Treatment drugs for diseases caused by intestinal immune disorders

Abstract

The purpose of the present invention is to control the number of peripheral regulatory T cells in the intestinal tract to thereby establish a novel means for treating a disease relating to the cells. For this purpose, provided is a therapeutic drug for a disease, said drug comprising a substance capable of controlling the number of peripheral regulatory T cells in the intestinal tract and being characterized in that the aforesaid substance is an activator or suppressor of the hepatic branch of vagal afferent pathway, an activator or suppressor of left vagal efferent pathway, or an agonist or antagonist of muscarinic acetylcholine receptor.

Description

[Technical Field]

[0001] This invention relates to a novel therapeutic agent for a disease, a novel screening method for a therapeutic agent for a disease, and a novel treatment method for a disease. [Background technology]

[0002] The intestines function as a vital organ responsible for digestion and absorption, and are protected from the outside world (lumen) by a single layer of columnar epithelial cells. Within the lumen, they are constantly exposed to foreign substances such as over 100 trillion intestinal bacteria and dietary antigens, but intestinal homeostasis is maintained by the function of peripheral regulatory T cells (pTregs) in the intestines, preventing excessive inflammatory responses. Until now, specific intestinal bacteria, components derived from intestinal bacteria, short-chain fatty acids, cytokines, etc., have been considered important for the differentiation and maintenance of peripheral regulatory T cells. On the other hand, the relatively high incidence of inflammatory bowel disease in depression and irritable bowel syndrome, which have been considered neurological diseases, has suggested that the autonomic nervous system may be deeply involved in intestinal immune abnormalities (Non-patent documents 1, 2, 3, and 4). Recent reports have suggested the possibility that the nervous system is involved in the intestinal immune mechanism, but the relationship between the nervous system and intestinal peripheral regulatory T cells has long been unclear. Furthermore, previous research reports on the brain-gut axis have not specifically shown the neural circuits connecting the brain and the gut, leaving many mysteries from an anatomical perspective. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Godinho-Silva, C., Cardoso, F. & Veiga-Fernandes, H. Neuro-Immune Cell Units: A New Paradigm in Physiology. Annu. Rev. Immunol. 37, 19-46 (2019). [Non-Patent Document 2] Chavan, SS, Pavlov, VA & Tracey, KJ Mechanisms and Therapeutic Relevance of Neuro-immune Communication. Immunity 46, 927-942 (2017). [Non-Patent Document 3] Huh, JR & Veiga-Fernandes, H. Neuroimmune circuits in inter-organ communication. Nat Rev Immunol 20, 217-228 (2019). [Non-Patent Document 4] Chu, C., Artis, D. & Chiu, IM Neuro-immune Interactions in the Tissues. Immunity 52, 464-474 (2020). [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] If the relationship between the nervous system and peripheral regulatory T cells in the intestines is clarified, it will become possible to artificially regulate the number of peripheral regulatory T cells, which could lead to the development of new treatment methods for diseases related to peripheral regulatory T cells (e.g., inflammatory bowel disease).

[0005] This invention was made against this background and aims to regulate the number of peripheral regulatory T cells in the intestines and provide a new therapeutic means for diseases related to these cells. [Means for solving the problem]

[0006] As a result of diligent research to solve the above problems, the inventors have found that 1) peripheral regulatory T cells in the intestines are influenced by parasympathetic nerve signals from the brain, 2) the liver collects and integrates information about the intestinal environment and transmits this information to the brain via the vagus nerve, and 3) antigen-presenting cells, which are considered to be extremely important for the differentiation and maintenance of peripheral regulatory T cells in the intestines, are located in close proximity to nerves in the lamina propria of the intestinal mucosa, and that muscarinic acetylcholine receptor subtype 1 is strongly expressed in these intestinal antigen-presenting cells.

[0007] This invention was completed based on the above findings. In other words, the present invention provides the following [1] to

[20] . [1] A therapeutic agent for a disease containing a substance that has the effect of regulating the amount of peripheral regulatory T cells in the intestinal tract, characterized in that the substance is a substance that activates or inhibits the afferent pathway of the hepatic branch of the vagus nerve, a substance that activates or inhibits the efferent pathway of the left vagus nerve, or an agonist or antagonist of a muscarinic acetylcholine receptor.

[0008] [2] A therapeutic agent for the disease described in [1], characterized in that the disease is inflammatory bowel disease, autoimmune disease, allergy, cancer, depression, or gastrointestinal infection.

[0009] [3] A therapeutic agent for the disease described in [1], characterized in that the disease is inflammatory bowel disease.

[0010] [4] A therapeutic agent for any of [1] to [3], characterized in that the substance that regulates the amount of peripheral regulatory T cells in the intestinal tract is a muscarinic acetylcholine receptor agonist.

[0011] [5] A therapeutic agent for the disease described in [4], characterized in that the agonist of a muscarinic acetylcholine receptor is bethanechol, muscarine, pilocarpine, or cevimeline.

[0012] [6] A therapeutic agent for any of [1] to [3], characterized in that the substance that regulates the amount of peripheral regulatory T cells in the intestinal tract is an antagonist of muscarinic acetylcholine receptors.

[0013] [7] A therapeutic agent for the disease described in [6], characterized in that the antagonist of a muscarinic acetylcholine receptor is atropine, tropicamide, oxybutynin, propiverine, tolterodine, solifenacin, or imidafenacin.

[0014] [8] A screening method for therapeutic drugs for a disease, comprising the steps of co-culturing intestinal antigen-presenting cells and CD4-positive T cells in the presence of a test substance, and detecting the induction of regulatory T cells.

[0015] [9] The screening method according to [8], characterized by detecting the induction of regulatory T cells by detecting the expression of FoxP3.

[0016]

[10] The screening method according to [8] or [9], characterized in that the disease is inflammatory bowel disease, autoimmune disease, allergy, cancer, depression, or gastrointestinal infection.

[0017]

[11] The screening method according to [8] or [9], characterized in that the disease is inflammatory bowel disease.

[0018]

[12] A method for treating a disease by regulating the amount of peripheral regulatory T cells in the intestinal tract, characterized by comprising activating or inhibiting the afferent vagus nerve hepatic branch of the target of treatment, activating or inhibiting the efferent left vagus nerve of the target of treatment, or administering a muscarinic acetylcholine receptor agonist or antagonist to the target of treatment.

[0019]

[13] A method for treating the disease described in

[12] , characterized in that the disease is inflammatory bowel disease, autoimmune disease, allergy, cancer, depression, or gastrointestinal infection.

[0020]

[14] A method for treating the disease described in

[12] , characterized in that the disease is inflammatory bowel disease.

[0021]

[15] A method for treating any of the diseases described in

[12] to

[14] , characterized by comprising administering a muscarinic acetylcholine receptor agonist to the patient.

[0022]

[16] A method for treating the disease described in

[15] , characterized in that the agonist of a muscarinic acetylcholine receptor is bethanechol, muscarine, pilocarpine, or cevimeline.

[0023]

[17] A method for treating any of the diseases described in

[12] to

[14] , characterized by comprising administering a muscarinic acetylcholine receptor antagonist to the target of treatment.

[0024]

[18] A method for treating the disease described in

[17] , characterized in that the antagonist of the muscarinic acetylcholine receptor is atropine, tropicamide, oxybutynin, propiverine, tolterodine, solifenacin, or imidafenacin.

[0025]

[19] A method for treating any of the diseases described in

[12] to

[18] , characterized in that the target of treatment is an animal other than a human.

[0026]

[20] A method for operating a cuff electrode to regulate the amount of peripheral regulatory T cells in the intestinal tract by stimulating the hepatic branch of the vagus nerve.

[0027] This specification includes the content described in the specification and / or drawings of the Japanese Patent Application No. 2020-095241, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0028] This invention provides a novel therapeutic agent for a disease, a novel screening method for therapeutic agents for a disease, and a novel treatment method for a disease. [Brief explanation of the drawing]

[0029] [Figure 1] The potential interaction between APCs and nerve cells in the gut. [Figure 2] The hepatic vagal sensory afferent pathway is essential for NTS activation during colitis. [Figure 3] The liver-brain-gut axis controls the homeostasis of colonic Tregs via muscarinic signaling from APCs. [Figure 4] Disruption of the hepatovagal pathway exacerbates colitis in mice in a muscarinic signaling-dependent manner. [Figure 5] Muscarinic signaling in the colonic APC activates Treg induction. [Figure 6] Colitis activates the hepatocerebral axis. [Figure 7] Anatomical diagram of the hepatic vagus nerve in a mouse. [Figure 8] The effect of vagus nerve transection on the maintenance and stability of pTregs in the large intestine. [Figure 9] Maintaining homeostasis of Treg cells in the large intestine involves afferent vagus nerves from the liver, rather than the spinal cord. [Figure 10] Hemiphrenic vagus nerve sectioning revealed functional asymmetry of the vagus nerve. [Figure 11] The effects of VGx and HVx on endogenous enteric neurons. [Figure 12] The effects of mAChR and α7nAChR on maintaining Treg cells in the colon. [Figure 13] The effect of gut microbiota on maintaining colonic Tregs in the liver-brain-gut axis. [Figure 14] The effect of HVx on colitis. [Figure 15] Schematic diagram of vagus nerve hepatic branch electrical stimulation (VHNS). [Figure 16] Vagus nerve hepatic branch electrical stimulation (VHNS) suppresses the pathogenesis of colitis in mice. [Figure 17] Two-bottle preference assays using vagus nerve-resected mice. [Figure 18] Regulation of lung immune cells by the vagus nerve's hepatic branches. [Figure 19] Control of intestinal peristalsis by the vagus nerve. [Modes for carrying out the invention]

[0030] The present invention will be described in detail below. (1) Therapeutic drugs The present invention provides a therapeutic agent for a disease that contains a substance that regulates the amount of peripheral regulatory T cells in the intestinal tract, characterized in that the substance is a substance that activates or inhibits the afferent pathway of the hepatic branch of the vagus nerve, a substance that activates or inhibits the efferent pathway of the left vagus nerve, or an agonist or antagonist of a muscarinic acetylcholine receptor.

[0031] In this specification, "regulating the amount of peripheral regulatory T cells in the intestines" means increasing or decreasing the amount of peripheral regulatory T cells in the intestines. By increasing the amount of peripheral regulatory T cells in the intestines, excessive immune responses can be suppressed, and therapeutic effects against inflammatory diseases can be expected. On the other hand, by decreasing the amount of peripheral regulatory T cells in the intestines, immune responses can be strengthened, and therapeutic effects against gastrointestinal infections can be expected.

