Application of ubiquitin-specific protease 33 in regulation and control of polarization of macrophages and anti-tumor and / or anti-infection

Abstract

The invention provides application of ubiquitin specific protease 33 in regulation and control of polarization of macrophages and anti-tumor and / or anti-infection, and belongs to the technical field of biological medicine manufacturing. The invention discloses ubiquitin specific protease 33 serving as a new immune activation target to participate in polarization of M1 type macrophages. The invention reveals that USP33 regulates and controls the protein stability of the IRF2 factor through a ubiquitination modification way for the first time, and further regulates and controls the expression of downstream inflammatory cytokines such as INF-gamma. The USP33 is knocked out or knocked down to degrade the IRF2, so that the inhibition effect of the IRF2 on a proinflammatory signal is relieved, the polarization of macrophages to M1 type is promoted, the expression of proinflammatory factors is up-regulated, the proliferation of tumor cells is inhibited, and the growth of tumors is remarkably inhibited; and meanwhile, the defense capability of the host to the Listeria monocytogenes can be enhanced by virtue of myeloid specific knockout of the USP33.

Description

Technical Field

[0001] This invention belongs to the field of biomedical manufacturing technology, specifically relating to the application of ubiquitin-specific protease 33 in regulating macrophage polarization, anti-tumor and / or anti-infection. Background Technology

[0002] Macrophage polarization plays a crucial role in infection and the tumor microenvironment. In the tumor microenvironment, macrophages generally tend to promote tumor progression and deterioration. Tumor-associated macrophages (TAMs) are mostly M2 type (pro-tumor type), whose recruitment and functional activation are driven by a variety of key regulatory factors. They promote tumor cell invasion and metastasis by secreting various factors and inhibit T cell activity to form an immunosuppressive microenvironment. In contrast, M1 type macrophages exhibit significant anti-tumor activity by directly phagocytosing, releasing toxic molecules, secreting pro-inflammatory cytokines and chemokines, presenting tumor antigens, inhibiting tumor angiogenesis, and counteracting the immunosuppressive microenvironment. In the infection microenvironment, M1 macrophages are the primary bactericidal type, clearing bacteria through various mechanisms such as metabolic shifts, synergistic action with neutrophils, and feedback activation by pro-inflammatory cytokines to produce highly reactive intermediates. M2 macrophages promote eosinophil recruitment and Th2 immune responses in parasitic infections, but also suppress antiviral immunity. While they also possess bactericidal activity, it is relatively weak, and excessive inflammatory responses or suppression of antiviral immunity may lead to adverse consequences. However, there are currently no reports on effective components for regulating macrophage polarization.

[0003] Ubiquitin-specific protease 33 (USP33), as a deubiquitinating enzyme, primarily prevents proteasome degradation within cells by deubiquitinizing substrate proteins, thereby regulating various intracellular activities, including signaling pathways, autophagy, centrosome amplification, and tumorigenesis. Although aberrant expression of USP33 has been reported to be associated with various diseases, particularly tumorigenesis, its role in immune cells remains unreported in the literature. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of USP33 in the preparation of products that regulate macrophage polarization.

[0005] This invention provides the application of USP33 in the preparation of products that regulate macrophage polarization.

[0006] This invention provides the application of a reagent that inhibits the expression level of ubiquitin-specific protease 33 and / or a reagent that inhibits the expression level of the gene encoding ubiquitin-specific protease 33 in the preparation of a product that promotes the polarization of macrophages into M1 macrophages.

[0007] Preferably, the reagent that inhibits the expression level of the gene encoding ubiquitin-specific protease 33 includes at least one of the following: siRNA, shRNA, and sgRNA.

[0008] This invention provides a method for promoting macrophage polarization into M1 macrophages by inhibiting or interfering with the expression of the encoding gene for ubiquitin-specific protease 33 or ubiquitin-specific protease 33 in macrophages, thereby obtaining M1 macrophages specifically knocked out by USP33.

[0009] Preferably, the method for inhibiting or interfering with the expression of ubiquitin-specific protease 33 in macrophages is to target and bind to ubiquitin-specific protease 33 in macrophages using small interfering RNA fragments siUSP33-1, siUSP33-2, siUSP33-3, and siUSP33-4.

[0010] This invention provides the use of reagents that inhibit USP33 expression or monocytes that interfere with USP33 expression in the preparation of drugs that are antitumor, antibacterial, and antiviral.

[0011] Preferably, the tumor in the antitumor includes at least one of the following: colon cancer, breast cancer, pancreatic duct adenocarcinoma, gastric adenocarcinoma, liver cancer, lung adenocarcinoma, and neuroblastoma;

[0012] The anti-tumor treatment includes inhibiting tumor growth and / or inhibiting tumor metastasis.

[0013] Preferably, the cell types of the colon cancer include HCT116 cells or MC38 cells; the cell types of the breast cancer include MDA-MB-231 cells or MCF-7 cells; the cell types of the pancreatic duct adenocarcinoma include CFPAC-1; the cell types of the gastric adenocarcinoma include MKN45; the cell types of the liver cancer include HEPG2; the cell types of the lung adenocarcinoma include NCI-H1975; and the cell types of the neuroblastoma include SH-SY5Y.

[0014] Preferably, the bacteria in the antibacterial infection treatment includes Listeria monocytogenes; the virus in the antiviral treatment includes at least one of the following: human cytomegalovirus, measles virus, and influenza A virus.

[0015] Preferably, the antibacterial infection includes at least one of the following diseases: sepsis, meningitis, and mononucleosis.

