Application of substances that inhibit CLEC5A in the preparation of drugs for the treatment and / or prevention of hepatitis B.

Abstract

This invention relates to the field of biomedicine, and particularly to the application of substances that inhibit CLEC5A in the preparation of drugs for the treatment and / or prevention of hepatitis B. Experimental results show that knocking down CLEC5A in THP-1 and PBMC-induced macrophages significantly reduced the levels of HBV DNA, HBV RNA, HBsAg, and HBeAg in HepG2.2.15 cells, while significantly increasing the levels of inflammatory cytokines such as IL-1β. Simultaneously, HBV clearance efficiency was higher in Clec5a gene knockout mice, and HBV replication levels were significantly reduced in HepG2.2.15 cells after Clec5a knockout in selected mouse macrophages.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of substances that inhibit CLEC5A in the preparation of medicaments for the treatment and / or prevention of hepatitis B. Background Technology

[0002] HBV infection and persistent HBV: HBV is a heterologous virus that can cause liver diseases, including acute hepatitis, cirrhosis, liver cancer, and hepatocellular carcinoma (HCC). Globally, approximately 250 million people suffer from chronic HBV infection, resulting in nearly one million deaths annually. In my country, the HBV infection rate is 60%. Therefore, HBV infection remains a long-standing public health problem in my country that threatens the health and lives of its people.

[0003] Most newly infected healthy adults can effectively clear HBV, but about 5% of adults and most newborns cannot effectively clear the invading HBV, resulting in persistent HBV infection. Approximately 25% of those with persistent HBV infection eventually develop severe liver disease, eventually leading to liver cancer. Persistent HBV infection or clearance is influenced by viral, environmental, and genetic factors. Differences in the HBV genome, host age and sex, and co-infection with hepatitis C virus (HCV), hepatitis D virus (HDV), and human immunodeficiency virus (HIV) are important factors contributing to persistent HBV infection. However, twin studies and segregation analyses strongly support the important role of host genetic factors in the development of persistent HBV infection. Multiple genome-wide association studies (GWAS) based on case-control populations have indicated that several genes, including HLA-DP, HLA-DQ, HLA-C, EHMT2, TCF19, and UBE2L3, are considered susceptibility genes influencing persistent HBV infection. However, these genes may only represent a small subset of genes associated with persistent HBV infection and are insufficient to fully explain the molecular mechanisms of persistent HBV infection. Therefore, identifying and characterizing genes related to persistent HBV infection and studying their functional mechanisms are essential and of great significance for elucidating the molecular mechanisms of persistent HBV infection.

[0004] It is well known that both host and viral factors determine the outcome of HBV infection in the host, and impaired immune response is mainly the cause of chronic, latent, reactive, and exacerbating HBV infection. The innate immune system recognizes viral components through pattern recognition receptors (PRRs) and acts as the first line of defense to limit viral replication in host cells. Pattern recognition receptors (PRRs) are mainly expressed on the surface, cytoplasm, and serum of innate immune cells such as monocytes, macrophages, and dendritic cells. They can directly recognize pathogen components and their products such as teichoic acid, peptidoglycan, endotoxins, mannose, DNA, or RNA, as well as certain common specific molecular structures on the surface of apoptotic and senescent host cells. Common PRRs include the Toll-like receptor family (TLRs), the nucleotide-binding oligomerization domain-like receptor family (NLRs), the retinoic-acid-inducible gene I-like receptor family (RLRs), C-type lectin receptors (CLRs), DNA-dependent activation of IRF3 (DAI), and Absent in melanoma 2 (AIM2).

[0005] Myeloid C-type lectin receptors (CLRs) in innate immunity are glycan-binding receptors that specifically recognize glycolipids and glycoproteins in pathogens and host cells. Since many pathogens, including bacteria, parasites, fungi, and viruses, are encapsulated in glycans, host CLR recognition of glycans is crucial for inducing an immune response. CLRs may influence immunity at multiple levels, from phagocytosis to the production of effector cytokines and chemokines. Furthermore, various CLRs act as endocytic receptors on antigen-presenting cells (APCs), thus participating in the uptake of antigens by pathogens and subsequent T cell activation. The role of CLRs in viral recognition is complex. CLRs expressed by APCs, such as dendritic cells (DCs) and macrophages, can recognize a variety of viruses and rapidly respond to them, thereby forming an antiviral immune response. However, some viruses have evolved mechanisms that utilize CLRs to trigger cell entry, disrupt intracellular signaling pathways, or inhibit APC effector function.