[0032] When used to increase the amount of peripheral regulatory T cells in the intestinal tract, the therapeutic agent for the disease of the present invention contains a substance that activates the vagus nerve hepatic branch afferent pathway, a substance that activates the left vagus nerve efferent pathway, or a muscarinic acetylcholine receptor agonist. Conversely, when used to decrease the amount of peripheral regulatory T cells in the intestinal tract, the therapeutic agent for the disease of the present invention contains a substance that inhibits the vagus nerve hepatic branch afferent pathway, a substance that inhibits the left vagus nerve efferent pathway, or a muscarinic acetylcholine receptor antagonist.

[0033] The types of diseases treated are not particularly limited, as long as they can be treated by regulating (increasing or decreasing) the amount of peripheral regulatory T cells in the intestines. Specifically, diseases that can be treated by increasing the amount of peripheral regulatory T cells in the intestines include diseases caused by intestinal immune abnormalities (inflammatory bowel disease, autoimmune diseases, allergies, etc.), cancer, and depression. Diseases that can be treated by decreasing the amount of peripheral regulatory T cells in the intestines include gastrointestinal infections and cancer. Examples of gastrointestinal infections include norovirus infection, rotavirus infection, and pathogenic E. coli enteritis.

[0034] Substances that activate or inhibit the afferent pathway of the hepatic branch of the vagus nerve, substances that activate or inhibit the efferent pathway of the left vagus nerve, and agonists or antagonists of muscarinic acetylcholine receptors are not particularly limited as long as they have the effect of regulating the amount of peripheral regulatory T cells in the intestinal tract. Specific examples of muscarinic acetylcholine receptor agonists include bethanechol, muscarine, pilocarpine, and cevimeline, while examples of muscarinic acetylcholine receptor antagonists include atropine, tropicamide, oxybutynin, propiverine, tolterodine, solifenacin, and imidafenacin, but are not limited to these.

[0035] The therapeutic agent for the disease of the present invention can be prepared by formulating a substance that regulates the amount of peripheral regulatory T cells in the intestinal tract using known pharmaceutical methods. Specifically, it can be prepared as an injectable (intraperitoneal injection, subcutaneous injection, intravenous injection, intramuscular injection), drip infusion, capsule, liquid, suspension, emulsion, etc. Other components such as pharmacologically acceptable carriers may be included in the formulation. Examples of other components include sterile water, physiological saline, solvent, base material, emulsifier, vegetable oil, suspension agent, surfactant, stabilizer, preservative, binder, diluent, isotonic agent, analgesic agent, disintegrant, lubricant, buffer, coating agent, colorant, and other additives, which can be used in appropriate combinations.

[0036] The therapeutic agent for the disease of the present invention is primarily intended for use in humans, but may also be used in animals other than humans. Examples of animals other than humans include mice, rats, hamsters, rabbits, cats, dogs, cattle, horses, pigs, sheep, and monkeys.

[0037] The dosage of the therapeutic agent for the disease of the present invention can be appropriately determined depending on the type of substance that regulates the amount of peripheral regulatory T cells in the intestinal tract, the type of disease, the form of administration, the method of administration, and the age and weight of the patient being treated. For example, when administering a muscarinic acetylcholine receptor agonist to a human, it is preferable to administer 0.1 to 100 g per adult per day, and more preferably 0.1 to 10 g.

[0038] The method of administering the therapeutic agent for the disease of the present invention is not particularly limited, but examples include intraperitoneal injection, subcutaneous injection, intralymphatic injection, intravenous injection, and intravenous drip infusion.

[0039] (2) Screening method The present invention relates to a screening method for a drug for a disease, characterized by comprising the steps of co-culturing intestinal antigen-presenting cells and CD4-positive T cells in the presence of a test substance, and detecting the induction of regulatory T cells. The method for detecting the induction of regulatory T cells is not particularly limited, but it is preferably carried out by a method for detecting FoxP3 expression. The disease may be the same as that of the drug described above.

[0040] (3) Treatment method The present invention relates to a method for treating a disease by regulating the amount of peripheral regulatory T cells in the intestinal tract, and is characterized by comprising activating or inhibiting the afferent vagus nerve hepatic branch of the target of treatment, activating or inhibiting the efferent left vagus nerve of the target of treatment, or administering a muscarinic acetylcholine receptor agonist or antagonist to the target of treatment.

[0041] Activation or inhibition of the vagus nerve hepatic afferent pathway can be achieved by administering substances with such effects to the patient, but it can also be achieved by applying electrical stimulation to the vagus nerve hepatic afferent pathway or by transection of the vagus nerve hepatic afferent pathway. Similarly, activation or inhibition of the left vagus nerve efferent pathway can be achieved by administering substances with such effects to the patient, applying electrical stimulation to the left vagus nerve efferent pathway, or by transection of the left vagus nerve efferent pathway.

[0042] The agonists and antagonists of muscarinic acetylcholine receptors, the diseases they target, and the target populations for treatment can be the same as those for the aforementioned therapeutic drugs.

[0043] (4) Operation method of the cuff electrode The cuff electrode operation method of the present invention involves stimulating the vagus nerve hepatic branch to regulate the amount of peripheral regulatory T cells in the intestinal tract. As shown in Figure 15, by activating the cuff electrode placed on the vagus nerve hepatic branch and applying electrical stimulation to the vagus nerve hepatic branch, the amount of peripheral regulatory T cells in the intestinal tract can be increased, thereby enabling the treatment of diseases such as inflammatory bowel disease. [Examples]

[0044] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0045] [Example 1] Foxp3 + Peripheral regulatory T cells (pTregs) are most abundant in mucosal tissue, particularly the lamina propria (LP) of the large intestine, and maintain immune homeostasis in the intestinal tract. 5,6 pTregs are generated by a combination of signals from microorganisms and food, including cytokines such as TGF-β and RA, Clostridia clusters IV, XIVa, XVIII, Bacteroides fragilis, microbiota-associated molecular patterns (MAMPs), and short-chain fatty acids (SCFAs). 5-13In addition to these numerous environmental stimuli, recent research has made great progress in showing that immune cells are under the control of the autonomic and enteric neurons. 3,14-16 This suggests that the differentiation of pTregs at mucosal sites is governed by mechanisms that have not been recognized until now.

[0046] In fact, the gastrointestinal tract is not only highly innervated but also contains a large number of adaptive and innate immune cells. 14,17 In the LP of the large intestine, immunohistochemical analysis of the localization of neurons (β-tubulin III + ) and MHC-II + APCs (mainly CX3CR1 + mononuclear phagocytes (MNPs)) revealed that neurons and APCs are in close proximity (Figure 1a, b, Figure 5a, b). Intestinal APCs, particularly CX3CR1 + MNPs and CD103 + dendritic cells (DCs) produce RA and preferentially support the generation of pTregs in the intestinal microenvironment rich in TGF-β. 8,18-23 Although the immunomodulatory role of the autonomic nervous system is known 1-3,15-17 , it is not yet fully understood how the vagus nerve affects intestinal homeostasis by controlling intestinal APCs and pTregs. To investigate the immunological function of the vagus nerve, subdiaphragmatic trunk vagotomy (hereinafter, VGx) was performed on wild-type (WT) C57BL / 6 (B6) mice (Figure 5c, d). Interestingly, vagotomized mice had a significant decrease in the number of Foxp3 + T helper cells, particularly Helios-RORγt + pTregs in the large intestine compared with sham-operated mice (Figure 1c, d, Figure 5e, f). In addition to the decrease in pTregs in the large intestine, significant decreases were observed in the levels of Aldh1a1 and Aldh1a2 encoding RA synthase RALDH1 and RALDH2, and the aldehyde dehydrogenase activity of large intestinal APCs (Figure 1e, f).

[0047] To identify the neurotransmitters that transmit signals from intestinal nerve cells to colonic APCs, mRNA-seq was performed on APCs collected from the spleen and intestine. Intestinal APCs showed higher expression levels of the Chrm1 gene, which encodes the muscarinic ACh receptor, compared to splenic APCs, suggesting that neurotransmitters are involved in the regulation of intestinal APCs in a tissue-specific manner (Figure 1g, Figure 5g). Additionally, CX3CR1 was identified. + MNP and CD103 + It is noteworthy that in DC-rich APC fractions, the expression of Chrm1, Aldh1a1, and Aldh1a2 is common to genes that define a typical subset of APC, such as Itgae (CD103), Cx3cr1, and Irf8 (Figure 1h, i, Figure 5h). Furthermore, this finding was confirmed by quantitatively evaluating the expression of Aldh1a1 and Aldh1a2 in APCs of the colon stimulated with multiple neurotransmitters, including Ach, muscarin, adrenaline, neuropeptide Y, substance P, serotonin (5-HT), and neuromedin U (Figure 1j). Furthermore, muscarinic and intestinal neurospheroids induced the expression of Aldh1a1 and Aldh1a2 in colonic APCs obtained from WT mice and human intestines (Figure 1k, l), but co-culture of APCs lacking Chrm1, 2, and 4 (mAChR TKO) with neurospheroids failed (Figure 5i, j). In addition, in colonic APCs of WT mice pre-cultured with muscarinic or neurospheroids, Foxp3 + Treg production was promoted in APCs, but not in APCs of mAChR TKO mice (Figure 5k-n). These results suggest that ACh-mAChR signaling in APCs contributes to maintaining a pTreg population in the gut.

[0048] To test this hypothesis, we evaluated whether vagus nerve-mediated signaling is necessary to prevent intestinal inflammation. VGx increased susceptibility to a dextran sulfate sodium (DSS)-induced colitis model (Figure 6a-c). Since VGx reduced the number of pTregs and induced a localized inflammatory environment, we then investigated which afferent neurons of the vagus nerve are involved in regulating and maintaining the pTreg pool in the intestines. The vagus nerve innervates most of the gastrointestinal tract, and its afferent neurons transmit sensory input to bilateral nodular ganglions (NGs). 24 In colitis, these sensory inputs are further projected to the solitary nuclei (NTS) of the brainstem (Figure 6d-f). Notably, when acute colitis develops, the sensory afference of the liver is activated in vivo, which is achieved by selectively surgically separating the common hepatic branch of the vagus nerve (hereinafter referred to as "HVx"). 25 It disappeared as a result (Figures 2a, b, 7a-c). The liver is constantly exposed to nutrients, bacterial products, toxins, and metabolites from the intestines, and this intestinal-liver axis, connected by portal circulation, has been shown to be a cause of liver disease. 26,27 Furthermore, nutrients and bacterial products activate the vagus nerve via mTORC1 (mechanistic target of rapamycin complex 1) signaling. 28 (Figure 6g-i) It is thought that the hepatic afferentity of the vagus nerve is activated during colitis. In fact, retrograde tracing of the liver supported the idea that the liver senses the intestinal microenvironment, activates the hepatic afferentity of the vagus nerve, and transmits this signal to the brain via the left NG (Figure 2c, d). Importantly, the common hepatic branch of the branching vagus nerve in HVx mice was found to be related to the sympathetic nerve TH, according to electrophysiological and immunohistochemical evaluation. + Neuron-free capsaicin-sensitive TRPV1 +It is mainly composed of sensory afferent nerves (Figure 7d, e). When the common hepatic branch of the vagus nerve was blocked with capsaicin, the number of pERK-positive cells was significantly reduced in the left NG rather than the right NG (Figure 2b). Furthermore, the number of retrograde labeled cells was significantly reduced in the left NG rather than the DRG in HVx mice, indicating that the common hepatic branch of the vagus nerve transmits signals via the left NG, not the right NG or DRG (Figure 2c, Figures 7f-h). These results suggest that sensory information from the gut environment is transmitted to the brain by crossing the ascending pathway of the left vagus nerve from the liver to the brain.