[0016] This invention provides the application of ubiquitin-specific protease 33 (USP33) in the preparation of products that regulate macrophage polarization. This invention uses the deubiquitinating enzyme USP33 as a novel target for immune activation, participating in macrophage phenotypic remodeling. Specifically, USP33 regulates the stability of the IRF2 factor protein through ubiquitination, thereby regulating the expression of downstream inflammatory cytokines such as Interferon-γ; simultaneously, knocking out or downregulating USP33 degrades IRF2, relieving its inhibitory effect on pro-inflammatory signaling, promoting macrophage polarization towards the M1 type, upregulating the expression of pro-inflammatory factors, and inhibiting tumor cell proliferation. In vivo experiments have demonstrated that knocking out myeloid USP33 in colon cancer significantly activates M1 macrophage differentiation, enhances macrophage phagocytosis, antigen presentation, and cytokine secretion capabilities, as well as T cell infiltration and anti-tumor immune response functions, and significantly inhibits tumor growth; simultaneously, myeloid-specific knockout of USP33 also enhances the host's defense against Listeria monocytogenes. Attached Figure Description

[0017] Figure 1 The results show the detection of USP33's participation in and response to inflammatory responses in macrophages. A represents the detection results of USP33 expression in various organ tissues; B represents the effect of different concentrations of TNFα on the expression levels of proteins related to the NFκB signaling pathway and the IRF3 signaling pathway in macrophages; C represents the detection results of proteins related to the NFκB signaling pathway in macrophages after USP33 interference; and D represents the expression levels of cytokines in macrophages after USP33 interference.

[0018] Figure 2 The results of flow cytometry immunofluorescence detection of surface markers in M1 macrophages after interference with USP33;

[0019] Figure 3 The results show the interaction between USP33 and IRF2. A represents the results of the immunoprecipitation assay for the interaction between USP33 and endogenous IRF2, and B represents the results of the endogenous USP33 pull-down assay.

[0020] Figure 4 The results show the effect of suppressing IRF2 expression levels on macrophage differentiation into the M1 type;

[0021] Figure 5 The results of detection of ubiquitination modification of IRF2 regulated by USP33;

[0022] Figure 6 The results of the detection of USP33 regulating the stability of IRF2 protein;

[0023] Figure 7Results of USP33 and IRF2 protein detection in M1 and M2 macrophages;

[0024] Figure 8 To interfere with the effects of USP33 on the proliferation of various tumor cells;

[0025] Figure 9 The results show the effect of USP33 knockout on tumor growth in a mouse model of colorectal cancer induced by azomethane / dextran sulfate sodium (AOM / DSS); A is a schematic diagram of colorectal cancer mouse model induction; B is a curve showing the weight change of the colorectal cancer mouse model; C is the morphological results of colorectal cancer; D is the tumor size measurement results; E is the HE staining results.

[0026] Figure 10 The results of single-cell transcriptomic analysis of tumor macrophages in USP33 knockout colorectal tumors are shown in Figure A, which represents mouse colorectal tumor cell typing based on specific markers; Figure B shows the characteristic analysis of M1 and M2 macrophages in myeloid USP33 knockout tumors before and after knockout; Figure C shows the macrophage clustering diagram; Figure D shows the characteristic analysis results of M1 and M2 macrophage clusters in myeloid USP33 knockout tumors before and after knockout; and Figure E shows the proportion of different macrophage clusters in the tumor before and after myeloid USP33 knockout.

[0027] Figure 11 The results of single-cell transcriptome analysis of T cells in USP33 knockout colorectal tumors are shown in Figure A, where mouse T cells are genotyped according to specific markers, B shows the changes in the proportion of different T cells in the tumor before and after myeloid USP33 knockout, and C shows the results of KEGG signaling pathway enrichment analysis of T cells before and after myeloid USP33 knockout.

[0028] Figure 12 The results show the subcutaneous tumorigenicity of MC38 cells in a mouse model of colon cancer caused by myeloid USP33 knockout.

[0029] Figure 13 The results show the survival rate of mice infected with Listeria monocytogenes with myeloid USP33 deletion.

[0030] Figure 14 The results show the effect of myeloid USP33 deletion on bacterial clearance in infected mice. Detailed Implementation

[0031] This invention provides the application of ubiquitin-specific protease 33 in the preparation of products that regulate macrophage polarization.

[0032] In this invention, the USP33 protein in macrophages participates in and responds to the inflammatory response, specifically by interfering with USP33 protein expression. Both the NFκB signaling pathway and the IRF3 signaling pathway within macrophages are significantly activated. Simultaneously, the levels of intracellular inflammation and immune-related cytokines, such as TNFα, IL6, CCL2, IFNα, and IFNβ, are also significantly increased. These cytokines and signaling pathways possess multiple biological functions, including antiviral, antitumor, and immunomodulatory effects, suggesting that USP33 may be involved in the polarization process of macrophages.

[0033] In this invention, macrophage polarization includes two directions: one for M1 macrophages and the other for M2 macrophages. The expression level of USP33 is significantly reduced in M1 macrophages and significantly increased in M2 macrophages, indicating that USP33 responds to macrophage polarization.

[0034] In this invention, USP33 promotes macrophage polarization towards M1-type macrophages through negative regulation, upregulates the expression of pro-inflammatory factors, and inhibits tumor cell proliferation. In an embodiment of this invention, after interfering with the expression level of USP33 in macrophages and inducing treatment, the macrophage type was determined. The results showed that the proportion of M1-type macrophages was effectively increased, and the activation of M1-type macrophage signaling pathways was achieved. These signaling pathways preferably include those associated with M1-type macrophage polarization, such as those involving NF-κB, TNF, Toll receptor, interferon-α signaling pathway, cytokine-cytokine receptor interactions, and chemokine signaling pathways. Activation of cytokine-cytokine receptor interactions and chemokine signaling pathways enhanced the anti-tumor function of macrophages. This indicates that USP33 participates in regulating M1-type macrophage differentiation.

[0035] In this invention, the USP33 protein interacts with the RF2 protein to form the USP33-IRF2 axis, playing a crucial role in regulating macrophage polarization. USP33 regulates the stability of the IRF2 factor protein through ubiquitination, thereby controlling the expression of downstream inflammatory cytokines such as INF-γ. Knockout or knockdown of USP33 leads to IRF2 degradation, relieving IRF2's inhibitory effect on pro-inflammatory signaling and promoting macrophage polarization towards the M1 type.

[0036] In this invention, the product comprises at least one of the following: a differentiation promoter, a cell culture medium, a reagent kit, and a drug. This invention does not impose any particular limitation on the preparation method of the product; any preparation method well known in the art may be used.