[0006] Several macrophage receptors (CLRs) have been reported to participate in HBV virus recognition and play important roles in antiviral immune responses. Both DC-SIGN and L-SIGN can recognize HBV, but HBV's α-mannosidase I, which modifies the N-linked oligosaccharide structure, prevents both from recognizing HBV, allowing HBV to evade DC signal recognition and thus disrupting potential immune activation responses. Liu et al. used mice with LSectin deficiency during adenovirus infection and HBV replication as a mouse model of viral hepatitis, demonstrating that LSectin promotes the reduction of liver inflammation by delaying HBV clearance, and that this process may be hijacked by HBV as an immune evasion mechanism. Macrophage mannose receptors (MMRs) are involved in the recognition of HBV surface antigen (HBsAg) and the uptake of DCs. Since HBsAg-positive DCs are frequently found in the livers of HBV patients, MMR-mediated HBsAg-DC interactions may exist within the liver, making it a major site of HBV infection.

[0007] Myeloid C-type lectin 5A (CLEC5A), also known as myeloid DAP12-associated lectin 1 (MDL-1), is a type II transmembrane receptor primarily expressed in monocytes, macrophages, and neutrophils. The short cytoplasmic domain of CLEC5A interacts with the ITAM-containing transmembrane linker DAP-12, leading to DAP-12 phosphorylation, which subsequently transmits signals via Syk kinase. CLEC5A lacks complete Ca2+. 2+The CLEC5A complex, with coordination and carbohydrate-binding sites, exhibits a homodimeric structure on the cell surface. To date, CLEC5A has been shown to recognize a variety of viral pathogens, including dengue virus (DV), Japanese encephalitis virus (JEV), and influenza virus. CLEC5A binds to DV and activates a signaling cascade, simultaneously releasing pro-inflammatory cytokines that contribute to the pathophysiological changes in DV-infected patients. Upon DV recognition, DAP-12 is phosphorylated, leading to Syk activation; subsequently, pro-inflammatory cytokines IL-1β and IL-18 are induced, activating the NLRP3 inflammasome and caspase-1, ultimately triggering pyroptosis. Furthermore, DV can upregulate CLEC5A expression, thereby activating nuclear factor Nrf2 in mouse mononuclear phagocytes, resulting in enhanced tumor necrosis factor (TNF)-α production. Antibody-mediated CLEC5A blockade attenuates pro-inflammatory cytokine production by DV-infected macrophages, suggesting that CLEC5A targeting can improve tissue damage. CLEC5A, after binding to DAP-12 and DAP-10 in bone marrow-derived osteoclasts, is also essential for osteoclastogenesis and bone remodeling. Osteoclasts are multinucleated giant cells, unlike macrophages, involved in bone remodeling. DV infection of osteoclasts has recently been shown to upregulate osteolytic activity. In CLEC5A... - / - A decrease in DV-induced osteolytic activity was observed in mice, and sustained administration of a Clec5a antagonist to wild-type mice also inhibited DV-activated osteolytic activity. JEV binds to CLEC5A and induces DAP-12 phosphorylation in macrophages. Antibody-mediated CLEC5A blockade inhibited JEV-induced release of pro-inflammatory cytokines from microglia and prevented peripheral tissue damage to neurons. Furthermore, CLEC5A antibody treatment reduced the infiltration of leukocyte-containing JEVs into the central nervous system, alleviated neuroinflammation, and decreased JEV-induced lethality in mice. In a recent study, CLEC5A was shown to interact with the hemagglutinin (HA) protein of influenza virus. Antibody-mediated CLEC5A blockade or silencing led to reduced levels of pro-inflammatory cytokines produced by human macrophages. Compared to wild-type mice, Clec5a... - / - Decreased levels of pro-inflammatory cytokines, reduced lung immune cell infiltration, and improved survival rates in mice suggest that CLEC5A plays a crucial role in the inflammatory response, thereby contributing to influenza pathogenicity. However, the association between CLEC5A and HBV infection remains unclear. Summary of the Invention

[0008] In view of this, the present invention provides the use of substances that inhibit CLEC5A in the preparation of medicaments for the treatment and / or prevention of hepatitis B.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] The experimental results of this invention show that:

[0011] In a first aspect, the experimental results of the present invention demonstrate the application of knocking out or knocking down CLEC5A in the preparation of formulations or drugs that reduce the levels of one or more of HBV DNA, HBV RNA, HBsAg, or HBeAg in tumor cells.