[0049] Based on the anatomical lateral orientation of the vagus nerve, we investigated how the sensory afference of the hepatic vagus nerve affects intestinal Tregs and characterized the effects of HVx on the intestine and spleen. As a result, in APCs obtained from the large intestine of HVx and VGx mice, CD4 +The proportion of pTregs among T cells, as well as the expression and activity of aldehyde dehydrogenase, were significantly reduced compared to mice that underwent sham surgery (Figure 2e, Figures 8a-d). This reduction in colonic pTregs by HVx occurred rapidly on day 2 and was consistently observed regardless of the sex, strain, or species of the mice (Figure 8e-i). Since pTregs generated in vivo have demethylated Treg-specific demethylation regions (TSDRs), the rapid decrease in pTregs in HVx mice is thought to be due to the vagus nerve altering the DNA methylation status of TSDR29-34, thereby epigenetically influencing the maintenance and stability of intestinal pTregs. Similarly, HVx impaired the differentiation and stability of pTregs in T cell-reconstituted mice and unleashed the suppression of the RA-mediated Th17 differentiation program (Figure 8j-l). The importance of hepatic afferent sensation of the left ngeal nerve (NG) in maintaining an adequate reservoir of intestinal Tregs was supported by the results showing that perturbation of hepatic vagal afferentness to the left NG by capsaicin administration, rather than disinhibition of DRGs by capsaicin or RTX, leads to a decrease in the number of colonic Tregs and a decrease in APC aldehyde dehydrogenase activity (Figure 9). This suggests that the function of the vagus nerve is asymmetric (Figure 2f, Figure 10a-c). The maintenance of colonic Tregs is less dependent on the sympathetic nervous system compared to MM and ILC2, as has already been reported in relation to viral and parasitic infections. 35,36 (Figure 10d-h). In contrast to the effects on the small and large intestines, the frequency of splenic Tregs was normal in HVx mice. However, surgical resection of the CG-SMG or chemical blockade of β2-adrenergic receptors and 7-nicotinic Ach receptors (7-nAchR) significantly reduced splenic Tregs (Figure 10i-o). It has been previously reported that splenic nerves, which are mainly composed of adrenaline fibers originating from the CG-SMG, suppress T cell activation via 7-nAchR and inhibit systemic cytokine production from splenic macrophages. 37-42It was predicted that severance of the CG-SMG or splenic nerve, rather than vagus nerve transection itself, would affect the splenic Treg population. These results support the idea that the liver-brain-gut neural arc monitors the intestinal microenvironment and regulates intestinal pTreg levels by sending Ach signals to control colonic APCs.

[0050] Therefore, the inventors decided to investigate the role of muscarinic Ach signaling in intestinal APCs in vivo. Genetically removing mAChR reduced the expression of Aldh1a1 and Aldh1a2 in colonic APCs, resulting in a decrease in colonic pTregs (Figure 3a-d). In VGx and HVx mice, c-Fos present in the colonic myoplexus was reduced. +Although the number of enteric nerve cells was found to be lower in VGx and HVx mice compared to sham surgery mice, there was no effect on the expression of Hand2, a transcription factor necessary for terminal differentiation of enteric nerve cells (Figure 11a-f). Furthermore, in VGx and HVx mice, intestinal small molecule peptide neurotransmitters of the parasympathetic nervous system (Ach), rather than those of the sympathetic nervous system (norepinephrine) or sensory nervous system (calcitonin gene-related peptide (CGRP)), were reduced compared to sham surgery mice (Figure 11g-h). Since hepatic selective vagus nerve transection and trunk vagus nerve transection mainly reduce local Ach levels in the intestine, we investigated whether mAChR activation restores the expression and activity of aldehyde dehydrogenase in the intestinal APC. Administration of bethanechol, an mAChR agonist, restored the expression of Aldh1a1 and Aldh1a2 in the colonic APC of HVx mice compared to mice that underwent sham surgery. However, 7-nAchR agonists and antagonists, as well as 7-nAchR43 gene deficiency, made little contribution (Figure 12a-j). Furthermore, bethanechol-treated HVx WT mice showed an increased frequency of pTregs, rather than in mAChR TKO mice, suggesting that the hepatocerebral-enteric nerve arc stimulates the colonic APC and forms a pTreg niche (Figure 3e, Figure 12k, l). Taken together, these results support the idea that neural input from hepatic sensory afferent nerves is essential for initiating this vagus-vagus hepatocerebral-enteric nerve arc, and that this reflex arc is independent of the sympathetic nervous system and axonal reflexes.

[0051] Since the generation and maintenance of pTregs are highly dependent on the microbiome and metabolites, the inventors investigated the role of the microbiome in the generation and maintenance of pTregs. The gut microbiomes of HVx mice and sham surgical control mice showed no significant differences in composition or diversity, and transplantation of fecal bacteria from these mice induced equivalent amounts of gut pTregs in germ-free mice (Figures 3 and 13a-e). This suggests that the hepatocerebral enteric nerve arc maintains a pTreg pool independently of changes in the gut microbiota and metabolites caused by HVx. Furthermore, even after sterilization of the gut of HVx mice, pTregs, particularly microorganism-independent pTregs, were induced.13 No decrease was observed (Figure 13l, m). Taken together, these data suggest that the liver-brain-gut neural arc maintains the baseline level of pTregs in the gut, and that this level depends on the input of tonic microorganisms.

[0052] The role of the liver-brain-gut neural circuit in regulating intestinal pTregs was previously unexpected, so we investigated whether this is related to the development of colitis. In mice with surgically or chemically severed hepatovagal nerve branches, the frequency of pTregs decreased (Figures 3a, 8a, b, 5c, d), and as a result, they became more susceptible to colitis induced by DSS and 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Figures 4a-c, 14a-c). Similarly, Rag2 - / - Unlike mice with T cell deficiency, HVx did not exacerbate colitis in mice (Figure 14d-f). Furthermore, splenectomy was used in an endotoxemia model. 37-39 Unlike the previously observed group, HVx had little effect on the severity of colitis in HVx mice (data not shown). Furthermore, because HVx did not significantly alter the composition of the gut microbiota (Figure 13a-c), HVx mice exhibited more severe colitis than cohabiting sham-surgical mice (Figure 14g, h). In addition, HVx did not increase susceptibility to DSS-induced colitis in antibiotic-treated mice or MyD88-deficient mice (Figure 14il). This suggests that sustained microbial input is necessary to maintain the gut pTreg pool and to activate the liver-brain-gut neural arc. On the other hand, the exacerbation of DSS-induced colitis in HVx mice was suppressed by cholinergic agents (Figure 4g-i, Figure 14m-o). Taken together, these data suggest that the liver-brain-gut neural circuit functions as a feedback loop to protect the gut from excessive inflammation (Figure 14p).

[0053] Based on the above, this study concluded that the exogenous vagal reflex connecting the hepatic vagal sensory afferent pathway, brainstem, vagal efferent pathway, and enteric neurons is related to mAChR. +The study revealed an interesting activity: stimulating APCs and maintaining the reservoir of peripheral regulatory T cells. Recent retrospective cohort studies have reported that patients newly diagnosed with depression are at higher risk of developing IBD. 44 An imbalance in the autonomic nervous system is highly likely to contribute to the pathogenesis of IBD. Direct and reciprocal gut-brain neural reflexes control appetite, food reward, cancer, fatty liver, Parkinson's disease, and other neurological disorders. 45-48 In addition, the inventors' findings offer a unique perspective on tissue-specific immune cell adaptation mediated by both the liver and the central nervous system. Dysfunction of this liver-brain-gut neural circuit makes the gut more susceptible to inflammation. In other words, the suppression of tumorigenesis by denervation may be due to a reduction in the number of pTregs in the colon. This study revealed that the liver-brain-gut neural circuit plays a crucial role in identifying immunomodulatory niches and fine-tuning the immune response in the gut. Interventions targeting this liver-brain-gut neural arc could lead to IBD 49 It has the potential to be widely applied to the treatment of infectious diseases, intestinal cancer, and other conditions.

[0054] [Experimental Method] animal C57BL / 6(WT) mice, BALB / c mice, and Jcl:Wistar rats were purchased from CREA Nippon (Tokyo, Japan). Five-week-old male germ-free (GF) mice (C57BL / 6 background strain) were purchased from Sankyo Lab Service Co., Ltd. and reared at the Keio University School of Medicine GF Facility. Ly5.1 mice, Foxp3 CreERT2 Mouse, Cx3cr1 GFP / GFP Transgenic (Cx3cr1 gfp ) Mouse, Rag2 knockout (Rag2 - / - ) Mouse, Myd88 knockout (Myd88 - / -The mice were obtained from The Jackson Laboratory (Maine, USA). The Chrm1 / Chrm2 / Chrm4 triple knockout (mAChR TKO) mice were obtained from the Animal Resources Development Center (Kumamoto, Japan). The Wnt1 promoter / enhancer (Wnt1-Cre) was crossed with the EGFP reporter mouse (CAG-CATloxP / loxP-EGFP) to create the Wnt1-Cre / Floxed-EGFP double transgenic mouse. 50 I obtained Foxp3. CreERT2 Mouse as a floxed-tdTomato reporter mouse 51 These mice were crossbred with other mice to obtain Foxp3-reporter mice. All experiments used mice aged 6-8 weeks. All mice were reared under SPF conditions at the animal housing facility of Keio University School of Medicine. All experiments were approved by the regional animal experimentation committee (Keio University, Tokyo, Japan) and conducted in accordance with institutional guidelines and home office regulations.

