KR102677330B1 - Composition for preventing and treating placental inflammation comprising TBK1 inhibitor - Google Patents

Abstract

The present invention relates to Amlexanox and a composition containing it for preventing or treating high-risk pregnancy and maternal infection, including placental dysfunction. In this study, we investigated for the first time the potential effect of TBK1 inhibition by Amlexanox, originally a treatment for allergic rhinitis, on NLRP3 inflammasome activation in LPS-treated human trophoblast cell lines and pregnant mice. In other words, Amlexanox has excellent effects in improving and treating placental dysfunction and maternal infection.

Description

Composition for preventing and treating placental inflammation comprising TBK1 inhibitor}

The present invention relates to a pharmaceutical composition for preventing or treating placental inflammation, comprising a TBK1 inhibitor and a pharmaceutically acceptable excipient, and a method for screening the TBK1 inhibitor.

During pregnancy, when there is resistance between mother and fetus, physiological inflammation is helpful during implantation and early placental stages. An appropriate response of the immune system in the placenta is essential to protect the fetus from various stimuli such as infections and pathogens. However, pathological inflammatory conditions can cause placental insufficiency, which can lead to a variety of pregnancy-related disorders, including preterm birth, intrauterine growth restriction, and preeclampsia. Placental vegetative cells identify and respond to pathogen-associated molecular types, such as bacterial cell wall components, by expression of toll-like receptors (TLRs). Activation of Toll-like receptors promotes intracellular signaling, leading to the activation of NF-κB (NF-γB/p65) and pro-cytokines, including interleukin-1, interleukin-6, and tumor necrosis factor-alpha, resulting in abnormal placental dysfunction. Affects the expression of inflammatory cytokines. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome is a large cytosolic multiprotein complex composed of the NLRP3 scaffold, the apoptosis-associated particle-like protein (ASC) adapter, and caspase-1 as interleukin-1 beta convertase. These are initiated by a wide range of pathogen-associated molecule types, such as lipopolysaccharide (LPS), a Toll-like receptor 4 ligand, or damage-associated molecule types, such as adenosine triphosphate, and act in inflammation. In response to stimulation, the NLRP3 inflammasome activates caspase-1 and cleaves the inactive form of pro-interleukin-1 beta into the active form. It is associated with pregnancy dysfunction, including preterm birth, fetal growth restriction, recurrent miscarriage and preeclampsia. Abnormal interleukin-1 beta production in the placenta and human vegetative cells is associated with NLRP3 inflammasome activation following placental inflammation. TANK binding kinase 1 (TBK1) is an innate immune kinase related to the non-canonical IκB kinase that plays an important role in the innate immune system. TBK1 can be stimulated by phosphorylation in response to stimulation of Toll-like receptor3, Toll-like receptor4, and pathogens. However, the molecular mechanisms involved in the phosphorylation of TBK1 have not been fully elucidated. Our previous studies showed that activated TBK1 causes obesity-induced ubiquitinated protein addition in liver tissue and that inhibiting the TBK1 pathway prevents liver fibrosis in a mouse model of obesity and non-alcoholic steatohepatitis. TBK1 is linked to stimulus-dependent NF-κB activation, affecting various diseases such as inflammatory diseases and cancer. However, the importance of TBK1 in LPS-induced NLRP3 inflammasome activation in trophoblasts and placenta has not yet been elucidated.

In this study, we investigated for the first time the potential effect of TBK1 inhibition on NLRP3 inflammasome activation in LPS-treated human trophoblast cell lines and pregnant mice. Additionally, the molecular mechanism by which TBK1 regulates LPS-induced NLRP3 inflammasome activation was also studied. The results suggest that TBK1 phosphorylation is stimulated in placental tissue and trophoblast cells of mice treated with LPS. Both genetic deletion and pharmacological inhibition of TBK1 improved LPS-induced placental inflammation and NLRP3 inflammasome activation. We also found that TBK1 deficiency inhibits LPS-induced NLRP3 inflammasome activation through an mTORC1-dependent mechanism. Therefore, TBK1 inhibition may serve as a potential therapeutic target for placental dysfunction associated with the NLRP3 inflammasome and inflammation.

Republic of Korea Patent No. 10-1660863 (registered on September 22, 2016)

(Research Paper 0001) Perry, A. K., E. K. Chow, J. B. Goodnough, W. C. Yeh and G. Cheng (2004). “Differential requirement for TANK-binding kinase-1 in type I interferon responses to toll-like receptor activation and viral infection.” J Exp Med 199(12): 1651-1658 (Research Paper 0002) Cho, C. S., H. W. Park, A. Ho, I. A. Semple, B. Kim, I. Jang, H. Park, S. Reilly, A. R. Saltiel and J. H. Lee (2018). “Lipotoxicity induces hepatic protein inclusions through TANK binding kinase 1-mediated p62/sequestosome 1 phosphorylation.” Hepatology 68(4): 1331-1346

As a result of studying the importance of TBK1 in NLRP3 inflammasome activation in trophoblast cells and the placenta, the present inventors confirmed that intrauterine placental inflammation can be prevented or treated using a TBK1 inhibitor, thereby completing the present invention.

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating intrauterine placental inflammation and a method for screening substances capable of inhibiting TBK1 activity to search for drugs in the pharmaceutical composition.

