Therapeutic agent for diseases caused by intestinal immune disorder

Abstract

To regulate the number of peripheral regulatory T cells in the intestinal tract and to provide a new means for treating diseases associated with the cells.SOLUTION: A therapeutic agent for a disease, comprising a substance having an effect of regulating the amount of peripheral regulatory T cells in the intestinal tract, wherein the substance is a substance that activates or suppresses the hepatic vagal afferent pathway, a substance that activates or suppresses the left vagal efferent pathway, or an agonist or antagonist of a muscarinic acetylcholine receptor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel therapeutic agent for a disease, a novel method for screening a therapeutic agent for a disease, and a novel method for treating a disease. [Background technology]

[0002] The intestinal tract functions as an important organ responsible for digestion and absorption, and is confronted with the external environment (lumen) by a single layer of columnar epithelial cells. Although the intestinal lumen is constantly exposed to over 100 trillion intestinal bacteria, dietary antigens, and other foreign substances, the intestinal homeostasis is maintained by the function of peripheral regulatory T cells (pTregs) in the intestinal tract, preventing excessive inflammatory responses. Previously, specific intestinal bacteria, intestinal bacterial components, short-chain fatty acids, and cytokines have been considered important factors in the differentiation and maintenance of peripheral regulatory T cells. Meanwhile, the relatively high incidence of inflammatory bowel disease in depression and irritable bowel syndrome, which have been considered neurological disorders, has suggested that the autonomic nervous system may be deeply involved in intestinal immune disorders (Non-Patent Documents 1, 2, 3, and 4). Recent reports have suggested the possible involvement of the nervous system in the intestinal immune system, but the relationship between the nervous system and peripheral regulatory T cells in the intestinal tract has long been unclear. Furthermore, research reports on the brain-gut correlation that have been published so far have not specifically shown the neural circuits connecting the brain and the gut, and many mysteries remain 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). Summary of the Invention [Problem to be solved by the invention]

[0004] If the relationship between the nervous system and intestinal peripheral regulatory T cells is clarified, it will be possible to artificially regulate the number of intestinal peripheral regulatory T cells, which will lead to the development of new treatments for diseases associated with intestinal peripheral regulatory T cells (e.g., inflammatory bowel disease).

[0005] The present invention has been made against this background, and aims to provide a new means of treating diseases associated with intestinal peripheral regulatory T cells by regulating the number of these cells. [Means for solving the problem]

[0006] As a result of extensive research to solve the above problems, the present inventors have found that: 1) intestinal peripheral regulatory T cells 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 intestinal peripheral regulatory T cells, are located in close proximity to nerves in the intestinal lamina propria, and that muscarinic acetylcholine receptor subtype 1 is strongly expressed in these intestinal antigen-presenting cells.

[0007] The present invention was completed based on the above findings. That is, the present invention provides the following [1] to

[20] . [1] A therapeutic agent for a disease, comprising a substance that has the effect of regulating the amount of peripheral regulatory T cells in the intestinal tract, wherein 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] The therapeutic agent for a disease according to [1], wherein the disease is inflammatory bowel disease, autoimmune disease, allergy, cancer, depression, or gastrointestinal infection.

[0009] [3] The therapeutic agent for the disease according to [1], wherein the disease is inflammatory bowel disease.

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

[0011] [5] A therapeutic agent for the disease according to [4], wherein the agonist of the muscarinic acetylcholine receptor is bethanechol, muscarine, pilocarpine, or cevimeline.

[0012] [6] A therapeutic agent for a disease described in any one of [1] to [3], characterized in that the substance having the effect of regulating the amount of peripheral regulatory T cells in the intestinal tract is an antagonist of a muscarinic acetylcholine receptor.

[0013] [7] A therapeutic agent for the disease according to [6], wherein the muscarinic acetylcholine receptor antagonist is atropine, tropicamide, oxybutynin, propiverine, tolterodine, solifenacin, or imidafenacin.

[0014] [8] A screening method for a therapeutic agent 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 in that induction of regulatory T cells is detected by detecting the expression of FoxP3.