[0037] This invention provides the application of a reagent that inhibits the expression level of ubiquitin-specific protease 33 and / or a reagent that inhibits the expression level of the gene encoding ubiquitin-specific protease 33 in the preparation of a product that promotes the polarization of macrophages into M1 macrophages.

[0038] In the present invention, the amino acid sequence of ubiquitin-specific protease 33 is as shown in SEQ ID NO:1 (MTGSNSHITILTLKVLPHFESLGKQEKIPNKMSAFRNHCPHLDSVGEITKED LIQKSLGTCQDCKVQGPNLWACLENRCSYVGCGESQVDHSTIHSQETKHYLTVNLTTLRVWCYACSKEVFLDRKLGTQPSLPHVRQPHQIQENSVQDFKIPSNTTLKTPLVAVFDDLDIEADEEDELRARGLTGLKNIGNTCYMNAALQALSNCPPLTQFFLDCGGLARTDKKPAICKSYLKLMTELWHKSRPGSVVPTTLFQGIKTVNPTFRGYSQQDAQEFLRCLMDLLHEELKEQVMEVEEDPQTITTEETMEEDKSQSDVDFQSCESCSNSDRAENENGSRCFSEDNNETTMLIQDDENNSEMSKDWQKEKMCNKINKVNSEGEFDKDRDSISETVDLNNQETVKVQIHSRASEYITDVHSNDLSTPQILPSNEGVNPRLSASPPKSGNLWPGLAPPHKKAQSASPKRKKQHKKYRSVISDIFDGTIISSVQCLTCDRVSVTLETFQDLSLPIPGKEDLAKLHSSSHPTSIVKAGSCGEAYAPQGWIAFFMEYVKRFVVSCVPSWFWGPVVTLQDCLAAFFARDELKGDNMYSCEKCKKLRNGVKFCKVQNFPEILCIHLKRFRHELMFSTKISTHVSFPLEGLDLQPFLAKDSPAQIVTYDLLSVICHHGTASSGHYIAYCRNNLNNLWYEFDDQSVTEVSESTVQNAEAYVLFYRKSSEEAQKERRRISNLLNIMEPSLLQFYISRQWLNKFKTFAEPGPISNNDFLCIHGGVPPRKAGYIEDLVLMLPQNIWDNLYSRYGGGPAVNHLYICHTCQIEAEKIEKRRKTELEIFIRLNRAFQKEDSPATFYCISMQWFREWESFVKGKDGDPPGPIDNTKIAVTKCGNVMLRQGADSGQISEETWNFLQSIYGGGPEVILRPPVVHVDPDILQAEEKIEVETRSL).The reagent for inhibiting the expression level of ubiquitin-specific protease 33 preferably includes small interfering RNA fragments siUSP33-1, siUSP33-2, siUSP33-3, and siUSP33-4. The reagent for inhibiting the expression level of the gene encoding ubiquitin-specific protease 33 preferably includes at least one of the following: siRNA, shRNA, and sgRNA. The reagent also includes derivatives containing one of the siRNA, shRNA, and sgRNA. The derivatives preferably include recombinant vectors, gene editing systems, and recombinant viruses. The types of products are the same as those described in the above technical solutions and will not be elaborated upon here.

[0039] This invention provides a method for promoting macrophage polarization into M1 macrophages by inhibiting or interfering with the expression of the encoding gene for ubiquitin-specific protease 33 or ubiquitin-specific protease 33 in macrophages, thereby obtaining M1 macrophages specifically knocked out by USP33.

[0040] In this invention, the method for inhibiting or interfering with the expression of ubiquitin-specific protease 33 in macrophages is preferably to use small interfering RNA to target and bind to ubiquitin-specific protease 33 in macrophages. In embodiments of this invention, the reagents for inhibiting the expression level of the gene encoding ubiquitin-specific protease 33 include siUSP33-1 and siUSP33-2. The nucleotide sequence of siUSP33-1 is shown in SEQ ID NO:2 (CAAUGUUAAUUCAGGAUGA). The nucleotide sequence of siUSP33-2 is shown in SEQ ID NO:3 (GGCUUGGAUCUUCAGCCAU). Experiments show that both siUSP33-1 and siUSP33-2 can effectively reduce the expression level of ubiquitin-specific protease 33, and the effect of siUSP33-2 is better than that of siUSP33-1.

[0041] Given that interfering with the expression of USP33 in macrophages promotes M1 macrophage polarization, this invention provides the use of reagents that inhibit USP33 expression or monocytes that interfere with USP33 expression in the preparation of drugs that are antitumor, antibacterial, and antiviral.

[0042] In this invention, the reagent for inhibiting USP33 gene expression preferably includes at least one of the following: siRNA, shRNA, and sgRNA, such as small interfering RNA fragments siUSP33-1, siUSP33-2, siUSP33-3, etc. The reagent also includes derivatives containing one of the siRNA, shRNA, and sgRNA. The derivatives preferably include recombinant vectors, gene editing systems, and recombinant viruses. The recombinant vector includes a recombinant vector containing one of the siRNA, shRNA, and sgRNA. This invention does not impose any particular limitation on the backbone vector of the recombinant vector; any backbone vector well-known in the art can be used. The gene editing system preferably includes a CRISPR-Cas9 gene editing system or a CRISPR-Cas12 gene editing system. The host virus of the recombinant virus includes lentivirus or adenovirus. The source of USP33 preferably includes macrophages or progenitor cells of macrophages. The progenitor cells of macrophages preferably include monocytes. The method for preparing the monocytes interfering with USP33 expression is preferably using THP1 cells or extracting them from bone marrow.

[0043] In this invention, the anti-colon cancer treatment preferably includes inhibiting the growth and / or metastasis of colon cancer. The cell types of the colon cancer preferably include HCT116 cells or MC38 cells. Knocking out the expression of the USP33 gene or protein promotes macrophage polarization to form M1 macrophages, and the increased number of M1 macrophages exerts an anti-tumor effect.

