Cytokine production adjustment agent

Abstract

The purpose of the present invention is to provide an agent for adjusting cytokine production by lipopolysaccharide derived from Escherichia coli, or an agent for adjusting TLR4 activation by lipopolysaccharide derived from Escherichia coli. The present invention provides: an agent for adjusting cytokine production by lipopolysaccharide derived from Escherichia coli, the agent including lipopolysaccharide derived from bacteria belonging to genus Paracoccus; or an agent for adjusting TLR4 activation by lipopolysaccharide derived from Escherichia coli, the agent including lipopolysaccharide derived from bacteria belonging to genus Paracoccus.

Description

Cytokine production regulators

[0001] The present invention relates to an agent or method for regulating cytokine production, or an agent or method for regulating TLR4 activation.

[0002] Lipopolysaccharide (hereinafter also referred to as "LPS" or "lipopolysaccharide") is a component that constitutes the cell wall of Gram-negative bacteria such as Escherichia coli. When Gram-negative bacteria are killed or destroyed, LPS released from the bacteria induces reactions such as fever in mammals, and in large amounts can be fatal. Therefore, LPS is also called endotoxin.

[0003] The basic structure of LPS comprises a lipid portion (lipid A) and a polysaccharide portion. The polysaccharide portion is further composed of a core and an O-antigen polysaccharide side chain. Normally, LPS is incorporated into the cell wall of Gram-negative bacteria with the lipid A embedded in the outer membrane and the O-antigen polysaccharide side chain protruding from the cell wall, and is not easily released from the cell wall. When Gram-negative bacteria die and the cells are lysed, LPS is released and acts on animal cells, for example, to exert various physiological activities.

[0004] Released LPS exerts its effects via TLR4 (toll-like receptor 4) on the cell membrane of target cells. Binding of LPS to TLR4 induces the production of inflammatory cytokines such as TNFα, IL-6, and IL-12, as well as type I interferon (IFN), via the intracellular signaling pathway (Non-Patent Document 1). In particular, LPS from E. coli and other bacteria simultaneously and potently activates these signals, thereby inducing a strong inflammatory response.

[0005] Thus, LPS promotes cytokine production via the TLR4 signaling pathway. Although cytokine production generally leads to strong immunostimulatory activity and plays a role in the host's defense response to protect against bacterial infection, excessive cytokine production can cause toxicity and even sepsis.

[0006] In addition to LPS, TLR4 is known to recognize pathogen-associated molecular patterns (PAMPs), such as heat-shock proteins (HSPs) produced by some viruses and Chlamydia pneumoniae. Furthermore, TLR4 has been shown to recognize endogenous factors present in the body (damage-associated molecular patterns (DAMPs)), including high mobility group box protein 1 (HMGB1) and HSPs, which are released when autologous cells are damaged, as well as extracellular matrix degradation products such as fibronectin and hyaluronic acid, and fatty acids, and to induce the production of various inflammatory cytokines and IFNs from various cells (Non-Patent Document 2).

[0007] Non-Patent Document 3 discloses that lipid A derived from bacteria belonging to the genus Rhodobacter reduces the cytokine-inducing activity of LPS. However, this finding is limited to certain species, and no evidence is provided for LPS from bacteria belonging to the genus Paracoccus.

[0008] Biochemistry, 79, 769-776, 2007. Folia Pharmacol. Jpn. 144, 167-171 (2014). Lipid A-like Molecules That Antagonize the Effects of Endotoxins on Human Monocytes, J. Biol. Chem., 266, 29, 1991, 19490-19498.

[0009] An object of the present invention is to provide an agent that regulates cytokine production or TLR4 activation.

[0010] The present inventors have conducted extensive research and found that lipopolysaccharides derived from bacteria belonging to the genus Paracoccus regulate cytokine production and TLR4 activation. Specifically, the present invention is as follows: [1] An agent for regulating cytokine production, comprising lipopolysaccharides derived from bacteria belonging to the genus Paracoccus. [2] An agent for regulating TLR4 activation, comprising lipopolysaccharides derived from bacteria belonging to the genus Paracoccus. [3] The agent according to [1] or [2], wherein cytokine production or TLR4 activation induced by bacteria is regulated by lipopolysaccharides derived from bacteria belonging to the genus Paracoccus. [4] The agent according to [3], wherein cytokine production or TLR4 activation induced by Escherichia coli lipopolysaccharide is regulated by lipopolysaccharides derived from bacteria belonging to the genus Paracoccus. [5] The agent according to any of [1] to [4], wherein the regulation includes inhibition. [6] The agent according to any one of [1] to [5], which has a lipid A portion of a lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus, in which four 3-hydroxyacyl chains having 8 to 16 carbon atoms are bound to the glucosamine backbone of the lipid A, and an acyl chain having 8 to 13 carbon atoms is further bound to the hydroxyl group at the 3-position of one or two of the 3-hydroxyacyl chains. [7] The agent according to any one of [1] to [5], wherein the lipid A portion has a structure represented by formula (I): The agent according to [6], which has the formula: [8] A feed composition comprising the agent according to any one of [1] to [7]. [9] A pharmaceutical or food composition comprising the agent according to any one of [1] to [7].

[10] A method for regulating cytokine production, which comprises administering lipopolysaccharide derived from bacteria belonging to the genus Paracoccus to a subject.

[11] A method for regulating TLR4 activation, which comprises administering lipopolysaccharide derived from bacteria belonging to the genus Paracoccus to a subject.

[12] The method according to

[10] or

[11] , wherein cytokine production or TLR4 activation induced by bacteria is regulated by lipopolysaccharide derived from bacteria belonging to the genus Paracoccus.

[13] The method according to

[12] , wherein cytokine production or TLR4 activation induced by Escherichia coli lipopolysaccharide is regulated by lipopolysaccharide derived from bacteria belonging to the genus Paracoccus.

[14] The method according to any one of

[10] to

[13] , wherein the regulation comprises inhibition.

[15] The method according to any one of

[10] to

[14] , which comprises a lipid A portion of a lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus, wherein four 3-hydroxyacyl chains having 8 to 16 carbon atoms are bound to the glucosamine backbone of the lipid A, and an acyl chain having 8 to 13 carbon atoms is further bound to the hydroxyl group at the 3-position of one or two of the 3-hydroxyacyl chains.

[16] The lipid A portion has a structure represented by formula (I):

[15] The method according to

[17] , wherein the lipopolysaccharide is derived from a bacterium belonging to the genus Paracoccus for use in regulating cytokine production.

[18] The lipopolysaccharide is derived from a bacterium belonging to the genus Paracoccus for use in regulating TLR4 activation.

[19] The lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus according to

[17] or

[18] , wherein cytokine production or TLR4 activation induced by the bacterium is regulated by the lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus.

[20] The lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus according to

[19] , wherein cytokine production or TLR4 activation induced by E. coli lipopolysaccharide is regulated by the lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus.

[21] The lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus according to any of

[17] to

[20] , wherein the regulation includes inhibition.

[22] The lipopolysaccharide derived from bacteria belonging to the genus Paracoccus according to any one of

[17] to

[21] , which has a lipid A portion of lipopolysaccharide derived from bacteria belonging to the genus Paracoccus, wherein four 3-hydroxyacyl chains having 8 to 16 carbon atoms are bound to the glucosamine backbone of the lipid A, and an acyl chain having 8 to 13 carbon atoms is further bound to the hydroxyl group at the 3-position of one or two of the 3-hydroxyacyl chains.