[0016]

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

[0017]

[11] The screening method according to [8] or [9], wherein 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 in that it comprises activating or inhibiting the afferent pathway of the hepatic branch of the vagus nerve in the subject to be treated, activating or inhibiting the efferent pathway of the left vagus nerve in the subject to be treated, or administering an agonist or antagonist of a muscarinic acetylcholine receptor to the subject to be treated.

[0019]

[13] The method for treating a disease according to

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

[0020]

[14] The method for treating a disease according to

[12] , wherein the disease is inflammatory bowel disease.

[0021]

[15] A method for treating a disease according to any one of

[12] to

[14] , comprising administering to a subject an agonist of a muscarinic acetylcholine receptor.

[0022]

[16] The method for treating the disease according to

[15] , wherein the agonist of the muscarinic acetylcholine receptor is bethanechol, muscarine, pilocarpine, or cevimeline.

[0023]

[17] A method for treating a disease according to any one of

[12] to

[14] , comprising administering to a subject a muscarinic acetylcholine receptor antagonist.

[0024]

[18] The method for treating the disease according to

[17] , wherein the muscarinic acetylcholine receptor antagonist is atropine, tropicamide, oxybutynin, propiverine, tolterodine, solifenacin, or imidafenacin.

[0025]

[19] The method for treating a disease according to any one of

[12] to

[18] , wherein the subject of treatment is an animal other than a human.

[0026]

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

[0027] This specification includes the contents disclosed in the specification and / or drawings of Japanese Patent Application No. 2020-095241, which is a priority document of this application. [Effects of the Invention]

[0028] The present invention provides a novel therapeutic agent for a disease, a novel method for screening a therapeutic agent for a disease, and a novel method for treating a disease. [Brief explanation of the drawings]

[0029] [Figure 1] Possible interactions between APCs and neurons in the intestine. [Figure 2] Hepatic vagal sensory afferents are essential for NTS activation during colitis. [Figure 3] The liver-brain-gut axis regulates colonic Treg homeostasis via muscarinic signaling in APCs. [Figure 4] Disruption of the hepatic vagal pathway exacerbates colitis in mice in a muscarinic signaling-dependent manner. [Figure 5] Muscarinic signaling in colonic APCs activates Treg induction. [Figure 6] Colitis activates the liver-brain axis. [Figure 7] Anatomy of the mouse hepatic vagus nerve. [Figure 8] Effect of vagotomy on the maintenance and stability of colonic pTregs. [Figure 9] Vagal afferents from the liver, but not the spinal cord, are involved in the maintenance of colonic Treg homeostasis. [Figure 10] Hemidiaphragmatic vagotomy revealed functional asymmetry of the vagus nerve. [Figure 11] Effects of VGx and HVx on intrinsic enteric neurons. [Figure 12] Effects of mAChRs and α7nAChRs on the maintenance of colonic Tregs. [Figure 13] Effect of gut microbiota on colonic Treg maintenance in the liver-brain-gut axis. [Figure 14] Effect of HVx on colitis. [Figure 15] Schematic diagram of hepatic vagus nerve stimulation (VHNS). [Figure 16] Electrical stimulation of the hepatic branch of the vagus nerve (VHNS) suppresses the pathology of colitis in mice. [Figure 17] Two-bottle preference assays using vagotomized mice. [Figure 18] Regulation of pulmonary immune cells by the hepatic branch of the vagus nerve. [Figure 19] Vagus nerve control of intestinal peristalsis. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be described in detail below. (1) Therapeutic drugs The therapeutic agent for a disease of the present invention is 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, and is 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] As used herein, "regulating the amount of peripheral regulatory T cells in the intestinal tract" means increasing or decreasing the amount of peripheral regulatory T cells in the intestinal tract. By increasing the amount of peripheral regulatory T cells in the intestinal tract, excessive immune responses can be suppressed, and therapeutic effects against inflammatory diseases and the like can be expected. On the other hand, by decreasing the amount of peripheral regulatory T cells in the intestinal tract, immune responses can be strengthened, and therapeutic effects against gastrointestinal infections and the like can be expected.

