WO2019208897A1 - METHOD FOR SCREENING INFLAMMATORY RESPONSE REGULATOR TARGETING PKCα-LSD1-NFκB PATHWAY AND USE THEREOF - Google Patents

Abstract

The present application discloses a method for screening an inflammatory response regulator targeting PKCα-LSD1-NF-κB. In response to excessive inflammatory stimuli, PKCα translocates to the nucleus and phosphorylates LSD1. LSD1 phosphorylation is required for p65 binding and facilitates p65 demethylation, leading to enhanced stability of the p65 protein. Therefore, the method according to the present application can be advantageously used for developing a therapeutic agent for treating systemic inflammatory diseases including sepsis by regulating PKCα or LSD1 activity.

Description

Screening method for inflammatory response modulators targeting the PKCα-LSD1-NFκB pathway

It is a technical field related to the development of therapeutic agents based on inflammation control mechanisms.

Inflammatory responses mediated by NF-κB signals are essential for the host's defense against pathogen invasion. The regulatory mechanisms of NF-κB signaling are well studied, but epigenetic regulation of this inflammatory response is not well known.

In particular, sepsis due to an inflammatory response is a serious disease with a mortality rate of 70%, accounting for 20% of ICU inpatients and more than 1500 deaths per day worldwide. However, despite the relatively high mortality rate compared to other diseases, no special treatment has been developed. The only treatment for sepsis currently on sale is Xigris, a multinational pharmaceutical company, Lilly CO. Ziglis is a substance similar to activated protein C that lowers inflammation and relieves blood clotting. The drug was approved for use in adult patients at high risk of death due to infection in 2001. However, pediatric clinical trials have shown that Xigris is not effective for children with severe blood infections and has severe side effects such as head bleeding. Causing clinical trials to cease.

In recent years, the initial excessive innate immune response seen in sepsis induces immunoparalysis and, in the majority of deaths, deaths due to inability to fight primary or secondary pathogens rather than death due to initial excess immune responses. (Docke, WD et al. Monocyte deactivation in septic patients: restoration by IFN-γ treatment. Nature Med. 3, 678-81 (1997)). These results indicate that the treatment of sepsis is limited only by the early inflammatory cytokine suppression technology.

Therefore, there is an urgent need to develop new drugs based on various mechanisms to obtain safe and high therapeutic effects.

Sepsis begins when bacterial products such as endotoxin bind to Toll-like receptors, signaling is relayed through adapter molecules, and nuclear factor kappa-light-chain-enhancer of activated Bcells) is known to follow the signal up to and including the transcription factor (TF) (Abraham, J. Infect. Dis. 2003; 187: S364-S369; Tiruppathi et al., Nat. Immunol. 2014; 15: 239247). NF-κB signaling, one of the inflammation-related molecules in systemic inflammatory diseases, must be carefully regulated to fine tune the inflammatory response (Caldwell et al., Genes Dev. 2014; 28: 2120-2133).

US Patent Publication No. 2009-0317833 relates to a method for screening a substance that binds to a TLR4 (toll-like receptor) intracellular region, a signaling pathway for TLR4 by binding to TLR4, whose transcription is regulated by NF-κB. Blocking mechanisms to initiate inflammation.

However, there is a need for the development of therapeutics based on molecular mechanisms of how the immune system detects the intensity and duration of exposure of pathogens, or which signal transduction pathways or signal cascades determine additional activation.

The present application seeks to provide a therapeutic screening method based on a novel inflammatory trigger mechanism.

In one aspect provided herein is a method of providing a cell expressing PCKa, LSD1 and NF-κB; Treating the cells with a stimulus capable of inducing an NF-κB mediated inflammatory response, wherein the treatment causes an inflammatory response by the PCKα->LSD1-> NF-κB pathway in the cells, Treating a test substance that is expected to inhibit an inflammatory response by the pathway; And when the inflammatory response by the pathway is inhibited in the cells treated with the test substance as a result of the treatment, compared with the control group not treated with the test substance, selecting the test substance as an inflammatory inhibition candidate. Inhibition of the inflammatory response by the pathway may include reducing phosphorylation of LSD1, demethylation of p65 subunit of NF-κB or binding of LSD1 and p65 subunit. Provided are methods for screening inflammatory response inhibitors mediated by NF-κB signals, measured in at least one of the decreases.

In one embodiment, the stimuli that can induce NF-κB mediated inflammatory responses by the PCKα-> LSD1-> NF-κB pathway are different receptors that accept stimuli, but the PCKα-> LSD1-> NF-κB pathway NF Stimulation that activates, induces, or triggers and amplifies pathways by -κB, such as tumor necrosis factorα (TNFα), interleukin 1-beta (IL-1β), pathogen-associated molecular pattern (PAMP), or bacterial LPS (lipopolysaccharides) It is a stimulus that works specifically for the immune situation.

In one embodiment the cells that can be used in the method according to the present invention are used macrophage, such as mouse leukmic monocyte-macrophage (264.7) or bone marrow-derived macrophage (BMDM) derived from mouse macrophages.

In one embodiment instead of the cells expressing PCKa, LSD1 and NF-κB in the method according to the invention, or the cells may be provided as an animal model except human.

Candidates selected by the method according to the invention can be used for the treatment of NF-κB mediated inflammatory diseases such as chronic inflammatory disease sepsis, autoimmune diseases or rheumatoid arthritis.

Inflammatory responses mediated by NF-κB signals are essential for the host's defense against pathogen invasion. The regulatory mechanism of NF-κB signaling is well studied, but epigenetic regulation of the inflammatory response is not well known. In the present invention, it was confirmed that a new signal transduction axis called PKCα-LSD1-NF-κB is important for the activation and amplification of the inflammatory response. In response to excessive inflammatory stimuli, PKCα migrates to the nucleus to phosphorylate LSD1. LSD1 phosphorylation is required for p65 binding and promotes demethylation of p65 to enhance the stability of p65 protein. Removal of LSD1 phosphorylation using Lsd1 ^{SA / SA} mice and inhibition of PKCα or LSD1 activity in wild-type mice has been shown to reduce inflammatory lung injury and mortality caused by sepsis. This indicates that the PKCα-LSD1-NF-κB signaling cascade is important for the control of the inflammatory response, and the development of drugs that target such signaling can be useful in the development of therapeutic agents for systemic inflammatory diseases including sepsis.

1 shows that LSD1 is phosphorylated in response to inflammatory signals by PKCα (A) H & E staining of lungs after 6 hours of intraperitoneal injection of PBS or LPS (10 mg / kg body weight) into wild type and Lsd1 ^{SA / SA} mice One picture (6 experiments in each group). Scale bar, 100 μm. The image shown is representative of three independent experiments. (B) Survival was monitored for 72 hours after LPS injection into wild-type (11 mice) and Lsd1 ^{SA / SA} mice (10 mice) of similar age and weight. (** p <0.01, log-rank test) (C) BMDM was extracted from wild-type and Lsd1 ^{SA / SA} mice, treated with LPS for 2 hours (or not treated with LPS) or immunized with the indicated antibody. Blot. (D) Immune blot data of lung tissue extracted from wild type and Lsd1 ^{SA / SA} mice (6 per group). Mice were injected with Go6976 (1 mg per kg body weight, dissolved in corn oil) or corn oil, a solvent of Go6976, 1 hour before LPS intraperitoneal injection (10 mg injection per kg body weight, tested 6 hours after injection). (E) Immunoblot in wild-type BMDM after LPS treatment for the indicated time. Tubilin antibody was used as a control for loading of cytoplasmic proteins. laminA / C antibodies were used as loading controls for nuclear proteins. (F) BMDM cells were treated with LPS for 2 hours with or without Go6976 for 6 hours. After that, only nuclear protein was isolated and extracted and immunoprecipitation experiments of PKCα and LSD1 were performed. (G) Immunoblot experiments were performed with the indicated antibodies by separating LPS from BMDM for the indicated time. (H) After treatment with LPS for 2 hours according to the indicated concentration, only the nuclear protein of BMDM was isolated and immunoblot experiments were performed with the indicated antibody.

2 shows that phosphorylation of LSD1 induced by LPS is required for activation of NF-κB target genes at the genome-wide level. (A) Flowchart showing strategy of RNA sequencing analysis. (B) hierarchical clustering result, 3,558 differently expressed genes (Differentially Expressed Gene or less, DEG) were identified. Transcriptional Sart Site (TSS) The presence or absence of the p65 peak around +/- 2.5kbps was drawn. (C) Gene ontology (GO) analysis of the cluster 1 genes revealed that genes involved in cytokine production and inflammatory responses were significantly distributed in the cluster 1 gene pool. (D) De novo motif analysis for p65 peak near TSS of gene in cluster 1. Hypergeometric p-values were calculated. (E) Analyzing quantitative RT-PCR results of LSD1 phosphorylation dependent gene with or without LPS 2 hours treatment in BMDM of wild type and Lsd1 ^{SA / SA} mice (F) Lungs of wild type and Lsd1 ^{SA / SA} mice (6 per group) Analysis of quantitative RT-PCR results of LSD1 phosphorylation dependent gene with or without LPS 6-hour intraperitoneal injection in tissues (1mg per kg body weight, dissolved in corn oil) or corn oil, a solvent of Go6976 (6 per group). To analyze the quantitative RT-PCR results of LSD1 phosphorylation-dependent genes in lung tissue of mice. Data are expressed as mean ± standard deviation; n = 3, * p <0.05, *** p <0.001 (Two-way ANOVA).

Figure 3 shows that LPS-induced LSD1 phosphorylation is required for recruiting p65 to the promoter of the target gene (A) 2 hours treatment of LPS in BMDM cells, immunoprecipitation experiment after extraction of p65 and LSD1 binding only nuclear protein It was performed through. (B) In vitro kinase experiments using wild and dominant negative forms of PKCα. GST-LSD1 and GST-p65 were purified from E. coli and used as substrate. (C) In vitro GST pulldown experiments were performed using phosphorylated GST-LSD1 (which preceded the kinase experiment with PKCα prior to the GST pulldown experiment) and p65. At this time, the experiment was performed in the presence or absence of λ-posphatase of 1,000 units. (D) ChIP experiment in LSD1 phosphorylation dependent target promoter following LPS 2-hour treatment in BMDM of wild type and Lsd1 ^{SA / SA} mice. (E) LPS 2 hours in Raw264.7 cells treated with Go6976 for 6 hours in advance and ChIP experiments on LSD1 phosphorylation dependent target promoter. Data are expressed as mean ± standard deviation; n = 3, ** p <0.01, *** p <0.001 (Two-way ANOVA) (D, E).