[0012] Secondly, the experimental results of this invention demonstrate the application of knocking out or knocking down CLEC5A in the preparation of formulations or drugs that increase the level of inflammatory cytokines in tumor cells.

[0013] In some specific embodiments of the present invention, the inflammatory cytokines include one or more of IL6, IL1β, or TNFα.

[0014] Thirdly, the experimental results of this invention demonstrate the application of knocking out or knocking down CLEC5A in the preparation of formulations or drugs that improve HBV clearance efficiency.

[0015] Fourthly, the experimental results of this invention demonstrate the application of knocking out or knocking down CLEC5A in the preparation of formulations or drugs that reduce HBV replication levels.

[0016] Fifthly, the experimental results of this invention demonstrate the application of substances that inhibit CLEC5A in the preparation of medicaments for the treatment and / or prevention of hepatitis B.

[0017] Sixthly, experimental results of the present invention demonstrate the application of the substance for inhibiting CLEC5A in combination with any other active ingredient in the preparation of pharmaceutical compositions for treating and / or preventing hepatitis B.

[0018] In some specific embodiments of the present invention, the tumor cells include HepG2.2.15 cells.

[0019] In summary, the experimental results of this invention show that: knocking down CLEC5A in THP-1 and PBMC-induced macrophages significantly reduced the levels of HBV DNA, HBV RNA, HBsAg, and HBeAg in HepG2.2.15 cells, while significantly increasing the levels of inflammatory cytokines such as IL-1β; conversely, overexpression of CLEC5A in THP-1 and PBMC-induced macrophages significantly increased the levels of HBV DNA, HBV RNA, HBsAg, and HBeAg in HepG2.2.15 cells, while significantly decreasing the levels of inflammatory cytokines such as IL-1β. Furthermore, HBV clearance efficiency was higher in Clec5a knockout mice, and HBV replication levels were significantly reduced in HepG2.2.15 cells after Clec5a knockout in selected mouse macrophages. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0021] Figure 1 This demonstrates a co-culture system of THP1-induced macrophages and HepG2.2.15 cells.

[0022] Figure 2 Knockdown of CLEC5A in THP-1 cells significantly reduced HBV replication levels in HepG2.2.15 cells; among which, Figure 2 a shows that after stable knockdown of CLEC5A in THP1 cells, the mRNA level of CLEC5A was significantly reduced; Figure 2 b shows that after stable knockdown of CLEC5A in THP1 cells, the protein level of CLEC5A was significantly reduced; Figure 2 c shows that knocking down CLEC5A in THP-1 cells significantly reduced the level of HBV DNA in HepG2.2.15 cells co-cultured with it; Figure 2 d shows that knocking down CLEC5A in THP-1 cells significantly reduced the level of HBV pgRNA in HepG2.2.15 cells co-cultured with it; Figure 2 The study showed that knocking down CLEC5A in THP-1 cells significantly reduced the level of HBV preS / S RNA in HepG2.2.15 cells co-cultured with it. Figure 2 f shows that knocking down CLEC5A in THP-1 cells significantly reduced the total level of HBV RNA in HepG2.2.15 cells co-cultured with it; Figure 2 g showed that knocking down CLEC5A in THP-1 cells significantly reduced the level of HBsAg in HepG2.2.15 cells co-cultured with it; Figure 2h showed that knocking down CLEC5A in THP-1 cells significantly reduced the level of HBeAg in HepG2.2.15 cells co-cultured with it.

[0023] Figure 3 Overexpression of CLEC5A in THP-1 cells significantly increased HBV replication levels in HepG2.2.15 cells; among which, Figure 3 a shows that after stable overexpression of CLEC5A in THP1 cells, the mRNA level of CLEC5A was significantly increased; Figure 3 b shows that after stable overexpression of CLEC5A in THP1 cells, the protein level of CLEC5A increased significantly. Figure 3 c shows that after overexpression of CLEC5A in THP-1 cells, the level of HBV DNA in HepG2.2.15 cells co-cultured with it was significantly increased; Figure 3 d shows that after overexpression of CLEC5A in THP-1 cells, the level of HBV pgRNA in HepG2.2.15 cells co-cultured with CLEC5A was significantly increased; Figure 3 The study showed that after overexpression of CLEC5A in THP-1 cells, the level of HBV preS / S RNA in HepG2.2.15 cells co-cultured with CLEC5A was significantly increased. Figure 3 f shows that after overexpression of CLEC5A in THP-1 cells, the total level of HBV RNA in HepG2.2.15 cells co-cultured with it was significantly increased; Figure 3 g showed that after overexpression of CLEC5A in THP-1 cells, the level of HBsAg in HepG2.2.15 cells co-cultured with CLEC5A was significantly increased; Figure 3 h showed that after overexpression of CLEC5A in THP-1 cells, the level of HBeAg in HepG2.2.15 cells co-cultured with it was significantly increased.