In order to achieve the above problems, the present invention provides a pharmaceutical composition for preventing or treating placental inflammation comprising Amlexanox, a TBK1 inhibitor, and a pharmaceutically acceptable additive. In the present invention, we evaluated the effect of TBK1 inhibition on lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation and the underlying mechanism in human trophoblast cell lines and mouse placenta. TBK1 phosphorylation was upregulated in response to LPS in trophoblasts and placenta. Pharmacological and genetic inhibition of TBK1 in trophoblasts improved LPS-induced NLRP3 inflammasome activation, placental inflammation, and subsequent interleukin 1 production, and maternal administration of the TBK1 inhibitor Amlexanox reduced LPS-induced pregnancy side effects. improved. In particular, TBK1 inhibitors can block LPS-induced NLRP3 inflammasome activation by targeting mTORC1 (mammalian target of rapamycin complex 1).

According to one embodiment of the present invention, the TBK1 inhibitor is Amlexanox, but is not limited to this, and is any substance that can bind to the TBK1 protein and inhibit its activation, such as a peptide, antibody, or compound that binds to the TBK1 protein. There is no limit to anything. Additionally, it may be selected from antisense oligonucleotides, peptide mimetics, polynucleotides, siRNA, shRNA, or compounds that can bind to TBK1 mRNA or inhibit gene expression. In particular, the compound that binds to the TBK1 protein and inhibits its activation may be selected from the known Amlexanox, BX795, MRT67307, AZ13102909, SR8185, and MPI-0485520 (Myrexis), but is not limited thereto.

According to the present invention, inflammation in the placenta during pregnancy is a response to stimulation, and the NLRP3 inflammasome activates caspase-1 and cleaves the inactive form of pre-interleukin-1β into the active form. This causes placental insufficiency in the uterus due to placental inflammation, leading to premature birth, fetal growth restriction, preeclampsia, pre-eclampsia, placental abruption, congenital disorders, preterm labor, repeated miscarriage, and intrauterine growth retardation (IUGR) due to placental insufficiency. It could be the cause. Therefore, the present invention can provide information on preventing or treating placental inflammation by administering a pharmaceutical composition containing a TBK1 inhibitor to a pregnant woman.

According to one embodiment of the present invention, culturing TBK1-expressing cells; Adding a candidate drug to the culture medium and culturing it; and analyzing the level of TBK1 activity in the cells to select a candidate substance that inhibits the activity of the TBK1 protein. In particular, the activity of the TBK1 protein can be confirmed by analyzing gene expression, protein synthesis, or protein phosphorylation.

The present invention relates to Amlexanox, a TBK1 inhibitor, and a composition containing the same for preventing or treating placental inflammation. In this study, we investigated for the first time the potential effect of TBK1 inhibition by Amlexanox, originally a treatment for allergic rhinitis, on NLRP3 inflammasome activation in LPS-treated human trophoblast cell lines and pregnant mice. As a result, Amlexanox was found to reduce placental inflammation. It was confirmed that it has an excellent effect in preventing, improving, or treating.