4 shows that demethylation of p65 by LSD1 enhances protein stabilization of p65. (A) Raw264.7 cells transfected wild-type (WT), SA, and KA mutations with a flag tag of LSD1. After 4 hours of MG132 treatment, LPS was further treated for 2 hours. Only nuclear proteins of these cells were extracted and immunoprecipitation experiments were performed between p65 and WT, SA and KA. (B) Raw264.7 cells were treated with MG132 for 4 hours and LPS was added for 2 hours to extract only nuclear proteins, followed by in vivo demethylation experiments. After immunoprecipitation experiments with p65 antibodies, methylation of p65 was detected using methyl specific antibodies of p65. (C) Purified flag-p65 substrate in cell lysate, GST-SET7 / 9 as methylase, purified in E. coli, and demethylase His-LSD1 purified in E. coli, in vitro demethylation experiment. Was performed. Prior to demethylation, in vitro kinase experiments using PKCα were used to obtain phosphorylated LSD1. The reaction detected the methylation of p65 after SDS-PAGE using an antibody that recognizes p65 K314 / 315 methylation. (D) Immunoblot experiments were performed using the indicated antibodies after extracting only the nuclear protein by treating LMG for 2 hours with BMDM of wild type and Lsd1 ^{SA / SA} mice pretreated with MG132 for 4 hours. (E) Raw264.7 cells were transfected with wild-type (WT), SA, and KA mutants carrying the LSD1 flag tag, and further treated with LPS for 2 hours after MG132 treatment for 4 hours. This protein extract was pulled down using Ni ²⁺ -NTA bead. Ubiquitination of p65 was detected through the p65 antibody. (F) 4 hours of MG132 followed by immunoprecipitation using p65 antibody in Raw264.7 cells further treated with LPS for 2 hours. Ubiquitination of p65 was detected via FK2 antibody. (G) Scheme of the demethylation of p65 mediated by LSD1 phosphorylation leads to stabilization of p65.

Figure 5 shows that the PKCα-LSD1 phosphorylation axis modulates a sustained inflammatory response (A) mRNAs of Mcp-1, Il-6, Cebpd by treatment of LPS according to the time indicated in BMDM of wild type and Lsd1 ^{SA / SA} mice Was detected via quantitative RT-PCR. (B) Go6976 was pretreated with BMDM in wild-type mice for 6 hours, followed by LPS treatment according to the indicated time, and mRNAs of Mcp-1, Il-6 and Cebpd were detected via quantitative RT-PCR. (C) LPS was treated according to the indicated time in BMDM, and the nucleoprotein was extracted to confirm the endogenous binding state of p65 and LSD1. (D) LPS was treated at the time zone indicated in BMDM of wild type and Lsd1 ^{SA / SA} mice. Perform ChIP experiments at LSD1 phosphorylation dependent target promoters. (E) ChIP experiments run on LSD1 phosphorylation dependent target promoters using the antibodies indicated in BMDM. Go6976 was pretreated for 6 hours and then LPS treated for the designated time prior to cell collection. Data are expressed as mean ± standard deviation; n = 3, * p <0.05, ** p <0.01, *** p <0.001 (Two-way ANOVA) (A, B, D, E). (F) Schematic diagram of when and how excessive LPS promotes p65 stabilization in the nucleus during sustained inflammatory responses.

6 shows that sustained expression of p65 in the nucleus and hence activation of the inflammatory response is via PKCα-LSD1-NFκB signaling. (A) Immunoblots were performed using LPS-treated and nucleoproteins with designated antibodies according to the time indicated in BMDM of wild-type and Lsd1 ^{SA / SA} mice. (B) Representative images of immunofluorescence assays. Immunofluorescence experiments were performed by transfecting WT, SA, and KA mutant forms with HA tags on Lsd1 ^{− / −} MEF cells, followed by treatment with LPS for a designated time. HA (green); p65 (red); DAPI (blue)., Scale bar, 20 μm. Immunoblotting experiments were performed using antibodies designated in the nuclear proteins of (C, D) BMDM (C) and Raw264.7 (D) cells. Go6976 or GSK-LSD1 was pretreated for 6 hours and LPS was treated for a specified time before cell collection. (E) Immunoblotting experiments were performed using antibodies designated in the nucleoprotein of Raw264.7 cells. Go6976 and GSK-LSD1 were pretreated for 6 hours and LPS was further treated for 2 hours. At this time, MG132 was treated 4 hours before cell collection.

7 shows that inhibition of PKCα or LSD1 activity in mice reduces mortality from sepsis (A) Immunoblot experiments in lung tissue of wild type mice (3 in each group). Mice were injected with LPS intraperitoneal (10 mg per kg body weight, tested 6 hours after injection) 1 hour prior to Go6976 ((CAS 136194-77-9), selective PKC inhibitor, 1 mg per kg body weight) or GSK-LSD1 (1mg per kg of body weight) is injected into the abdominal cavity. (B) Wild-type (blue line), Lsd1 ^{SA / SA} (red line) mice underwent CLP surgery (20 in each group). Survival of the animals was monitored every 6 hours for 144 hours after CLP surgery. (C, D) 1 mg of Go6976 per kg of body weight (green lines, 20 animals each) (C) or GSK-LSD1 (in purple line, respectively) via intravenous injection at 12 and 50 hours after CLP surgery, respectively. 20 animals). Survival was monitored every 6 hours for 144 hours after CLP surgery. ** p <0.01 (log-rank test) (BD) (E) Representative picture of H & E in lung tissue after CLP surgery. Wild type and Lsd1 ^{SA / SA} mice. Alternatively, wild type mice were injected intravenously with Go6976, GSK-LSD1 or vehicle at 1 mg / kg of body weight 12 hours and 50 hours after surgery (6 per group), respectively. Mice were euthanized 72 hours after CLP surgery. Scale bar, 200 μm. (F, G) Concentrations of cytokines (MCP-1, IL-6, TNF-α) in blood 72 hours after CLP surgery. Wild type and Lsd1 ^{SA / SA} mice (F) or wild type mice were injected intravenously with Go6976, GSK-LSD1 or vehicle at 1 mg / kg of body weight 12 hours and 50 hours after surgery (G), respectively. (H, I) Sepsis markers ALT, LDH, BUN were measured 72 hours after CLP surgery. Wild type and Lsd1 ^{SA / SA} mice (H) or wild type mice were intravenously injected with Go6976, GSK-LSD1 or vehicle at 1 mg / kg of body weight 12 hours and 50 hours after surgery (I), respectively. Data are expressed as mean ± standard deviation; n = 5, * p <0.05, ** p <0.01, *** p <0.001 (Two-way ANOVA) (F, H). Data are expressed as mean ± standard deviation; n = 5, * p <0.05, ** p <0.01 (unpaired two-tailed Student's t-test) (G, I). (J) Schematic showing that blocking PKCα or LSD1 activity decreases protein stability of p65 and induces resistance to acute systemic inflammation, thereby increasing the survival rate of sepsis in Lsd1 ^{SA / SA} mice compared to wild type.

The present application is based on the discovery that a new signal transduction axis leading to PKCα-> LSD1-> NF-κB is important in the activation and amplification of inflammatory responses following excessive inflammatory stimuli. Specifically, LSD1 (lysine specific histone demethylase 1) is an important epigenetic factor of the inflammatory response, which is phosphorylated by PKCα (Protein Kinase C α), which has moved to the nucleus in response to excessive inflammatory stimuli, to demethylate p65 of NF-κB. By activating the inflammatory response. Demethylation by LSD1 stabilizes the p65 protein and sustains NFKB activity, amplifying the inflammatory response. Therefore, in one embodiment, as shown in FIG. 5 and FIG. 6, and as shown schematically in FIG. 7J, when the protein stability of p65 constituting NF-κB is reduced by inhibiting the demethylation activity of PKCα or LSD1 thereby, acute systemic It can increase resistance by inducing resistance to inflammation.

In one aspect thereof, the present invention relates to a method for screening an inflammatory response inhibitor targeting the PCKa-> LSD1-> NF-κB pathway, which plays an important role in inducing an inflammatory response as disclosed herein.

The method according to the present invention comprises the steps of providing a cell expressing PCKa, LSD1 and NF-κB; Treating a stimulus capable of inducing an NF-κB mediated inflammatory response in the cell, wherein the treatment causes, triggers, or initiates an inflammatory response by the PCKα-> LSD1-> NF-κB pathway in the cell, Treating the cell with a test substance that is expected to inhibit the pathway or inhibit the inflammatory response caused by the pathway; And inhibiting the test substance when the pathway is inhibited or when the inflammatory response by the pathway is inhibited in cells treated with the test substance as compared with the control group not treated with the test substance. Screening for candidates.

The regulation of LSD1 by protein kinase C alpha (PKCα) is known in light-cycle rhythm regulation, and NF-κB is known as an inflammatory factor, but the PCKa-> LSD1-> NF-κB pathway is described herein. Was first identified in. PKCα has been reported to amplify the expression of inflammatory cytokines in the epidermis and induce an acute inflammatory response in mice treated with phorbol 12-myristate 13-acetate (PMA), which are activators of PKCα, PKCβ1 / β2 and PKCγ. et. al., Inflamm. Res. 1996; 45: 289292). PKC signaling also plays a critical role in the activation of the inflammatory response (Langlet et al., Eur. J. Immunol. 2010; 40: 505515), but the molecule for the target gene associated with the substrate of PKC and activation of the inflammatory response by PKC. The mechanism is not yet clear. It was found herein that the direct substrate of PKCα is LSD1, which phosphorylates LSD1 and does not phosphorylate (see FIG. 1, etc.).