[0024] Figure 4 This demonstrates a co-culture system of PBMC-induced macrophages and HepG2.2.15 cells.

[0025] Figure 5 Knockdown of CLEC5A in PBMC-induced macrophages significantly reduced HBV replication levels in HepG2.2.15 cells; among which, Figure 5 a shows that knocking down CLEC5A in PBMC cells significantly reduced the mRNA level of CLEC5A. Figure 5 b shows that knocking down CLEC5A in PBMC cells significantly reduced the level of HBV DNA in HepG2.2.15 cells co-cultured with it. Figure 5 c shows that after knocking down CLEC5A in PBMC cells, the level of HBV pgRNA in HepG2.2.15 cells co-cultured with it was significantly reduced; Figure 5 d shows that knocking down CLEC5A in PBMC cells significantly reduced the level of HBV preS / S RNA in HepG2.2.15 cells co-cultured with it; Figure 5 The study showed that knocking down CLEC5A in PBMC cells significantly reduced the level of HBsAg in HepG2.2.15 cells co-cultured with CLEC5A. Figure 5 f shows that knocking down CLEC5A in PBMC cells significantly reduced the level of HBeAg in HepG2.2.15 cells co-cultured with it.

[0026] Figure 6 Overexpression of CLEC5A in PBMC-induced macrophages significantly increased HBV replication levels in HepG2.2.15 cells; among which, Figure 6 a shows that overexpression of CLEC5A in PBMC cells significantly increased the mRNA level of CLEC5A. Figure 6 b shows that after overexpression of CLEC5A in PBMC cells, the level of HBV DNA in HepG2.2.15 cells co-cultured with CLEC5A was significantly increased; Figure 6 c shows that after overexpression of CLEC5A in PBMC cells, the level of HBV pgRNA in HepG2.2.15 cells co-cultured with CLEC5A was significantly increased; Figure 6 d shows that after overexpression of CLEC5A in PBMC cells, the level of HBV preS / SRNA in HepG2.2.15 cells co-cultured with CLEC5A was significantly increased; Figure 6 e shows that after overexpression of CLEC5A in PBMC cells, the level of HBsAg in HepG2.2.15 cells co-cultured with CLEC5A was significantly increased; Figure 6 f shows that after overexpression of CLEC5A in PBMC cells, the level of HBeAg in HepG2.2.15 cells co-cultured with it was significantly increased.

[0027] Figure 7 The study showed that CLEC5A in THP-1-induced macrophages inhibited the secretion of inflammatory cytokines such as IL-1β during HBV infection; among them, Figure 7 a shows that after knocking down CLEC5A in THP-1 cells, the level of IL6 in the system co-cultured with HepG2.2.15 cells was significantly increased; Figure 7 b shows that after knocking down CLEC5A in THP-1 cells, the level of IL-1β in the system co-cultured with HepG2.2.15 cells was significantly increased; Figure 7 c shows that after knocking down CLEC5A in THP-1 cells, the level of TNF-α in the system co-cultured with HepG2.2.15 cells was significantly increased; Figure 7d shows that after overexpression of CLEC5A in THP-1 cells, the level of IL6 in the system co-cultured with HepG2.2.15 cells was significantly reduced; Figure 7 The results showed that after overexpression of CLEC5A in THP-1 cells, the level of IL-1β in the system co-cultured with HepG2.2.15 cells was significantly reduced. Figure 7 f shows that after overexpression of CLEC5A in THP-1 cells, the level of TNF-α in the system co-cultured with HepG2.2.15 cells was significantly reduced.