Figure 1 shows the results confirming that phosphorylation of TBK1 occurs in mouse placenta and human trophoblast cells exposed to LPS. (A, B) Pregnant mice were injected intraperitoneally with LPS (200 μg/kg) at 17.5 days after pregnancy. After the indicated time, placental tissues were collected from mice and analyzed by immunoblot using anti-phospho-TBK1 and TBK1 antibodies. (Number = 4 per group). (CF) HTR8/SVneo cells were treated with the indicated concentrations of LPS for 45 min (C, D) and for 24 h (E, F). Cell lysates were analyzed by immunoblot using anti-phospho-TBK1 and TBK1 antibodies. (GJ) HTR8/SVeo cells were treated with LPS (1 μg/ml) for the indicated periods. Cell lysates were analyzed by immunoblot using anti-phospho-TBK1 and TBK1 antibodies. Alpha-tubulin was used as a loading control. The intensity of the bands was quantified and normalized to total protein concentration. Data are expressed as mean ± SEM. *p <0.05; **p <0.01; ***p <0.001 (Student's t test).
Figure 2 relates to the fact that TBK1 deficiency alleviates NLRP3 inflammasome activation by LPS in trophoblast cells. (AC) HTR8/SVneo cells were treated with 1 μg/ml LPS or 20 μM Amlexanox for 24 hours (A, B) or 12 hours (C), and then treated with 5 mM ATP for 45 minutes. (A, B) Cell lysates were analyzed by immunoblot using anti-NLRP3 and anti-Caspace1 antibodies. (C) Relative mRNA expression for NLRP3 and interleukin-1beta was measured by qRT-PCR. (D) HTR8/SVneo cells collected in conditioned medium were treated with 1 μg/ml LPS or 10 μM Amlexanox for 36 hours and then with 5mM ATP for 45 minutes as shown in the figure. Interleukin-1 beta levels were quantified by enzyme-linked immunosorbent assay. (EG) As shown, HTR8/SVneo cells were infected with lentivirus expressing shRNA targeting luciferase (sh-LUC) or TBK1 (sh-TBK1) and incubated for 24 h (E, F) or 12 h ( G) were treated with 1 μg/ml LPS, followed by 5mM ATP for 45 min. (EG) Cell lysates were analyzed by immunoblot using anti-NLRP3, anti-Caspace1 antibodies, and anti-TBK1 antibodies. (G) Relative mRNA expression for NLRP3 and interleukin-1beta was measured by qRT-PCR. Alpha-tubulin was used as a loading control. The intensity of the bands was quantified and normalized to total protein concentration. (H) HTR8/SVneo cells collected in conditioned medium were infected with sh-LUC or sh-TBK1 lentivirus as shown in the figure, treated with 1 μg/ml LPS for 24 hours, and then treated with 5mM ATP for 45 minutes. . Interleukin-1 beta levels were quantified by enzyme-linked immunosorbent assay. Data are expressed as mean ± SEM. *p <0.05; **p <0.01; ***p <0.001 (one-way analysis of variance, Tukey test)
Figure 3 relates to TBK1 deficiency inhibiting NF-κB/p65 activation by LPS in trophoblast cells. (A, B) HTR8/SVneo cells were treated with 1 μg/ml LPS or 100 μM Amlexanox for 1 hour. Cell lysates were analyzed by immunoblot using anti-phospho-IκBalpha, anti-IκBalpha, anti-phosphorylation-P65, and anti-P65 antibodies. Alpha-tubulin was used as a loading control. (C) Immunofluorescence staining for P65 in HTR8/SVneo cells treated with 1 μg/ml LPS or vehicle for 45 minutes in the presence or absence of 100 μM amlexanox. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (D, F) HTR8/SVneo cells were infected with lentivirus expressing shRNA targeting luciferase (sh-LUC) or TBK1 (sh-TBK1) as shown in the figure and incubated with 1 μg/ml LPS for 1 hour. It has been processed. Cell lysates were analyzed by immunoblotting with anti-phospho-IκBalpha, anti-IκBalpha, anti-phosphorylated-p65, and anti-p65 antibodies. Alpha-tubulin was used as a loading control. (E) Immunofluorescence staining for p65 (green) in HTR-8/SVneo cells infected with sh-LUC or sh-TBK1 lentivirus and treated with 1 μg/ml LPS or vehicle for 45 min. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. Data are expressed as mean ± SEM. *p <0.05; **p <0.01; ***p <0.001 (one-way analysis of variance, Tukey test).
Figure 4 shows the results confirming the effect of Amlexanox on the placental morphology of pregnant mice treated with LPS. (AE) As shown, pregnant mice were intraperitoneally injected with saline, LPS (200 μg/kg), or Amlexanox (2.5 mg/kg) at 17.5 days after pregnancy. (A) Representative images of the fetus and placenta for each maternal group. Scale bar, 0.5 cm (B, C) Average fetal and placental weight and length in each group. (Number = 33-50 per group). (D, E) Periodic acid-Schiff (PAS) staining of placental tissue from pregnant mice treated with vehicle, LPS, and LPS+amlexanox at 17.5 days post gestation. The length of the decidua (De), labyrinth zone (LZ), and junctional zone (JZ) of the midsection was quantified (number = 11 animals per group). Scale bar, 1 mm. Data are expressed as mean ± SEM. *p <0.05; **p <0.01; ***p <0.001, ns, not significant. (One-way analysis of variance, Fisher's LSD test).
Figure 5 shows that treatment with Amlexanox alleviates placental NLRP3 inflammasome activation in LPS-treated pregnant mice. (AD) Pregnant mice were intraperitoneally injected with saline, LPS (200 μg/kg), or Amlexanox (2.5 mg/kg) at 17.5 days after pregnancy. (A, B) Placental tissue was collected from mice and analyzed by immunoblot using anti-NLRP3 and anti-Cespace-1 antibodies (number = 9 per group). Alpha-tubulin was used as a loading control. The intensity of the bands was quantified and normalized to total protein concentration. (C) Placental tissues were collected from mice and analyzed by qRT-PCR for NLRP3 expression. (Number = 11-12 per group). (D) Immunohistochemical staining analysis for NLRP3 and interleukin-1beta was performed on placental tissue from pregnant mice treated with vehicle, LPS, and LPS+amlexanox at 17.5 days post gestation. Nuclei were stained with hematoxylin. The boxed area is an enlargement of the adjacent panel. LZ and JZ represent the Ravidinus zone and junction zone, respectively. Scale bar, 200 μm; Inset, 100 μm. *p <0.05; **p <0.01; ***p <0.001 (one-way analysis of variance, Tukey test).
Figure 6 shows that treatment with Amlexanox improves the inflammatory response caused by LPS in the mouse placenta. (AC) Pregnant mice were intraperitoneally injected with LPS (200 μg/kg) or Amlexanox (2.5 mg/kg) at 17.5 days after pregnancy. (A, B) Placental tissue was collected from mice and analyzed by immunoblot using anti-phosphorylated-p65 and anti-p65 antibodies (number = 9 per group). Alpha-tubulin was used as a loading control. The intensity of the bands was quantified and normalized to total protein concentration. (C) Placental tissues were collected from mice and qRT for expression of interleukin-1beta, interleukin-6, interleukin-10, TNF-alpha, TGF-beta1, MCP-1, and interferon-beta (10 per group). -Analyzed by PCR. Data are expressed as mean ± SEM. *p <0.05; **p <0.01; ***p <0.001, ns, not significant. (One-way analysis of variance, Fisher's LSD test).
Figure 7 shows that TBK1 deficiency inhibits LPS-induced NLRP3 inflammatory activation through an mTORC1-dependent mechanism. (A, B) Pregnant mice were injected intraperitoneally with LPS (200 μg/kg) or Amlexanox (2.5 mg/kg) at 17.5 days after pregnancy. Placental tissues were collected from mice and analyzed by immunoblot using anti-phospho-S6K and anti-S6K antibodies (number = 5 per group). (C, D) As shown, HTR8/SVneo cells were treated with 1 μg/ml LPS or 100 μM Amlexanox for 24 hours. Cell lysates were analyzed by immunoblot using anti-phospho-S6K and anti-S6K antibodies. Alpha-tubulin was used as a loading control. (E, F) HTR8/SVneo cells were infected with lentivirus expressing shRNA targeting luciferase (sh-LUC) or TBK1 (sh-TBK1) as shown in the figure and incubated with 1 μg/ml LPS for 24 hours. It has been processed. Cell lysates were analyzed by immunoblot using anti-phospho-S6K and anti-S6K antibodies. (GJ) HTR8/SVneo cells were treated with 1 μg/ml LPS, 100 nM rapamycin, or 250 nM Torin 1 for 24 hours. Cell lysates were analyzed by immunoblot using anti-NLRP3, anti-Caspace1, anti-phospho-S6K, and anti-S6K antibodies. Alpha-tubulin was used as a loading control. The intensity of the bands was quantified and normalized to total protein concentration. Data are expressed as mean ± SEM. *p <0.05; **p <0.01; ***p <0.001 (one-way analysis of variance, Tukey test).
Figure 8 shows that NLRP3 protein expression increased in the junctional area and placental labyrinth of LPS-treated mice. Immunohistochemical staining analysis for NLRP3 was performed on placental tissue from pregnant mice treated with vehicle, LPS, and LPS+amlexanox at 17.5 days post gestation. Nuclei were stained with hematoxylin. The boxed area is an enlargement of the adjacent panel. LZ and JZ represent the Ravidinus zone and junction zone, respectively. Scale bar, 200 μm; Inset, 100 μm.
Figure 9 shows that the NLRP3 inflammasome was activated in HTR8/SVneo and Sw.71 cells when stimulated with LPS and ATP. (A, B) HTR8/SVneo cells and Sw.71 cells were treated with 1㎍/㎖ LPS for 24 hours and then treated with 5mM ATP for 45 minutes. Cell lysates were analyzed by immunoblot using anti-NLRP3 and anti-Caspace1 antibodies. Alpha-tubulin was used as a loading control.
Figure 10 shows the results of in vitro cytotoxicity analysis, measuring the cell viability of HTR-8/SVneo and Sw.71 cells treated with different concentrations of Amlexanox. (A, B) HTR8/SVneo cells and Sw.71 cells were treated with the indicated concentrations of Amlexanox for 24 hours. Cytotoxicity was measured by the WST-1 method. (C) Sw.71 cells were treated with 1 μg/ml LPS for 24 hours and then treated with 5mM ATP for 45 minutes. Cell lysates were analyzed by immunoblot using anti-NLRP3 and anti-Caspace1 antibodies. (D) Sw.71 cells were infected with lentivirus expressing shRNA targeting luciferase (sh-LUC) or TBK1 (sh-TBK1) as shown in the figure and treated with 1 μg/ml LPS for 24 hours. Afterwards, it was treated with 5mM ATP for 45 minutes. Cell lysates were analyzed by immunoblot using anti-NLRP3, anti-Caspace1, and anti-TBK1 antibodies. Alpha-tubulin was used as a loading control.

