C for 15 s, 60°C for 60 s, and 95°C for 1 s.

[0143] 4. Construction of THP1-PLCG2(845) stable cell line

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

[0145] Construction of a PLCG2 mutant mouse model: Knockin mice were generated using CRISPR / Cas9 technology. This technology involves designing a guide RNA specific to the target gene to direct the Cas9 nuclease to modify the genome at the insertion site, thereby increasing the efficiency of homologous recombination in the modified gene region and homologously recombining the target fragment into the target site. The route for generating knockin mice using this technology is as follows:

[0146] 1. Design the guideRNA target sequence for the target site genome and perform in vitro transcription based on the sequence to obtain the guideRNA for the gene;

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

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

[0149] 4. Identify the genotype of the F0 generation mice obtained by PCR and sequencing;

[0150] 5. F0 generation mice were bred with WT mice to obtain positive F1 generation heterozygous mice;

[0151] 6. Deliver 6-8 week old positive F1 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 replacement without departing from the concept of the present invention is within the scope of protection of the present invention.

Claims

1. Use of an inhibitor targeting the IFNβ signaling pathway in the preparation of a medicament for treating APLAID, characterized in that: The inhibitor targeting the IFNβ signaling pathway is Anifrolumab.

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