[0028] Figure 8 The study showed that CLEC5A in PBMC-induced macrophages inhibited the secretion of inflammatory cytokines such as IL-1β during HBV infection; among them, Figure 8 a shows that after knocking down CLEC5A in PBMC cells, the level of IL6 in the system co-cultured with HepG2.2.15 cells was significantly increased; Figure 8 b shows that after knocking down CLEC5A in PBMC cells, the level of IL-1β in the system co-cultured with HepG2.2.15 cells was significantly increased; Figure 8 c shows that after knocking down CLEC5A in PBMC cells, the level of TNF-α in the system co-cultured with HepG2.2.15 cells was significantly increased; Figure 8 d shows that after overexpression of CLEC5A in PBMC cells, the level of IL6 in the system co-cultured with HepG2.2.15 cells was significantly reduced; Figure 8 e shows that after overexpression of CLEC5A in PBMC cells, the level of IL-1β in the system co-cultured with HepG2.2.15 cells was significantly reduced; Figure 8 f shows that after overexpression of CLEC5A in PBMC cells, the level of TNF-α in the system co-cultured with HepG2.2.15 cells was significantly reduced.

[0029] Figure 9 This demonstrates the construction of C57BL / 6J mice with Clec5a gene knockout; among them, Figure 9 a shows the construction of Clec5a gene knockout C57BL / 6J mice using CRISPR / Cas9 technology; Figure 9 Western blotting analysis showed that Clec5a protein was indeed not expressed in the liver tissue of C57BL / 6J mice with Clec5a gene knockout.

[0030] Figure 10 The Clec5a gene knockout showed a higher HBV clearance efficiency; among them... Figure 10 The positive rate of HBsAg in C57BL / 6J mice was significantly reduced after Clec5a gene knockout. Figure 10b shows that the serum HBsAg level in C57BL / 6J mice was significantly reduced after Clec5a gene knockout; Figure 10 c shows that the copy number of HBV DNA in the serum of C57BL / 6J mice was significantly reduced after Clec5a gene knockout.

[0031] Figure 11 Clec5a is mainly expressed in macrophages in mouse liver tissue;

[0032] Figure 12 The knockout of Clec5a in mouse macrophages significantly reduced the HBV replication level in HepG2.2.15 cells; Figure 12 The results showed that after co-culturing Clec5a gene knockout macrophages with HepG2.2.15 cells, the level of HBV DNA in HepG2.2.15 cells was significantly reduced, while after co-culturing Clec5a gene knockout non-macrophages with HepG2.2.15 cells, the level of HBV DNA in HepG2.2.15 cells did not change significantly. Figure 12 b shows that after co-culturing Clec5a gene knockout macrophages with HepG2.2.15 cells, the level of HBV pgRNA in HepG2.2.15 cells was significantly reduced, while after co-culturing Clec5a gene knockout non-macrophages with HepG2.2.15 cells, the level of HBV pgRNA in HepG2.2.15 cells did not change significantly. Figure 12 c shows that after co-culturing Clec5a gene knockout macrophages with HepG2.2.15 cells, the level of HBV preS / S RNA in HepG2.2.15 cells was significantly reduced, while after co-culturing Clec5a gene knockout non-macrophages with HepG2.2.15 cells, the level of HBV preS / S RNA in HepG2.2.15 cells did not change significantly. Figure 12 The results showed that after co-culturing Clec5a gene knockout macrophages with HepG2.2.15 cells, the level of HBsAg in HepG2.2.15 cells was significantly reduced, while after co-culturing Clec5a gene knockout non-macrophages with HepG2.2.15 cells, the level of HBsAg in HepG2.2.15 cells did not change significantly. Figure 12 The study showed that co-culturing Clec5a gene knockout macrophages with HepG2.2.15 cells significantly reduced HBeAg levels in HepG2.2.15 cells, while co-culturing Clec5a gene knockout non-macrophages with HepG2.2.15 cells did not significantly change HBeAg levels. Detailed Implementation

[0033] This invention discloses the application of CLEC5A inhibitors in the preparation of drugs for the treatment and / or prevention of hepatitis B. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0034] To systematically discover new CNVs / genes / pathways associated with HBV chronicity, we previously conducted a copy number-based genome-wide association study on the genomes of 356 individuals with chronic HBV infection and 671 healthy individuals with chronic HBV infection. We found that the deletion domain of the human chromosome 7q34 CNV region was significantly associated with HBV chronicity, and the C-type lectin receptor CLEC5A gene is an effector gene of this CNV region, suggesting that the CLEC5A gene may be a susceptibility gene for HBV chronicity.