[23] The lipid A portion has a structure represented by formula (I): The lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus according to

[22] , having the formula:

[0011] According to the present invention, it is possible to regulate cytokine production in cells using lipopolysaccharide derived from bacteria belonging to the genus Paracoccus. Because lipopolysaccharide derived from bacteria belonging to the genus Paracoccus can regulate cytokine production, an agent containing lipopolysaccharide derived from bacteria belonging to the genus Paracoccus is useful as a cytokine production regulator. Furthermore, lipopolysaccharide derived from bacteria belonging to the genus Paracoccus or a cytokine production regulator is also useful as a feed composition, food composition, or pharmaceutical composition, and may be useful, for example, as a pharmaceutical composition for preventing or treating cytokine-related diseases.

[0012] In another aspect, the present invention makes it possible to regulate TLR4 activation using lipopolysaccharide derived from bacteria belonging to the genus Paracoccus. Because lipopolysaccharide derived from bacteria belonging to the genus Paracoccus can regulate TLR4 activation, an agent containing lipopolysaccharide derived from bacteria belonging to the genus Paracoccus is useful as a regulator of TLR4 activation. Furthermore, lipopolysaccharide derived from bacteria belonging to the genus Paracoccus or a regulator of TLR4 activation is also useful as a feed composition, food composition, or pharmaceutical composition, and may be useful, for example, as a pharmaceutical composition for the prevention or treatment of diseases associated with the TLR4 signaling system.

[0013] This figure shows the absorbance (mean value of N=6) when HEK-293 hTLR4 cells were cultured for 24 hours in the presence of LPS. The control (N) indicates a group to which HEK-Blue Detection medium (20 μl) was added instead of LPS. The control (P) indicates a group to which E. coli-derived LPS (0.1 μg / mL) and HEK-Blue Detection medium (10 μl) were added. P indicates a group to which Paracoccus LPS and E. coli-derived LPS (0.1 μg / mL) were added. R indicates a group to which Rhodobacter LPS and E. coli-derived LPS (0.1 μg / mL) were added. The results of ELISA assays of TNF-α (left) and IL-6 (right) in the supernatant after 47 hours of culture of macrophages in the presence of LPS are shown. GC charts of fatty acid analysis are shown. The top graph shows the analysis results of fatty acids derived from Paracoccus LPS, and the bottom graph shows the analysis results of the fatty acid standard mix. This shows a GC-MS spectrum (retention time 9.440 minutes). The top figure shows the analysis results of fatty acids derived from Paracoccus LPS, and the bottom figure shows database data for C12:1 (C5-C6 cis). This shows a GC-MS spectrum (retention time 14.417 minutes). The top figure shows the analysis results of fatty acids derived from Paracoccus LPS, and the bottom figure shows database data for 3-OH C10:0. This shows a GC-MS spectrum (retention time 23.010 minutes). The top figure shows the analysis results of fatty acids derived from Paracoccus LPS, and the bottom figure shows database data for 3-OH C14:0. This shows the MALDI-TOF MS measurement results when the lipid A portion of LPS derived from bacteria belonging to the genus Paracoccus was measured in negative ion mode.

[0014] The present invention will be described in more detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be appropriately modified and implemented without departing from the spirit of the present invention. All references cited in this specification, including publications, patent applications, and patents, are incorporated herein by reference in their entirety.

[0015] The present invention relates to an agent for regulating cytokine production or TLR4 activation, comprising lipopolysaccharide derived from bacteria belonging to the genus Paracoccus (hereinafter collectively referred to as "regulators of the present invention"). The present invention also relates to a feed composition, food composition, or pharmaceutical composition (hereinafter collectively referred to as "compositions of the present invention") comprising lipopolysaccharide derived from bacteria belonging to the genus Paracoccus or the regulator of the present invention.

[0016] 1. LPS Derived from Bacteria Belonging to the Genus Paracoccus The regulator of the present invention or the composition of the present invention contains LPS (lipopolysaccharide) derived from bacteria belonging to the genus Paracoccus.

[0017] In this specification, the bacterium belonging to the genus Paracoccus is not particularly limited. As the bacterium belonging to the genus Paracoccus, Paracoccus carotinifaciens, Paracoccus marcusii, Paracoccus haeundaensis, or Paracoccus zeaxanthinifaciens is preferably used, Paracoccus carotinifaciens or Paracoccus zeaxanthinifaciens is more preferably used, and Paracoccus carotinifaciens is particularly preferably used. Specific strains of bacteria belonging to the genus Paracoccus include, for example, Paracoccus carotinifaciens strain E-396 (FERM BP-4283), Paracoccus bacterium strain A-581-1 (FERM BP-4671), Paracoccus marcusii strain DSM 11574, Paracoccus bacterium strain N-81106, Paracoccus haeundaensis strain BC 74171, Paracoccus zeaxanthinifaciens strain ATCC 21588, and Paracoccus sp. PC-1, and mutant strains of these are also preferably used in the present invention. The E-396 and A-581-1 strains have been internationally deposited with the National Institute of Technology and Evaluation (NITE) International Patent Organism Depositary (NITE-IPOD) (2-5-8 Kazusa Kamatari, Kisarazu, Chiba 292-0818) as the international depositary authority as follows: E-396 strain: Identification number: E-396, Accession number: FERM BP-4283, Original deposit date: April 27, 1993 A-581-1 strain: Identification number: A-581-1, Accession number: FERM BP-4671, Original deposit date: May 20, 1994

[0018] As used herein, the LPS derived from bacteria belonging to the genus Paracoccus is not particularly limited as long as it is an LPS obtained from bacteria belonging to the genus Paracoccus. In the present specification, the LPS derived from bacteria belonging to the genus Paracoccus may be used in a purified state or in the form of a mixture with other components.

[0019] For example, LPS derived from bacteria belonging to the genus Paracoccus may be contained in the regulator or composition of the present invention in the form of an LPS-containing extract of bacteria belonging to the genus Paracoccus, etc. For example, bacteria belonging to the genus Paracoccus may be appropriately cultured, and the resulting culture may be extracted to obtain an extract, which can be used as LPS derived from bacteria belonging to the genus Paracoccus.

[0020] The culture of bacteria belonging to the genus Paracoccus may be subjected to a single or a combination of treatments such as concentration, drying, dilution, crushing, heating, etc. before or after the extraction treatment. Drying treatments include spray drying, freeze drying, vacuum drying, and drum drying. Therefore, the extract includes an extract of a culture of bacteria belonging to the genus Paracoccus and / or an extract of a processed culture of bacteria belonging to the genus Paracoccus.

[0021] LPS can be extracted using a hot water phenol method (O. Westphal, K. Jann, "Methods in Carbohydrate Chemistry", ed. by R. Whistler, Vol. 5, p. 83, Academic Press, New York (1965)) or a phenol-chloroform-petroleum ether extraction (PCP) method (C. Galanos, O. Luderitz, O. Westphal, Eur. J. Biochem., 9, 245 (1969)).

[0022] Those skilled in the art can further purify the extracted LPS derived from bacteria belonging to the genus Paracoccus, using conventionally known methods.

[0023] LPS derived from bacteria belonging to the genus Paracoccus has lipid A. In the present invention, lipid A has a backbone consisting of two glucosamine molecules linked together via a β(1→6) bond. In this specification, this backbone of lipid A is referred to as the "glucosamine backbone."

[0024] In one embodiment of the present invention, a phosphate group is bound to position 1 of the glucosamine at the reducing end of the glucosamine backbone, and a phosphate group is bound to position 4 of the glucosamine at the non-reducing end. In another embodiment of the present invention, the number of phosphates bound to the glucosamine backbone may be 1 to 3, and the phosphate may be modified. In another embodiment of the present invention, a hydroxyl group may be used instead of the phosphate group.