[0055] Subphrenic vagus nerve sectioning and hepatic selective vagus nerve sectioning Subphrenic vagus nerve sectioning was performed bilaterally or unilaterally (left or right), as previously reported (Figure 5c,d). 52 Male mice anesthetized with a combination of medetomidine, midazolam, and butorphanol were given a midline incision to broadly expose the upper abdominal organs. The bilateral subdiaphragmatic trunks of the vagus nerve along the esophagus were exposed and transected. In the sham surgery group, these vagus nerve trunks were exposed but not transected. Liver-selective vagus nerve transsection (HVx) was performed as described (Figure 7). 25 The ventral subphrenic vagus trunk was exposed under anesthesia as described above. Since the common hepatic branch of the vagus nerve forms a neurovascular bundle, this branch was selectively ligated with silk sutures and cut using a microscope. In the sham surgery group, the common hepatic branch was exposed but not cut.

[0056] Selective blockade of hepatic vagal afferent pathways by applying capsaicin around the vagus nerve. After separating the hepatic branch of the vagus nerve trunk from the surrounding tissue using paraffin paper, it was wrapped for 30 minutes with a cotton swab soaked in either vehicle (Tween80:olive oil = 1:9) alone or 10 mg / ml of capsaicin dissolved in vehicle solution. After 30 minutes, the cotton string was removed and the abdominal incision was closed. 28 .

[0057] Laparoscopic surgery and superior mesenteric ganglion transection A midline incision was made to widely expose the upper abdominal organs of mice anesthetized with isoflurane. The celiac ganglion (CG) is connected to the superior mesenteric ganglion (SMG) via a short nerve trunk (Figure 10d). The celiac ganglion-superior mesenteric ganglion (CG-SMG) complex was exposed and removed along the superior mesenteric artery. In the sham surgery group, the superior mesenteric artery was exposed but not removed. 53 .

[0058] Intrathecal administration of resiniferatoxin (RTX) and capsaicin DRG or spinal TRPV1 + To perform neuronal ablation, resiniferatoxin (RTX) (25 ng / mouse, vehicle; 0.25% DMSO / 0.02% Tween-80 / 0.05% ascorbic acid in PBS) or capsaicin (10 μg / mouse, vehicle; 10% EtOH / 10% Tween 80 in PBS) was injected intrathecally into mice using a 25-liter Hamilton syringe with a 28-gauge needle. Control mice were administered only the vehicle. The phenotype of immune cells in the colon was analyzed 7 days after injection. TRPV1 in DRG and spinal cord was analyzed. + Neuronal depletion was confirmed by immunohistochemistry.

[0059] Parabiosis The parabiosis surgery was performed using the method described above. 54After shaving the corresponding temporal region of each mouse, a matching skin incision was made from the forelimb to the base of the hindlimb, and the subcutaneous fascia was bluntly dissected to create approximately 1 / 2 cm of free skin. The corresponding free skin was then tightly sutured with surgical clips to attach the parabion. Two weeks after surgery, the mice were subjected to either sham or HVx.

[0060] T cell reconfiguration model The T cell rearrangement model was performed as previously described. 54 Rag2 - / - Mice were selected using FACS to obtain wild-type naive CD4 + CD45Rb hi Cells 3 × 10 5 The drug was injected intraperitoneally into each mouse. The mice's body weight was monitored weekly. At the end of the experiment, colonic Treg cells were analyzed by FACS.

[0061] DSS-induced colitis model Mice were given a 2% sodium dextran sulfate (DSS) solution to induce colitis. The mice's body weight was measured daily, and the presence of diarrhea and rectal bleeding was visually checked. DAI was assessed in a blinded mouse group (maximum total score of 12). Histological activity score (maximum total score of 40) was assessed as the sum of three parameters: extent, inflammation, and cryptographic damage. 55 .

[0062] TNBS-induced colitis model 2,4,6-trinitrobenzenesulfonic acid (TNBS) was obtained from Sigma-Aldrich. To pre-sensitize the mice, a 1.5 × 1.5 cm area of ​​abdominal skin was scraped and 150 μl of 1% (w / v) TNBS solution was applied. Seven days after pre-sensitization, the mice were re-administered 150 μl of 2.5% TNBS in 50% ethanol rectally under general anesthesia with isoflurane. 56 Colon tissue sections were stained with H&E, and histological scores were determined in the same manner as in the DSS model.

[0063] Administration of antibiotics To evaluate the potential contribution of gut microbiota to the exacerbation of DSS colitis following vagus nerve transection, mice were administered broad-spectrum antibiotics (6.7 g / L ampicillin, 6.7 g / L neomycin, 3.3 g / L vancomycin, 6.7 g / L metronidazole) three times a week via a nasogastric tube for three weeks (500 μL / mouse). As a control, the same amount of distilled water was administered via a nasogastric tube.

[0064] In vivo administration of betanethyl, salbutamol, propranolol, methyl licaconitine, and GTS-21 Bethanechol (BETH) was dissolved in water. After 12 hours of surgery, mice were given water or BETH (300 μg per mouse) daily. 57 The drugs were injected intraperitoneally. To evaluate the effect of adrenaline signaling on the homeostasis of colonic Tregs, salbutamol (β2-agonist) and propranolol (β-blocker) were used. Salbutamol and propranolol were dissolved in PBS before use. After 12 hours of surgery, mice were injected intraperitoneally daily with PBS (200 μl per mouse), salbutamol (30 μg per mouse), or propranolol (300 μg per mouse). 58 The role of α7 nicotinic acetylcholine receptors in maintaining colonic Tregs was evaluated using methyl licaconitine (MLA, an α7 nicotinic acetylcholine receptor antagonist) and GTS-21 (an α7 nicotinic acetylcholine receptor agonist). MLA and GTS-21 were dissolved in PBS. After 12 hours of surgery, mice were injected intraperitoneally daily with PBS (200 μl per mouse), MLA (150 μg per mouse), or GTS-21 (300 μg per mouse). 59 .

[0065] Retrograde tracing from the liver 1 μl of Alexa Fluor 488-labeled wheat germ aglutinin (WGA488) (5 mg / ml) was injected into the liver in 40 spots using a 30-gauge needle connected to a Hamilton syringe. One week after WGA488 injection, mice were first perfused with PBS, and then with 4% PFA in PBS. The separated NG and DRG were post-processed after 2 hours and cryoprotected by immersion in 30% sucrose in PBS for 24 hours. Sections of the frozen NG and DRG were cut to a thickness of 6 mm using a cryostat, collected on slides, and immediately dried. The slides contained DAPI in ProLong TM I mounted it using Diamond Antifade Mountant.

[0066] Electrophysiological recording of sympathetic nerve activity Sympathetic nervous system activity was measured using the previously described method. 57 The common hepatic branch of the vagus nerve (CG-SMG) was identified and exposed for measurement of neuronal activity. The electrical activity of each nerve was amplified 50,000–100,000 times through a 100–1,000 kHz bandpass filter and monitored with an oscilloscope. The amplified and filtered neuronal activity was converted to a standard pulse by a window discriminator, and then the discharge and electrical background noise were separated. Both discharge rates and neurograms were sampled using a PowerLab analog-to-digital converter and recorded and analyzed on a computer. Background noise measured 30–60 minutes after euthanasia of the animals was subtracted. Neuronal activity was rectified and integrated, normalized to 100% of baseline neuronal activity.

[0067] Isolation of mononuclear cells from the lamina propria of the colonic mucosa of mice. Mucosal mononuclear cells (LPMCs) were isolated using the method described above. 6 The dissected colonic mucosa was cut into 5 mm pieces. The tissue contained calcium, including 1 mM DTT and 5 μM EDTA. 2+ Mg 2+Cells were incubated in free HBSS at 37°C for 30 minutes, followed by digestion with collagenase and DNase for 45 minutes. Cells were then separated using a Percoll density gradient. The number of viable cells was measured using a Countess II (Thermo Fisher Scientific) scale.

[0068] Isolation of spleen cells from mice The spleen was crushed into 100 μm nylon particles, and then the red blood cells were lysed in a 0.84% ​​ammonium chloride solution.

[0069] Nerve cells derived from intestinal neurospheres Whole intestines of Wnt1-Cre / Floxed-EGFP double transgenic cells at embryonic day 13.5 (E14.5) were digested with 0.1% trypsin / EDTA trypsin at 37°C for 30 minutes. After mechanically lysing and washing the cells, they were cultured for 7 days in a CO2 incubator at 37°C in ultra-low adhesion T-25 cell culture flasks (CORNING) with supplemented DMEM / F12 (25 μg / ml insulin, 100 μg / ml transferrin, 20 nM progesterone, 30 nM sodium selenite, 60 nM putrescine, 100 ng / ml recombinant human EGF, 100 ng / ml recombinant human FGF, 20 ng / ml B27 50). After neurosphere formation, intestinal neurospheres were plated onto uncoated cell culture plates and cultured for 7 days in differentiation medium (DMEM / F12 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS)). Cells differentiated from intestinal neurospheres were dissociated using trypsin and stained on ice for 30 minutes with antibodies: PE conjugate anti-mouse CD24 antibody (30F-1), APC conjugate anti-mouse CD184 antibody (L276F12), PE / Cy7 conjugate anti-mouse / human CD44 antibody (IM7), and Brilliant Violet 510 conjugate anti-mouse CD45.2 antibody (104). Cell sorting was performed using FACS aria II, and neurons derived from intestinal neurospheres (GFP) were identified. + CD45.2-CD184-CD44-CD24 +The cells were collected. For co-culture, sorted and purified colonic APCs were added to the culture medium.

[0070] Fluorescence-activated cell sorting (FACS) analysis After blocking with anti-mouse CD16 / CD32 antibody for 20 minutes, the cells were incubated with specific fluorescent-labeled monoclonal antibodies at 4°C for 30 minutes, then permeabilized with Permeabilization Buffer, and in the case of Treg staining, the intracellular part was stained with anti-Foxp3 mAb. The following monoclonal antibodies were used for FACS analysis: anti-mouse CD45.2, CD3e, CD4, CD11b, CD11c, MHC-II, NK1.1, TCRβ, B220, NKp46, Gata3, IL-17A, IL-22, Foxp3, Helios, RORγt antibodies. Dead cells were excluded using 7-AAD staining or Fixable Viability Dye eFluor. Events were acquired using a FACS Canto II (BD Biosciences) and analyzed using FlowJo software (BD Biosciences). Colonic APCs (CD45.2 + CD3 - NK1.1 - B220 - MHC - II + cells, CD45 + CD3 - B220 - NK1.1 - CD11c + CD11b - 、CD45 + CD3 - B220 - NK1.1 - CD11c + CD11b + and CD45 + CD3 - B220 - NK1.1 - CD11c - CD11b +Cells were sorted using a BD FACS Aria-II (BD Bioscience). Colonic APCs were cultured overnight in RPMI-1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin and then stimulated with muscarine.