[0035] Our study found that knocking down CLEC5A in THP-1 and PBMC-induced macrophages significantly reduced the levels of HBV DNA, HBV RNA, HBsAg, and HBeAg in HepG2.2.15 cells, while significantly increasing the levels of inflammatory cytokines such as IL-1β. Conversely, overexpression of CLEC5A in THP-1 and PBMC-induced macrophages significantly increased the levels of HBV DNA, HBV RNA, HBsAg, and HBeAg in HepG2.2.15 cells, while significantly decreasing the levels of inflammatory cytokines such as IL-1β. Furthermore, HBV clearance efficiency was higher in Clec5a knockout mice, and HBV replication levels were significantly reduced in HepG2.2.15 cells after Clec5a knockout in selected mouse macrophages.

[0036] The raw materials and reagents used in the experiments of this invention can all be purchased from the market.

[0037] The present invention will be further illustrated below with reference to the embodiments:

[0038] Example 1: CLEC5A in THP1 cells promotes HBV replication in HepG2.2.15 cells.

[0039] To investigate the function of CLEC5A in macrophages during chronic HBV infection, such as... Figure 1As shown, we constructed a non-contact co-culture system of THP-1-induced macrophages and HepG2.2.15 cells. First, THP-1 cells with CLEC5A interference or control THP-1 cells were induced into M2-type macrophages: THP-1 cells were collected and counted, and 2 × 10⁶ cells were cultured... 5 One THP-1 cell was seeded in a 12-well plate and induced for 24 h with PMA (final concentration 100 ng / mL). Then, IL-4 (final concentration 20 ng / mL) and IL-13 (final concentration 20 ng / mL) were added for further induction for 48 h. Next, 1×10⁶ cells were seeded in the wells. 5 HepG2.2.15 cells were seeded into the upper chamber of a co-culture system and co-cultured non-contactly for 48-72 cells. Finally, the cell culture supernatant was collected from the co-culture system, centrifuged at 1000g, and the supernatant was used to quantitatively detect HBV DNA levels, and HBsAg and HBeAg levels were measured using ELISA. RNA was extracted from HepG2.2.15 cells in the upper chamber, and HBV RNA levels were detected using q-PCR and Northern blotting techniques. THP-1 macrophages in the lower chamber were collected and stored at -80℃ for later use.

[0040] To investigate the function of CLEC5A in HBV infection, the inventors first designed two pairs of shRNA sequences targeting CLEC5A (as shown in Table 1), constructed them into the lentiviral vector PLVshRNA-EGFP(2A)Puro, packaged the lentivirus, and then infected THP-1 cells. After selection using Puro, cell lines with stable CLEC5A knockdown were obtained. After obtaining stable cell lines, RNA was extracted for qRT-PCR identification, and protein was extracted and Western blotting was performed. It was found that both shRNA fragments could knock down the expression level of CLEC5A by more than 50% (e.g., ...). Figure 2 a, b) can be used for subsequent experiments.

[0041] Table 1. shRNA sequence information of CLEC5A

[0042] serial number Serial Number Sequence information (from 5' to 3') Knock down CLEC5A-#1 SEQ ID No.1 CTTATTGTGGTAGTGCTTA Knock down CLEC5A-#2 SEQ ID No.2 GTAGTGCTTAAAGTTGTTG

[0043] CLEC5A has been reported to play an important role in the infection of RNA viruses such as dengue virus (DV), Japanese encephalitis virus (JEV), and influenza virus. However, its function in the infection of the DNA virus HBV has not been reported. We induced macrophages from THP-1 cells with stable CLEC5A knockdown and control cells, and then co-cultured them with HepG2.2.15 cells for 72 hours. After collecting the HepG2.2.15 cells, quantitative analysis revealed that HBV DNA levels were significantly reduced after CLEC5A knockdown. Figure 2 c); After RNA extraction, quantitative PCR and Northern blotting techniques were used to detect that HBV RNA levels were significantly reduced after CLEC5A knockdown. Figure 2 df); Collect cell culture supernatant from the co-culture system, centrifuge at 1000g, and take the supernatant. ELISA detection showed that knocking down CLEC5A significantly reduced the levels of HBsAg and HBeAg. Figure 2 g, h).

[0044] Meanwhile, we constructed THP-1 cells stably overexpressing CLEC5A using lentiviral infection. After obtaining stable cell lines, RNA was extracted for qRT-PCR identification, and protein was extracted and analyzed by Western blotting. The results showed that the overexpression of CLEC5A was significantly enhanced (e.g., Figure 3 (a, b) can be used for subsequent experiments. After THP-1 cells stably overexpressing CLEC5A and control cells were induced into macrophages, they were co-cultured with HepG2.2.15 cells for 72 hours. HepG2.2.15 cells were collected, and quantitative analysis revealed a significant increase in HBV DNA levels after CLEC5A overexpression. Figure 3 c); After RNA extraction, quantitative PCR and Northern blotting techniques were used to detect that HBV RNA levels were significantly reduced after CLEC5A overexpression. Figure 3 df); Collect cell culture supernatant from the co-culture system, centrifuge at 1000g, and take the supernatant. ELISA detection showed that HBsAg and HBeAg levels significantly increased after CLEC5A overexpression. Figure 3 g, h).