[0025] The lipid A of the present invention has four acyl chains each having 8 to 16 carbon atoms directly bonded to a glucosamine backbone, and the acyl chains have a hydroxyl group at the 3-position. Herein, an acyl chain having a hydroxyl group at the 3-position is also referred to as a 3-hydroxyacyl chain. That is, in lipid A, two molecules of glucosamine are bonded, with a 3-hydroxyacyl chain amide-linked to the 2-position of glucosamine and a 3-hydroxyacyl chain ester-linked to the 3-position. The two molecules of glucosamine may be the same glucosamine or may be two glucosamine molecules bonded to different 3-hydroxyacyl chains.

[0026] In the present invention, the 3-hydroxyacyl chains bonded to the 2- and 3-positions of glucosamine have 8 to 16 carbon atoms, preferably 10 to 14 carbon atoms, more preferably 10, 11, 12, 13, or 14 carbon atoms, and even more preferably 10 or 14 carbon atoms.

[0027] The number of double bonds in the 3-hydroxyacyl chain directly bonded to the glucosamine backbone is 0, 1 or 2, preferably 0.

[0028] In the present invention, the 3-hydroxyacyl chain bound to the glucosamine backbone is preferably 3-OH10:0 (a 3-hydroxyacyl chain having 10 carbon atoms and no double bonds) or 3-OH14:0 (a 3-hydroxyacyl chain having 14 carbon atoms and no double bonds).

[0029] In one embodiment of the present invention, the 3-hydroxyacyl chain attached to the 2-position of glucosamine is 3-OH14:0, and the 3-hydroxyacyl chain attached to the 3-position of glucosamine is 3-OH10:0.

[0030] In the lipid A of the present invention, one or two of the four 3-hydroxyacyl chains directly bonded to the glucosamine backbone are further ester-linked to the hydroxyl group at position 3 of the 3-hydroxyacyl chain with an acyl chain having 8 to 13 carbon atoms. In one embodiment of the present invention, an acyl chain having 8 to 13 carbon atoms is further ester-linked to the hydroxyl group at position 3 of one or two 3-hydroxyacyl chains bonded to position 2 of the glucosamine backbone. Due to the attachment of such additional acyl chains, lipid A has five or six acyl chains.

[0031] In the present invention, the acyl chain bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain directly bonded to the glucosamine backbone has 8 to 13 carbon atoms, preferably 8 to 12 carbon atoms, and more preferably 12 carbon atoms.

[0032] In the present invention, the number of double bonds in the acyl chain bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain directly bonded to the glucosamine backbone is 0, 1 or 2, and preferably 1.

[0033] In the present invention, the acyl chain bonded to the hydroxyl group at the 3-position of the 3-hydroxyacyl chain directly bonded to the glucosamine backbone is preferably a C12:1 (a 3-hydroxyacyl chain having 12 carbon atoms and one double bond), more preferably a C12:1 chain having a double bond (cis) at the 5th carbon (C5-6) counting from the carbonyl group (C12:1(Δ ５ )).

[0034] In one embodiment of the present invention, the acyl chain bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain directly bonded to the glucosamine backbone is bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain bonded to position 2 of the glucosamine on the non-reducing end side, but is not bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain bonded to position 2 of the glucosamine on the reducing end side.

[0035] Examples of lipid A of the present invention are given below: In one embodiment of the present invention, lipid A has a structure in which four 3-hydroxyacyl chains each having 8 to 16 carbon atoms are bound to a glucosamine backbone, and an acyl chain having 8 to 13 carbon atoms is further bound to the hydroxyl group at the 3-position of one or two of the 3-hydroxyacyl chains.

[0036] In another aspect of the present invention, lipid A has a structure in which four 3-hydroxyacyl chains having 10 to 14 carbon atoms are bound to a glucosamine backbone, and one or two of the 3-hydroxyacyl chains having 10 to 14 carbon atoms bound to the 2-position of the glucosamine backbone are further bound to the 3-position hydroxyl group of an acyl chain having 8 to 13 carbon atoms.

[0037] In another embodiment of the present invention, lipid A has a structure in which four 3-hydroxyacyl chains having 10 to 14 carbon atoms are bound to a glucosamine backbone, and one or two of the 3-hydroxyacyl chains having 10 to 14 carbon atoms bound to the 2-position of the glucosamine backbone are further bound to the 3-position hydroxyl group of a 12-carbon acyl chain.

[0038] In another embodiment of the present invention, lipid A has a structure in which four 3-hydroxyacyl chains having 10 to 14 carbon atoms are bound to a glucosamine backbone, and a 12-carbon acyl chain is further bound to the hydroxyl group at position 3 of the 10 to 14-carbon 3-hydroxyacyl chain bound to position 2 of the glucosamine backbone, and the 12-carbon acyl chain is bound to the hydroxyl group at position 3 of the 14-carbon 3-hydroxyacyl chain bound to position 2 of the glucosamine at the non-reducing end, but is not bound to the hydroxyl group at position 3 of the 14-carbon 3-hydroxyacyl chain bound to position 2 of the glucosamine at the reducing end.

[0039] In another embodiment of the present invention, lipid A has a structure in which four 3-hydroxyacyl chains having 10 or 14 carbon atoms are bound to a glucosamine backbone, and a 12-carbon acyl chain is further bound to the hydroxyl group at position 3 of the 14-carbon 3-hydroxyacyl chain bound to position 2 of the glucosamine backbone, and the 12-carbon acyl chain is bound to the hydroxyl group at position 3 of the 14-carbon 3-hydroxyacyl chain bound to position 2 of the glucosamine at the non-reducing end, but is not bound to the hydroxyl group at position 3 of the 14-carbon 3-hydroxyacyl chain bound to position 2 of the glucosamine at the reducing end.

[0040] In one aspect of the present invention, lipid A has a structure in which 3-OH14:0 is bound to the 2-position of glucosamine, 3-OH10:0 is bound to the 3-position of glucosamine, and C12:1, which contains a double bond (cis) between C5 and C6, is bound to 3-OH14:0 bound to the 2-position of glucosamine on the non-reducing end side.

[0041] In one embodiment of the present invention, the LPS derived from a bacterium belonging to the genus Paracoccus has the structure of formula (I): It contains a lipid A moiety having the formula:

[0042] 2. Regulator and Composition of the Present Invention As shown in the Examples, LPS derived from bacteria belonging to the genus Paracoccus can regulate cytokine production. Furthermore, LPS derived from bacteria belonging to the genus Paracoccus can regulate TLR4 or the TLR4 signaling pathway. TLR4 activation includes activation of TLR4 itself and activation of the TLR4 signaling pathway.

[0043] Cytokines are proteins secreted primarily by immune system cells and are physiologically active substances involved in cell-cell interactions. For example, LPS is known to initiate signal transduction by binding to TLR4 present on the cell membrane, enhance cytokine-inducing activity, and induce inflammatory responses. That is, LPS induces cytokine production via TLR4, resulting in endotoxin shock, fever, and other symptoms. Therefore, regulators or compositions containing LPS derived from bacteria belonging to the genus Paracoccus that regulate cytokine production are expected to regulate physiological activity via the regulation of cytokine production, and are therefore useful as feeds, medicines, and foods.

[0044] In one aspect of the present invention, bacterially induced cytokine production or TLR4 activation is regulated by LPS derived from bacteria belonging to the genus Paracoccus. That is, in one aspect of the present invention, the target regulated by the regulator of the present invention is bacterially induced cytokine production or TLR4 activation. The bacterium may be a bacterium that induces cytokine production or TLR4 activation. Examples of bacteria that induce cytokine production or TLR4 activation include enterobacteria, such as Escherichia coli, Bacteroides, Enterobacter, and Salmonella.