[0071] Measurement of aldehyde dehydrogenase activity Aldehyde dehydrogenase (ALDH) activity was measured using an ALDEFLUOR staining kit according to the manufacturer's protocol. Cells were suspended at a concentration of 10 6 cells / ml in ALDEFLUOR assay buffer containing either the activated ALDEFLUOR substrate (final concentration 1.5 μM) with or without diethylaminobenzaldehyde (DEAB) (final concentration 15 μM), an ALDH inhibitor, and incubated at 37 °C for 30 minutes. FACS analysis was performed using a BD Biosciences FACS CantoII.

[0072] In vitro Treg induction assay Naive CD4 + cells were isolated from the spleens of WT mice using a naïve CD4 + T cell isolation kit. Naive CD4 + cells (1 × 10 5 ) were cultured in 96-well plates using RPMI-1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 U / ml penicillin, 100 lg / ml streptomycin, and 55 μM 2-mercaptoethanol. For Treg induction, naïve T cells were stimulated with muscarine or neurons derived from neurospheroids (1 × 10 5 ) 60 in the presence or absence of colonic APCs (2 × 10 4 ) using 2 μl / well of anti-CD3 / CD28 microbeads and 2 ng / ml of TGF-β for 3 days.

[0073] Organizational sample Normal intestinal mucosa was collected from areas other than the affected area of ​​colorectal cancer patients. All experiments were approved by the Institutional Review Board of Keio University School of Medicine, and written informed consent was obtained from all patients.

[0074] Isolation of human colon LP cells The large intestine was dissected, and the mesentery fat and connective tissue were washed in situ. The entire large intestine was cut into 0.5 cm pieces and digested. These fragments were first washed with HBSS, then incubated in PBS containing 1 mM DTT and 5 mM EDTA at 37°C for 20 minutes. The supernatant containing the IEL fraction was discarded. The remaining LP fraction was washed twice with PBS and then digested with 1.0 mg / ml collagenase and 0.05 mg / ml DNase at 37°C for 60 minutes. The LP suspension was passed through a 70-μm filter. Cells were then separated by Percoll density gradient. The interface was collected, and the cells were washed before staining and cell sorting. CD45 was used for cell sorting. + CD3 - CD19 - CD56 - HLA - DR hi Human colon APCs were gated on by sorting cells using BD FACS Aria-II. Human colon APCs were cultured overnight in RPMI-1640 containing 10% FSB and 1% penicillin-streptomycin, and then stimulated with muscarine.

[0075] RNA sequencing analysis RNA sequencing (RNA-seq) was performed and analyzed using the method previously described. 61Total RNA was prepared from approximately 20,000–50,000 cells using TRIzol. Subsequently, mRNA-seq libraries were generated from the total RNA according to the protocol using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490S), NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads (NEB, E7765S), and NEBNext Multiplex Oligos for Illumina (Index Primers Set 1 and 2) (NEB, E7335S and E7550S). The libraries were sequenced using Illumina with 150 bp (paired-end reads). To quantify the amount of transcript, kallisto (v0.44.0, options: -b 100) was used. 62 RNA-seq reads were pseudo-aligned to ENSEMBL transcripts (release 95 GRCm38) using [tool name]. The expression levels of APC subset signature genes containing neurotransmitter receptor genes (expression >1 TPM in at least one sample) were visualized by creating a heatmap (MORPHEUS; https: / / software.broadinstitute.org / morpheus / ) with hierarchically clustered rows and columns, and a ternary plot (ggtern v3.1.0).

[0076] Collection of fecal samples and isolation of bacterial DNA Fecal samples were collected from the same mouse on day 0 and day 2 postoperatively. In this experiment, each mouse was housed in a separate cage. Bacterial DNA was prepared as previously described. 63Bacterial DNA was isolated by enzymatic lysis using lysozyme and achromopeptidase. The DNA samples were purified by treatment with ribonuclease A and precipitation with 20% polyethylene glycol solution (PEG6000 in 2.5M sodium chloride). The DNA was then centrifuged, washed with 75% ethanol, and dissolved in trisethylenediaminetetraacetic acid (trisEDTA) buffer.

[0077] Sequencing and processing of bacterial 16S rRNA genes in fecal DNA The V3-V4 region, the hypervariable region of the 16S gene, was amplified using Ex Taq Hot Start (Takara Bio) and then purified using AMPure XP (Beckman Coulter). Approximately equal amounts of each amplified DNA were pooled to prepare a mixed sample, which was then sequenced using Miseq Reagent Kit V3 (600 Cycles) and Miseq sequencer (Illumina) according to the manufacturer's instructions. Sequence analysis was performed using QIIME software package version 1.9.1. 64,65 The paired-end sequences were joined using the fastq-join tool from the ea-utils software package (https: / / doi.org / 10.2174 / 1875036201307010001). High-quality sequences per sample (15,000) were randomly selected from sequences that passed the quality filter. Both primer sequences were truncated using cutadapt (https: / / doi.org / 10.14806 / ej.17.1.200), followed by chimera detection using the de novo method of USEARCH66, and then the UCLUST algorithm. 67Using a sequence identity threshold of 96%, sequences were assigned to operational taxonomic units. Similarity searches were performed using the GLSEARCH program against the publicly available 16S (RDP version 10.27 and CORE update 2 September 2012) and NCBI genome databases, and each operational taxonomic unit was assigned. The data was diluted to 10,000 sequences per sample, based on the dilution curve. The relative presence of community members was determined using the diluted data. UniFrac analysis was performed as previously described. 68 .

[0078] Sterile mice Fecal samples were collected from Sham and HVx mice. The fecal samples were suspended in an equal volume (w / v) of PBS containing 40% glycerol, snap-frozen, and stored at -80°C until use. The frozen stock was thawed, suspended in five times the volume of PBS, and passed through a 100 μm cell strainer. GF mice were orally administered 200 μl of the suspension using a sterile stainless steel needle. After 3 weeks of colony formation, the phenotype of colonic immune cells was analyzed.

[0079] Quantitative reverse transcription (qRT-) PCR analysis RNA was isolated and purified from colon tissue and cells using the RNeasy Mini Kit. Reverse transcription was performed using the iScript cDNA Synthesis Kit. Real-time PCR amplification was performed using the Thermal Cycler Dice Real Time System (Takara Bio). Gene expression levels were normalized using 18S ribosomal mRNA.

[0080] Histological and immunohistochemical examinations The liver, colon, NG, and DRG were fixed in 10% formalin and embedded in paraffin. The spinal cord (Th4-7 and 13) and colon were cryoprotected for 24 hours in 30% sucrose solution and stored in OCT compound. The paraffin-embedded colon sections were stained with H&E and then examined. For immunohistochemistry, antigens were activated by autoclaving and blocked with Block Ace. Primary antibody reactions were performed at room temperature for 4 hours (dilution ratios: PGP 9.5 (1 / 1000), pERK 1 / 2 (1 / 500), TUBB3 (1 / 200), TRPV1 (1 / 1000), TH (1 / 1000)) or overnight at 4°C (IA / IE (1 / 200), TUBB3 (1 / 200)). After washing with PBS, sections were incubated with a secondary antibody (1 / 400) labeled with Alexa Fluor 488 or Alexa Fluor 647 at room temperature for 2 hours. Tissue samples were observed using a BX53 microscope (Olympus) and an LSM 710 confocal laser scanning microscope (Carl Zeiss). Images were analyzed using Imaris (Oxford Instruments), ZEN (Carl Zeiss), and ImageJ (NIH).

[0081] Immunohistochemical testing of pERK1 / 2 and c-Fos pEKR1 / 2 and c-Fos expression were analyzed immunohistochemically as reported. 1DSS-treated mice were perfused transcardially with PBS containing 4% paraformaldehyde and 0.2% picric acid under anesthesia. Nodular ganglions and brain tissue were collected and post-fixed at 4°C for 2 hours to overnight with the same fixative, followed by incubation in phosphate buffer containing 30% sucrose for 48 hours. Longitudinal sections (8 μm) of NG were cut at 48 μm intervals using a precision cryostat (Leica Microsystems, IL). Coronal sections (40 μm) of the hindbrain were cut at 120 μm intervals using a cryostat. Rabbit polyclonal antibody (1 / 500) against pERK1 / 2 and Alexa 488-labeled goat anti-rabbit IgG (1 / 500) were used. Fluorescence images were acquired using a BX50 microscope and a DP50 digital camera (Olympus). For c-Fos staining, anti-c-Fos antiserum (1 / 10,000) was used as the primary antibody. Nickel diaminobenzidine (DAB) was used for color development. Neurons that responded to pERK1 / 2 and c-Fos in the medial NTS were counted.

[0082] c-Fos immunohistochemical staining in the enteric nervous plexus To prepare the enteric plexus, the large intestine of fed Sham, VGx, and HVx mice was cut longitudinally into 3 cm sections and immersed in ice-cold plastic plates containing PBS. The mucosal layer was removed, and the enteric plexus was immersed overnight in 4% PFA and washed with cold PBS at room temperature. The samples were blocked with blocking solution at room temperature for 1 hour. Subsequently, the samples were incubated overnight at room temperature with primary antibodies (HuC / HuD, 1 / 500; c-Fos, 1 / 500) diluted with antibody diluent, washed three times with PBS, and then incubated with secondary antibodies (1 / 400) at room temperature for 90 minutes, washed three times with PBS. The samples were mounted with fluorescent mounting media. Fluorescence of different tissues was measured using a confocal Zeiss Laser Scanning Microscope LSM-710.

[0083] Measurement of CGRP, ACH, and NE levels in the large intestine The levels of neurotransmitters in the large intestine were measured using the method described above. 69-71Colon tissue was washed with PBS and homogenized. The homogenate was centrifuged at 15,000 xg for 10 minutes at 4°C, and the supernatant was collected. The samples were stored at -80°C until use. Protein concentrations were measured by BCA assay (Thermo Fisher Scientific). The concentrations of CGRP (Phoenix Pharmaceuticals), acetylcholine (Abcam), and norepinephrine (LsBio) in the homogenate were measured by ELISA.

[0084] Western blot analysis Proteins were extracted from liver tissue using T-PER and PhosSTOP (Sigma), which contain protease inhibitors. Western blotting was performed using Clarity Western ECL Substrate and ChemiDoc Imaging System (Bio-Rad) as previously described. 72 .