Placental inflammation, both infectious and non-infectious, causes a variety of adverse pregnancy outcomes, including preterm birth, IUGR, and pre-eclampsia. TBK1 is widely expressed in animals and is involved in activation of the NF-κB pathway. However, it is unclear what role TBK1 plays in infections during pregnancy. The present inventors observed phosphorylation of TBK1 in mouse placental tissue and human trophoblast cells after inducing inflammation in the placenta with LPS, and found that increased production of interleukin-1 beta is associated with placental inflammation and its onset during pregnancy. Confirmed. These results of the present invention biologically support evidence that abnormal activity of interleukin-1 beta mediates NLRP3 inflammasome activity in trophoblast cells of the maternal placenta.

In the present invention, it has been proven that LPS stimulates the inflammasome in trophoblast cells, but we investigated the role of TBK1 in LPS-induced NLRP3 inflammasome activity, and as a result, the present inventors found that LPS-induced placental inflammation is caused by TBK1. We demonstrated that phosphorylation and the activity of the NLRP3 inflammasome are related. In addition, LPS inhibits the development of the fetus and placenta, as evidenced by the weight and length of the fetus and placenta, suggesting that phosphorylation of TBK1 is involved in placental inflammation and NLRP3 inflammasome activity.

In a previous study, the present inventors confirmed that BX795, a TBK1 inhibitor, inhibits the expression of inflammatory cytokines such as interleukin-6 and interleukin-10 in a non-alcoholic steatohepatitis mouse model, but the association between placental inflammation caused by LPS and activation of TBK1 has not been studied before.

In the present invention, Amlexanox, a TBK1 inhibitor, inhibits inflammatory cytokines and chemokines, including interleukin-1β, interleukin-10, TNF-α, TGF-β1, MCP-1, and interferon-beta, in the placenta of mice affected by LPS. It was discovered that it causes a decrease in gene expression. Interferon-beta expression can be induced by a type of pathogen-associated molecule in trophoblast cells through the TLR4/IRF3 signaling pathway. Previous studies have shown that abnormal production of interferon-beta and the associated activation of interferon-stimulated genes cause fetal death. Consistent with this, gene expression of interferon-beta was increased in the placenta of LPS-treated mice compared to control mice, and was significantly attenuated in mice treated with amlexanox after exposure to LPS.

In the present invention, pretreatment with Amlexanox clearly shows a decrease in the expression of interleukin-1β beta in the placenta of mice treated with LPS, and that blocking TBK1 activation can also affect the expression of interferon-beta by activating IRF3. It shows. Given that TBK1 mediates the phosphorylation and nuclear translocation of IRF3, which contributes to the induction of type I interferons, the present invention suggests that TBK1 inhibition in LPS-treated pregnant mice may be partially mediated through changes in the production of interferon-beta. It suggests that there is. TBK1 has been implicated in the activation of the atypical NF-κB pathway. However, TBK1 will also inhibit the activity of the NF-κB pathway.