[0045] Cytokines whose production is regulated by LPS derived from bacteria belonging to the genus Paracoccus include cytokines produced by TLR4 activation or cytokines produced via TLR4 signaling. LPS initiates signaling by binding to TLR4, resulting in cytokine production. Therefore, in one aspect of the present invention, cytokines whose production is regulated by LPS derived from bacteria belonging to the genus Paracoccus are cytokines whose production is induced by LPS (e.g., LPS from Escherichia coli). The LPS that induces cytokine production may be any LPS that binds to TLR4 and induces cytokine production, and the species from which it is derived is not limited. For example, LPS that induces cytokine production is LPS derived from Escherichia coli. In another aspect of the present invention, TLR4 activation regulated by LPS derived from bacteria belonging to the genus Paracoccus includes activation by LPS, for example, activation of TLR4 by LPS binding. The LPS that induces TLR4 activation may be any LPS that binds to and activates TLR4, and the species from which it is derived is not limited. For example, the LPS that induces TLR4 activation is LPS derived from Escherichia coli.

[0046] In the present invention, the cytokine whose production is regulated by LPS derived from bacteria belonging to the genus Paracoccus may be any cytokine produced by activation of TLR4 or via signal transduction of TLR4, such as TNF-α, IL-6, IL-12, INF-β, or a combination thereof.

[0047] In the present specification, the type of E. coli is not particularly limited. The E. coli-derived LPS is not particularly limited as long as it is derived from E. coli, and examples thereof include LPS released from E. coli in the internal environment of an organism, LPS obtained by extracting E. coli, and processed products or extracts of E. coli containing LPS.

[0048] Modulation of cytokine production by LPS derived from bacteria belonging to the genus Paracoccus includes inhibition of cytokine production. Modulation of cytokine production by LPS derived from bacteria belonging to the genus Paracoccus can be achieved by contacting cells with LPS derived from bacteria belonging to the genus Paracoccus. Contact may be in vivo, such as by administration, ingestion, or feeding, or in vitro, such as by adding LPS to a cell culture medium and culturing. Modulation of cytokine production by LPS derived from bacteria belonging to the genus Paracoccus includes, for example, a decrease in the amount of cytokine produced by cells compared to when LPS derived from bacteria belonging to the genus Paracoccus is absent, a decrease in the rate of cytokine production compared to when LPS derived from bacteria belonging to the genus Paracoccus is absent, or no cytokine is produced at all. Furthermore, an example of a target that can be modulated by LPS derived from bacteria belonging to the genus Paracoccus is cytokine production induced by LPS (e.g., LPS derived from Escherichia coli). That is, modulation of cytokine production by LPS derived from bacteria belonging to the genus Paracoccus includes suppression of cytokine production induced by LPS (e.g., LPS derived from Escherichia coli). Modulation of cytokine production includes, for example, a decrease in the amount of cytokine production induced by LPS (e.g., LPS derived from Escherichia coli) compared to when LPS derived from bacteria belonging to the genus Paracoccus is not present, a decrease in the rate of cytokine production induced by LPS (e.g., LPS derived from Escherichia coli), or no induction of cytokine production by LPS (e.g., LPS derived from Escherichia coli).

[0049] Modulation of TLR4 activation by LPS derived from bacteria belonging to the genus Paracoccus includes inhibition of TLR4 activation. Modulation of TLR4 activation by LPS derived from bacteria belonging to the genus Paracoccus can be achieved by contacting cells expressing TLR4 with LPS derived from bacteria belonging to the genus Paracoccus. Contact may be in vivo, such as by administration, ingestion, or feeding, or in vitro, such as by adding LPS to a cell culture medium and culturing it. Modulation of TLR4 activation by LPS derived from bacteria belonging to the genus Paracoccus includes, for example, suppression of cytokine production from cells compared to the absence of LPS derived from bacteria belonging to the genus Paracoccus (including a decrease in the amount of cytokine production, a decrease in the rate of cytokine production, or no cytokine production), a decrease in the level of TLR4 signaling, or a decrease in the level of TLR4 activation (e.g., the proportion of dimerized TLR4) compared to the absence of LPS derived from bacteria belonging to the genus Paracoccus. Furthermore, an example of a target regulated by LPS derived from bacteria belonging to the genus Paracoccus is TLR4 activity induced by LPS (e.g., LPS derived from Escherichia coli). That is, modulation of TLR4 activation by LPS derived from bacteria belonging to the genus Paracoccus includes suppression of TLR4 activation by LPS (e.g., LPS derived from Escherichia coli). Modulation of TLR4 activation includes, for example, suppression of cytokine production induced by LPS (e.g., LPS derived from Escherichia coli) compared to the absence of LPS derived from bacteria belonging to the genus Paracoccus (including a decrease in the amount of cytokine production, a decrease in the rate of cytokine production, or no cytokine production), a decrease in the level of TLR4 signaling induced by LPS (e.g., LPS derived from Escherichia coli), or a decrease in the level of TLR4 activation (e.g., the proportion of dimerized TLR4) induced by LPS (e.g., LPS derived from Escherichia coli).

[0050] Furthermore, the LPS derived from bacteria belonging to the genus Paracoccus of the present invention exhibits not only antagonistic activity (inhibitory effect) against cytokine production or TLR4 activation, but also weak agonistic activity. Therefore, in another aspect of the present invention, the regulation of cytokine production by LPS derived from bacteria belonging to the genus Paracoccus includes both suppression of cytokine production and weak cytokine production. Furthermore, in another aspect of the present invention, the regulation of TLR4 activation by LPS derived from bacteria belonging to the genus Paracoccus includes both suppression of TLR4 activation and weak TLR4 activation. For example, LPS derived from bacteria belonging to the genus Paracoccus is expected to suppress strong inflammatory effects caused by cytokines induced in an environment containing Escherichia coli or the like through its antagonistic activity, while imparting or maintaining immunostimulatory activity through its weak agonistic activity.

[0051] Cytokine production can be evaluated by measuring the amount of cytokine contained in or secreted from cells using a conventionally known method. For example, the amount of cytokine contained in the cell supernatant or cells can be measured by ELISA, Western blot, or HPLC.

[0052] The level of TLR4 activation can be assessed by detecting the amount or state of a substance involved in the TLR4 signal transduction pathway. For example, this includes measuring the amount of cytokines produced by TLR4 activation in TLR4-expressing cells. Alternatively, TLR4 activation may be assessed using an experimental system in which a reporter gene is introduced and TLR4 activation can be assessed by reporter gene activity. Alternatively, the expression level of mRNA translated into cytokines may be measured by RT-PCR. For example, the level of activated TLR4 may be assessed by measuring the amount of dimerized TLR4.

[0053] For the regulator of the present invention, LPS derived from bacteria belonging to the genus Paracoccus can be used as is, but it may also contain a pharmaceutically acceptable carrier, a carrier acceptable for food, a carrier acceptable for feed, etc. The regulator of the present invention can be used as a pharmaceutical composition, a food composition, or a feed composition (the composition of the present invention).

[0054] In the composition of the present invention, LPS derived from bacteria belonging to the genus Paracoccus can be used as is, but it may also contain a pharmaceutically acceptable carrier, a carrier acceptable for use as a food, a carrier acceptable for use as a feed, or the like.

[0055] The content of LPS derived from bacteria belonging to the genus Paracoccus in the regulator or composition of the present invention is not particularly limited and can be selected appropriately depending on, for example, the dosage form.