[0044] In one embodiment of the present invention, co-culturing macrophages with USP33-interfered cells with HCT116 tumor cells significantly reduced the number of monoclonal HCT116 tumor cells. This indicates that USP33 interference promotes macrophage polarization towards the M1 type, thereby inhibiting tumor cell proliferation.

[0045] In one embodiment of the present invention, USP33 expression was knocked out in monocytes by genetic engineering to obtain genetically engineered mice with USP33 knockout. A tumor mouse model was then constructed by inducing colon cancer using AOM / DSS. The USP33 knockout mouse model showed smaller tumors and fewer tumor-infiltrating cells, and also demonstrated the ability to inhibit the subcutaneous tumorigenesis of MC38 cells.

[0046] In this invention, the antibacterial infection is applicable to infections caused by bacteria, and in some embodiments, the bacteria preferably include Listeria monocytogenes. The anti-infection preferably includes at least one of the following diseases: sepsis, meningitis, and mononucleosis. The antibacterial infection manifests as prolonging patient survival and / or reducing bacterial levels in the patient's body.

[0047] In this invention, the antiviral virus includes at least one of the following: human cytomegalovirus, measles virus, and influenza A virus.

[0048] The following examples illustrate the application of ubiquitin-specific protease 33 provided by the present invention in regulating macrophage polarization, anti-tumor activity, and / or anti-infection, but these should not be construed as limiting the scope of protection of the present invention.

[0049] The biological materials, reagents, and primer sequences used in the experiment are shown in Tables 1 to 4.

[0050] Table 1 Cell line information

[0051] cell lines Chinese name Cultivation conditions 293T Human embryonic kidney cells DMEM + 10% FBS HCT116 Human colon cancer cells 1640+10%FBS MC38 mouse colon cancer cells 1640+10%FBS THP1 Human mononuclear cells (acute monocytic leukemia cells) 1640+10%FBS U2OS Human osteosarcoma cells DMEM + 10% FBS MDA-MB-231 Human breast cancer cells DMEM + 10% FBS MCF-7 Human breast cancer cells DMEM + 10% FBS SH-SY5Y Human neuroblastoma cells DMEM / F12+10%FBS CFPAC-1 Pancreatic ductal adenocarcinoma cells DMEM + 10% FBS MKN45 Human gastric adenocarcinoma cells 1640+10%FBS NCI-H1975 Human lung adenocarcinoma cells DMEM + 10% FBS HEPG2 Human liver cancer cells DMEM + 10% FBS

[0052] Table 2 siRNA sequences

[0053]

[0054]

[0055] Table 3 Antibody Information

[0056]

[0057]

[0058] Table 4. Main Reagents and Kits

[0059] name company Item number lactic acid Solarborg L8630-5g Crystal violet staining solution Azure Sky C0121-100ml Listeria monocytogenes chromogenic agar plates (LA) land bridge PBE006 <![CDATA[Red Blood Cell Lysis Buffer Hybri-Max TM > Sigma-Aldrich R7757 EDTA anticoagulant tube 1.5ml Solarborg YA1461-1pk V-52 Veterinary Blood Cell Analysis Reagent Kit Mindray V-52 Proteinase K Full Gold Bio GE201-01 5-Bromo-4-chloro-3-indole-β-D-glucoside (X-Gluc) JSENB JS0286-5MG Lithium chloride JSENB JS0123-100G Thioglycolate culture medium (thioglycolate broth) Solarborg LA8740-250 Moon syllable Sunwater 50107 Anti-Flag Affinity Gel Bimake B23102-5mL Anti-HAmagneticbeads Selleck B26201 NP40 Aggregated Beauty MF430-100ml Cell scraper Falcon 353085-1 MurineIL-4 Pipettek 214-14-5 RecombinantMurineM-CSF Pipettek 315-02-10ug eBioscience Intracellular Fixation and Perturbation Buffer Invesco Great Wall 88-8824-00 KOD-Plus-Neo Toyobo / Toyobo KOD-401 200 mesh filter BIO-NEW NLW08 300 mesh filter BIO-NEW NLW12

[0060] Example 1

[0061] USP33 participates in and responds to inflammatory responses in macrophages.

[0062] Mouse heart, liver, stomach, lung, kidney, muscle, colon, spleen, and thymus tissues were collected, and Western blotting was used to detect USP33 expression. The results showed that USP33 was not only highly expressed in the colon, but also highly expressed in some immune organs such as the liver, spleen, and thymus. Figure 1The study used THP-1 cells to induce macrophage differentiation with 100 ng / ml PMA (Phorbol12-myristate 13-acetate). Two small interfering fragments were used to interfere with USP33 expression, and the protein levels of P-P65 and P-IRF3 were measured. The results showed that after interfering with USP33 expression, both the NFκB signaling pathway and the IRF3 signaling pathway within macrophages were significantly activated. Simultaneously, the levels of intracellular inflammation and immune-related cytokines, such as TNFα, IL6, CCL2, IFNα, and IFNβ, were also significantly increased. According to previous literature, TNFα has a direct tumor-killing effect; IL6 is an important pro-inflammatory cytokine involved in regulating immune responses and inflammatory reactions; CCL2 can recruit monocytes and macrophages to inflammatory sites, promoting the development of inflammatory responses; IFNα and IFNβ are members of the interferon family and have multiple biological functions, including antiviral, antitumor, and immunomodulatory effects. These molecules all point to the possibility that macrophages may polarize into M1-type macrophages after USP33 interference.

[0063] Example 2

[0064] USP33 knockdown promotes macrophage differentiation into M1 type.

[0065] Macrophages induced by the TLR-like receptor agonist PMA in the THP1 cell line were subjected to interference with USP33 using two small interfering fragments. The surface marker CD80 of M1 macrophages was then detected by flow cytometry immunofluorescence.

[0066] The results showed that the proportion of M1 macrophages was significantly increased after THP1-induced macrophages (see...). Figure 2 This indicates that USP33 is involved in regulating the differentiation of M1 macrophages.

[0067] Example 3

[0068] USP33 regulates the activation of M1 macrophage signaling pathways.