[0032] When used for the purpose of increasing the amount of peripheral regulatory T cells in the intestinal tract, the therapeutic agent for a disease of the present invention contains a substance that activates the hepatic branch of the vagus nerve afferent pathway, a substance that activates the left vagal nerve efferent pathway, or a muscarinic acetylcholine receptor agonist.In contrast, when used for the purpose of decreasing the amount of peripheral regulatory T cells in the intestinal tract, the therapeutic agent for a disease of the present invention contains a substance that suppresses the hepatic branch of the vagus nerve afferent pathway, a substance that suppresses the left vagal nerve efferent pathway, or a muscarinic acetylcholine receptor antagonist.

[0033] The type of disease is not particularly limited as long as it can be treated by regulating (increasing or decreasing) the amount of peripheral regulatory T cells in the intestinal tract. Specific examples of diseases that can be treated by increasing the amount of peripheral regulatory T cells in the intestinal tract include diseases caused by intestinal immune abnormalities (inflammatory bowel disease, autoimmune disease, allergies, etc.), cancer, depression, etc., while examples of diseases that can be treated by decreasing the amount of peripheral regulatory T cells in the intestinal tract include gastrointestinal infections, cancer, etc. Examples of gastrointestinal infections include norovirus infection, rotavirus infection, and pathogenic Escherichia coli enteritis.

[0034] The substances that activate or inhibit the hepatic branch afferent pathway of the vagus nerve, the substances that activate or inhibit the left vagus nerve efferent pathway, and the 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 agonists of muscarinic acetylcholine receptors include bethanechol, muscarine, pilocarpine, and cevimeline, and examples of antagonists of muscarinic acetylcholine receptors include, but are not limited to, atropine, tropicamide, oxybutynin, propiverine, tolterodine, solifenacin, and imidafenacin.

[0035] The therapeutic agents for diseases 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, they can be prepared as injections (intraperitoneal, subcutaneous, intravenous, or intramuscular injections), drip infusions, capsules, liquids, suspensions, emulsions, and the like. When formulated, other components such as pharmacologically acceptable carriers may be included. Examples of other components include sterile water, physiological saline, solvents, bases, emulsifiers, vegetable oils, suspending agents, surfactants, stabilizers, preservatives, binders, diluents, isotonicity agents, soothing agents, disintegrants, lubricants, buffers, coating agents, colorants, and other additives, which may be used in appropriate combinations.

[0036] The therapeutic target of the present invention for treating diseases is primarily humans, but may also be animals other than humans, such as mice, rats, hamsters, rabbits, cats, dogs, cows, horses, pigs, sheep, and monkeys.

[0037] The dosage of the therapeutic agent for a 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 dosage form, the administration method, the age and body weight of the subject to be treated, etc. Specific dosages, for example, when an agonist of a muscarinic acetylcholine receptor is administered to a human, are preferably 0.1 to 100 g, and more preferably 0.1 to 10 g, per adult per day.

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

[0039] (2) Screening method The screening method of the present invention is a method for screening therapeutic agents for diseases, and is 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 is preferably carried out by a method for detecting FoxP3 expression. The diseases may be the same as those for the therapeutic agents described above.

[0040] (3) Treatment method The disease treatment method of the present invention is 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 pathway of the hepatic branch of the vagus nerve in the subject to be treated, activating or inhibiting the efferent pathway of the left vagus nerve in the subject to be treated, or administering an agonist or antagonist of a muscarinic acetylcholine receptor to the subject to be treated.

[0041] The activation or inhibition of the hepatic branch of the vagus nerve afferents can be achieved by administering substances with such effects to the subject, but also by electrically stimulating the hepatic branch of the vagus nerve afferents or by severing the hepatic branch of the vagus nerve.Similarly, the activation or inhibition of the left vagal nerve efferents can be achieved by administering substances with such effects to the subject, electrically stimulating the left vagal nerve efferents, or by severing the left vagal nerve efferents.

[0042] The agonists and antagonists of muscarinic acetylcholine receptors, the diseases, and the targets of treatment may be the same as those of the therapeutic agents described above.

[0043] (4) How the cuff electrode operates The method of operating the cuff electrode of the present invention stimulates the hepatic branch of the vagus nerve 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 hepatic branch of the vagus nerve and providing electrical stimulation to the hepatic branch of the vagus nerve, 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. [Example]

[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 tissues, particularly the lamina propria (LP), and maintain immune homeostasis in the intestinal tract. 5,6 The generation of pTregs is promoted by a combination of cytokines, such as TGF-β and RA, and microbial and dietary signals, including Clostridia clusters IV, XIVa, and 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 shown that immune cells are under the control of autonomic and enteric nerve cells, making significant progress. 3,14-16 This suggests that pTreg differentiation at mucosal sites is governed by previously unrecognized mechanisms.