[0056] The form of the regulator or composition of the present invention is not particularly limited, and may be, for example, a powder, emulsion, aqueous solution, etc. The LPS derived from bacteria belonging to the genus Paracoccus may be contained in the regulator or composition of the present invention in an amount of 0.05% or more, 0.1% or more, 1% or more, 2% or more, 5% or more, 10% or more, or 90% or more.

[0057] The regulator or composition of the present invention can be formulated by adding additives. Any additives acceptable in the art may be used, and conventionally known additives can be used as appropriate. Examples of additives include excipients, binders, stabilizers, disintegrants, lubricants, flavorings, suspending agents, coating agents, etc., and the regulator or composition of the present invention may contain, for example, one or more of these.

[0058] The carrier used in the present invention is appropriately selected from excipients, diluents, binders, bulking agents, lubricants, disintegrants, stabilizers, wetting agents, emulsifiers, buffers, suspending agents, preservatives or antioxidants, pH adjusters, gelling agents, solubilizers, colorants, flavors, sweeteners, etc., and those commonly used for formulation can be used in producing the regulator or composition of the present invention. Examples include starches, sugars such as dextrose, lactose, sucrose, and glucose, sugar alcohols such as mannitol, xylitol, erythritol, sorbitol, and maltitol, acacia gum, gum arabic, gelatin, magnesium aluminometasilicate, hydrotalcite, inorganic compounds such as anhydrous calcium phosphate, calcium carbonate, calcium silicate, and light anhydrous silicic acid, polyvinylpyrrolidone, hydroxypropylmethylcellulose, microcrystalline cellulose, talc, silica, magnesium stearate, sodium starch glycolate, sodium lauryl sulfate, cellulose derivatives, methyl-p-hydroxybenzoate, sorbic acid, water, and mineral oils. However, the carriers that can be used in the present invention are not limited to these examples, and examples include gum arabic, maltodextrin, tocopherol, medium-chain fatty acids, and ascorbic acid.

[0059] When used as a pharmaceutical composition or for medical purposes, it can be made into a dosage form for oral administration or parenteral administration (intravenous, intraarterial, intraperitoneal, rectal, subcutaneous, intramuscular, sublingual, intranasal, intravaginal, etc.) Examples of such dosage forms include, but are not limited to, solutions, tablets, orally rapidly disintegrating tablets, powders, granules, capsules, syrups, injections, suppositories, sprays, ointments, patches, drinks, etc.

[0060] When used as a food composition or in food applications, LPS derived from bacteria belonging to the genus Paracoccus can be formulated as a functional food, health food, or supplement, or can be incorporated into conventional foods. When used as a food composition or in food applications, LPS derived from bacteria belonging to the genus Paracoccus can be formulated in the form of, but not limited to, functional foods, health foods, supplements, confectioneries (jelly, gummies, gum, yogurt, pudding, biscuits, cookies, chocolate, candy, cake, ice cream, chewing gum), retort pouch foods, soups, drinks (juices, teas, jelly drinks, powdered drinks), dairy products, and the like. Furthermore, sweeteners, seasonings, emulsifiers, suspending agents, preservatives, and the like can be added as needed. Furthermore, the food composition of the present invention can also be used as a food additive.

[0061] When used in a feed composition or for feed applications, LPS derived from bacteria belonging to the genus Paracoccus can be formulated as a compound feed or can be added to a basal feed used in normal rearing. When used in a feed composition or for feed applications, the LPS may be in any form, which can be appropriately selected depending on the type and growth stage of the subject to be fed. Examples include, but are not limited to, powder, pellets, flakes, paste, and liquid. Subjects to be fed include mammals including poultry, fish, shellfish, and crustaceans.

[0062] The dosage (including the amount of food intake or feeding) of the regulator or composition of the present invention is not particularly limited and can be selected appropriately depending on, for example, the severity of the patient's symptoms, the type of disease, etc. For example, the daily dosage for a subject such as a patient is typically 0.3 to 3000 μg / kg, preferably 3 to 3000 μg / kg, more preferably 10 to 1000 μg / kg, and even more preferably 3 to 300 μg / kg, calculated as LPS derived from bacteria belonging to the genus Paracoccus, and this amount is administered to a subject such as a patient once a day or in appropriate divided doses.

[0063] The pharmaceutical composition is useful for preventing or treating diseases associated with cytokines or diseases associated with the TLR4 signaling system, and the subjects for prevention or treatment are mammals, birds, or seafood, preferably humans.

[0064] As used herein, cytokines refer to cytokines produced by TLR4 activation or cytokines produced via TLR4 signaling, preferably cytokines whose production is induced by LPS (e.g., Escherichia coli LPS) or cytokines produced when LPS binds to TLR4 and activates TLR4. Examples of such cytokines include TNF-α, IL-6, IL-12, and INF-β. As used herein, "cytokine-related diseases" include diseases associated with abnormal or excessive production of these cytokines. Examples of such diseases include sepsis, rheumatoid arthritis, autoimmune diseases, intravascular coagulation syndrome, myocardial infarction, and hypoxemia.

[0065] As used herein, "diseases associated with the TLR4 signaling system" include diseases associated with abnormal or excessive production of cytokines due to activation of the TLR4 signaling system. "Diseases associated with the TLR4 signaling system" are, for example, "diseases associated with cytokines." Examples of such diseases include sepsis, rheumatoid arthritis, autoimmune diseases, intravascular coagulation syndrome, myocardial infarction, and hypoxemia.

[0066] The present invention also relates to a method for preventing or treating a disease associated with cytokines produced by Escherichia coli-derived LPS, or a method for preventing or treating a disease associated with the TLR4 signaling pathway activated by Escherichia coli-derived LPS, comprising administering to a patient LPS derived from a bacterium belonging to the genus Paracoccus, or the regulator of the present invention, or the composition of the present invention. The above-mentioned preventive or therapeutic method is in detail similar to the description of the pharmaceutical composition of the present invention.

[0067] Furthermore, LPS derived from bacteria belonging to the genus Paracoccus, or the regulator of the present invention, or the composition of the present invention can also be used for the prevention or treatment of diseases associated with cytokines produced by LPS derived from Escherichia coli, or for the prevention or treatment of diseases associated with the TLR4 signaling pathway activated by LPS derived from Escherichia coli. The above-mentioned prevention or treatment method is in detail similar to the description for the pharmaceutical composition of the present invention.

[0068] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0069] Test 1. Modulation of TLR4-Mediated Signal Transduction [Test Method] The HEK-hTLR4 cells used in this test are HEK (human embryonic kidney) cells expressing human TLR4. These cells were labeled HEK-Blue hTLR4 (catalog number hkb-htlr4) from InvivoGen. HEK-Blue hTLR4 cells were obtained by transfecting HEK293 cells with human TLR4, MD-2, and CD14 coreceptor genes, and an inducible SEAP (secreted fetal alkaline phosphatase) reporter gene. The SEAP reporter gene was under the control of the IL-12p40 minimal promoter fused to five NF-κB and AP-1 binding sites. Stimulation of TLR4 with a ligand activates NF-κB and AP-1, which induce SEAP production, resulting in increased SEAP production. When SEAP is secreted, a dye contained in the cell culture medium, HEK-Blue Detection (InvivoGen), reacts and changes color from purple to blue, and the color change was measured at ABS 620 nm.

[0070] The tests used LPS derived from Escherichia coli, LPS derived from Paracoccus (a bacterium belonging to the Paracoccus genus), and LPS derived from Rhodobacter. The LPS derived from Escherichia coli was lipopolysaccharide, derived from Escherichia coli O55 (ultracentrifuged product) (Fujifilm Wako Pure Chemical Industries, Ltd., product code 128-05171), and the LPS derived from Rhodobacter was LPS-RS Ultrapure (Invivogen, product code tlrl-prslps).