[0085] Raptor knockdown in the liver via RNA interference Si-negative control (Si-Cont) and Si-Raptor (In-VivoReady grade) were conjugated with Invivofectamine 2.0 Reagent (Invitrogen) precisely according to the manufacturer's protocol. Subsequently, 200 μl of the conjugated siRNA was intravenously injected into the tail vein of male WT mice (body weight 22-25 g) at a dose of approximately 7 mg of siRNA per kg of body weight.

[0086] Statistics information All values ​​are expressed as mean ± sem. Statistical analysis was performed using unpaired two-tailed Student's t-tests or Tukey's post-hoc test and one-way ANOV for multiple comparisons.

[0087] Data acquisition All raw and processed sequencing data presented herein are available through NCBI GEO under accession number GSE140952. All computer code for RNA-seq analysis is also available at https: / / github.com / mikamiy / liver-brain-gut-neural-arc.

[0088] [Explanation of the diagram] Figure 1. Potential interaction between APCs and nerve cells in the gut. a. Representative immunofluorescence staining images of CX3CR1-GFP (green) and β-tubulin III (red) in the large intestine of mice. b. CD45.2 of Cx3cr1Gfp mice. + TCRβ - Representative CD11c and MHC-II staining of CD3-B220-NK1.1 gated squamous epithelial mononuclear cells of the colon. cf, Male B6 mice (WT mice) at 8 weeks of age underwent sham surgery or trunk vagus nerve transection (VGx). Colonic T cell phenotype and gene expression in the colon were analyzed 2 days later (n = 12 / group). c, CD4 in the lamina propria (LP) of the colon. + Among T cells, Foxp3 + Frequency of cells (Treg). d, Foxp3 in the large intestine. + e. Expression of RORγt in Treg. f. Expression of Aldh1a1 and Aldh1a2 mRNA in APCs of the colon. f. MHC-II of the colon. + APC (CD45 + TCRβ-CD3-B220-NK1.1-MHC-II + ALDH in ) + Cell frequency. Left panel, ALDH in APC + Cell histogram. Colonic mononuclear cells were incubated with ALDEFLUOR in the absence (solid) or presence (dotted) of DEAB (ALDH inhibitor). Aldefluor is shown above the horizontal line indicating the positive gate. +The percentage of cells is shown. Right panel, quantification. g, Heatmap of the expression of genes encoding neurotransmitter receptors classified by APC in the colon and spleen, selected by RNA-seq analysis. h, Colon CD11b + CD11c - (CD11b SP), CD11b + CD11c + (DP), CD11b - CD11c + Heatmap of macrophage and dendritic cell marker genes in (CD11c SP) cells. The sorting strategy for the experiment is shown in Figure 5. i. Three-tiered plot of gene expression in colonic CD11b SP, DP, and CD11c SP cells. The color scale shows mRNA concentration. Representative markers for neurotransmitter receptors, macrophages, and dendritic cells are shown. j. mRNA expression levels of Aldh1a1 and Aldh1a2 in colonic APCs treated with PBS (control), 10 μM acetylcholine (Ach), 10 μM muscarine (Mus), 100 nM adrenaline (Adre), 100 μM neuropeptide Y (NPY), 100 nM substance P (Sub-P), 10 μM serotonin (5-HT), or 100 ng / ml neuromedin U (NMU) for 12 hours (n = 5 / group). k, Expression of Aldh1a1 and Aldh1a2 in WT and mAChR TKO colonic APCs. Colonic APCs were isolated from WT or Chrm1,2,4-deficient (mAChR TKO) mice and treated with 10 μM Mus or left for 12 hours (n = 6 / group). l, mRNA levels of ALDH1A1 and ALDH1A2 in human colonic APCs. Colonic APCs were treated with 10 μM Mus or left for 12 hours (n = 7 / group). These are representative of three independent experiments (a, b, jl) or a pool of three independent experiments (cf). P values ​​were obtained by unpaired two-tailed Student's t-test (cf, k) or one-way ANOVA with Tukey's post-hoc test (jl). Error bars represent mean ± sem.

[0089] Figure 2 shows that the hepatic vagal sensory afferent pathway is essential for NTS activation during colitis. a, b, WT mice underwent sham surgery or HVx, and were administered DSS for 7 days starting on postoperative day 2 (n = 4 / group). NTS represents the nucleus tractus solitarius, DMV represents the dorsal motor nucleus of the vagus nerve, AP represents the occipital lobe, and NG represents the nodular ganglion. a, Representative image of immunohistochemical staining of c-Fos in the medulla oblongata (upper panel, bar: 200 μm), and section-by-section count of c-Fos expression in NTS and DMV (lower panel). b, Representative image of immunohistochemical staining of pERK in the nodular ganglion (NG) (upper panel, bar: 100 μm). c, d, Retrograde tracing of WGA. Alexa Fluor 488 in the NG one week after WGA injection into the liver. + Representative fluorescence images (c) and quantitative images (d) of neurons (green) and DAPI (blue). White arrows indicate Alexa Fluor 488 during NG. + Neurons are shown. e, WT mice underwent Sham, VGx, or hepatic vagal nerve section (HVx) (n = 9 / group). f, WT mice underwent Sham, ventral subphrenic vagal nerve section (LVx), or dorsal subphrenic vagal nerve section (RVx) (n = 4 / group). e, f, CD4 in the colon 2 days postoperatively. + Foxp3 inside cells + Cell frequency. Representative of two independent experiments (ad, f) or a pool of three independent experiments (e). P-values ​​were obtained by unpaired two-tailed Student's t-test (a, b, d) or one-way ANOVA (e, f) with Tukey's post-hoc test. Error bars represent mean ± sem.

[0090] Figure 3 shows that the liver-brain-gut axis controls colonic Treg homeostasis via muscarinic signaling from APCs. ad, WT and mAchR TKO mice were subjected to sham or HVx. e, WT and mAchR TKO mice were subjected to sham or HVx, and then injected with betanethyl (BETH; ip 300 μg / day) daily. The phenotype of colonic immune cells was analyzed on postoperative day 2 (n = 5 / group). a, MHC-II + ALDH in colonic APC +Cell frequency. ALDH + Histograms of APCs in cells and the colon (left panel). Quantification (right panel). b. mRNA expression of Aldh1a1 and Aldh1a2 in colonic APCs. c, e. CD4 in the colon + Foxp3 in cells + Cell frequency. d, Foxp3 in the large intestine + RORγt in Tregs + Cell frequency. P-values ​​were obtained by unpaired, one-way ANOVA using Tukey's post-hoc test. Error bars represent mean ± sem.

[0091] Figure 4 shows that disruption of the hepatovagal pathway exacerbates colitis in mice in a muscarinic signaling-dependent manner. ac, WT mice were subjected to Sham or HVx, followed by administration of DSS for 7 days starting on postoperative day 2. The graph summarizes data obtained from three independent experiments (n = 15 / group). df, mAchR TKO mice were subjected to Sham or HVx, followed by administration of DSS for 7 days starting on postoperative day 2. The graph summarizes data obtained from three independent experiments (n = 12 / group). d, Relative body weight change during acute colitis. b, e, DAI c, f, Representative HE staining of colon sections (left panel, bars: 200 μm) and histological scores (right panel). P-values ​​were obtained by unpaired two-sided Student's t-test. Error bars represent mean ± sem.

[0092] Figure 5. Muscarinic signaling in the colonic APC activates Treg induction. a, CX3CR1 in the large intestine of mice + Three-dimensional reconstruction of APC (green) and intestinal neurons (purple). b, MHCII + APC (green), intestinal Tuj + Neuron (purple), Foxp3 + Three-dimensional reconstruction of Tregs (yellow). c, d, Anatomical diagram (c) and surgical field (d) of subphrenic vagus nerve trunk transection. e, f, Phenotype of colonic T cells in mice after VGx surgery. CD4 in colonic LP+ Foxp3 in T cells + Cell frequency (e) and colon Foxp3 + RORγt in Tregs + Representative contour plot of pTreg frequency (f). g, Expression of Chrm1, Adrb2, Htr7, and Chrna7 mRNA in APCs of the colon and spleen. h, CD11b-CD11c of the colon by FACS + (CD11c SP), CD11b + CD11c + (DP), CD11b + CD11c - (CD11b SP) Subset Selection Method. i, j, Expression of retinol metabolism-related genes by enteric nerve cell-derived neurons in colorectal APCs was dependent on muscarinic signaling. i, Schematic diagram of the experiment. j, mRNA levels of Aldh1a1 and Aldh1a2 in colorectal APCs (n = 6 / group). k, l, Muscarinic signaling in colorectal APCs promoted Treg induction. k, Schematic diagram of the experiment. CD4 + Foxp3 in T cells + Frequency of Tregs (left panel). Representative contour plots (right panel). m, n, Intestinal neurospheroid-derived neurons activate Foxp3 via muscarinic signaling in the colon APC. + It promoted the induction of Treg cells. m, schematic diagram of the experiment. n, CD4 + Foxp3 in T cells + Treg frequency (left panel). Representative contour plot (right panel). Quantification (n = 6 / group). Representative of two (a, b, ik) or three (e, f) independent experiments. P-values ​​were obtained by one-way ANOVA with Tukey's post-hoc test. Data are shown mean ± SEM (j, l, n).

[0093] Figure 6: Colitis activates the hepatocerebral axis. ac, WT mice were subjected to Sham or VGx, and then DSS was administered for 7 days starting on postoperative day 2. The graph is a pooled representation of data from three independent experiments (n = 15 / group). a, Relative body weight change during colitis. ** b indicates P<0.01. b, DAI. c, Representative HE staining of colon sections (left panel, bar: 200 μm) and histological score (right panel). df, WT mice administered DSS or water for 6 days (n=6 / group). d, Representative image of immunofluorescence staining of pERK1 / 2 (green) in NG (upper panel, bar: 100 μm). Quantification of neurons expressing pERK1 / 2 (lower panel). e, Representative image of c-Fos immunoreaction in NTS (left panel, bar: 200 μm). Number of neurons showing c-Fos immunoreaction (right panel). f, Representative image of double immunofluorescence staining of pERK1 / 2 (green) and PGP9.5 (red) in mouse liver sections. Co-stained areas are shown in yellow (left panel). Scale bar indicates 10 μm. Quantification of pERK1 / 2 expression sites in PGP9.5-positive nerve fibers (right panel). g. Liver phosphor-mTOR and total mTOR protein levels. WT mice were administered Abx-cocktail for 3 weeks, followed by DSS for 4 days. h,i, WT mice were intravenously injected with Si-negative control (Si-Contin) or Raptor (Si-Raptor), and Sham or HVx was performed 3 days later (n = 6 / group). Colon T cell phenotype was analyzed 2 days postoperatively. h, CD4 + Foxp3 in T cells + Frequency of Tregs (left panel). Representative contour plots (right panel). i, Foxp3 in the colon. + RORγt during Treg + Cell frequency. Representative contour plot (left panel). Quantification (right panel). Representative example from two independent experiments (di). P-values ​​were obtained by unpaired two-tailed Student's t-test (af) and one-way ANOVA (h,i) with Tukey's post-hoc test. Data are shown mean ± SEM (bf, h, i).