[0069] The functional characteristics of macrophages after USP33 interference were further investigated. USP33 expression in macrophages was knocked down using siRNA. After validating the interference efficiency, RNA-seq sequencing was performed on the control group and the USP33-interference group. Total RNA was extracted using the Trizol method, and genomic DNA contamination was removed by DNase treatment. RNA integrity was detected using the Agilent Bioanalyzer. mRNA was isolated using polyA enrichment, fragmented to 150-300 bp, reverse transcribed into cDNA, ligated with Illumina sequencing adapters, and amplified by PCR to construct a strand-specific library. 150 bp paired-end sequencing was performed using the Illumina NovaSeq platform. Low-quality reads (Q30 > 90%) were removed using FastQC, and adapter sequences were removed using Trimmomatic. Hisat2 was used to align clean reads to the GRCm38 reference genome, FeatureCounts was used to calculate gene expression levels, and DESeq2 and GFOLD were used for normalization and differential analysis to screen for significant pathways.

[0070] The results are shown in Table 5. The results indicate that functional changes in macrophages are mainly concentrated in signaling pathways involved in M1 macrophage differentiation. For example, NF-κB, TNF, Toll receptor, and interferon-α signaling pathways play key roles in M1 macrophage differentiation. Furthermore, cytokine-cytokine receptor interactions and chemokine signaling pathways were also upregulated in USP33-interfered macrophages, and activation of these pathways enhanced the anti-tumor function of macrophages. Recently, IL-17 signaling has also been found to play a dual inhibitory role in colorectal cancer. In macrophages, the IL17RA signaling pathway participates in promoting the release of IL18, thereby enhancing the anti-tumor immune response of CD8+ T cells.

[0071] Table 5. Enrichment analysis of KEGG signaling pathways

[0072] describe p.adjust q value Cytokine-cytokine receptor interactions 6.5393E-11 4.8737E-11 NF-κB signaling pathway 3.0775E-08 2.2936E-08 Viral proteins interact with cytokines and receptors 4.0483E-07 3.0172E-07 TNF signaling pathway 7.4763E-07 5.572E-07 Chemokine signaling pathway 2.7734E-06 2.067E-06 IL-17 signaling pathway 3.157E-06 2.3529E-06 Rheumatoid Arthritis 5.4485E-05 4.0607E-05 African trypanosomiasis 0.0003277 0.00024423 Alcoholic liver disease 0.00048262 0.00035969 Lipids and Atherosclerosis 0.00048262 0.00035969 Legionnaires' disease 0.0013385 0.00099758 NOD-like receptor signaling pathway 0.00154964 0.00115494 Human cytomegalovirus infection 0.00426074 0.0031755 Chagas disease (South American trypanosomiasis) 0.00982266 0.00732077 Toll-like receptor signaling pathway 0.0098577 0.00734688 malaria 0.01154 0.00860069 Cytoplasmic DNA sensing pathway 0.02115653 0.01576783 measles 0.0238392 0.01776721 Epithelial cell signaling in Helicobacter pylori infection 0.02554723 0.01904019 Salmonella infection 0.02881464 0.02147538 pertussis 0.02881464 0.02147538 Nonalcoholic fatty liver disease 0.02881464 0.02147538 Necrotic apoptosis 0.03016902 0.02248479 H1N1 influenza 0.03735371 0.0278395

[0073] Example 4

[0074] USP33 interacts with the interferon inhibitor IRF2.

[0075] To preliminarily verify whether there is an interaction between USP33 and IRF2, the Flag-USP33 plasmid was constructed and transfected into HEK293 cells to achieve overexpression of Flag-USP33. Subsequently, co-immunoprecipitation (Co-IP) was performed using a Flag-tagged antibody. Co-IP results clearly showed that Flag-USP33 can interact with endogenous IRF2, forming a complex that is co-precipitated. A pull-down assay was performed using endogenous USP33. Endogenous USP33 was enriched using a specific antibody, and then the proteins bound to it were detected. The specific experimental steps included: transfecting the FLAG-IRF2 plasmid into HEK293 cells in a 10cm culture dish; 48 hours after transfection, adding 1ml of RIPA lysis buffer containing PMSF, DTT, Aprotinin, Leupeptin, and Na3VO4 and lysing for 30 minutes, taking 50μl as the input control, and using the remaining 950μl for IP; taking a 1.5ml test tube, adding 1ml of buffer containing protease inhibitors and 16μl of FLAG-Beads, rotating in a mixer for 5 minutes, centrifuging at 3000rpm for 5 minutes and repeating 3 times to pre-treat the magnetic beads; discarding the supernatant, adding 950μl of lysis buffer, and incubating overnight at 4℃ in a mixer; discarding the supernatant, washing 3 times with 1ml of buffer containing protease inhibitors, finally adding 50μl of 1×loading buffer, mixing well, boiling to elute the sample for Western blotting to detect IRF2 binding.

[0076] See results Figure 3 The experimental results show that there is an interaction between endogenous USP33 and endogenous IRF2, which further strengthens the conclusion that USP33 and IRF2 interact within cells, indicating that this interaction is real under physiological cellular conditions.

[0077] Example 5

[0078] Inhibition of IRF2 promotes macrophage differentiation into M1 type

[0079] IRF2 is a core regulator of antiviral immunity. In macrophages, IRF2 maintains precise regulation of the antiviral immune response by balancing IRF1-mediated interferon signaling activation and exerting negative feedback inhibition. IRF2 plays a pro-cancer role in various tumors. In nasopharyngeal carcinoma, it drives tumor glycolysis and proliferation by activating the CENP-N / AKT signaling axis; in esophageal cancer, it inhibits the IFNγ receptor signaling pathway, leading to tumor cell resistance to interferon-γ therapy. Furthermore, IRF2 is highly expressed in the tumor microenvironment, such as in colorectal cancer, and regulates T cell function, affecting anti-tumor immune responses. However, no literature has yet explored whether IRF2 regulates macrophage polarization and affects its function in tumor immunity. This study investigated whether IRF2 has the ability to regulate M1 macrophage differentiation after discovering the binding interaction between IRF2 and USP33.