[0071] The paracoccus bacteria used was Paracoccus carotinifaciens. The LPS derived from Paracoccus was a highly purified product (high purity product) purified by the PCP (phenol / chloroform / petroleum ether) method. Specifically, the purification of Paracoccus-derived LPS was carried out in the following four steps.

[0072] Step 1: Degreasing of dried Paracoccus cells. Panaferd-AX (ENEOS Techno Materials Corporation) was used as the dried cells. 60 g of dried cells were dispensed into 12 50 mL conical tubes (approximately 5 g per tube). Washing was performed in the following order (40 mL per tube) with ethanol (4 times), acetone (3 times), and diethyl ether (3 times). After washing, the cells were freeze-dried. The dry weight was 55.998 g.

[0073] Step 2: Extraction of LPS by PCP Method Approximately 56 g of washed and defatted bacterial cells were placed in a 500 mL wide-mouth glass reagent bottle. 300 mL of a PCP mixture consisting of liquid phenol (90 g + 11 mL water), chloroform, and petroleum ether (2:5:8) was added and stirred with a Polytron for 2 minutes on ice. The suspension was transferred to eight stainless steel centrifuge tubes and centrifuged at 5,000 × g for 15 minutes in a high-speed refrigerated centrifuge, Model 7780. The supernatant (150 mL) was collected in a 50 mL conical tube. The residue was extracted twice more with the PCP mixture, and the supernatants were collected. 110 mL was recovered the second time, and 180 mL the third time.

[0074] Step 3: LPS Precipitation and Purification (Obtaining Crudely Purified LPS) Six milliliters of supernatant was dispensed into 16 glass test tubes, and the solvent was removed under reduced pressure (40°C) using a centrifugal evaporator. The supernatant was added to the glass tubes from which the solvent had been removed, and the entire volume of the supernatant was evaporated. Water was added to the supernatant (phenol solution) after solvent removal, in an amount equivalent to 1 / 10 the volume of the phenol solution. Centrifugation (3,500 rpm x 20 minutes) was performed, and the supernatant was decanted into another tube. A precipitate was observed at the bottom of the tube. The precipitate was washed twice with 80% phenol (3 mL each time) and then four times with ether (approximately 3 mL each time). After removing the ether, the mixture was dried under reduced pressure. 447 mg of crudely purified LPS was obtained.

[0075] Step 4: High-level purification of crude LPS 440 mg of crude LPS was suspended and dissolved in water for injection to a concentration of 10 mg / mL. 1 M Tris-HCl pH 8.0 was added to a concentration of 10 mM. 1 M MgCl ２Aqueous solution was added to the tube to a concentration of 2 mM. 13.2 μL of benzonase solution was added to a final concentration of 10 U / mL. Nuclease treatment was performed by incubating at 37°C for 6 hours. Proteinase K (20 mg / mL) was added to the nuclease-treated solution to a final concentration of 100 μg / mL, and the mixture was then incubated at 37°C for 16 hours. The crude LPS solution after nuclease and protease treatment was divided into four tubes and ultrafiltered using four centrifugal ultrafiltration tubes with a molecular weight cutoff of 10 kDa. After ultrafiltration, the treated solution was ultrafiltered four times with water for injection. The internal solution of the ultrafiltration was recovered and washed with water for injection. Water for injection was added to a total volume of 88 mL to achieve a crude LPS concentration of 5 mg / mL. Triethylamine (TEA) and sodium deoxycholate (DOC) were added to a final concentration of 0.2% and 0.5%, respectively, and the mixture was dissolved and suspended. The solution was dispensed into eight 50 mL conical tubes, 11 mL each. An equal volume (11 mL) of water-saturated phenol was added to each tube, and the tubes were left in ice water for 10 minutes. Then, the tubes were centrifuged (3,500 rpm x 20 minutes). The aqueous layer (upper phase) was collected in another 50 mL conical tube. An equal volume of 0.2% TEA and 0.5% DOC aqueous solution was added to the phenol layer and mixed. After leaving the tubes for 5 minutes, the tubes were left in ice water for 10 minutes, and then centrifuged (3,500 rpm x 20 minutes). The aqueous layer (upper phase) was collected in another 50 mL conical tube. The collected aqueous layer was dispensed into eight 50 mL conical tubes, and an equal volume of water-saturated phenol was added. After leaving the tubes for 5 minutes, the tubes were left in ice water for 10 minutes, and then centrifuged (3,500 rpm x 20 minutes). The aqueous layer (upper phase) was collected in another 50 mL conical tube. The collected aqueous layer was dispensed into eight tubes, and 3M aqueous sodium acetate (pH 5.2) was added to a final concentration of 30 mM. 33 mL of ethanol was added to each tube to a final concentration of 75%. After thorough mixing, the mixture was stored at -20°C for 16 hours. The liquid from the centrifuge tube was dispensed into eight centrifuge tubes and centrifuged (4°C, 10,000 g x 30 minutes) using a high-speed refrigerated centrifuge. The supernatant was decanted, and the resulting precipitate was washed four times with 20 mL of 100% ethanol. The resulting precipitate was lyophilized and weighed to yield 67.2 mg of highly purified LPS.

[0076] The purity of LPS was calculated using the following formula: LPS amount = highly purified LPS amount - impurity amount LPS (purity) (%) = LPS amount / highly purified LPS amount x 100 Highly purified LPS contains nucleic acids and proteins as impurities. The amount of nucleic acids in the highly purified LPS was measured by UV absorbance, and the amount of protein was measured by the Lowry method.

[0077] The purity of LPS after step 4 is as follows: LPS (%) Nucleic acid (%) Protein (%) Total (%) 97.82 1.35 0.82 100.00

[0078] HEK-293 hTLR4 cells were cultured until they reached 80-90% confluence. The cells were cultured in HEK-Blue Detection medium at 3 × 10 5 The cells were suspended at a concentration of 200, 20, 2, or 0.2 μg / mL and seeded in 180 μL aliquots onto a 96-well plate. Paracoccus LPS ("P." in Figure 1) or Rhodobacter LPS ("R." in Figure 1) was suspended in HEK-Blue Detection medium at 200, 20, 2, or 0.2 μg / mL, and 10 μL of the suspension was added to each well. E. coli LPS was dissolved in HEK-Blue Detection medium at 2 μg / mL and 10 μL of the suspension was added to each well. The 96-well plate was incubated at 37°C under 5% CO2 in a CO2 incubator for 24 hours. The absorbance (Abs) at 630 nm was measured using a plate reader. The absorbance indicates the degree of TLR4-mediated signaling; higher absorbance indicates more activated TLR4-mediated signaling.

[0079] In the control (N) group, HEK-Blue Detection medium (20 μl) was added instead of E. coli LPS and Paracoccus LPS. In the control (P) group, E. coli LPS (0.1 μg / mL) and HEK-Blue Detection medium (10 μl) were added. In the Paracoccus LPS (P. in Figure 1) group, E. coli LPS (0.1 μg / mL) and Paracoccus LPS (0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, or 10 μg / mL) were added. In the Rhodobacter LPS (R. in Figure 1) group, E. coli LPS (0.1 μg / mL) and Rhodobacter LPS (0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, or 10 μg / mL) were added.

[0080] [Results] The absorbance measured for each group (average value of N=6) is shown in FIG.