Lysine specific demethylase 1 (also known as LSD1, AOF2 or BHC110) acts as a histone demethylase via a FAD dependent amine oxidase reaction (Metzger et al., Nature. 2005; 437: 436-439). The other LSD1 demethylates the lysine group of the nonhistone protein methylated by SET7 / 9. There is no known association between LSD1 and NF-κB in the inflammatory response, and LSD1 binds to p65 constituting NF-κB and demethylates it, as described below. Demethylation was found to stabilize p65 and eventually amplify the inflammatory response by regulating the activity of transcription factors involved in NF-κB mediated inflammatory responses (see FIG. 4, etc.).

NF-κB is an inflammatory factor consisting of subunits of p65 / RelA, p50 / NF-κB1, p52, RelB and c-Rel (Oeckinghaus and Ghosh, Cold Spring Harb. Perspect. Biol. 2009; 1: a000034). They have a common N-terminal Rel homology region, a DNA binding region and a sequence capable of entering the nucleus at the C-terminus, which are important for dimer formation. The p65-p50 heterodimer is the most abundant and known to have the highest activity. In unstimulated cells, IκB proteins bind to NF-κB subunits and sequester them cytoplasmically. In the presence of inflammatory stimuli, phosphorylation dependent proteasome degradation of IκB occurs, and the NF-κB subunit migrates from the cytoplasm to the nucleus (Fuchs et al., Oncogene. 1999; 18: 2039-2046). The NF-κB subunit migrated to the nucleus binds to the κB element of DNA and activates the expression of target genes involved in the inflammatory response. The recruitment of coactivators, for example CREB binding protein (CBP), is known to be critical for further activation of the target gene. Continuous LPS stimulation induces the expression of C / EBPδ by NF-κB, and the induced C / EBPδ works with NF-κB to further stimulate the transcription of cytokine coding genes so that C / EBPδ amplifies the inflammatory response. It is known to play a role. In one embodiment according to the present invention, phosphorylation of LSD1 in the LPS-induced PCKα-> LSD1-> NF-κB pathway is required for activation of NF-κB target genes at the genome-wide level (see FIG. 2) and is due to LPS treatment. LSD1 phosphorylation is required to recruit NF-κB p65 to the promoter of the target gene as a promoter (see FIG. 3), showing that the binding of LSD1 and p65 and thus p65 demethylation increases the protein stabilization of p65 (FIG. 4). Reference). This protein stabilization of NF-κB p65 eventually causes an inflammatory response.

PKCα, LSD1 and NF-κB (p65) used in the method according to the present invention are known genes and protein sequences, for example human protein sequence is LSD1: NP_001343496.1, PKCα: NP_035231.2, NF-κB (p65) ) Is known as NP_033071.1. Once the protein sequence is known, it will be apparent to those skilled in the art that the nucleic acid sequence can be readily derived therefrom.

In the method according to the present invention, a full-length or fragment having a variety of origins and protein sequences derived from each and a sequence substantially identical thereto may be used as long as they have such a function. Substantially the same is applied to both protein and nucleic acid sequence levels, and compared to a reference or reference sequence, there may be one or more substitutions, deletions, or additions to base or amino acid residues, but overall differences in function It means having a level of functionality that doesn't exist or that degrades functionality. Homology is at least 61% homology, more preferably 70% phase when the target sequence is aligned to the maximum correspondence and the aligned sequence is analyzed using algorithms commonly used in the art. By homology, even more preferably 80% homology, most preferably at least 90%, in particular at least 95% homology. Alignment methods for sequence comparison are known in the art. For example, Smith and Waterman, Adv. Appl. Math. (1981) 2: 482; Needleman and Wunsch, J. Mol. Bio. (1970) 48: 443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73: 237-44; Higgins and Sharp, CABIOS (1989) 5: 151-3; Corpet et al., Nuc. Acids Res. (1988) 16: 10881-90; Huang et al., Comp. Appl. BioSci. (1992) 8: 155-65 and Pearson et al., Meth. Mol. Biol. (1994) 24: 307-31. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. (1990) 215: 403-10) is accessible from NBCI and the like, and can be used with blast, blastp, blasm, blastx, tblastn and tblastx. It can be used in conjunction with a sequence analysis program. BLSAT is accessible at www.ncbi.nlm.nih.gov/BLAST/ and a method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

In one embodiment the PKCα, LSD1 and NF-κB proteins may be provided in the form of cells expressing it. For example, mammalian cells that express proteins (transient or stable transduction or endogenous expression), such as, but not limited to, PKCα, LSD1 and NF-κB are expressed and representative cells that can act include macrophages. Can be mentioned. Macrophages may be from animal or established cell lines. In particular, Raw 264.7 cell line or bone marrow-derived macrophage (BMDM) may be used. In one embodiment, a plasmid expressing the protein in a Raw 264.7 cell line can be used by transfection using a known method or a method described in the Examples herein.

PKCα-> LSD1-> NF-κB pathway is activated in the method according to the present invention, the substrate of PKCα is LSD1, PKCα phosphorylates LSD1, and phosphorylated LSD1 demethylates the p65 subunit of NF-κB Let's do it. Thus, in one embodiment, in the method according to the present disclosure, inhibition of the PKCα-> LSD1-> NF-κB signaling pathway results in a decrease or inhibition of phosphorylation of LSD1 by the PKCα, and / or of the p65 subunit of the NF-κB. Inhibition of demethylation, and / or inhibition or reduction of binding of LSD1 and p65. In addition, since activation of the signaling pathway causes an inflammatory response, inhibition of the pathway may be referred to as inhibition of the inflammatory response by the pathway. Inhibition of this pathway or inflammatory response may be achieved by culturing cells expressing the proteins constituting the mechanism described above in a cell culture plate, and then adding a test substance thereto, Total protein can be extracted to determine the degree of phosphorylation of LSD1, LSD1 demethylation activity, the binding of LSD1 and NF-κB p65, or the degree of NF-κB p65 demethylation by LSD1. Compared to the control (untreated test material), reduced phosphorylation of LSD1, inhibition of demethylation of p65 subunit of NF-κB or binding of LSD1 and p65 subunit by NF-κB signal It can be selected as a mediated inflammatory response candidate.

The degree of phosphorylation inhibition in the method according to the present application can be measured using a known method, for example, can be confirmed using a protein blot method. There are antibodies available for protein identification and antibodies that identify phosphorylated LSD1 as well as full-length LSD1 are commercially available. Such antibodies may be used to specifically identify the total expression level of LSD1 or the amount of LSD1 that is not or phosphorylated, but is not limited thereto. Compared with the control (untreated test material), inhibiting the degree of phosphorylation of LSD1 protein can be selected as a candidate for the treatment of sepsis. In this case, the protein may be labeled using a variety of markers such as protein tags, biotin, fluorescent materials, acetylation, radioisotopes or commercially available protein labeling kits for ease of detection, It can be detected using a detector suitable for the labeled material.

Methylation or demethylation in the methods according to the invention can be carried out using known methods, including, but not limited to, the use of antibodies that specifically trafficize methylated proteins, see also Examples herein. can do.

In the method according to the present invention, the binding or interaction of LSD1 and the p65 subunit can be measured using various methods known in the art. For example, the East to Hybrid method and the Confocal Microscopy method for confirming the binding / interaction of intracellular proteins. Co-immunoprecipitation, surface plasma resonance (SPR) and spectroscopy methods include, but are not limited to, further references on comparisons and detailed experimental methods on these methods. Berggard et al., (2007) "Methods for the detection and analysis of protein-protein interactions ", PROTEOMICS Vol7: pp 2833-2842.

The method according to the present invention comprises the step of inducing an inflammatory response mediated by the NF-κB signal in the cell, thereby activating the pathway consisting of PCKα, LSD1 and NF-κB as described above.

In one embodiment a variety of stimuli that can elicit an inflammatory response mediated by an NF-κB signal to a cell can be used herein, for example, a stimulus that can elicit an NF-κB mediated inflammatory response is TNFα (tumor necrosis). factorα), IL-1β (interleukin 1-beta), pathogen-associated molecular pattern (PAMP) or bacterial lipopolysaccharides (LPS) (Courtois G, Gilmore TD (2006) Mutations in the NF-kappaB signaling pathway: implications for human disease.Oncogene.2006 Oct 30; 25 (51): 6831-43; Gutierrez H, Davies AM (2011) Regulation of neural process growth, elaboration and structural plasticity by NF-κB. Trends Neurosci. 2011 Jun; 34 (6 ): 316-25.).

An inflammatory or inflammatory response herein is a protective response involving immune cells, blood vessels, and molecular mediators as a complex biological response of tissue to stimuli that is harmful to tissue, such as pathogens, stimulants, or damaged cells. The inflammatory response is to eliminate the initial cause of cell damage, remove damaged cells or tissues, and initiate tissue repair. Inflammatory reactions are accompanied by symptoms such as fever, pain, redness and swelling.

The inflammatory response mediated by the NF-κB signal herein means a case where the NF-κB protein is activated by the aforementioned immune signal stimulation. In particular herein, among the pathways activating NF-κB, in particular the pathway by demethylation of the p65 subunit by LSD1. When the inflammatory response is activated, expression of cytokines at the molecular level is increased, for example, IL-1β, IL-6, MCP-1, TNF-α, and the like, thereby detecting the activation of the inflammatory response.

In the method according to the invention the cells are treated with test substances which are expected to inhibit the PCKα-> LSD1-> NF-κB pathway.

Inhibition of this pathway can be measured by reducing phosphorylation of LSD1 and / or demethylation of p65 subunit of NF-κB and / or reducing or inhibiting binding of LSD1 and p65. Such reduction or inhibition means inhibition or reduction compared to a control not treated with the test substance, and one of ordinary skill in the art will readily be able to determine the extent of inhibition or reduction based on the disclosure herein and / or common sense in the art. In determining inhibition or reduction, one may also measure changes in the amount of cytokine expression that increases with activation of the inflammatory response.