[0080] THP-1 cells were induced to differentiate into macrophages using 100 ng / ml PMA. USP33 cells were interfered with using two small interfering fragments. After 48 hours, flow cytometry was used to detect the M1 marker CD80 and the M2 marker CD206. Results showed that inhibition of IRF2 significantly led to macrophage differentiation into M1 macrophages. Figure 4 ).

[0081] Example 6

[0082] Deubiquitinase USP33 regulates the ubiquitination modification of IRF2.

[0083] Overexpression of USP33 significantly reduced IRF2 ubiquitination levels. Conversely, interference with USP33 significantly increased IRF2 ubiquitination levels. HEK293 cells were co-transfected with FLAG-IRF2, HA-UB, and GFP-USP33, treated overnight with 5 mg / ml MG132, and the collected proteins were incubated overnight with FLAG-Beads. Western blotting was used to detect ubiquitination levels. The results showed that overexpression of USP33 significantly reduced IRF2 ubiquitination levels. This result directly demonstrates that USP33 can remove ubiquitin molecules from the IRF2 protein, thereby exerting a deubiquitination effect. After successfully interfering with USP33 expression, we again used immunoprecipitation to detect IRF2 ubiquitination levels. The results showed that when USP33 expression was interfered with, the ubiquitination level of IRF2 significantly increased. This indicates that endogenous USP33 can inhibit IRF2 ubiquitination modification under normal conditions. When USP33 expression is inhibited, IRF2 loses this deubiquitination protection, leading to an increase in its ubiquitination level. Combining the experimental results of overexpression and interference with USP33, we can clearly conclude that USP33 can indeed exert its deubiquitination function and regulate the ubiquitination level of IRF2. (See results below) Figure 5

[0084] Example 7

[0085] USP33 regulates the protein stability of IRF2.

[0086] To further investigate whether USP33 affects the protein stability of IRF2, a cycloheximide (CHX) assay was performed. Cycloheximide inhibits protein synthesis. K293 cells were then routinely cultured to 80% confluence and then injected at a rate of 1-3 × 10⁻⁶. 5Cells were seeded at a density of 1 / well in 6-well plates. Using the Lipomax transfection reagent system, cells were transfected with a negative control siRNA and two USP33-specific siRNAs. The siRNAs were diluted with serum-free medium and mixed with the transfection reagent, incubated for 15 minutes, and then added to cells for 48 hours to achieve interference. Reinforcement experiment design: 24 hours after interference transfection, the interference group was additionally transfected with the GFP-USP33 plasmid. The plasmid was mixed with Lipomax in serum-free medium and incubated for 30 minutes before being added to cells, which were then cultured for another 48 hours to express the exogenous protein. After transfection, the medium was replaced with complete medium containing 50 μg / ml CHX. Cells were collected at 0, 2, 4, 8, and 12 hours, washed with ice-cold PBS, and lysed in RIPA lysis buffer containing protease inhibitors for 30 minutes on ice. The supernatant was collected after centrifugation at 12,000g for 10 minutes at 4°C. Protein concentration was determined by BCA method, and after adjustment, it was denatured at 95℃ for 5 minutes; 30 μg of protein was taken for 10% SDS-PAGE electrophoresis, and transferred to PVDF membrane by wet transfer method (100V, 60 minutes); blocked with 5% skim milk for 1 hour, and incubated overnight at 4℃ with anti-IRF2 (1:1000), anti-USP33 (1:2000 verification and correction) and anti-GAPDH (1:5000 internal control) primary antibodies in sequence, and then incubated at room temperature for 1 hour with HRP secondary antibody (1:5000) for ECL development.

[0087] See results Figure 6 The results showed that the half-life of IRF2 protein was significantly shortened after USP33 interference, indicating that USP33 interference inhibited the stability of IRF2 protein. After USP33 was reintroduced, the inhibited IRF2 protein stability was restored. All of these experiments demonstrate that USP33 regulates the stability of IRF2 protein.

[0088] Example 8

[0089] USP33 responds to macrophage polarization

[0090] There are two main types of macrophage polarization: M1 and M2. To better investigate the role of USP33 in this polarization, THP-1 cells were first seeded at 8 × 10^5 cells / well in 6-well plates and induced to form M0 macrophages for 24 hours with complete medium containing 100 ng / ml PMA. The M1 polarized group was then stimulated for 48 hours with fresh medium containing 100 ng / ml LPS and 20 ng / ml IFN-γ, while the M2 polarized group was stimulated for 48 hours with medium containing 20 ng / ml IL-4 and 20 ng / ml IL-13. Before flow cytometry, the samples were washed with PBS and then incubated with CD80-PE (M1 marker) and CD206-FITC (M2 marker) antibodies for 30 minutes in the dark, with an isotype control also included. For Western blotting, total protein was extracted with RIPA lysis buffer, quantified using the BCA method, and 30 μg of protein was loaded onto a membrane. The membrane was then transferred by SDS-PAGE electrophoresis, blocked with 5% skim milk for 1 hour, and incubated with primary antibody overnight at 4°C and secondary antibody for 1 hour at room temperature. Finally, the membrane was developed by ECL.

[0091] See results Figure 7 The results showed that both M1 and M2 macrophages were successfully induced. We then examined the expression of USP33 in M1 and M2 macrophages, and the results showed that USP33 was significantly decreased in M1 macrophages and significantly increased in M2 macrophages.

[0092] Example 9

[0093] Macrophages that interfere with USP33 inhibit tumor cell proliferation.

[0094] To investigate whether USP33 knockout-induced M1 macrophages have a direct tumor-killing effect, we first induced macrophages in cell culture plates in advance and promoted M1 macrophage differentiation by interfering with USP33. Then, we placed Transwell chambers with 0.4 μm pores on the cell plates and seeded 2000 different types of tumor cells into each chamber for co-culture.