In the present invention, considering that TBK1 is involved in cell- and stimulus-dependent activation of NF-κB, we directly investigated the effect of TBK1 deficiency on the LPS-induced NF-κB pathway and found that Amlexanox using mouse and human trophoblast cells. It clearly showed that the treatment reduced the LPS-induced NF-κB/p65 pathway. It can be seen that inhibition of TBK1 is closely related to the negative regulation of the NF-κB/p65 pathway. In conclusion, we provide evidence for a potential role of TBK1 in LPS-induced activation of the placental NLRP3 inflammasome. LPS treatment upregulates activation of the placental NLRP3 inflammasome and phosphorylation of TBK1, which mediates inflammation.

Inflammation within the placenta during pregnancy is a response to stimulation, and the NLRP3 inflammasome activates caspase-1 and cleaves the inactive form of pre-interleukin-1β into the active form. This causes placental inflammation within the uterus, which can cause premature birth, fetal growth restriction, preeclampsia, pre-eclampsia, placental abruption, and congenital disorders. Therefore, the present invention can provide information on preventing or treating intrauterine inflammation by administering a pharmaceutical composition containing a TBK1 inhibitor to a pregnant woman.

The pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration. When formulated, diluents such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants, or It is prepared using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing simvastatin with at least one excipient, such as starch, calcium carbonate, gelatin, etc. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspension solvents include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The present invention will be described in detail below based on examples. The terms, examples, etc. used in the present invention are merely illustrative to explain the present invention in more detail and aid the understanding of those skilled in the art, and the scope of rights of the present invention should not be construed as limited thereto.

Technical terms and scientific terms used in the present invention, unless otherwise defined, represent meanings commonly understood by those skilled in the art in the technical field to which this invention pertains.

[Example]

reagent

Protein immunoblots showed phospho-TBK1, TBK1, NLRP3, caspase-1, phospho-p65, p65, phospho-IκB, IκB, phospho-S6K, S6K (Cell Signaling Technology, Danvers, MA, USA), and caspase-1. (Santa Cruz Biotechnology, Dallas, TX, USA), and antibodies against alpha-tubulin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Lipopolysaccharide (Escherichia coli LPS, serotype 0127: B8), adenosine triphosphate, and rapamycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amlexanox and Torin 1 were purchased from Cayman Chemical Company, Ann Arbor, MI, USA.

animal

Female C57BL/6 mice, 6–8 weeks old, were purchased from Samtaco (Osan, Korea). All mice were raised in an environmentally controlled room where temperature (20-25 °C) and humidity (50 ± 5%) were controlled with a 12-hour light/dark cycle. The female mouse mated with the male mouse, and successful mating was confirmed by checking the vaginal plug the next day. Pregnant mice received an intraperitoneal injection of LPS (200 μg/kg) or saline at 17.5 days after pregnancy. In the Amlexanox + LPS group, pregnant mice were intraperitoneally injected with Amlexanox (2.5 mg/kg) 27 hours and 3 hours before intraperitoneal administration. Pregnant mice were sacrificed at various times (4, 6, 8, 10, and 12 hours) after LPS treatment, and placental samples were collected for molecular analysis. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Konyang University Animal Ethics Committee (P-21-19-A-01).

Cell culture and processing

Human early pregnancy trophoblast cell lines HTR-8/SVneo (American Type Culture Collection, Rockville, MD, USA) and Sw.71 (gifted by Dr. Gil Mor, Yale University School of Medicine, New Haven, CT, USA) were used in the experiment. It was used. HTR-8/SVneo cells were incubated with 10% fetal bovine serum (Welgene), 4500 mg/l D-glucose, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvic acid, and 100 U/ml penicillin-streptomycin ( The cells were cultured in RPMI 1640 medium (Welgene, Gyeongsan, Korea) supplemented with Welgene. Sw.71 cells were cultured in Dulbecco's modified Eagle's medium (Welgene) containing 10% fetal bovine serum, L-glutamine, 4500 mg/l D-glucose, and 100 U/ml penicillin-streptomycin. All cultures were maintained at 37 °C in a humidified 5% carbon dioxide atmosphere. For LPS treatment, cells were cultured in the presence of LPS or treated with LPS and then treated with 5 mM adenosine triphosphate for 45 minutes as previously described. An equal volume of phosphate buffered saline was used as a vehicle control. In addition, the same amount of phosphate buffered saline was added to the cells to which the TBK1 inhibitor Amlexanox was added to serve as a vehicle control.

Cytotoxicity assay

HTR-8/SVneo cells were measured using the WST-1 (EZ-CytoX Enhanced Cell Viability Assay Kit, Daeil Lab Service, Seoul, Korea) assay according to the manufacturer's protocol. Briefly, HTR-8/SVneo cells (1 × 10 ⁴ cells/well) were seeded in a 96-well plate and treated with Amlexanox according to the known concentration for 24 hours. Afterwards, 10 μl of WST-1 reagent was added to each well and incubated for 30 minutes at 37 °C in a 5% carbon dioxide incubator (Thermo Scientific, Waltham, MA, USA). Absorbance was measured at 450 nm using an Epoch 2 microplate reader, and the absorbance values of treated cells were expressed as a percentage of the control absorbance value.