Modulation herein includes activation of a particular biological function, stimulation or upregulation, or degradation or downregulation, or both, and regulation in an in vitro state, regulation in an in vivo state, in an ex vivo state, It includes all of the adjustments. In one embodiment the regulation is the inhibition of the inflammatory response.

Test agents used in the methods herein are expected to act on the PCKα-> LSD1-> NF-κB pathway signaling system to reduce phosphorylation of LSD1, thereby regulating and specifically inhibiting demethylation of the p65 subunit of NF-κB. As a substance, low molecular weight compounds, high molecular weight compounds, mixtures of compounds (eg, natural extracts or cell or tissue cultures), or biopharmaceuticals (eg, proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, apps) Tammers, RNAzyme and DNAzyme) or sugars and lipids, and the like.

In one embodiment according to the present application, a low molecular weight compound may be used as a test substance. The test substance can be obtained from a library of synthetic or natural compounds and methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA) and Available from MycoSearch (USA). Test materials can be obtained by a variety of combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution Required by the synthetic library method, the "1-bead 1-compound" library method, and the synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994 and the like. For example, for the purpose of screening drugs, compounds having a low molecular weight therapeutic effect may be used. For example, compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely illustrative. It is not limited to this.

Biologics can also be used for screening, for example. Biologics refers to cells or biomolecules, and biomolecules refer to proteins, nucleic acids, carbohydrates, lipids or substances produced using cellular systems in vivo and ex vivo. Biomolecules may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, proteins or biological organics found in polynucleotides, peptides, antibodies, or other plasma.

As a result, candidates are expected to inhibit phosphorylation of LSD1 and / or demethylation of p65 subunit of NF-κB and / or binding of LSD1 and p65 in the presence of the test substance as compared to the control group not in contact with the test substance. Selected by substance. At least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about Candidates may be selected to have an increase or decrease of at least 100% or higher.

Proteins as used herein can be prepared using methods known in the art. In particular, the use of genetic recombination technology. For example, the plasmid containing the corresponding gene encoding the protein may be delivered to prokaryotic or eukaryotic cells such as insect cells and mammalian cells, overexpressed and purified. The plasmid may be used, for example, after transfection into an animal cell line, such as used in the exemplary embodiments herein, to purify the expressed protein, but is not limited thereto. In this case, the protein may be labeled using a variety of markers such as biotin, fluorescent material, acetylation, radioisotopes, or commercially available protein labeling kits for ease of detection, and labeled materials. Can be detected using a detector suitable for.

Or by expressing the DNA or RNA sequence encoding the screening protein in a suitable host cell to make the cell lysate or by translating the mRNA of the screening protein in vitro and purifying the screening protein by protein isolation methods known in the art. Can be. Usually, in order to remove cell debris and the like, the cell lysate or the result of in vitro translation is centrifuged, followed by precipitation, dialysis, and various column chromatography. Ion exchange chromatography, gel-permeation chromatography, HPLC, reverse phase-HPLC, preparative SDS-PAGE, affinity columns and the like are examples of column chromatography. Affinity columns can be made, for example, using anti-screening protein antibodies.

In addition, the type of test substance used in the method may refer to the aforementioned.

Inflammatory response modulators, in particular inhibitory substances, selected by the method according to the invention can be used as therapeutic agents for inflammatory diseases.

Inflammatory diseases herein include various symptoms or diseases caused by an inflammatory or inflammatory response, such as sepsis, allergy, asma, autoimmune diseases, hepatitis, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury and transplant rejection, etc. Various symptoms or diseases.

Diseases caused in particular by the NF-κB-mediated inflammatory response include, but are not limited to, sepsis, rheumatoid arthritis or autoimmune diseases.

In one embodiment according to the invention the method according to the invention is used in particular for the screening of sepsis therapeutic material. In another embodiment the substances screened by the method according to the invention are also useful as therapeutic agents for sepsis.

As used herein, the term “septicemia” refers to Enterococcus spp., Staphylococcus spp., Streptococcus spp, Enterobacteriacae family, Providencia spp. And Pseudomonas spp. A systemic inflammatory response resulting from bacterial or parasitic infections, such as, and the like, refers to a disease exhibiting symptoms such as increased heart rate, hypotension, hypothermia, hyperthermia, rapid breathing, and an increase or decrease in white blood cell count. In particular, in one embodiment according to the present application, Lsd1 ^{SA / SA} mice showed higher survival rates with lower inflammatory cytokine production than WT mice under conditions that mimic sepsis. These data demonstrate that targeting PKCα-LSD1 signaling may be a powerful therapeutic strategy for inflammatory diseases such as sepsis.

As used herein, the term “treatment” is a concept that includes inhibiting, eliminating, alleviating, alleviating, ameliorating, and / or preventing a disease or condition or condition resulting from the disease.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example

Experiment Method and Materials

Cell Culture: Raw264.7 (KCLB), L929, MEFs cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Welgene) with ZelShield (Minerva Biolabs GmbH) 1%, 10% FBS. To ensure conditions without mycoplasma, mycoplasma testing of all cells was routinely performed.

BMDM Preparation: Mice were euthanized via CO ₂ oversuction and mice obtained femur and tibia. After washing with 70% ethanol and cold PBS, the bone marrow was separated from the femur and tibia. Bone marrow cells were cultured in RPMI-1640 medium (Welgene, 1% ZelShield, 10% FBS) at a concentration of 1 × 10 ⁶ to 2 × 10 ⁶ / ml. Since then, Macrophage colony stimulating factor (10ng / ml, sigma) and 10% L929-conditioned medium were added to the cells and differentiated for 7-8 days.

Antibodies and reagents: Santa Cruz Biotechnology product: p65 antibody (sc-372, diluted 1: 1000 for immunoblot, diluted 1: 200 for immunofluorescence), LaminA / C antibody (sc-6215 , Diluted 1: 1000 for immunoblot, GFP antibody (sc-9996, diluted 1: 5000 for immunoblot), GST antibody (sc-459, diluted 1: 5000 for immunoblot) Use); Cell Signaling Products: LSD1 antibody (# 2139, diluted 1: 1000 for immunoblot), PKCα antibody (# 2056, diluted 1: 1000 for immunoblot); Abcam products: p65 antibody (ab7970), LSD1 antibody (ab17721), H3K9Ac antibody (ab4441), H3K9me2 antibody (ab1220), H3K4me2 antibody (ab32356); Novus product: LSD1 antibody (NB-100-1762), PKCα antibody (NB-110-57356, diluted 1: 1000 for immunoblot), C / EBPδ antibody (NB-110-85519, for immunoblot Dilute to 1: 1000 and use); Millipore product: p-PKCa S657 antibody (# 06-822, diluted 1: 1000 for immunoblot) p-LSD1 antibody (ABE 1462, diluted 1: 200 for immunoblot); Sigma products: β-actin antibody (A1978, diluted 1: 5000 for immunoblot), Flag antibody (F3165, diluted 1: 5000 for immunoblot), Lipopolysaccharides (LPS) from E. coli O127 : B8 (L3129), GSK-LSD1 (SML1072); Other third party products: HA antibody (MMS-101R, diluted 1: 5000 for immunoblot, diluted 1: 200 for immunofluorescence, Covance product), Tubulin antibody (LF-PA0146A, immunoblot Diluted 1: 1000 for use, Abfrontier product), mono-methyl-p65 (K314 / 315) antibody (ENH006, diluted 1: 500 for immunoblot, Elabscience Biotechnology) FK2 antibody (BML-PW8810) , Diluted 1: 1000 for immunoblot, Enzo Life Sciences), Go6976 (13310, Cayman), MG132 (M-1157, AG Scientific), TNT T7 Quick Coupled Transcription / Translation System (L1170, Promega) Product), λ-phosphatase (P0753, from NEB).

Animal Breeding : A method made by Lsd1 ^{SA / SA} mice with a C57BL / 6J background has already been described (Nam et al., Mol. Cell. 2014; 53: 791-805). Wild-type and Lsd1 ^{SA / SA} mice of 8-10 weeks of age were used for the experiment. Mice were maintained at 22-23 ° C. room temperature and light conditions (12 hours light: 12 hours dark, light on at 8 am). Food and water were provided for free eating. All animal experiments were approved by the Institutional Animal Care and Use Committee.

LPS Treatment : After the cells were grown in the absence of FBS for 24 hours, LPS (1 μg / ml or the indicated concentration) was treated for the indicated time. Cells were collected and lysed according to each experimental method for use in immunoblot or quantitative RT-PCR or ChIP experiments.

Quantitative RT-PCR : Total RNA was isolated from lung tissue, BMDM, or Raw264.7 cells using TRIzol (Invitrogen). RNA was reverse transcribed using oligo dT primers and M-MLV Reverse Transcriptase (Enzynomics). The resulting cDNA was PCR with TOPreal ^™ qPCR 2X PreMix (SYBR Grenn with high ROX, Enzynomics) and gene specific primers. mRNA amount was detected by ABI-7500. Primer information is as follows.

mouse Mcp-1 Forward 5’-GGCTCAGCCAGATGCAGTTAAC-3 ’,

mouse Mcp-1 Reverse 5’-AGCCTACTCATTGGGATCATCTTG-3 ’,

mouse Il-6 Forward 5’-CATAAAATAGTCCTTCCTACCCCAAT-3 ’,

mouse Il-6 Reverse 5’-CACTCCTTCTGTGACTCCAGCTTA-3 ’,

mouse Il-1b Forward 5’-GATGATAACCTGCTGGTGTGTGA-3 ’,

mouse Il-1b Reverse 5’-GTTGTTCATCTCGGAGCCTGTAG-3 ’,

mouse Cebpd Forward 5’-CTCCACGACTCCTGCCATGT-3 ’,

mouse Cebpd Reverse 5’-GAAGAGGTCGGCGAAGAGTTC-3 ’,

mouse RelA Forward 5’-TGTGGAGATCATCGAACAGCCG-3 ’,

mouse RelA Reverse 5'-TTCCTGGTCCTGTGTAGCCATTGAT-3 '.