[0095] See results Figure 8The results showed that when Transwell chambers with 0.4 μm pores were added to cell plates and tumor cells were seeded into the chambers for co-culture, the cells, unable to pass through the chambers, formed monoclonal colonies on the upper layer. Crystal violet staining revealed a significant reduction in monoclonal colonies formed in human colon cancer cell lines HCT116, human lung adenocarcinoma cells NCI-H1975, human neuroblastoma cells SH-SY5Y, human gastric adenocarcinoma cells MKN45, liver cancer cell line HEPG2, human breast cancer cells MDA-MB-231, MCF-7, and pancreatic ductal adenocarcinoma cells CFPAC-1 after co-culturing with macrophages that interfered with USP33. These results indicate that interference with USP33 promotes macrophage polarization towards the M1 type, thereby inhibiting the proliferation of various tumor cell types.

[0096] Example 10

[0097] Myeloid USP33 knockout can inhibit AOM / DSS-induced colorectal cancer progression.

[0098] It is known that in the initial stages of tumor development, tumor-associated macrophages (TAMs) enter the tumor under the attraction of chemokines such as CCL2 released by cancer cells. To better investigate the role of USP33 in the tumor immune microenvironment in mice, we selected another tumor model mouse: the AOM / DSS-induced colon cancer mouse model. Litterctomies of different genotypes aged 6–8 weeks were collected and weighed. After abdominal disinfection with 75% alcohol, AOM was injected intraperitoneally at a dose of 10 mg / kg, not exceeding 200 μL. The sham-operated group received an equal volume of physiological saline intraperitoneally. Day 0 was defined as the AOM injection date. After 7 days, the mice were fed drinking water containing 2% sodium dextran sulfate (DSS) for one week. After another 7 days, the drinking water was replaced with normal drinking water for two weeks. This process was repeated twice. Mouse weight changes were monitored twice weekly, and mortality rates during the modeling process were recorded. After modeling is completed, stop feeding the mice 12 hours in advance to ensure there is no feces in the colon and rectum that could affect subsequent experiments. If there is feces, it must be completely removed before proceeding. Euthanize the mice by cervical dislocation and remove the entire colon and rectum. Photograph the colon tumors to obtain images, and collect and analyze the colon tumors. Perform HE staining on the colon tumors and observe the tumor sections.

[0099] See results Figure 9 The results showed that, under the same tumor induction conditions, compared with wild-type USP33... + / + Mice, USP33 - / - Myeloid-specific knockout mice have smaller tumors and fewer tumor-infiltrating cells.

[0100] Example 11

[0101] Single-cell transcriptome analysis of tumor macrophages in USP33 knockout colon tumors

[0102] To further clarify the role of USP33 deficiency in M1 macrophage polarization and tumor growth in the immune microenvironment, USP33 + / + and USP33 - / - In mice, colon tumors were induced using AOM / DSS, and then the intact colon tumors were sequenced using 10×scRNA-seq single-cell sequencing. After thawing, the samples were digested with a mixture of collagenase IV / DNase I at 37°C with shaking for 30 minutes, and then filtered through a 40μm filter to obtain a single-cell suspension with a viable cell rate >90%. Through quantitative and quality control, we obtained high-quality single-cell sequencing data of wild-type and USP33-specific knockout colon tumors. Analysis was performed in RStudio (v4.3.2) using the Seurat (v5.0.1) workflow: After importing the data using the Read10X function, samples from the same group were merged; cells with <200 or >8500 genes and >20% mitochondrial gene content were strictly quality controlled and removed; after LogNormalization standardization, 2000 hypervariable genes were selected using FindVariableFeatures for PCA dimensionality reduction (npcs=50), and the top 15 principal components were selected using the Jack Straw test. Unsupervised clustering was performed using FindNeighbors (dims=1:15, k.param=20) and FindClusters (resolution=1.2) based on the PCA results. After visualization with UMAP / t-SNE, 10 major cell populations were manually annotated based on mouse colon cancer feature markers.

[0103] like Figure 10 As shown, the cells were divided into 10 major groups, including B cells, monocytes, endothelial cells, epithelial cells, fibroblasts, macrophages, giant cells, neutrophils, NKT cells, and ILC / T cells. Differential analysis was performed using FindAllMarkers (min.pct=0.25, logfc.threshold=0.25) to screen for differentially expressed genes between groups, and GO / KEGG enrichment analysis was conducted using ClusterProfiler (v4.10.0), focusing on genes related to M1 macrophage pathways (such as IFN-γ response and antigen presentation) and immune microenvironment regulation. For ease of subsequent data analysis, the wild-type was named USP33. + / + The group named the single-core, macrophage system-specific knockout USP33 as USP33. - / - The study comprised a total of 36,168 cells.

[0104] Mouse macrophages were further extracted from single-cell sequencing results, and then the AddModuleScore function was used to analyze wild-type USP33 cells using M1 and M2 macrophage markers. + / + USP33, specifically knocked out by the mononuclear macrophage system - / - Enrichment scoring was performed on mouse macrophages.

[0105] like Figure 11 As shown, compared to the wild type, USP33 - / - The overall percentage of M1 macrophages in mice remained unchanged, but the M1 trait scores were higher. We then further subdivided the macrophages into six cell clusters: Mac0, Mac1, Mac2, Mac3, Mac4, and Mac5. The results showed that the proportion of Mac4 in Usp33- / - mice increased from 2.58% to 35.5%, while Mac0 and Mac1 significantly decreased. To further explore the phenotypes of these clusters, we then compared the M1 and M2 traits of these six cell clusters. The results showed that, except for Mac3, which exhibited a significant M2 trait, Mac1 and Mac4 showed a significant M1 trait.