[0045] Example 2: CLEC5A in PBMC cells promotes HBV replication in HepG2.2.15 cells.

[0046] To further confirm the function of CLEC5A in promoting HBV replication, we repeated the experiment in human peripheral blood mononuclear cells (PBMCs). Figure 4As shown, we constructed a non-contact co-culture system of PBMC-induced macrophages and HepG2.2.15 cells. First, fresh human peripheral blood was collected and PBMCs were isolated. 2 × 10⁶ cells were then cultured with PBMCs. 5 PBMC cells were seeded in 12-well plates and induced for 4 days with hrM-CSF (final concentration 20 ng / mL) to obtain macrophages. Macrophages induced from PBMCs were infected with lentiviruses that stably knocked down or overexpressed CLEC5A, or with control lentiviruses. After 48 hours, green fluorescence-positive cells were sorted by flow cytometry, and RNA and protein were extracted to detect the knockdown and overexpression effects of CLEC5A. The sorted cells were then co-cultured with HepG2.2.15 cells in a non-contact manner for 48-72 hours. Finally, the cell culture supernatant from the co-culture system was collected, centrifuged at 1000g, and the supernatant was used to quantitatively detect HBV DNA levels and HBsAg and HBeAg levels using ELISA. RNA was extracted from HepG2.2.15 cells in the upper chamber, and HBV RNA levels were detected using q-PCR and Northern blotting techniques. THP-1 macrophages in the lower chamber were collected and stored at -80℃ for later use.

[0047] First, we co-cultured PBMC-induced macrophages with CLEC5A knockdown and control cells with HepG2.2.15 cells for 72 hours. We then collected the HepG2.2.15 cells and quantitatively analyzed them to find that CLEC5A knockdown occurred in PBMC-induced macrophages. Figure 5 a) The level of HBV DNA in HepG2.2.15 cells was significantly reduced after this procedure. Figure 5 b); Quantitative PCR was used to detect RNA extraction and found that knocking down CLEC5A in PBMC-induced macrophages significantly reduced the level of HBV RNA in HepG2.2.15 cells. Figure 5 c, d); The cell culture supernatant from the co-culture system was collected, centrifuged at 1000g, and the supernatant was used for ELISA detection. It was found that the levels of HBsAg and HBeAg in HepG2.2.15 cells were significantly reduced after CLEC5A knockdown in PBMC-induced macrophages. Figure 5 e, f).

[0048] Subsequently, we co-cultured PBMC-induced macrophages overexpressing CLEC5A and control cells with HepG2.2.15 cells for 72 hours. We then collected the HepG2.2.15 cells and quantitatively detected that CLEC5A was overexpressed in the PBMC-induced macrophages. Figure 6 a) The level of HBV DNA in HepG2.2.15 cells was significantly increased after treatment. Figure 6b); After RNA extraction, quantitative PCR was used to detect that the level of HBV RNA in HepG2.2.15 cells was significantly increased after PBMC-induced macrophage overexpression of CLEC5A. Figure 6 c, d); The cell culture supernatant from the co-culture system was collected, centrifuged at 1000g, and the supernatant was used for ELISA detection. It was found that the levels of HBsAg and HBeAg in HepG2.2.15 cells were significantly increased after PBMC-induced macrophage overexpression of CLEC5A. Figure 6 e, f).

[0049] Example 3: CLEC5A in macrophages inhibits the secretion of inflammatory cytokines such as IL-1β during HBV infection.

[0050] Furthermore, we used ELISA to detect the levels of IL6, IL1β, and TNFα in the cell culture supernatant of the co-culture system. Firstly, after knocking down CLEC5A in THP-1-induced macrophages, the levels of IL6, IL1β, and TNFα in the co-culture supernatant significantly increased. Figure 7 (ac); After overexpression of CLEC5A in THP-1 induced macrophages, the levels of IL6, IL1β and TNFα in the supernatant of the co-culture system were significantly reduced. Figure 7 ef).