RNA-seq analysis : RNA-seq libraries were prepared using TruSeq RNA Sample prep kit v2 (Illumina) according to the producer's instructions. RNA-seq libraries were pair-end sequencing in Illumina Hi-seq 3000/4000 SBS kit v3 (MACROGEN Inc.). All RNA-seq data was mapped in the mouse genome (mm9) using the Tophat package (Kim et al., Genome Biol. 2013; 14: R36). Differential analysis was performed using EdgeR packages with a false discovery rate (FDR) cutoff of 1 × 10 ⁻⁴ (Kim et al., Genome Biol. 2013; 14: R36; Robinson et al., Bioinformatics. 2010; 26: 139-140). Was performed. Hierarchical clustering analysis was performed using gene expression values of DEG. In particular, Ward's criterion was used for genes with 1- (correlation) as the distance measure. Cluster heatmaps were drawn using the z score in the sample for each gene. ChIP-seq data was mapped to the mouse genome using Bowtie. Peaks for p65 were performed with Homer's findPeaks command using the matching input control. In each DEG, the p65 peak located within 10 kbps of TSS was examined. De novo p65 peaks were performed using the findMotifsGenome command in Homer. Global run-on sequencing (GRO-seq) was used before and after LPS treatment in macrophages to obtain the amount of nascent transcript. The GRO-seq fastq file trimmed trim_galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to discard the polyA and adapter sequences (Hah et al., Proc. Natl. Acad. Sci. USA; 2015; 112: E297-E302). The trimmed read values were mapped with bowtie (v1.0.0) using the -best -v 2 -m 1 option. The bowtie file is tagged with the makeTagDirectory feature of HOMER. The average profile was obtained after normalizing the mapped read value to 1 × 10 ⁻⁷ .

ChIP experiment : Cells were cross- linked in 1% formaldehyde for 10 minutes and washed twice with cold PBS. The cells were placed in 1 ml of harvest buffer (0.1 M Tris-HCl [pH 9.4], added 10 mM DTT before the experiment), scraped with a scraper, placed in a 1.5 ml tube, placed at 30 ° C. for 15 minutes, and then centrifuged at 6000 rpm for 3 minutes. It was. After washing the pallet of cells with cold PBS, buffer I (0.25% Triton X-100, 10 mM EDTA, 10 mM HEPES [pH 6.5], and 0.5 mM EGTA) and buffer II (200 mM NaCl, 1 mM EDTA, 10 mM HEPES [pH 6.5], and 0.5 mM EGTA) were added sequentially and washed. Add ChIP lysis buffer (50 mM Tris-HCl [pH 8.1], 1% SDS, 10 mM EDTA [pH 7.6], add protease inhibitor cocktail before the experiment) and prepare chromatin sections via sonication. Chromatin extract made from DNA fragments of 250 bp in average length was diluted with dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl [pH 8.1], added protease inhibitor cocktail before the experiment). The antibody was added and immunoprecipitated overnight at 4 ° C. The next day, 40 μl of protein A / G sepharose beads were added and rotated at 4 ° C. for 2 hours. Beads were TSE I buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], and 150 mM NaCl), TSE II buffer (0.1% SDS, 1% Triton X- 100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], and 500 mM NaCl), buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 10 mM Tris-HCl [pH 8.1] and 1 mM EDTA), three times TE buffer (10 mM Tris-HCl [pH 8.0], and 1 mM EDTA). Elution buffer (1% SDS, 0.1 M NaHCO ₃ ) was used to elute from the beads. The eluted extracts were left at 65 ° C. overnight for reverse cross-linking and DNA was purified using QIA quick Gel Extraction Kit (QIAGEN). Purified DNA was analyzed via quantitative RT-PCR. 2 μl of a total of 50 ul of DNA was used for PCR. Sequence information of the PCR primer is as follows.

mouse Mcp-1 Forward 5’-CACCCCATTACATCTCTTCCCC-3 ’,

mouse Mcp-1 Reverse 5’-TGTTTCCCTCTCACTTCACTCTGTC-3 ’,

mouse Il-6 Forward 5’-AGCTACAGACATCCCCAGTCTC-3 ’,

mouse Il-6 Reverse 5'-TGTGTGTCGTCTGTCATGCG-3 '.

ChIP-seq analysis : ChIP-seq libraries were prepared using a KAPA library preparation kit according to the manufacturer's instructions. The ChIP-seq library was single-ended sequenced in Illumina Hi-seq 4000 SBS kit v3 (MACROGEN Inc.). ChIP-seq reads were aligned to the mouse reference genome (NCBI build 37, mm9) using Bowtie (Langmead et al., Genome Biol. 2009; 10: R25). Only unique peaks were used by HOMER for peak calling and annotating (Heinz et al., Mol. Cell. 2010; 38: 576-589). After LPS treatment, the p65 peak overlapping with LSD1 was considered. The BedGraph file was created and viewed in the Santa Cruz Genome Browser at the University of California. P65 (SC-372) and LSD1 (ab1772) antibodies were used for ChIP-seq.

Preparation of Tissue Lysate : Lung tissue was washed with cold PBS before homogenizing, to remove blood. Lungs were added to RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl [pH 7.5], and 2 mM EDTA [pH 8.0], protease inhibitor cocktail before the experiment). ), And then, centrifuged at 4 ℃, 14,000g, clean supernatant was used for immunoblot experiment.

Whole cell lysate and intracellular fractionation : All cells were washed with cold PBS. Resuspend in RIPA buffer containing protease inhibitor to make whole cell lysate, and output 3, duty cycle with 30 and 5 pulses using Branson Sonifier 450. Cells were lysed with Buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, pre-test DTT, PMSF, protease inhibitors added) and separated on ice for 15 minutes. 0.5% NP-40 was added and centrifuged for 1 minute at 120 g 4 ° C. Supernatant (cytoplasmic part) was transferred to a new tube. The pellet, which is the nucleus part, was centrifuged at 120 g 4 ° C for 1 minute. Discard the supernatant and re-dissolve the pellet in buffer C (20 mM HEPES [pH 7.9], 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, pre-test DTT, PMSF, protease inhibitors added) and then sonicate as whole cell lysate. do. All lysates were quantified by Bradford and analyzed via SDS-PAGE.

In vitro kinase experiment : Kinases were prepared by immunoprecipitation of PKCα in HEK293T cell lysast, and the substrates GST-LSD1 and GST-p65 were purified from E. coli. PKCα, GST-LSD1, and GST-p65 were reacted with kinase assay buffer (40 mM Tris-HCl [pH 7.5], 10 mM MgCl ₂ , 1mMDTT, and 5 μCiof [γ- ³² P] ATP) for 30 minutes. 5X sample buffer was added to the reaction and boiled for 10 minutes. SDS-PAGE experiments were conducted with the samples, and phosphorylation was detected by autoradiography.

In vitro GST pull-down experiments : GST fusion constructs were expressed in Rosetta E. coli bacteria (Novagen). Crude extracts were prepared by sonication in GST binding buffer (125 mM NaCl, 20 mM Tris-HCl [pH 7.8], 10% Glycerol, 0.1% NP-40, 0.5 mM DTT added before protease inhibitors), 30 at 13000 rpm. Lysates were centrifuged for minutes. Only the supernatant was separated and 100ul of glutathione-Sepharose beads (GE Healthcare) was added and rotated overnight at 4 ° C. p65 protein was prepared using cold methionine of TNT T7 Quick Coupled Transcription / Translation system (Promega, L1170) as directed by the producer. In vitro kinase experiments with cold ATP were performed to produce phosphorylated GST-LSD1 before GST pull-down experiments. Phosphorylated LSD1 was divided into two tubes by the same amount, and one tube was treated with 1000 units of λ-phosphatase (NEB, P0753) and reacted for 30 minutes at 30 ° C. GST fusion proteins bound to beads were washed with buffer (150 mM NaCl, 25 mM Tris-HCl [pH 8.0], 10% Glycerol, 0.1% NP-40, and 1 mM EDTA) and mixed with p65 protein synthesized in vitro Rotation was performed at 4 ° C. for 1 hour in GST binding buffer. Beads were washed 7 times with GST binding buffer, and the sample buffer was boiled for 10 minutes and analyzed by SDS-PAGE and immunoblot.

In vitro methylation demethylation experiments : In vitro methylation experiment methods have been previously reported (Kim et al., Nat. Commun. 2016; 7: 10347). Flag-p65 was purified from HEK293T extract expressing p65 with Flag-tag using Flag M2 agarose beads (Sigma, A2220). After overnight incubation at 4 ° C., BC500 buffer (20 mM Tris-HCl [pH 7.9], 15% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.2 mM PMSF, 0.05% Nonidet P40, to remove the protein bound to the beads) And 500 mM KCl). After a clean wash, the beads were washed three times with methylation assay buffer (50 mM Tris-HCl [pH 8.5], 20 mM KCl, 10 mM MgCl ₂ , 10 mM Mb-mercaptoethanol, and 250 mM sucrose). In vitro methylation experiments were performed with flag-p65 bound to beads, GST-SET7 / 9 protein isolated from beads (L-Glutathione reduced, Sigma G4251) and SAM (Sigma, A7007) in methylation assay buffer Was performed overnight. Wash buffer (50 mM NaH ₂ PO ₄ [pH8.0], 10 mM Tris-HCl [pH8.0] to completely remove SET7 / 9 protein bound to bead-defected flag-p65 prior to in vitro demethylation experiments ], 500 mM NaCl, and 0.5% TritonX-100) and clean with demethylation buffer (50 mM Tris-HCl [pH 8.5], 50 mM KCl, 5 mM MgCl ₂ , 5% glycerol, and 0.5 mM PMSF). After the change, LSD1 protein or phosphorylated LSD1 protein was added. After reacting at 37 ° C. overnight, 2X sample buffer was added, boiled for 10 minutes, SDS-PAGE, and analyzed by immunoblot.