[0046] Indeed, the gastrointestinal tract is not only highly innervated but also highly populated with adaptive and innate immune cells. 14,17 In the colonic LP, neurons (β-tubulin III + ) and MHC-II + APC (mainly CX3CR1 + Immunohistochemical analysis of the localization of mononuclear cells (MNPs) revealed that neurons and APCs were in close proximity (Fig. 1a, b, Fig. 5a, b). Intestinal APCs, especially CX3CR1 + MNPs and CD103 + Dendritic cells (DCs) produce RA and preferentially support the development of pTregs in the TGF-β-rich intestinal microenvironment. 8,18-23 The immunomodulatory effect of the autonomic nervous system is known. 1-3,15-17 However, how the vagus nerve influences intestinal homeostasis by regulating intestinal APCs and pTregs remains poorly understood. To investigate the immunological function of the vagus nerve, wild-type (WT) C57BL / 6 (B6) mice underwent subdiaphragmatic truncal vagotomy (hereafter referred to as VGx) (Fig. 5c, d). Interestingly, vagotomized mice showed significantly lower levels of Foxp3 in the colon compared to sham-operated mice. + T helper cells, especially Helios-RORγt + The number of pTregs was significantly reduced (Fig. 1c, d; Fig. 5e, f). In addition to the reduction in colonic pTregs, there was a significant decrease in the levels of Aldh1a1 and Aldh1a2, which encode the RA synthases RALDH1 and RALDH2, and in the aldehyde dehydrogenase activity of colonic APCs (Fig. 1e, f).

[0047] To identify the neurotransmitters that transmit signals from enteric neurons to colonic APCs, we performed mRNA-seq on APCs collected from the spleen and intestine. The expression levels of Chrm1, a gene encoding the muscarinic ACh receptor, were higher in intestinal APCs than in splenic APCs, suggesting that neurotransmitters are involved in the tissue-specific regulation of intestinal APCs (Fig. 1g, Fig. 5g). CX3CR1 + MNP and CD103 + It is also noteworthy that the DC-enriched APC fraction shares expression of Chrm1, Aldh1a1, and Aldh1a2 compared with genes defining typical subsets of APCs, such as Itgae (CD103), Cx3cr1, and Irf8 (Figures 1h, i, and 5h). We further confirmed this finding by quantitatively assessing the expression of Aldh1a1 and Aldh1a2 in colonic APCs stimulated with multiple neurotransmitters, including Ach, muscarinic, adrenaline, neuropeptide Y, substance P, serotonin (5-HT), and neuromedin U (Figure 1j). Furthermore, muscarinic acid and intestinal neurospheroids induced the expression of Aldh1a1 and Aldh1a2 in colonic APCs derived from WT mice and human intestines (Fig. 1k, l). However, co-culture of neurospheroids with APCs lacking Chrm1, 2, and 4 (mAChR TKO) failed (Fig. 5i, j). Furthermore, colonic APCs from WT mice pre-cultured with muscarinic acid or neurospheroids did not express Foxp3. + The generation of Tregs was promoted in APCs from mAChR TKO mice, but not in APCs from mAChR TKO mice (Fig. 5k-n). These results suggest that ACh-mAChR signaling in APCs contributes to maintaining the pTreg population in the intestine.