Plasmid and virus production

Using polyethylenimine reagent, HEK293T cells were transduced with the following lentiviral constructs along with packaging plasmids: sh-luciferase (sh-LUC) and sh-TBK1 constructs (obtained from the RNAi Consortium collection at the Broad Institute). Lentivirus supernatants were collected and filtered after 48 and 72 hours. HTR8/SVneo cells and Sw.71 cells were cultured for 2-3 days with lentiviral medium containing 4 μg/ml polybrene.

Quantitative real-time PCR

Total RNA was extracted from placental tissues and HTR8/SVneo cells using TRizol reagent (Takara, Shiga, Japan) according to the manufacturer's instructions. Complementary DNA was synthesized by reverse transcription from RNA using a cDNA synthesis kit (BioFact, Daejeon, Korea). Quantitative real-time reverse transcription PCR (qRT-PCR) was performed in triplicate with SYBR Green Real-Time PCR Master Mix Reagent (BioFact) using the QuantStudio 3 Real-time PCR system (Life Technologies, Carlsbad, CA, USA). Relative mRNA expression was calculated from critical cycle (Ct) values by comparison to mouse or human cyclophilin. Primers are as follows;

mouse Nlrp3: forward 5′- AGCCTTCCAGGATCCCTCTTC-3′,

reverse 5′- CTTGGGCAGCAGTTTCTTTC-3′;

mouse Tnf-α: forward 5′-TCCCAGGTTTCTCTTCAAGGGA-3′,

reverse 5′-GGTGAGGAGCACGTAGTCGG-3′;

mouse Tgf-β1: forward 5′- CTCCCGTGGCTTCTAGTGC-3′,

reverse 5′-GCCTTAGTTTGGACAGGATCTG-3′;

mouse Il-6: forward 5′- TAGTCTTCCTACCCCAATTTCC-3′,

reverse 5′-TTGGTCCTTAGCCACTCCTTC-3′;

mouse Il-10: forward 5′-GCTCTTACTGACTGGCATGAG-3′,

reverse 5′- CGCAGCTCTAGGAGCATGTG-3′;

mouse Mcp-1: forward 5′-CATCCACGTGTTGGCTCA-3′,

reverse 5′-GATCATCTTGCTGGTTGAATGAGT-3′;

mouse Il-1β: forward 5′- TCTTTGAAGTTGACGGACCC-3′,

reverse 5′-TGAGTGATACTGCCTGCTG-3′;

mouse IFN-β: forward 5′-GCACTGGGTGGAATGAGACT-3′,

reverse 5′-AGTGGAGAGCAGTTGAGGAC-3′;

mouse Cyclophilin A: forward 5′-GAGCTGTTTGCAGACAAAAGTTC-3′,

reverse 5′- CCCTGGCACATGAATCCTGG-3′;

human NLRP3: forward 5′-GATCTTCGCTGCCGATCAACAG-3′,

reverse 5′-CGTGCATTATCTGAACCCCAC-3′;

human IL-1β: forward 5′- AAAGAGGCACTGGCAGAA-3′,

reverse 5′-AGCTCTGGCTTGTTCCTCAC-3′;

human Cyclophilin A: forward 5′-GCAAAGTGAAAGAAGGCATGAA-3′,

reverse 5′- CCATTCCTGGACCCAAAGC-3′.

Immunoblotting

Placental tissue, HTR8/SVneo cells, and Sw.71 cells were isolated in radioimmunoassay buffer containing Complet protease inhibitor cocktail (Roche, Basel, Switzerland). Lysates were placed on ice for 20 min and then centrifuged at 18,000 × g for 15 min at 4 °C. Protein concentration was measured using the bicinchoninic acid protein assay (Pierce, Rockford, 1L, USA). Lysates were boiled in sodium dodecyl sulfate (SDS) and Lemli sample buffer for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA) for phospho-TBK1, TBK1, NLRP3, Caspase-1, phospho-p65, p65, We investigated with primary antibodies against phospho-IκBα, IκBα, phospho-S6K, S6K, and alpha-tubulin. After attaching a secondary antibody coupled to HRP (Bio-Rad, Hercules, CA, USA), chemiluminescence was detected using the Fusion Solo system (Vilber Lourmat, Marne-le-Vallee, France). Densitometric analysis of the blot was performed using ImageJ (National Institutes of Health, USA).

Histological analysis

Mouse placental tissue was fixed in 10% neutral buffered formalin for 24 hours, dehydrated, and then fixed in paraffin. Fixed tissue sections (5 μm) were prepared and stained with hematoxylin and eosin (H&E) dye or periodic acid Schiff dye. For immunohistochemistry, paraffin sections were deparaffinized, rehydrated, and then underwent antigen retrieval. Internally derived peroxidase was treated using 3% hydrogen peroxide. After blocking non-specific antigens, the slides were reacted with anti-NLRP3 antibody or anti-IL-1β antibody at 4°C for one day, followed by biotinylated secondary antibody (Vector Laboratories, Bullingame, CA, USA). Antibodies were visualized with Streptavidin-HRP (BD Pharmingen, San Diego, CA, USA) using diaminobenzidine (Sigma-Aldrich). Hematoxylin was used to visualize the nuclei. Samples were analyzed by light microscopy (DM2500; Leica, Wetzlar, Germany).

Enzyme-linked immunosorbent assay (ELISA)

Cell supernatants collected from HTR-8/SVneo cells were analyzed for levels of the secreted inflammatory cytokine interleukin-1 beta using the DuoSet ELISA development kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The optical density of the final color reaction product was measured at 450 nm using an Epoch2 microplate reader (Bio-Tek Instruments).