Ubiquitination Experiment : Cells were transfected with DNA plasmids with Hismax-ubiquitin. 48 hours after transfection, MG132 (5 μg / ml) was treated for 4 hours. Cells were then disrupted with buffer A (6 M guanidium-HCl, 0.1 M Na ₂ HPO ₄ / NaH ₂ PO ₄ , 0.01 M Tris-HCl [pH8.0], 5 mM imidazole and 10 mM β-mercaptoethanol), followed by Ni ² at room temperature with ⁺ -NTA beads (QIAGEN) it was incubated for 4 hours. Beads were buffer A, buffer B (8 M urea, 0.1 M Na ₂ PO ₄ / NaH ₂ PO ₄ , 0.01 M Tris-Cl [pH8.0], and 10 mM β-mercaptoethanol), buffer C (8 M urea, After washing with 0.1 M Na ₂ PO ₄ / NaH ₂ PO ₄ , 0.01 M Tris-Cl [pH6.3], and 10 mM β-mercaptoethanol, the proteins attached to the beads were buffer D (200 mM imidazole, 0.15 M). Tris-Cl [pH 6.7], 30% glycerol, 0.72 M β-mercaptoethanol, and 5% SDS). And analyzed by immunoblot.

Denatured immunoprecipitation for endogenous ubiquitination assay : Raw264.7 cells were broken in denaturing buffer (50 mM Tris-HCl [pH 7.5], 70 mM β-Mercaptoethanol, and 1% SDS) and boiled for 5 minutes at 95 ° C. After dilution with non-denaturing lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100), immunoprecipitation was carried out using p65 antibody. Samples were analyzed by immunoblot using FK2 antibody.

Immunofluorescence Assay: Lsd1 ^{− / −} MEFs were grown on cover slips coated with 1% gelatin. Cells were washed twice with PBS for immunofluorescence experiments and fixed for 2 minutes with 2% formaldehyde. The cells were washed twice with PBS dissolved in 0.1% triton-X 100. Cells were incubated in 0.5% triton-X 100 dissolved PBS for 5 minutes to infiltrate the antibody and then blocked with blocking buffer (5% BSA in 0.1% PBS-T) to prevent nonspecific binding of the antibody. . Primary antibody (HA, MMS-101R Covance, diluted 1: 200 with blocking buffer; anti-p65, sc-372 from Santa Cruz, 1: 200 diluted with blocking buffer) was added to the blocking solution and incubated with the cells. . Thereafter, the mixture was washed 8 times with 0.1% PBS-T solution and incubated with secondary antibody (diluted 1: 200 with Invitrogen, molecular probes, blocking buffer) for 1 hour. After washing 8 times with 0.1% PBS-T solution again, it was mounted with a vectashield (vectashield) (H-1000), and obtained by a confocal microscope (Carl Zeiss, LSM700).

Induction of sepsis: For cecal ligation and puncture (CLP), male mice are given oxygen via a small rodent gas anesthesia machine (RC2, Vetequip, Pleasanton), 2% isoflurane (Forane). , JW pharmaceutical). Anesthesia was first anesthetized in the breathing chamber and through the facial mask to allow for natural breathing during surgery. CLP-induced sepsis was performed as previously described (Wang et al., Nat. Med. 2004; 10: 1216-1221). In short, a 2 cm midline incision was placed to expose the cecum and adjacent intestines. The cecum was then tightly bound to 5.00 mm from the end of the cecum using 3.0-silk sutures and punctured using a 22 gauge needle. The cecum was then gently squeezed out and a small amount of feces was drained out of the perforation and returned to place in the abdominal cavity. The open surgical site was closed with a 4.0-silk suture.

Hematoxylin and eosin (H & E) staining: Lung samples were extracted from each mouse to analyze histological changes in mouse lungs, washed in PBS three times to remove blood and 4 ° C. in 4% formaldehyde solution (Junsei) Fixed for 20 hours. After fixation the samples were dehydrated with ethanol series and paraffin embedded. Sections were made 4 μm thick and placed on slides. Slides were deparaffinized and rehydrated in a 60 ° C. oven and then stained with hematoxylin (Sigma). Slides were soaked three times in acidic alcohol to remove excess staining and counterstained with eosin (Sigma). Then washed with ethanol series, soaked in xylene and mounted. Blind analysis was performed by light microscopy to evaluate lung structure, tissue edema and infiltration of inflammatory cells in lung tissue.

Clinical Chemistry and Cytokine Secretion Level Determination in Sepsis Mouse Plasma : Alanine transaminase (ALT), blood urea nitrogen (BUN), and LDH were measured with biochemical kits (Mybiosource) using fresh serum. To check the concentrations of IL-6, MCP-1, and TNF-α, an ELISA kit (R & D Systems) was used according to the manufacturer's instructions. Values were measured using an ELISA plate reader (Tecan, GmbH).

Statistical Analysis: Using GraphPad Prism software, data were analyzed by Student t-test and log rank test to see the difference of group, and two-way ANOVA to see the difference between group and condition. ^* p <0.05, ^** p <0.01, ^*** p <0.001.

Example 1 Significance Analysis of In Vivo Inflammatory Response of LSD1 Phosphorylation by PKCα

In order to determine whether phosphorylation of LSD1 is important for the inflammatory response in vivo, it was confirmed whether LPS-induced inflammatory response occurred properly in Lsd1 ^{SA / SA} mice. Wild-type (WT) and Lsd1 ^{SA / SA} mice were used to analyze LPS-induced inflammation and acute lung injury mouse models. Histopathological examination confirmed that WT mice injected with LPS showed severe lung and alveolar damage, but the response was significantly weakened in Lsd1 ^{SA / SA} mice (FIG. 1A). 80% of WT mice died within 66 hours of LPS administration, but only 30% of Lsd1 ^{SA / SA} mice died within the same time period (FIG. 1B). The inventors have found that Lsd1 ^{SA / SA} mice are less likely to develop LPS-induced inflammatory reactions, acute lung injury, and thus have low mortality. LPS treatment of bone marrow-derived macrophages (BMDM) from WT mice induced PKCα and LSD1 phosphorylation, whereas BMDM from Lsd1 ^{SA / SA} mice induced phosphorylation of PKCα but not LSD1 phosphorylation. It was confirmed (Fig. 1C). In addition to BMDM, phosphorylation of LPS-induced LSD1 was also detected in lung tissue lysate in WT mice. When mice were pre-injected with Go6976 and injected with LPS, it was also confirmed that phosphorylation of LSD1 was completely blocked (FIG. 1D).

To confirm how LPS induces phosphorylation of LSD1, immunoblot experiments were performed by fractionating BMDM cells into cytoplasm and nucleus. LPS treatment induced phosphorylation of PKCα, the active form of PKCα, and found that phosphorylated PKCα entered the nucleus (FIG. 1E). In addition, the migration of LPS-induced PKCα into the nucleus induced LSD1 and PKCα binding in the nucleus (FIG. 1F). To determine when phosphorylation occurred, the LPS was treated at each time period, and the phosphorylation of PKCα increased with phosphorylation of LSD1 from 60 minutes after LPS treatment was observed in BMDM (FIG. 1G). It was also confirmed that phosphorylation of PKCα and LSD1 was induced when high concentrations (> 100ng / ml) of LPS were treated (FIG. 1H). To identify the molecular mechanism of the inflammatory response associated with phosphorylation of LSD1, experiments were then conducted at high concentrations of LPS. In vivo mouse experiments showed that Lsd1 ^{SA / SA} mice were not prone to LPS-induced inflammatory responses, acute lung injury, and low mortality. Phosphorylated PKCα migrates to the nucleus to induce LSD1 phosphorylation only after treatment with high doses of LPS (> 100ng / ml) for more than 60 minutes.

Example 2 Phosphorylation of LPS-induced LSD1 Analyzes Necessity for Activation of NF-κB Target Gene at Genome-wide Level

Because LPS-induced transcriptional modules are precisely regulated for initiation and amplification of the inflammatory response (Medzhitov and Horng, 2009), we attempted to identify which gene sets depend on LSD1 phosphorylation in the transcriptional regulation of inflammatory responses. RNA-sequencing was performed after LPS treatment on BMDM extracted from WT and Lsd1 ^{SA / SA} mice (FIG. 2A). In LPS treatment, total transcription amounts of WT and Lsd1 ^{SA / SA} BMDM were compared to identify a total of 3,558 differently expressed genes (hereinafter referred to as DEGs) by unsupervised hierarchical cluster analysis (see experimental method). Of these genes, we were particularly interested in the gene pool (cluster1 in FIG. 2B) which is activated in LPS treatment in WT BMDM but not in Lsd1 ^{SA / SA} BMDM. It was thought that phosphorylated LSD1 would act as a coactivator of transcription. Unlike the genes of other clusters, the gene of Cluster 1 showed significantly higher p65 peak (hyper-geometric p-value <1e-130) around the transcription start site (hereinafter, TSS). This means that p65 is a major transcription factor (TF), which induces the expression of genes present in cluster 1 upon LPS treatment (FIG. 2B). Gene ontology (GO) analysis using Enrichr (http://amp.pharm.mssm.edu/Enrichr/) confirmed that many genes related to cytokine production and inflammatory responses existed in cluster 1 (FIG. 2C). . In addition, De novo motif analysis confirmed that p65 is the major TF of the cluster 1 gene (FIG. 2D). The results of the RNA-seq data were once again confirmed by quantitative RT-PCR analysis (eg Mcp-1, Il-6, Il-1β and Cebpd of Cluster 1) (FIG. 2E). In addition, as a result of confirming mRNA level of LSD1 phosphorylation dependent gene in cluster 1 by using mRNA extracted from lung tissue of mouse, it was confirmed that it was significantly lower in Lsd1 ^{SA / SA} compared to the control WT mouse at the mouse individual level. Consistently, pre-injection of Go6976 into WT mice confirmed that the expression of LSD1 phosphorylation dependent gene in lung tissue mRNA was lower than that of the control (FIG. 2G). This data indicates that phosphorylation of LSD1 induced by LPS is required for activation of the NF-κB target gene associated with p65.