[0106] Example 12

[0107] Single-cell transcriptome analysis of T cells in USP33 knockout colon tumors

[0108] In the tumor immune microenvironment, various T cells play distinct and indispensable roles. CD4 + After recognizing tumor antigens presented by antigen-presenting cells, T cells can differentiate into different effector subsets. For example, Th1 cells can secrete cytokines such as interferon-γ, activate macrophages, and enhance CD8+. + The killing activity of T cells effectively inhibits tumor growth and metastasis. CD8 +T cells are the main force in directly killing tumor cells. They specifically recognize antigenic peptide-MHC class I molecule complexes on the surface of tumor cells through their surface T cell receptors, releasing perforin, granzymes, and other substances to induce tumor cell apoptosis. Treg cells play an immunosuppressive role in tumor immunity. They can inhibit the activation and proliferation of effector T cells and maintain immune tolerance by secreting inhibitory cytokines such as IL-10 and TGF-β, and through direct cell-cell contact. However, this may also allow tumor cells to evade immune surveillance. Th2 cells mainly participate in humoral immune responses, and their role in tumor immunity is relatively complex. On the one hand, they can assist B cells in producing antibodies; on the other hand, they may promote tumor growth and metastasis in certain situations. Th17 cells play a role in regulating the inflammatory response in the tumor microenvironment and also have a certain influence on tumor development and progression. The results showed that compared with the control group, the USP33 knockout group exhibited significant changes in the tumor immune microenvironment, with CD4... + T cells and CD8 + The number of infiltrating T cells increased significantly, while the number of Treg cells decreased significantly.

[0109] See results Figure 11 Knockout of myeloid USP33 can reshape the tumor immune microenvironment, disrupt the original immune balance, and make the immune system more inclined to activate CD4, which has anti-tumor effects. + T cells and CD8 + T cells, while weakening the immunosuppressive effect of Treg cells.

[0110] Example 13

[0111] Myeloid USP33 knockout reduces the subcutaneous tumorigenicity of mouse colon cancer MC38 cells.

[0112] A subcutaneous tumorigenesis model of MC38 colon cancer cells was selected. USP33 cells from the same litter, aged 6-8 weeks, were chosen. + / + and USP33 - / - In mice, the hair at the tumor inoculation site was shaved, and after disinfection with 75% alcohol, 5 × 10⁵ mice were subcutaneously inoculated. 5 MC38 cells were used. Tumor size was measured using digital calipers after tumor cell injection. Mice were sacrificed 14 days after tumor cell injection, and tumors were excised and processed for other experiments. Mouse tumor volume = (length × width) 2 ) / 2.

[0113] like Figure 12 As shown, compared to wild-type mice, the tumor size in Lyz2-USP33-specific knockout mice was significantly reduced. These results indicate that myeloid macrophages can significantly inhibit tumor growth in vivo after USP33 knockout.

[0114] Example 14

[0115] Myeloid USP33 deficiency increases innate defenses following LM infection.

[0116] Wild-type and specifically knockout USP33 mice were intraperitoneally injected with 1 × 10 5 Sublethal dose of CFU of Listeria monocytogenes (LM) is an infectious dose that falls between clearing the bacteria and causing severe sepsis.

[0117] like Figure 13 As shown, the survival rate of mice was measured within 10 days after LM infection. Compared with wild-type mice, Lyz2-USP33 knockout mice showed a higher survival rate.

[0118] Example 15

[0119] Myeloid USP33 deletion increases post-infection LM clearance

[0120] Intraperitoneal injection of 1×10 5 Three days after inoculation with CFU of Listeria monocytogenes (LM), mice were euthanized by cervical dislocation, and their spleens were collected. After grinding, the spleens were added to PBS containing 0.05% Triton and allowed to stand for 5 minutes. The lysate was then aspirated and evenly spread onto PYG solid culture dishes.

[0121] See results Figure 14 The results showed that, compared with wild-type mice, LM bacteria in the spleen of Lyz2-USP33-specific knockout mice were cleared significantly faster.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of ubiquitin-specific protease 33 in the preparation of products that regulate macrophage polarization.

2. The use of a reagent that inhibits the expression level of ubiquitin-specific protease 33 and / or a reagent that inhibits the expression level of the gene encoding ubiquitin-specific protease 33 in the preparation of a product that promotes macrophage polarization into M1 macrophages.

3. The application according to claim 2, characterized in that, The reagents used to inhibit the expression level of the gene encoding ubiquitin-specific protease 33 include at least one of the following: siRNA, shRNA, and sgRNA.

4. A method for promoting macrophage polarization towards M1 macrophages, characterized in that, By inhibiting or interfering with the expression of the gene encoding ubiquitin-specific protease 33 or ubiquitin-specific protease 33 in macrophages, M1 macrophages induced by specific knockout of USP33 were obtained.

5. The method according to claim 4, characterized in that, The method for inhibiting or interfering with the expression of ubiquitin-specific protease 33 in macrophages is to target and bind to ubiquitin-specific protease 33 in macrophages using small interfering RNA fragments siUSP33-1, siUSP33-2, siUSP33-3, and siUSP33-4.

6. The use of reagents that inhibit USP33 expression or monocytes that interfere with USP33 expression in the preparation of at least one of the following drugs: antitumor, antibacterial infection, and antiviral infection.

7. The application according to claim 6, characterized in that, The tumors mentioned in the anti-tumor treatment include at least one of the following: colon cancer, breast cancer, pancreatic duct adenocarcinoma, gastric adenocarcinoma, liver cancer, lung adenocarcinoma, and neuroblastoma cells; The anti-tumor treatment includes inhibiting tumor growth and / or inhibiting tumor metastasis.

8. The application according to claim 7, characterized in that, The cell types for the colon cancer include HCT116 cells or MC38 cells; the cell types for the breast cancer include MDA-MB-231 cells or MCF-7 cells; the cell types for the pancreatic duct adenocarcinoma include CFPAC-1; the cell types for the gastric adenocarcinoma include MKN45; the cell types for the liver cancer include HEPG2; the cell types for the lung adenocarcinoma include NCI-H1975; and the cell types for the neuroblastoma include SH-SY5Y.

9. The application according to claim 6, characterized in that, The bacteria mentioned in the antibacterial infection treatment include Listeria monocytogenes; The antiviral agent includes at least one of the following viruses: human cytomegalovirus, measles virus, and influenza A virus.

10. The application according to claim 6, characterized in that, The anti-infective treatment includes at least one of the following diseases: sepsis, meningitis, and mononucleosis.

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