Statistical analysisData are expressed as mean ± standard error (s.m.m). Unless otherwise noted, data presented in figure panels are representative of at least three independent experiments. The significance of differences between the two experimental groups was determined using a two-tailed Student's t test. Multiple comparisons were performed using one-way analysis of variance followed by Turkey's or Fisher's least significant difference (LSD) post hoc test. P values less than 0.05 were considered statistically significant (***P < 0.001; **P < 0.01; *P < 0.05; ns, no statistically significant difference).

TBK1 phosphorylation is induced in placenta and trophoblast cells after LPS exposure.

Phosphorylation of TBK1 protein in mouse placenta was assessed in response to LPS-induced maternal inflammatory response. Immunoblot analysis showed that TBK1 phosphorylation was significantly increased 4, 6, 8, and 10 hours after LPS treatment in the placenta (Figure 1A and 1B). To measure the phosphorylation level of TBK1 in trophoblasts during maternal inflammation, human early pregnancy trophoblasts HTR8/SVneo and Sw.71 were stimulated with various LPS concentrations. Immunoblot analysis showed that LPS increased TBK1 phosphorylation in HTR-8/SVneo after short (Figure 1C and 1D) and long time treatment (Figure 1E and 1F) in a dose-dependent manner. The TBK1 phosphorylation process was further studied by treating cells with LPS (1 μg/ml), and the results showed that short- and long-term exposure to LPS significantly increased the level of phosphorylated TBK1 in HTR-8/SVneo cells in a time-dependent manner (Figure 1G-J). Similar to HDR-8/SVneo cells, expression of phosphorylated TBK1 was also stimulated by LPS treatment in Sw.71 cells.

TBK1 deficiency attenuates LPS-induced NLRP3 inflammasome activation in trophoblasts.

NLRP3 inflammasome activation in trophoblast cells is associated with the pathogenesis of placental inflammation. To investigate the effect of LPS on placental NLRP3 protein expression, placentas from mice exposed to LPS were analyzed by immunochemistry. The results showed that NLRP3 protein expression was increased in the junctional and placental labyrinths of LPS-treated mice compared with control placentas of saline-treated mice (Figure 8). Consistent with the above results, the NLRP3 inflammasome was activated in HTR8/SVneo and Sw.71 cells when stimulated with LPS and ATP (Figure 9).

We also investigated whether TBK1 is involved in LPS-induced NLRP3 inflammasome activation. Amlexanox, a TBK1 inhibitor, was used to explore the effect of TBK1 deficiency on LPS-induced NLRP3 inflammasome. Cell viability of HTR-8/SVneo and Sw.71 cells treated with different concentrations of Amlexanox was measured. As a result of in vitro cytotoxicity analysis, Amlexanox showed low cytotoxicity to cells up to 200 μM (Figure 10). Immunoblotting analysis showed that Amlexanox significantly reduced LPS-induced NLRP3 mRNA levels and NLRP3 and Kespace 1 protein levels (Figures 2A-2C and Figure 10). To confirm these results, the level of interleukin-1 beta was analyzed using enzyme-linked immunosorbent assay. Pretreatment of HTR-8/SVneo cells with Amlexanox significantly lowered the level of interleukin-1 beta compared to LPS-treated cells (Figure 2D).

To identify the specific signaling mechanism by which Amlexanox inhibits LPS-induced NLRP3 inflammasome activation in trophoblast cells, lentiviral vectors expressing short-hairpin RNA (shRNA) targeting human TBK1 were used in HTR-8/SVneo and introduced into Sw.71 cells. Knockdown of TBK1 was sufficient to block the upregulation of NLRP3 and cleaved-kespace-1 in LPS-treated cells (Figure 2E-G), consistent with the inhibitory effect of amlexanox on NLRP3 inflammasome activation. Enzyme-linked immunosorbent assay also showed that lentiviral knockdown of TBK1 reduced interleukin-1beta production (Figure 2H), suggesting that this was an inhibitory effect of TBK1 rather than a non-specific effect of amlexanox.

TBK1 deficiency attenuates LPS-induced NF-κB/p65 pathway in trophoblast cells.

NF-κB signaling mediates the priming signal of NLRP3 inflammasome activation and upregulates the expression levels of NLRP3 and pro-interleukin-1beta. LPS increased phosphorylation of NF-κB/p65 in a time-dependent manner, with the maximum effect observed at 1 hour after treatment with 1 μg/ml LPS (Figure 3). To investigate the effect of amlexanox on the NF-κB pathway, the phosphorylation levels of IκBα and NF-κB/p65 were measured in HTR-8/SVneo and Sw.71 cells. Amlexanox pretreatment reduced LPS-induced phosphorylation of IκBα and NF-κB/p65 (Figure 3).

We also examined the nuclear translocation of NF-κB/p65. LPS treatment resulted in a significant increase in NF-κB/p65 in HTR-8/SVneo cell nuclei, whereas amlexanox inhibited the nuclear translocation of NF-κB/p65 (Figure 3C). We also investigated whether knockdown of TBK1 reversed the effect of LPS on the NF-κB/p65 pathway. Lentiviral knockdown of TBK1 reduced LPS-induced phosphorylation of IκBα and NF-κB/p65 (Figures 3D,E). Immunofluorescence staining showed that knockdown of TBK1 was sufficient to inhibit nuclear translocation of NF-κB/p65 in LPS-treated cells (Figure 3F). These results indicate that TBK1 deficiency prevents LPS-induced nuclear translocation of NF-κB/p65 by phosphorylation of IκBα.