Example 3 Analysis of LSD-Induced LSD1 Phosphorylation is Important for Recruitment of NF-kb p65 to Target Promoter

Since p65 was identified as a major TF (transcription factor) regulated by phosphorylated LSD1, we investigated whether LSD1 binds to p65 in response to LPS. It was confirmed in BMDM cells that LSD1 binds to p65 in the nucleus upon LPS treatment (FIG. 3A). Since LSD1 was phosphorylated in response to LPS, it was assumed that phosphorylated LSD1 would bind to p65. In vitro kinase experiments were performed with p65 as a substrate because PKCα is likely to directly phosphorylate p65 as well as LSD1. PKCα did not phosphorylate p65 (FIG. 3B). This means that the direct substrate of PKCα in LPS-PKCα signaling is LSD1, not p65. To determine if LSD1 phosphorylation is important for binding to p65, GST pulldown analysis was performed using LSD1 phosphorylated in vitro. Prior to GST pulldown analysis, kinase experiments using PKCα were performed to obtain phosphorylated LSD1. GST pulldown analysis revealed that p65 binds directly to phosphorylated LSD1 and that treatment with phosphatase almost completely eliminates binding between LSD1 and p65 (FIG. 3C). This data indicates that LSD-induced LSD1 phosphorylation by PKCα is essential for binding to p65 in the nucleus.

As obtained in the mRNA-seq analysis (FIG. 2), the genes of Mcp-1 and Il-6 had LSD1 phosphorylation dependent expression. To determine if LSD1 phosphorylation affected the recruitment of LSD1 and p65 at the NF-κB target promoter, chromatin immunoprecipitation (ChIP) analysis was performed on WT and Lsd1 ^{SA / SA} BMDM cells. ChIP analysis showed that LPS1 and p65 recruitment increased significantly in the Mcp-1 and Il-6 promoters of WT BMDM when treated with LPS, but their recruitment was weakened in Lsd1 ^{SA / SA} BMDM (FIG. 3D). To investigate whether LPS treatment induced changes in histone methylation status, the histone H3K4 or H3K9 methylation status, which can be regulated by LSD1, was examined (Shi et al., Cell. 2004; 119: 941953). ChIP analysis of the Mcp-1 and Il-6 promoters of WT and Lsd1 ^{SA / SA} BMDM cells confirmed that histone H3K4me2 and H3K9me2 levels were similar. This indicates that target gene activation associated with LSD1 phosphorylation following LPS is independent of demethylation of histones. On the other hand, histone H3K9 acetylation levels increased on the Mcp-1 and Il-6 promoters in WT BMDM upon LPS treatment, but not on Lsd1 ^{SA / SA} BMDM (FIG. 3D). It is presumed that histone H3K9 acetylation did not increase because p65, the TF in Lsd1 ^{SA / SA} BMDM, was not recruited to the target promoter after LPS treatment. Furthermore, treatment of Go6976 in Raw264.7 macrophages almost completely blocked histone H3K9 acetylation, LSD1 and p65 recruitment to Mcp-1 and Il-6 promoters (FIG. 3E). These data indicate that phosphorylation of LPS-induced LSD1 is important for binding to p65, and that LSD1 and p65 recruitment of some NF-κB target promoters controls the phosphorylation of LSD1.

Example 4 Demethylation of p65 by LSD1 is Critical for Improving p65 Protein Stability

Because LSD1 phosphorylation is important for recruiting p65 to the target promoter properly in response to LPS, the study focused on how LSD1 regulates p65. It was found that p65 was methylated by the SET7 / 9 methylase by the K314 / 315 site and that methylation of p65 caused degradation of p65 (Yang et al., EMBO J. 2009; 28: 1055-1066). However, the identity of demethylases responsible for p65 demethylation and thus stabilization of p65 protein is unknown. We first investigated whether LSD1 is responsible for the demethylation of p65. Prior to in vivo demethylation analysis using the LSD1 K661A (KA) mutant lacking enzyme activity, the LSD1 KA mutant was first identified for its effect on binding to p65. LSD1 WT and LSD1 KA mutants showed similar binding to p65 upon LPS treatment, but LSD1 S112A (SA) mutants with phosphorylation defects failed to bind to p65 (FIG. 4A). Thereafter, demethylation analysis of p65 was performed and found that methylation of p65 by SET7 / 9 was eliminated by LSD1 WT while SA or KA mutants of LSD1 could not eliminate p65 methylation (FIG. 4B). The LSD1 SA mutant had demethylation enzyme activity similar to WT, but failed to demethylate p65 because binding to p65 requires phosphorylation of LSD1. We also performed in vitro demethylation assays using p65 methylated specific antibodies at the K314 / 315 site, and found that p65 methylation induced by SET7 / 9 was degraded by phosphorylated LSD1 (FIG. 4C). ). This data indicates that in response to LPS, LSD1 acts as a demethylase of p65, and phosphorylation of LSD1 is important for binding to p65. To clarify that LSD1 phosphorylation status affects p65 protein stabilization, the levels of p65 protein in the nuclei were compared in WT and Lsd1 ^{SA / SA} BMDM. Interestingly, after LPS treatment in Lsd1 ^{SA / SA} BMDM, no intranuclear p65 protein was detected, and treatment with 26S proteasome inhibitor MG132 restored the p65 protein expression in the nucleus of Lsd1 ^{SA / SA} BMDM (FIG. 4D). . It was also confirmed that the p65 protein recovered by MG132 treatment in the nucleus of Lsd1 ^{SA / SA} BMDM was methylated p65 (FIG. 4D). This means that phosphorylated LSD1 can bind to p65 and can prevent p65 methylation dependent proteolysis in the nucleus through the demethylase function of LSD1. Ubiquitination experiments were also performed for p65. Intracellular introduction of LSD1 SA or KA mutants increased p65 ubiquitination in the nucleus, but not in the cytoplasm (FIG. 4E). In addition, treatment with Go6976, which blocks the activity of PKCα, or GSK-LSD1, which blocks the activity of LSD1, significantly induced endogenous ubiquitination of p65 in the nucleus (FIG. 4F). Through a series of data we present evidence that both phosphorylation and demethylation activity of LSD1 is important for stabilization of p65 protein (FIG. 4G).

Example 5 PKCα-LSD1 Phosphorylation Axis Acts as an Axis for Controlling Persistent Inflammatory Responses

Transcription is regulated by sequential steps of a series of transcription factors. Induction of LPS dependent transcription modules is coordinated by many TFs (Litvak et al., Nat. Immunol. 2009; 10: 437443). Class I TF, including NF-κB, controls the onset of the inflammatory response within 2 hours of LPS induction. Class II TF, including C / EBPδ, is synthesized de novo after 2 hours of LPS stimulation and subsequently regulates the activation of inflammatory response genes for amplification of inflammatory signals. Class III TF, including PU.1 and C / EBβ, are lineage specific transcriptional regulators. All TFs cooperate with each other to regulate LPS-induced transcriptional responses. In RNA-seq analysis, de novo motif analysis using the Cluster1 gene confirmed PU.1 and C / EBP as well as p65 as the major TF (FIG. 2D). In addition, we identified Cebpd as one of the genes induced in LSD1 phosphorylation dependent manner in cluster 1. It was hypothesized that LSD1 phosphorylation regulates subsequent signal activation and amplification of the inflammatory response, since phosphorylation of LSD1 was induced 60 minutes after high doses of LPS, at which point Class I TF and Class II TF were relayed. Therefore, expression of inflammatory genes in WT and Lsd1 ^{SA / SA} BMDM was analyzed over time of LPS treatment. Interestingly, up to 30 minutes, the expression of Mcp-1 and Il-6 mRNAs in WT and Lsd1 ^{SA / SA} BMDM were similar, but from 60 to 120 minutes Mcp-1 and IL-6 mRNA expressions were significant compared to WT. Weakened in Lsd1 ^{SA / SA} BMDM (FIG. 5A). In addition, Cebpd mRNA expression was significantly attenuated in Lsd1 ^{SA / SA} BMDM compared to WT from 90 minutes after LPS treatment (FIG. 5A). Treatment of Go6976 with WT BMDM prior to LPS treatment showed a similar mRNA expression pattern to Lsd1 ^{SA / SA} BMDM treated with LPS (FIG. 5B). In addition, it was confirmed that p65 binds to LSD1 from 60 minutes after LPS treatment, which is the time when LSD1 is phosphorylated (FIG. 5C). This means that enhanced protein stabilization of p65 by LSD1 is important for the sustained activation of inflammatory genes. Since C / EBPδ functions as a TF for subsequent activation of inflammatory response genes, we performed ChIP analysis over LPS time course using LSD1, p65 and C / EBPδ antibodies to confirm the kinetics of promoter occupancy. From 60 minutes after LPS treatment, LSD1 was recruited to the Mcp-1 and Il-6 promoters of WT BMDM but not to Lsd1 ^{SA / SA} BMDM (FIG. 5D). Recruitment of p65 to the target promoter was detected in both WT and Lsd1 ^{SA / SA} BMDM 30 minutes after LPS treatment without LSD1 phosphorylation induction. However, 60 min after LPS treatment, p65 recruitment was maintained or increased in WT BMDM, whereas p65 recruitment was significantly decreased in Mcp-1 and Il-6 promoters of Lsd1 ^{SA / SA} BMDM (FIG. 5D). This data indicates that LSD1 phosphorylation plays an important role in maintaining p65 recruitment late in LPS treatment (after 60 minutes). C / EBPδ was also recruited at 120 after LPS treatment in WT BMDM, which leads to the relaying and amplification of the inflammatory response (FIG. 5D). In parallel, treatment with Go6976 yielded similar results to the ChIP results obtained with Lsd1 ^{SA / SA} BMDM (FIG. 5E). This data shows that the PKCα-LSD1-NF-κB signal cascade operates from 60 minutes after LPS treatment for transcriptional activation and subsequent amplification of inflammatory response genes (FIG. 5F).