[0081] When HEK-293 hTLR4 cells were stimulated with 0.1 μg / mL of E. coli LPS, the absorbance increased, indicating activation of TLR4-mediated signaling. This activation of TLR4 signaling by E. coli LPS was reduced in a concentration-dependent manner by Paracoccus LPS. That is, Paracoccus LPS inhibited E. coli LPS-induced TLR4 signaling. On the other hand, the addition of low concentrations (0.01 μg / mL or 0.1 μg / mL) of Rhodobacter LPS did not affect E. coli LPS-induced TLR4 signaling, whereas the addition of high concentrations (1 μg / mL or 10 μg / mL) of Rhodobacter LPS tended to reduce E. coli LPS-induced TLR4 signaling.

[0082] These results suggest that Paracoccus LPS acts as an antagonist to TLR4. Furthermore, the effect of Paracoccus LPS was shown to be more pronounced than that of Rhodobacter LPS. Furthermore, since E. coli LPS induces cytokine production via TLR4, it is suggested that Paracoccus LPS may regulate the cytokine-inducing activity of E. coli LPS.

[0083] Test 2. Site of action of Paracoccus-derived LPS and its TLR4 regulatory activity [Test method] THP-1 cells (a cell line derived from human acute monocytic leukemia) were treated with phorbol ester to differentiate them into macrophages, and then LPS was added. After 47 hours, the amounts of cytokines (TNF-α and IL-6) in the supernatant were measured by ELISA.

[0084] As controls, an untreated group ("Control" in Figure 2) and a group to which Escherichia coli-derived LPS was added ("E. coli LPS" in Figure 2) were set up, and as test groups, a group to which Paracoccus-derived LPS alone was added ("P. LPS" in Figure 2) and a group to which Paracoccus-derived LPS and anti-TLR4 antibody were added ("P. LPS + Antibody" in Figure 2) were set up.

[0085] Anti-TLR4 antibody (Abcam, product code: ab13556) was used as the anti-TLR4 antibody. The highly purified LPS obtained in Test 1 was used as the Paracoccus-derived LPS. Lipopolysaccharide derived from E. coli O55 (ultracentrifuged product) (Fujifilm Wako Pure Chemical Industries, Ltd., product code 128-05171) was used as the E. coli-derived LPS. LPS was mixed to a final concentration of 30 μg / mL.

[0086] For ELISA, Levis® Human TNF-α ELISA KIT (Fujifilm Wako Shibayagi Co., Ltd., product code 639-42331) or Levis® Human IL-6 ELISA KIT (Fujifilm Wako Shibayagi Co., Ltd., product code 635-42311) was used.

[0087] [Results] The results are shown in Figure 2. The left graph shows the amount of TNF-α (pg / mL) in the supernatant, and the right graph shows the amount of IL-6 (pg / mL) in the supernatant.

[0088] In the group containing only Paracoccus LPS (P. LPS), cytokine production (TNF-α and IL-6) was increased compared to the control group, but was lower than that produced by E. coli LPS. This indicates that Paracoccus LPS has some cytokine-inducing activity.

[0089] Furthermore, cytokine production was suppressed in the group containing Paracoccus LPS and anti-TLR4 antibody (P. LPS + Antibody) compared to the group containing Paracoccus LPS alone (P. LPS). The suppression of cytokine production in the "P. LPS + Antibody" group is thought to be due to the anti-TLR4 antibody present in the system blocking TLR4, thereby blocking the TLR4 signaling pathway. Therefore, this test example demonstrates that Paracoccus LPS acts on TLR4 and regulates the induction of cytokine production via the TLR4 signaling pathway.

[0090] LPS from any species basically initiates signal transduction and produces cytokines by binding to TLR4. This test example confirmed that LPS derived from bacteria belonging to the genus Paracoccus acts on TLR4, and therefore it can be said that LPS derived from bacteria belonging to the genus Paracoccus regulates the induction of cytokine production by LPS from any species, not just LPS derived from Escherichia coli.

[0091] Test 3. Identification of the Structure of Paracoccus LPS by Fatty Acid Analysis with GC-MS (1) Sample Preparation: Paracoccus LPS (highly purified product) prepared in Test 1 was used as the Paracoccus LPS. 1 mg of purified LPS was placed in a test tube and 5% HCl in methanol was added. The tube was then capped and incubated at 100°C for 2 hours. After cooling, 1 mL of water and 1 mL of a standard solution (10 μg / mL methyl palmitate-d31) were added and mixed by stirring. The mixture was centrifuged (3,000 rpm, 5 min), and the supernatant was collected as a sample. A fatty acid standard mix (Supelco® 37-component FAME Mix) was also analyzed.

[0092] (2) Apparatus and Measurement Conditions GC-MS: GCMS2010Ultra (SHIMADZU CORPORATION) was used as the measurement apparatus, and measurements were performed under the measurement conditions shown in Table 1.

[0093]

[0094] (3) Results The measurement results are shown in Table 2 and FIG.

[0095]

[0096] From the component amounts shown in Table 2, it was determined that the top three fatty acids, namely, C12:1, 3-hydroxy fatty acid-1, and 3-hydroxy fatty acid-2, were contained in the LPS composition. Furthermore, because the content of C18:1 and C18:0 was low, they were determined to be impurities. In this test, it is possible that not all ester and amide bonds were decomposed, so the component ratio between C12:1, 3-hydroxy fatty acid-1, and 3-hydroxy fatty acid-2 was not taken into consideration when identifying the lipid A structure.

[0097] (4) Identification of Fatty Acid Structure Next, the structures of C12:1, 3-hydroxy fatty acid-1, and 3-hydroxy fatty acid-2 were identified based on the MS fragment peaks.

[0098] The C12:1 fatty acid was identified as a C12:1 fatty acid with a cis double bond at C5-C6 by matching its GC retention time (9.440 min) with the MS fragment peak in the database (Figure 4).

[0099] 3-hydroxy fatty acid-1 was inferred to be 3-OH-10:0 from the MS fragment peak. That is, 3-hydroxy fatty acids usually have three characteristic peaks, [M-18], [M-50] and [M-92], and when 3-hydroxy fatty acid-1 is 3-OH-10:0, these three peaks are observed at [M-18] + 184, [M-50] + 152 and [M-92] + 110, respectively. 3-hydroxy fatty acid-1 was inferred to be 3-OH-10:0 because it has these peaks (Fig. 5).

[0100]

[0101] 3-hydroxy fatty acid-2 was also identified as 3-OH-14:0 by the same method as 3-hydroxy fatty acid-1 (FIG. 6).

[0102]

[0103] The fatty acid analysis method by GC-MS in this test example revealed that Paracoccus-derived LPS contains C12:1 fatty acids with cis double bonds at C5-C6, 3-OH-10:0, and 3-OH-14:0.

[0104] Test 4. Measurement of LPS and Lipid A by MALDI-TOF MS (1) Preparation of Lipid A As in Test 3, in Test 4, the Paracoccus-derived LPS (highly purified product) prepared in Test 1 was used as the Paracoccus-derived LPS. 4 mg of purified LPS was placed in a test tube and incubated in a 2% aqueous acetic acid solution at 100°C for 2 hours to hydrolyze the LPS. After cooling, the precipitate was collected by centrifugation (13,000 × g, 2 minutes), washed several times with water, and used as a sample for MALDI-TOF MS.

[0105] (2) Apparatus and Measurement Conditions MALDI-TOF MS: AXIMA Performance (Shimadzu Corporation) was used as the measurement apparatus, and measurements were performed under the measurement conditions shown in Table 3.