[0094] Figure 7: Anatomical diagram of the hepatic vagus nerve in a mouse. a. Anatomical diagram. b. Surgical field for hepatovagal transection. c. Schematic diagram showing the firing of the liver-brain-gut nerve arc in colitis. d. The common hepatic branch of the vagus nerve does not contain sympathetic nerves. Surgical field for electrical recording of hepatic sympathetic nerves (left panel). Electrical activity of the common hepatic branch of the vagus nerve and hepatic sympathetic nerves (middle panel). Representative images of immunofluorescence staining of tyrosine hydroxylase (TH) in hepatic branches and DRG (right panel, bar: 100 μm). e. TRPV1 in hepatovagal branches 2 days after capsaicin administration. + Fluorescent immunohistochemistry of neurons (bar: 200 μm). Retrograde tracing of f, g, h, and WGA. After sham or VGx in f, g, and WT mice, Alexa Fluor 488-labeled WGA was injected on postoperative day 2 (n = 3 / group). Alexa Fluor 488 in NG (f) and Th4 DRG (g) one week after WGA injection into the liver. + Fluorescence images of neurons (green) and DAPI (blue). Representative image (left panel, bars: 50 μm). h, Alexa Fluor 488 in DRG (Th4-7 and Th13) one week after WGA injection into the liver. + Fluorescence images of neurons (green) and DAPI (blue) (bars: 100 μm). Representative values ​​(dh) from two independent experiments. P-values ​​were obtained by unpaired two-tailed Student's t-test. Data are shown mean ± SEM (f, g).

[0095] Figure 8. Effects of vagus nerve transection on the maintenance and stability of pTregs in the large intestine. WT mice were treated with either Sham, VGx, or HVx (n = 9 / group). The phenotype and gene expression of colonic immune cells were analyzed on postoperative day 2. a, CD4 in the colon + Foxp3 in cells + Cell frequency. Representative contour plot. b. Foxp3 in the large intestine LP + RORγt during Treg +Cell frequency. Representative contour plot (left panel). Quantification (right panel). c, Expression of Aldh1a1 and Aldh1a2 mRNA in colonic APCs. d, MHC-II + ALDH in colonic APC + Cell frequency. ALDH + Histograms of cells and colonic APCs (left panel). Quantification (right panel). Sham or HVx was performed on e, f, and WT mice. Sham or HVx was performed on e, f, and WT mice, and the phenotype of colonic T cells was analyzed at the indicated time after HVx (n = 9 / group). CD4 in colonic LP + Foxp3 in T cells + Cell frequency (e), and Foxp3 in colonic LP + RORγt during Treg + Cell frequency (f). Representative contour plots (left panel) and quantification (right panel) are shown (e, f). g, h, i, CD4 in the colon 2 days postoperatively. + Foxp3 in cells + Cell frequency (g, h, i) and colonic Foxp3 + RORγt during Treg + Cell frequency (g, h). g, Sham or HVx performed on B6 mice (n = 10 / group). h, Sham or HVx performed on BALB / c mice (n = 4 / group). i, Sham or HVx performed on 6-week-old WT rats (n = 4 / group). j, k, Rag2 - / - Perform Sham or HVx on mice and CD4 + CD45RB hi T cells were transplanted (n = 8 / group). Four weeks after transplantation, mice were sacrificed and Treg cells in the colon were analyzed. j, CD4 in the colon + Foxp3 in cells + Cell frequency. Representative contour plot (left panel). Quantification (right panel). k, Foxp3 of the colon. + RORγt during Treg +Cell frequency. Representative contour plot (left panel). Quantification (right panel). Sham or HVx was administered to WT mice. The phenotype of immune cells in the colon was analyzed after 2 days. CD4 in the colon + IFN-γ in cells + Gata3 + , and IL-17A + Cell frequency (n = 9 / group). Representative values ​​from two independent experiments (e, f, h, i) or pooled values ​​from three independent experiments (ad, g, jl). P-values ​​were obtained by one-way ANOVA (bf) with Tukey's post-hoc test and unpaired two-tailed Student's t-test (gl). Data are shown as mean ± SEM.

[0096] Figure 9 shows that afferent vagus nerves from the liver, rather than the spinal cord, are involved in maintaining homeostasis of Treg cells in the large intestine. ae, WT mice were coated with corn oil (Oil) or capsaicin (Cap) on the hepatic vagal nerve branches. Phenotypic analysis of T cells and APCs in the colon was performed two days after capsaicin application (c, d, n = 16 / group; e, f, n = 8 / group). a, b, Representative fluorescence images of TRPV1 (red) and DAPI (blue) in NG (a) and Th4-DRG (b). Scale bar indicates 100 μm. c, CD4 in the colon + Foxp3 in cells + Cell frequency. Representative contour plot (left panel). Quantification (right panel). d, Foxp3 in the colon. + RORγt in Tregs + Cell frequency. Representative contour plot (left panel). Quantification (right panel). e, MHC-II + ALDH in colonic APC + Cell frequency. ALDH +Histograms of cells and colonic APCs (left panel). Quantification (right panel). f, mRNA expression of Aldh1a1 and Aldh1a2 in colonic APCs. gp, 8-week-old WT-type mice were intrathecally injected with capsaicin (gj, n = 5 / group) and resiniferatoxin (RTX) (kp, n = 4 / group). On day 7 after administration, TRPV1 in the spinal cord was measured. + Immune cells from nerves (Th4-7 and Th13) and the large intestine were analyzed. g, TRPV1 from the spinal cord. + Fluorescent immunohistochemical examination of nerves (bar: 200 μm). CD4 in the colon + Foxp3 in cells + Cell frequency (h, n) and colonic Foxp3 + RORγt in Tregs + Cell frequency (i, o). Effect of intrathecal RTX injection in k, l, DRG(k) and NG(l). Scale bar indicates 100 μm. m, CGRP level in the colon. Representative of two independent experiments (ab, go), or a pool of two (e) or three (c, d, f) independent experiments. P-values ​​were obtained by unpaired two-tailed Student's t-test. Data are shown mean ± sem.

[0097] Figure 10 reveals functional asymmetry of the vagus nerve after hemiphrenic vagus nerve section. ac, WT mice underwent sham, ventral (left) subphrenic vagal nerve sectioning (LVx), and dorsal (right) subphrenic vagal nerve sectioning (RVx) (n = 4 / group). Phenotypic analysis of T cells and APCs in the colon was performed on the second postoperative day. a, CD4 in the colon + Foxp3 in cells + Cell frequency. b. Foxp3 in colonic LP + RORγt during Treg + Cell frequency. Representative contour plot (left panel). Quantification (right panel). c, MHC-II + ALDH in colonic APC + Cell frequency. Colon APC and ALDH +Cell histogram (left panel). Quantification (right panel). dj, Blocking of sympathetic nerve signaling via CG / SMG does not affect the maintenance of Treg in the colon. d, Surgical field of CG / SMG ganglionectomy. e, Electrical activity of splenic nerves. The numbers in parentheses correspond to the nerves shown in d. fl, WT mice underwent sham (n = 4) or CG / SMG ganglionectomy (n = 5). j, WT mice were administered MLA (α7-agonist, 150 μg / day, ip) for 2 days, and splenic T cells were analyzed 12 hours after the last dose (n = 5 / group). f, i, j, CD4 in the colon (f) and spleen (i, j). + Foxp3 in cells + Cell frequency. g, Colon Foxp3 + RORγt in Tregs + Cell frequency. h, MHC-II + ALDH in colonic APC + Cell frequency. After Sham or HVx in km, WT mice, they were injected daily for 2 days with vehicle, salbutamol (30 μg / day, ip), or propanol (300 μg / day, ip) (n = 6 / group). CD4 levels in the colon 12 hours after the last injection were measured. + Foxp3 in cells + Cell (k) frequency, colon Foxp3 + RORγt in Tregs + Cell (l) frequency, MHC-II + ALDH in colonic APC + Cell (m) frequency. Sham or HVx was performed on O, WT mice. T cell phenotypes in the colon, small intestine, and spleen were analyzed on postoperative day 2. CD4 in the colon, small intestine, and spleen. + Foxp3 in T cells + Cell frequency (n = 4 / group). Representative value of two independent experiments (ao). P-values ​​were obtained by one-way ANOVA (b, c, ko) with Tukey's post-hoc test and unpaired two-tailed Student's t-test (fj). Data are shown as mean ± SEM.

[0098] Figure 11. Effects of VGx and HVx on endogenous enteric neurons. ac, g, WT mice were subjected to Sham (n=4) or VGx (n=5). df, h, WT mice were subjected to Sham (n=6) or HVx (n=6). Intrinsic enteric nerve cell activity was measured on postoperative day 2. a, d, Representative images of HuC / D (white) and c-Fos (red) immunofluorescence staining in the colon. Scale bar indicates 100 μm. b, e, c-Fos + Neuronal quantification. c, f, Hand2 mRNA expression in the colon. g, h, Acetylcholine, norepinephrine, and CGRP levels in the colon. * and ** These values ​​represent p < 0.05 and p < 0.01, respectively. The p-values ​​were obtained by unpaired two-tailed Student's t-tests. Data are shown as mean ± sem.

[0099] Figure 12. Effects of mAChR and α7nAChR on the maintenance of Treg cells in the colon. ag, WT mice were administered Sham or HVx, followed by daily injections of water or betanethyl (BETH; ip 300 μg / day) (ad) or GST-21 (ip 300 μg / day) (eg) for 2 days (a, b, n = 5 / group; c, d, n = 10 / group; eg, n = 6 / group). hj, WT mice received Sham or HVx, followed by daily injections of water, BETH only, or BETH + MLA was injected daily for two days (n = 6 / group). kl, WT, and mAchR TKO mice were injected with water or BETH daily for two days after receiving Sham or HVx (n = 4 / group). In relation to Figures 3c and d, the phenotype of colonic immune cells was analyzed 12 hours after the last injection. a, g, j, l, MHC-II + ALDH in colonic APC + b) Cell frequency, Aldh1a1 and Aldh1a2 mRNA expression in colonic APC, c, e, h, CD4 in the colon+ Foxp3 in cells + Cell frequencies d, f, i, k, Foxp3 in the large intestine + RORγt in Tregs + Cell frequency. P-values ​​were obtained by one-way ANOVA using Tukey's post-hoc test. Data are shown as mean ± sem.