Amlexanox alleviates pregnancy side effects in LPS-treated mice.

To evaluate whether maternal Amlexanox administration affects fetal-placental development in LPS-treated mice, we first measured fetal and placental weight and length in each group. Fetal and placental body weight and length were significantly reduced in LPS-treated mice compared to control mice, whereas amlexanox treatment prevented the reduction in fetal and placental body weight and length associated with LPS treatment (Figures 4A-C).

We also evaluated whether amlexanox affects placental growth and morphology in LPS-treated mice. Periodic acid-Schiff staining revealed that placentas exposed to LPS had significantly reduced decidua and junctional lengths, but no notable changes were observed in the length of the labyrinth layer. Interestingly, amlexanox treatment abolished LPS-induced placental morphological changes (Figures 4D,E). These study results suggest that maternal administration of Amlexanox alleviates LPS-induced morphological changes in the placenta.

Amlexanox reverses placental inflammation and NLRP3 inflammasome activation in LPS-treated mice.

To further determine whether maternal administration of Amlexanox affects placental NLRP3 inflammasome activation in vivo, the expression level of the NLRP3 inflammasome marker was measured in the placenta of LPS-treated mice. Immunoblot analysis showed that NLRP3 and cleaved caespace-1 protein levels were elevated in response to LPS treatment, and this increase was suppressed in amlexanox-treated mice exposed to LPS ( Fig. 5A, B ). Correspondingly, amlexanox treatment reduced NLRP3 mRNA levels in the placenta of LPS-treated mice (Figure 5C). Immunohistochemical staining analysis showed that NLRP3 and IL-1β staining was significantly increased in cytoplasmic globus cells in the junctional and labyrinth layers of LPS-treated mouse placenta, but expression was decreased with amlexanox treatment ( Figure 5D ).

To determine whether the effect of amlexanox on the NF-κB pathway could be observed in vivo, the phosphorylation level of NF-κB/p65 was measured in mouse placental tissue. Immunoblot results showed that phosphorylation of NF-κB/p65 increased in the LPS treatment group, while amlexanox treatment prevented the increase in phosphorylation levels of NF-κB/p65 (Figures 6A, B).

Additionally, qRT-PCR was used to evaluate whether maternal administration of Amlexanox affected gene expression of inflammatory cytokines and chemokines. Expression of interleukin-1beta, interleukin-6, interleukin-10, TNF-alpha, TGF-beta1, MCP-1, and interferon-beta genes was elevated in the placenta of LPS-treated mice compared to control mice. This increase was greatly attenuated when exposed to LPS followed by amlexanox treatment (Figure 6C). These data indicate that TBK1 inhibition by amlexanox alleviates placental inflammation and NLRP3 inflammasome activation.

TBK1 deficiency attenuates LPS-induced NLRP3 inflammasome activation through an mTORC1-dependent mechanism.

In response to LPS, TBK1 can directly activate mTORC1, but the function of TBK1 has been controversial. Accordingly, the present inventors confirmed the expression of target proteins of the mTORC1 sub-signal in placental tissues of control, vehicle, and Amlexanox-treated LPS mice. Immunoblot analysis showed that mice treated with amlexanox had lower levels of phosphorylated p70 ribosomal protein S6 kinase (p70S6K) compared to mice treated with LPS (Figures 7A, B). We also evaluated the effect of amlexanox on LPS-induced mTORC1 activation in HTR-8/SVneo cells and found decreased phosphorylation of p70S6K after amlexanox pre-treatment (Figures 7C,D). Consistent with the inhibitory effect of amlexanox on mTORC1 signaling, lantivirus-induced TBK1 knockdown blocked LPS-induced phosphorylation of P70S6K in HTR-8/SVneo cells (Fig. 7E,F), thereby inhibiting the activation of TBK1 by LPS. It indicates that it is involved in mediated mTORC1 activation.

To investigate whether mTORC1 inhibition affects LPS-induced NLRP3 inflammasome activation, we pretreated HTR-8/SVneo cells with the mTOR inhibitors rapamycin and Torin1. Immunoblot analysis showed that pre-treatment with rapamycin or Torin 1 inhibited the LPS-induced increase in protein levels of NLRP3 and degraded-kespace-1 (Figure 7G-J). Therefore, these data suggest that TBK1 deficiency alleviates LPS-induced NLRP3 inflammasome activation in an mTORC1-dependent manner.

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Claims (6)

A pharmaceutical composition for preventing or treating placental inflammation, comprising Amlexanox, a TBK1 inhibitor, and a pharmaceutically acceptable excipient.
According to claim 1,
A pharmaceutical composition for preventing or treating placental inflammation, characterized in that intrauterine placental inflammation causes any one of premature birth, fetal growth restriction, preeclampsia, pre-eclampsia, placental abruption, or congenital disorders.
delete Culturing TBK1-expressing cells;
Adding a candidate material to the culture medium and culturing it; and
Comprising the step of analyzing the level of TBK1 activity in the cells and selecting a candidate substance that inhibits the activity of the TBK1 protein,
Method for screening drugs to prevent or treat placental inflammation.
According to claim 4,
A method for screening a drug for preventing or treating placental inflammation, wherein the activity of the TBK1 protein is gene expression, protein synthesis, or protein phosphorylation.
According to claim 4,
The candidate material is selected from peptides, antibodies, or compounds that bind to the TBK1 protein, or is selected from antisense oligonucleotides, peptide mimetics, polynucleotides, siRNA, shRNA, or compounds that bind to the TBK1 gene. Method for screening drugs for prevention or treatment.