[0106]

[0107] (3) Results MALDI-TOF MS analysis (negative ion mode) is shown in Figure 7. From Figure 7, the negative ion peak of Lipid A was considered to be 1472. The three fragment peaks 1392, 1302, and 1222 were identified as 1392 (-HPO), 1302 (-3-OH-10:0), and 1222 (-HPO and -3-OH-10:0), respectively. This test example revealed that Paracoccus LPS contains Lipid A phosphate ester and 3-OH-10:0 fatty acid. The molecular weight of the entire Lipid A was also determined to be 1473 (Figure 7). When fatty acids (C12:1 fatty acid with a cis double bond at C5-C6, 3-OH-10:0, and 3-OH-14:0) are combined to match this molecular weight, it is understood that lipid A has one C12:1 fatty acid with a cis double bond at C5-C6, two 3-OH-10:0, and two 3-OH-14:0.

[0108] It is known that in LPS lipid A, the fatty acid directly bound to two glucosamine molecules is often a 3-hydroxyacyl chain with a hydroxyl group at position 3. Therefore, it is presumed that C12:1 fatty acids, which do not have a hydroxyl group at position 3, are not directly bound to glucosamine. Furthermore, 16S rRNA analysis showed that Paracoccus carotinifaciens shares 93% identity with Rhodobacter sphaeroides, suggesting that they are related species and that their lipid A structures are similar (FEMS Microbiology Reviews 4 (1988) 143-154). Considering the results of Experiments 3 and 4 and previous findings on lipid A, it was estimated that the lipid A of Paracoccus carotinifaciens has a structure in which 3-OH-14:0 is bound to the 2-amino group of glucosamine via an amide bond, 3-OH-10:0 is bound to the 3-hydroxyl group of glucosamine via an ester bond, and a C12:1 fatty acid with a cis double bond at C5-C6 is bound to the 3-hydroxyl group of 3-OH-14:0 via an ester bond (the structure is shown below). The above structure of lipid A of Paracoccus carotinifaciens is consistent with the findings on the LPS structure of Paracoccus denitrificans, which shares 97% identity with Paracoccus carotinifaciens in 16S rRNA analysis. That is, in the LPS of Paracoccus denitrificans, 3-OH-10:0 and C12:1 fatty acids are present in lipid A in the form of ester bonds, while 3-OH-14:0 and 3-oxo-14:0 (not detected in Tests 3 and 4) are present in lipid A in the form of amide bonds (FEMS Microbiology Letters 37 (1986) 63-67).

[0109]

[0110] According to the present invention, it is possible to regulate cytokine production by lipopolysaccharide derived from Escherichia coli. Lipopolysaccharide derived from bacteria belonging to the genus Paracoccus can regulate cytokine production by lipopolysaccharide derived from Escherichia coli, and therefore an agent containing lipopolysaccharide derived from bacteria belonging to the genus Paracoccus is useful as an agent for regulating cytokine production by lipopolysaccharide derived from Escherichia coli. Furthermore, the agent for regulating cytokine production is also useful as a feed composition, food composition, or pharmaceutical composition, and may be useful, for example, as a pharmaceutical composition for preventing or treating cytokine-related diseases.

[0111] In another aspect, the present invention makes it possible to regulate TLR4 activation by E. coli-derived lipopolysaccharide. Lipopolysaccharide derived from bacteria belonging to the genus Paracoccus can regulate TLR4 activation by E. coli-derived lipopolysaccharide, and therefore an agent containing lipopolysaccharide derived from bacteria belonging to the genus Paracoccus is useful as a regulator of TLR4 activation by E. coli-derived lipopolysaccharide. Furthermore, the cytokine production regulator is also useful as a feed composition, food composition, or pharmaceutical composition, and may be useful, for example, as a pharmaceutical composition for the prevention or treatment of diseases associated with the TLR4 signaling system.

[0112] [Rule 26, amended 23.10.2023]

Claims

1. An agent that regulates cytokine production, containing lipopolysaccharide derived from bacteria belonging to the genus Paracoccus.

2. An agent that modulates TLR4 activation, containing lipopolysaccharide derived from bacteria belonging to the genus Paracoccus.

3. The agent according to claim 1 or 2, wherein the production of cytokines induced by bacteria or the activation of TLR4 is regulated by lipopolysaccharides derived from bacteria belonging to the genus Paracoccus.

4. The agent according to claim 3, wherein the production of cytokines or activation of TLR4 induced by lipopolysaccharides of Escherichia coli is regulated by lipopolysaccharides derived from bacteria belonging to the genus Paracoccus.

5. The agent according to claim 1 or 2, wherein the adjustment includes inhibition.

6. The agent according to claim 1 or 2, comprising a lipid A portion of a lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus, wherein four 3-hydroxyacyl chains having 8 to 16 carbon atoms are bonded to the glucosamine skeleton of the lipid A, and an acyl chain having 8 to 13 carbon atoms is further bonded to the hydroxyl group at the 3 position of one or two of the 3-hydroxyacyl chains.

7. The lipid A portion has the structure of formula (I): 【Transformation 8】 The agent according to claim 6, having the following characteristics.

8. A feed composition comprising the agent according to claim 1 or 2.

9. A pharmaceutical composition or food composition comprising the agent described in claim 1 or 2.

10. A method for regulating cytokine production, comprising administering lipopolysaccharide derived from bacteria belonging to the genus Paracoccus to a subject.

11. A method for regulating TLR4 activation, comprising administering lipopolysaccharide derived from bacteria belonging to the genus Paracoccus to a subject.

12. The method according to claim 10 or 11, wherein the production of cytokines induced by bacteria or the activation of TLR4 is regulated by lipopolysaccharides derived from bacteria belonging to the genus Paracoccus.

13. The method according to claim 12, wherein the production of cytokines or the activation of TLR4 induced by lipopolysaccharides of Escherichia coli is regulated by lipopolysaccharides derived from bacteria belonging to the genus Paracoccus.

14. The method according to claim 10 or 11, wherein the adjustment includes suppression.

15. The method according to claim 10 or 11, wherein the lipopolysaccharide has a lipid A portion derived from a bacterium belonging to the genus Paracoccus, and four 3-hydroxyacyl chains having 8 to 16 carbon atoms are bonded to the glucosamine skeleton of the lipid A, and an acyl chain having 8 to 13 carbon atoms is further bonded to the hydroxyl group at the 3 position of one or two of the 3-hydroxyacyl chains.

16. The lipid A portion has the structure of formula (I): 【Chemistry 9】 The method according to claim 15, having the following characteristics.

17. A lipopolysaccharide derived from bacteria belonging to the genus Paracoccus, used for regulating cytokine production.

18. A lipopolysaccharide derived from bacteria belonging to the genus Paracoccus, used for regulating TLR4 activation.

19. The lipopolysaccharide derived from bacteria belonging to the genus Paracoccus according to claim 17 or 18, wherein the production of cytokines induced by bacteria or the activation of TLR4 is regulated by the lipopolysaccharide derived from bacteria belonging to the genus Paracoccus.

20. The lipopolysaccharide derived from bacteria belonging to the genus Paracoccus according to claim 19, wherein the production of cytokines or activation of TLR4 induced by lipopolysaccharides of Escherichia coli is regulated by lipopolysaccharides derived from bacteria belonging to the genus Paracoccus.

21. The lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus according to claim 17 or 18, wherein the regulation includes inhibition.

22. A lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus according to claim 17 or 18, wherein the lipopolysaccharide has a lipid A portion derived from a bacterium belonging to the genus Paracoccus, and four 3-hydroxyacyl chains having 8 to 16 carbon atoms are bonded to the glucosamine skeleton of the lipid A, and acyl chains having 8 to 13 carbon atoms are further bonded to the hydroxyl groups at the 3 position of one or two of the 3-hydroxyacyl chains.

23. The lipid A portion has the structure of formula (I): 【Chemistry 10】 A lipopolysaccharide derived from a bacterium belonging to the genus Paracoccus, as described in claim 22.

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