[0100] Figure 13. Effects of the gut microbiota on maintaining colonic Tregs in the liver-brain-gut axis. ac, WT mice were subjected to Sham or HVx (n = 4 / group). Fecal samples were collected from the same mice before treatment and 2 days post-treatment. a, α-diversity of microorganisms in feces. b, Principal coordinate analysis (PCoA) based on weighted UniFrac analysis of bacterial community structure (black = pre-treatment, red = Sham, blue = HVx). The two components of the weighted PCoA plot explained 45% and 22% of the variance. Dissimilarity between the two groups was evaluated by permutation multivariate analysis of variance (PERMANOVA). c, Taxonomic distribution at the phylum level. d, e, Fecal samples from Sham-treated or HVx mice were inoculated into 5-week-old male GF mice, and immunological phenotypes were measured 21 days post-inoculation (n = 5 / group). f, g, WT mice were subjected to Sham or HVx and co-fostered for 2 days at the inventor's SPF facility. The graph shows pooled data from three independent experiments (n = 14 / group). hk, Mice were sham or HVxed at 14 days postnatal (n = 10 / group). The phenotype of colonic immune cells was analyzed 2 days later (h). l, m, WT mice were treated with Abx-cocktail (metronidazole, vancomycin, ampicillin, neomycin) for 3 weeks and then sham or HVxed. The phenotype of colonic T cells was analyzed 2 days later. The graph shows pooled data from three independent experiments (n = 10 / group). d, f, i, l, CD4 in the colon + Foxp3 in T cells + Cell frequency. e, g, j, m, colon Foxp3 + RORγt in Treg + Expression of k, MHC-II +ALDH in colonic APC + Cell frequency. P-values ​​were obtained by one-way ANOVA (a, d, e, l, m) using Tukey's post-hoc test, or by unpaired two-tailed Student's t-test (f, g, ik). Data are shown as mean ± sem.

[0101] Figure 14. Effects of HVx on colitis. ac, WT mice were sensitized with TNBS. After 7 days, the mice underwent Sham or HVx and were simultaneously administered TNBS rectally. The graph shows pooled data from three independent experiments (n = 20 / group). df, 8-week-old male Rag2 - / -Mice underwent Sham surgery or HVx, and were administered DSS for 7 days starting 2 days post-surgery (n=12 / group). g, h, Mice that underwent Sham surgery and HVx were housed together and orally administered 2.0% DSS (w / v) for 7 days. The graph shows pooled data from two independent experiments (n = 8 / group). i, j, Abx-treated mice underwent Sham and HVx, and were orally administered 2.0% DSS (w / v) for 7 days starting 2 days later. The graph shows pooled data from two independent experiments (n = 10 / group). k, l, Myd88-deficient mice that underwent Sham surgery and HVx were orally administered 2.0% DSS (w / v) for 7 days (n = 13 / group). mo, Mice that underwent Sham surgery and hepatic vein resection were orally administered 2.0% DSS (w / v) and treated daily with BETH for 7 days. The graph shows pooled data from two independent experiments (n = 10 / group). a, d, g, I, k, m, Relative weight change during acute colitis. b, e, h, j, l, n, DAI. c, f, o, Representative HE staining of colon sections (left panel, bars: 200 μm) and histological scores (right panel). Each experiment was repeated at least twice, yielding similar results. P-values ​​were obtained by unpaired two-sided Student's t-test. Error bars indicate mean ± sem. p, Schematic diagram of the liver-brain-gut neural arc. A supine mouse is shown. The liver senses the intestinal microenvironment and transmits its sensory input to the left NTS in the brainstem, and ultimately to the parasympathetic nerves of the left vagus nerve and the nerve cells in the intestine. Intestinal APCs, activated by the hepatocerebral-gut neural arc, enhance ALDH expression and RA synthesis via mAChR, maintaining a reservoir of peripheral regulatory T cells.

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[0103] 〔Example 2〕 〔Description of Drawings〕 Figure 15 Schematic diagram of vagus nerve hepatic branch electrical stimulation (VHNS) C57BL6 / J mice (male, 10 weeks old) were purchased. After a one-week acclimatization period, the mice were laparotomyed under inhalation anesthesia and cuff electrodes (Figure 15a) were placed on the hepatic branch of the mouse vagus nerve (Figure 15b). To ensure that the current flowed from the liver to the brain, the liver side was set as the negative electrode and the brain side as the positive electrode. After electrode placement, a recovery period of about one week was allowed. Subsequently, the electrodes were connected to a modular stimulator, and electrical stimulation was administered to freely moving mice under the conditions of 10Hz, pulse width 500μs, ON 10s / OFF 90s, 3h / day. Mice with electrodes placed on the hepatic branch and connected to the modular stimulator, but without stimulation, were designated as the control group. After administering electrical stimulation for three consecutive days, the mice were sacrificed, and the colon and brain were collected. The composition ratio of regulatory T cells (Treg) in the colon was analyzed by FACS (Figure 15c). VHNS increased colonic Treg. Activation areas in the brain induced by electrical stimulation were evaluated using cFos, a neuronal activation marker. In situ hybridization revealed activation of cFos mRNA expression regions in the left nucleus tractus solitarius (NTS), the projection target of the hepatic branch of the vagus nerve, as well as the left dorsal nucleus of vagus nerve (DMV) and area postrema (AP), where efferent vagus nerve cell bodies are located (Figure 15d).

[0104] Figure 16 Vagus nerve hepatic branch electrical stimulation (VHNS) suppresses the pathogenesis of colitis in mice. C57BL6 / J mice (male, 10 weeks old) were purchased. After a one-week acclimatization period, the mice were laparotomyed under inhalation anesthesia and cuff electrodes were placed on the hepatic branch of the mouse vagus nerve. To ensure that the current flowed from the liver to the brain, the liver side was set as the negative electrode and the brain side as the positive electrode. After electrode placement, a recovery period of about one week was allowed. Subsequently, to induce colitis in the mice, a 2% dextran sulfate sodium (DSS) aqueous solution was administered to them in free-flowing water. Simultaneously with the start of DSS administration, the electrodes were connected to a modular stimulator, and electrical stimulation was administered to freely moving mice under the conditions of 10Hz, pulse width 500μs, ON 10s / OFF 90s, 3h / day. Mice with electrodes placed on the hepatic branch and connected to the modular stimulator, but without stimulation, were designated as the control group. Seven days after DSS administration, the colons were collected from the mice, and the state of colitis was evaluated using pathological specimens and intestinal length as indicators. When body weight was measured over a period of 7 days following the onset of colitis, VHNS suppressed weight loss due to DSS colitis (Figure 16a). In addition, intestinal shortening due to DSS colitis was suppressed by VHNS (Figure 16b). Pathological tissue specimens showed that the shedding of colonic epithelial cells due to colitis was suppressed by VHNS (Figure 16c).

[0105] Figure 17 Two-bottle preference assays using vagus nerve-resected mice C57BL6 / J mice (male, 10 weeks old) were purchased. After a one-week acclimatization period, the mice were laparotomyed under inhalation anesthesia and the vagus nerve was resected bilaterally (VGx), left side (LVx), or right side (RVx) (Figure 17a). Mice that underwent only laparotomy (Sham) were used as controls. After a recovery period of about one week, two-bottle preference assays were performed using these mice. Two water bottles were prepared; one was filled with a 600 mM glucose solution (Glu), and the other with a 30 mM acesulfame potassium solution (Ace K), an artificial sweetener. These were placed in a Rick analysis-based preference test apparatus (Figure 17b) and the experiment was conducted. The mice described above were kept in cages within the apparatus from 8 PM to 10 AM, and the number of times their mouths touched the water bottles was measured. This test was repeated for three days with the same individual, and the ratio of sucrose to Ace K in the total number of contacts was calculated each day. This ratio was evaluated as palatability. On the first day of the study (Day 1), the selectivity for sucrose and Ace K was almost the same in all mice. However, Sham and RVx mice selectively ingested sucrose as the days progressed. On the other hand, VGx and LVx mice selectively ingested sucrose and Ace K equally even on day 3 of the study (Figure 17c).

[0106] Figure 18 Regulation of lung immune cells by the vagus nerve's hepatic branch We purchased C57BL6 / J mice (male, 10 weeks old). After a one-week acclimatization period, we performed laparotomy under inhalation anesthesia and resected the mouse vagus nerve hepatic branch (HVx) (Figure 18a). Mice that underwent only laparotomy (Sham) were used as controls. After a recovery period of about one week, immune cells were collected from the lungs of these mice. The number of type 2 innate lymphoid cells (ILC2) in the lungs was analyzed by FACS. The number of lung ILC2 cells increased with HVx (Figure 18b).

[0107] Figure 19: Control of intestinal peristalsis by the vagus nerve. (Figure 19a) C57BL6 / J mice (male, 10 weeks old) were purchased. After a one-week acclimatization period, laparotomy was performed under inhalation anesthesia, and both sides of the mouse vagus nerve (HVx) were resected. Mice that underwent only laparotomy (Sham) were used as controls. After a recovery period of about one week, the intestinal peristalsis of these mice was evaluated. Overall gastrointestinal motility was evaluated using the intestinal transit time (ITT) test, gastric motility using the gastric empty test, and small intestinal motility using the small-bowel (SB) transit test. Vagus nerve resection reduced gastric and small intestinal motility and suppressed gastrointestinal peristalsis. (Figure 19b) C57BL6 / J mice (male, 10 weeks old) were purchased. After a one-week acclimatization period, the mice were laparotomyed under inhalation anesthesia and the proper hepatic branch (HVx) or gastroduodenal branch (GVx) of the mouse vagus nerve was resected (Figure 19b). Mice that underwent only laparotomy (Sham) were used as controls. After a recovery period of about one week, the intestinal peristalsis of these mice was evaluated. Overall gastrointestinal motility was evaluated using the intestinal transit time (ITT) test, gastric motility using the gastric empty test, small intestinal motility using the small-bowel (SB) transit test, and large intestinal motility using the colonic transit test. Neither HVx nor GVx affected gastric motility. HVx reduced small intestinal motility, while GVx reduced large intestinal motility. These results suggest that the vagus nerve controls peristalsis in different regions of the gastrointestinal tract depending on its branch.

[0108] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. [Industrial applicability]

[0109] This invention is applicable to industries related to pharmaceuticals.

Claims

[Claim 1] A pharmaceutical composition for use in a method for increasing the amount of peripheral regulatory T cells (pTregs) in the intestinal tract, comprising bethanechol or muscarine, wherein the method comprises administering bethanechol or muscarine and measuring aldehyde dehydrogenase activity and pTreg induction activity.

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