Example 6 Analysis of Sustained Expression of p65 and Activation of Inflammation is Dependent on the PKCα-LSD1-NF-κB Signaling Cascade

We have identified a nuclear endogenous p65 protein levels in accordance with Lsd1 ^{SA / SA} because observed in BMDM from 60 minutes after LPS treatment decreased the recruitment of p65 target promoters, the time elapsed from WT and Lsd1 ^{SA / SA} BMDM. The same p65 protein was detected in WT and Lsd1 ^{SA / SA} BMDM 30 minutes after LPS treatment, indicating that migration to LPS-induced p65 nuclei was not compromised at all in Lsd1 ^{SA / SA} BMDM (FIG. 6A). However, the p65 protein in the nucleus was not maintained in Lsd1 ^{SA / SA} BMDMs, and de novo synthesis of C / EBPδ also failed at 120 min after LPS treatment (FIG. 6A). In addition, the results of immunofluorescence analysis showed that Lsd1 ^{− / −} mouse embryonic fibroblasts (MEFs) to which LSD1 WT-, SA- or KA- were treated for 30 minutes and LPS1 were observed at the same level of p65. However, when processing the LPS 120 bun, LSD1 WT gave Lsd1 put ^{- / -} showed a p65 protein stabilization in the nucleus only MEFs LSD SA or semi Lsd1 put KA mutation ^{- / -} did not the MEFs (Fig. 6B). To further confirm that maintenance of p65 protein levels in the nucleus and sequential de novo synthesis of C / EBPδ is dependent on the PKCα-LSD1 signaling axis, immunoblot analysis was performed on nuclear fractions treated with Go6976 or GSK-LSD1. Treatment with Go6976 or GSK-LSD1 failed to stabilize p65 at 60 minutes after LPS treatment in WT BMDM (FIG. 6C) or Raw264.7 macrophages (FIG. 6D). In addition, treatment of MG132 restored the reduced p65 protein levels in the nucleus due to Go6976 or GSK-LSD1 (FIG. 6E). These data indicate that the PKCα-LSD1-NF-κB signaling cascade influences the maintenance of p65 protein levels in the nucleus and the sequential de novo synthesis of C / EBPδ, which is important for prolonging the inflammatory response.

Example 7 Reduction of Mortality from Sepsis Induction Due to Inhibition of PKCα Activity or LSD1 Activity in Mice

To investigate the in vivo effects of Go6976 or GSK-LSD1, mice were injected with Go6976 or GSK-LSD1, the lung tissue was nuclear fractionated, and immunoblot analysis was performed. When injected with Go6976 or GSK-LSD1, treatment with LPS did not stabilize p65 in the nucleus and did not newly synthesize C / EBPδ (FIG. 7A). Since LPS-induced systemic inflammation is much lower in Lsd1 ^{SA / SA} mice than in WT controls (FIGS. 1A and 1B), we sought to reaffirm previous findings in more severe sepsis mouse models. LPS infusion and surgery to bind and puncture the caecum (CLP) is a mouse model widely used to mimic human sepsis in rats. LPS injection mimics the early clinical characteristics of sepsis to induce systemic inflammation, but the CLP-induced sepsis model is a model that exhibits a cytokine profile similar to human sepsis, with a defecation secreting an immune response. Separation induced by various strains was induced through CLP surgery in WT and Lsd1 ^{SA / SA} mice of matching age, sex and weight. Interestingly, WT mice had a mortality rate of 100% within 90 hours after CLP surgery, whereas 50% of Lsd1 ^{SA / SA} mice survived 144 hours after CLP surgery (FIG. 7B). Next, the possibility of blocking PKCα or LSD1 activity reduced mortality and lung damage. Go6976, GSK-LSD1 or equivalent volume of solvent was injected twice at 12 and 50 hours after CLP surgery. Within 78 hours after CLP surgery, only solvent-injected mice had a 100% mortality, but only 40% of Go6976-injected mice and 50% of GSK-LSD1-injected mice died during the same period, which was more than 144 hours after CLP surgery. Survived (FIGS. 7C and 7D). Consistent with these data, histopathological examination revealed that WT mice showed severe lung and alveolar damage after CLP, but these responses were significantly significant in both Lsd1 ^{SA / SA} mice, mice injected with Go6976 or GSK-LSD1 in WT. It was shown to be reduced (FIG. 7E). We explored whether Go6976 or GSK-LSD1 treatment is effective in the regulation of inflammatory response genes in sepsis. Elevation of MCP-1, IL-6 and TNF-α by CLP was significantly reduced in Lsd1 ^{SA / SA} mice (FIG. 7F), Go6976 injected mice or GSK-LSD1 injected mice (FIG. 7G). Systemic inflammation in the onset of sepsis often leads to liver and kidney as the main target organs, causing multi-organ dysfunction. CLP significantly increased plasma levels of ALT (liver damage marker), LDH (tissue damage marker) and BUN (kidney damage marker). The concentrations of ALT, LDH and BUN in plasma were significantly reduced in Lsd1 ^{SA / SA} mice compared to WT mice (FIG. 7H), and also in plasma of mice injected with Go6976 or GSK-LSD1 (FIG. 7H). . 7I). Blocking PKCα activity or LSD1 activity, a sub-protein thereof, decreased the nuclear p65 protein stability, thus reducing acute systemic inflammation and increasing survival in sepsis (FIG. 7J).

Inflammatory responses are the main defense against invading pathogens and must be maintained until the pathogens are completely cleared to prevent the development of the disease caused by the pathogen. On the other hand, the inflammatory response also has a deleterious effect due to the activation of excessive inflammation, such as sepsis, so in order to avoid this, it must be terminated in a timely manner after the pathogen is removed. Therefore, understanding the molecular mechanisms of how excessive inflammatory responses are detected and maintained is essential to mitigate sepsis-induced mortality or organ damage. In this invention, the evidence that LSD1 phosphorylation by PKCα plays an important role in maintaining the protein stability of p65 and sustained inflammatory response after excessive LPS treatment. In vivo studies using Lsd1 ^{SA / SA} mouse mice have elucidated the function of LSD1 phosphorylation by PKCα in severe inflammatory responses. Lsd1 ^{SA / SA} mice that do not undergo phosphorylation of LSD1 significantly reduced excessive inflammatory response and tissue damage in sepsis shock. Go6976 or GSK-LSD1 treatment also significantly reduced excessive systemic inflammatory responses, demonstrating that PKCα activity and LSD1 activity are critical for activation of the inflammatory response. The PKCα-LSD1-NF-κB signaling cascade was found to play an important role in amplifying the inflammatory response and inducing inflammatory diseases including sepsis in response to excessive inflammatory stimuli.

It is important that LSD1 phosphorylation dependent genes responding to high doses of LPS include genes associated with sepsis and genes associated with Class II / III TF. The IL-6 and Mcp-1 genes of cluster 1 are well known genes associated with sepsis (Rincon, Trends Immunol. 2012; 33: 571-577). Interestingly, Cebpd ^{− / −} mice show less LPS-induced acute lung injury compared to WT and show reduced Il-6 gene expression, which is a phenotype similar to that of Lsd1 ^{SA / SA} mice.

Increased expression of Cebpd mRNA, which appears when the PKCα-LSD1-NF-κB signaling cascade works, means that this signaling cascade is crucial for subsequent amplification of the inflammatory response and ultimately leads to sepsis. Through the present invention, a functional link between excessive inflammatory stimulation and epigenetic regulation following an inflammatory response has been found. Acute and chronic inflammation induced by PMA, a PKC activator, has been previously reported, but the underlying PKC substrate and its target genes are not known. Since the present invention has discovered that PKCα enters the nucleus and phosphorylates LSD1 upon excessive inflammatory stimuli, PKCα connects the cytoplasm to the nucleus and recognizes and transmits an external excessive inflammatory stimulus for further activation of the inflammatory response. I think it functions as. Excessive inflammatory stimuli have found that LSD1 becomes a substrate of PKCα and functions as a demethylase of p65, increasing the stabilization of the p65 protein. In addition to phenotypic analysis of sepsis-induced Lsd1 ^{SA / SA} mice, inhibition of LSD1 activity with GSK-LSD1 or PKCα activity with Go6976 results in less tissue damage and higher survival by CLP surgery. Proved. Although antibiotic treatment for sepsis patients reduces mortality at an early stage, antibiotics cannot control the excessive inflammatory response itself during septic shock. In combination with antibiotics, a drug targeting the PKCα-LSD1-NF-κB signaling cascade, which causes the excessive inflammatory response described above, could be used to develop new therapeutics for sepsis patients with systemic inflammation. . In other words, the present invention indicates that the PKCα-LSD1-NF-κB signal transduction axis can be a potential therapeutic target in inflammatory diseases.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention. Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.

Claims (5)

1. Providing cells expressing PCKa, LSD1 and NF-κB;

   Treating the cells with a stimulus capable of inducing an NF-κB mediated inflammatory response, wherein the treatment causes an inflammatory response by the PCKα-> LSD1-> NF-κB pathway in the cells,

   Treating the cell with a test substance that is expected to inhibit the inflammatory response caused by the pathway; And

   If the result of the treatment, the inflammatory response by the pathway is inhibited in the cells treated with the test substance compared to the control group not treated with the test substance, the test substance includes the step of selecting an inflammatory response inhibitor candidate ,

   Inhibition of the inflammatory response by this pathway may be achieved by reducing phosphorylation of LSD1, inhibition of demethylation of p65 subunit of NF-κB or binding of LSD1 and p65 subunit A method of screening an inflammatory response inhibitor mediated by NF-κB, which is measured in at least one of the decreases.
2. The method of claim 1,

   The stimulus that can induce an NF-κB mediated inflammatory response is tumor necrosis factorα (TNFα), interleukin 1-beta (IL-1β), pathogen-associated molecular pattern (PAMP) or bacterial lipopolysaccharides (LPS).
3. The method according to claim 1 or 2,

   The cell is Raw 264.7 or bone marrow-derived macrophage (BMDM).
4. The method according to any one of claims 1 to 3,

   Instead of cells expressing the PCKa, LSD1 and NF-κB, the cells are provided as animal models except humans.
5. The method according to any one of claims 1 to 4, wherein the inflammation inhibitor is used as a therapeutic agent for sepsis, autoimmune disease or rheumatoid arthritis.

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