[0051] Secondly, after knocking down CLEC5A in PBMC-induced macrophages, the levels of IL6, IL1β, and TNFα in the supernatant of the co-culture system were significantly increased. Figure 8 (ac); After overexpression of CLEC5A in PBMC-induced macrophages, the levels of IL6, IL1β, and TNFα in the supernatant of the co-culture system were significantly reduced. Figure 8 ef).

[0052] Example 4: Increased HBV clearance efficiency in Clec5a gene knockout C57BL / 6J mice

[0053] To further verify the function of CLEC5A in HBV replication and infection, we used CRISPR / Cas9 technology to design two gRNAs to completely excise exons 2 to 7 of the Clec5a gene in C57BL / 6J mice, obtaining Clec5a gene knockout mice (the gene knockout mice were manufactured by Cyagen Biotech Ltd.). Figure 9 a). We randomly sacrificed 3 wild-type mice and 3 Clec5a gene knockout mice, removed liver tissue, ground it with liquid nitrogen to extract protein, and performed Western blotting to detect Clec5a protein levels. Figure 9 b).

[0054] We selected male mice weighing approximately 20g for subsequent experiments, including 25 wild-type mice and 25 Clec5a gene knockout mice. We established a chronic HBV infection model by high-pressure tail vein injection of pAAV-HBV1.2 plasmid (10ug / mouse). We collected approximately 300µL of blood weekly via orbital blood sampling, centrifuged at 4000rpm for 10 minutes after incubation at room temperature for 2 hours, and collected the supernatant as serum. We used quantitative methods to detect HBV DNA levels and immunoradioassay (IRMA) kits to detect HBsAg levels. We found that compared to the wild-type mouse group, the HBsAg positivity rate and HBsAg levels were significantly reduced after Clec5a gene knockout. Figure 10 a, b); at the same time, the level of HBV DNA also decreased significantly ( Figure 10 c).

[0055] It has been reported that CLEC5A is mainly expressed in monocytes, macrophages, and neutrophils. We also used flow cytometry to analyze whether CLEC5A is mainly expressed in macrophages in mouse liver tissue. We obtained fresh mouse liver tissue and used a mouse liver tissue dissociation kit and single-cell dissociation instrument from Medtronic to dissociate the liver tissue into single-cell suspensions. Mouse liver macrophages (Kupffer cells) were labeled with two antibodies: PE-labeled CD11b and APC-labeled F4 / 80. Clec5a protein was labeled with a FITC-labeled Clec5a antibody. We found that the positive rate of CLEC5A protein in macrophages was 90.1%, while the positive rate in non-macrophage cells was 1.57%, indicating that Clec5a is also mainly expressed in macrophages in mouse liver tissue.

[0056] We further separated CD11b and F4 / 80 double-positive macrophages and CD11b and F4 / 80 double-negative cells, and then co-cultured them with HepG2.2.15 cells in a non-contact manner for 72 hours. Afterwards, we collected the HepG2.2.15 cells, and quantitative analysis revealed that the level of HBV DNA in HepG2.2.15 cells was significantly reduced after Clec5a gene knockout in the macrophage-positive group. Figure 12 a) Quantitative PCR was used to detect RNA extraction and found that the level of HBV RNA in HepG2.2.15 cells was significantly reduced after Clec5a gene knockout in the macrophage-positive group. Figure 12 b, c); The cell culture supernatant from the co-culture system was collected, centrifuged at 1000g, and the supernatant was used for ELISA detection. It was found that the levels of HBsAg and HBeAg in HepG2.2.15 cells after Clec5a gene knockout in the macrophage positive group were significantly reduced. Figure 12d, e). However, in the non-macrophage group, there were no significant differences in the levels of HBV DNA, HBV RNA, HBsAg, and HBeAg in HepG2.2.15 cells after Clec5a gene knockout. Figure 12 (ae). The above results further demonstrate that CLEC5A plays a role in promoting HBV replication in macrophages.

[0057] 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. sequence list <110> Military Medical Research Institute of the Academy of Military Sciences of the Chinese People's Liberation Army <120> Application of CLEC5A inhibitors in the preparation of drugs for the treatment and / or prevention of hepatitis B. <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 cttattgtgg tagtgctta 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 gtagtgctta aagttgttg 19

Claims

1. The application of knocking out or reducing CLEC5A in macrophages in the preparation of drugs for treating hepatitis B, characterized in that, The CLEC5A in macrophages was knocked out or knocked down using shRNA, the nucleotide sequence of which is shown in SEQ ID No. 1 or SEQ ID No. 2.

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