[0048] To test this hypothesis, we assessed whether vagus nerve-mediated signals are required to prevent intestinal inflammation. VGx increased susceptibility to a dextran sulfate sodium (DSS)-induced colitis model (Fig. 6a-c). Given that VGx reduced the number of pTregs and induced a local inflammatory environment, we next investigated which vagus nerve afferent neurons are involved in regulating and maintaining the pTreg pool in the intestine. The vagus nerve innervates most of the gastrointestinal tract, and its afferent neurons transmit sensory input to the nodal ganglion (NG) on both sides. 24 During colitis, these sensory inputs are further projected to the solitary nucleus (NTS) in the brainstem (Figure 6d-f). Notably, acute colitis activates sensory afferents in the liver in vivo, which can be seen after selective surgical division of the common hepatic branch of the vagus nerve (hereafter referred to as "HVx"). 25 ) disappeared (Fig. 2a, b, Fig. 7a-c). Because the liver is constantly exposed to nutrients, bacterial products, toxins, and metabolites from the intestine, this gut-liver axis connected by the portal circulation has been demonstrated 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 (Fig. 6g-i), suggesting that hepatic sensory afferents of the vagus nerve are activated during colitis. Indeed, retrograde tracing of the liver supported the idea that the liver senses the intestinal microenvironment, activates hepatic sensory afferents of the vagus nerve, and transmits the signal to the brain via the left NG (Fig. 2c, d). Importantly, the common hepatic branch of the vagus nerve, which bifurcates in HVx mice, is involved in the sympathetic TH signaling, according to electrophysiological and immunohistochemical assessments. + Capsaicin-sensitive TRPV1 neurons not containing +The common hepatic branch of the vagus nerve is primarily composed of sensory afferents (Figure 7d, e). Blocking the common hepatic branch of the vagus nerve with capsaicin significantly reduced the number of pERK-positive cells in the left NG, but not in the right NG (Figure 2b). Furthermore, the number of retrogradely labeled cells was significantly reduced in the left NG, but not in the DRG, of HVx mice, indicating that the common hepatic branch of the vagus nerve sends signals through the left NG, but not the right NG or DRG (Figure 2c, Figure 7f-h). These results suggest that sensory information about the intestinal environment is transmitted to the brain via the ascending pathway of the left vagus nerve from the liver to the brain.

[0049] Considering the anatomical laterality of the vagus nerve, we explored how sensory afferents of the hepatic vagus nerve affect intestinal Tregs and characterized the effects of HVx on the intestine and spleen. As a result, APCs obtained from the colon of HVx and VGx mice were found to express CD4 +We confirmed that the proportion of pTregs among T cells, as well as the expression and activity of aldehyde dehydrogenase, were significantly reduced compared to sham-operated mice (Figure 2e, Figure 8a-d). This HVx-induced reduction in colonic pTregs occurred rapidly on day 2 and was consistent across rat sex, strain, and species (Figure 8e-i). Since in vivo-generated pTregs are demethylated at Treg-specific demethylated regions (TSDRs), the rapid reduction in pTregs in HVx mice is likely due to epigenetic effects of the vagus nerve on the maintenance and stability of intestinal pTregs by altering the DNA methylation status of TSDRs 29-34. Similarly, HVx impaired pTreg differentiation and stability in T cell-reconstituted mice, unleashing RA-mediated suppression of the Th17 differentiation program (Figure 8j-l). The importance of hepatic sensory afferents in the left NG for maintaining an adequate reservoir of intestinal Tregs was further supported by the finding that perturbation of hepatic vagal afferents to the left NG by vagal capsaicin administration, rather than DRG disinhibition by capsaicin or RTX, resulted in a decrease in colonic Treg numbers and reduced aldehyde dehydrogenase activity in APCs (Figure 9). This suggests asymmetric vagal function (Figure 2f, Figure 10a-c). It has already been reported that the maintenance of colonic Tregs is less dependent on the sympathetic nervous system than MMs and ILC2s in connection with viral and parasitic infections. 35,36 In contrast to the effects on the small and large intestines, the frequency of splenic Tregs was normal in HVx mice, but surgical removal of the CG-SMG or chemical blockade of β2-adrenergic receptors or 7-nicotinic ACh receptors (7-nAchR) significantly reduced splenic Tregs (Fig. 10i-o). It has previously been reported that the splenic nerves, primarily composed of adrenergic fibers emanating from the CG-SMG, suppress T cell activation via 7-nAchR and systemic cytokine production from splenic macrophages. 37-42Therefore, it was predicted that severing the CG-SMG or splenic nerve, but not vagotomy 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 the level of intestinal pTregs by transmitting ACh signals to control colonic APCs.

[0050] Therefore, we investigated the role of muscarinic ACh signaling in intestinal APCs in vivo. Genetic ablation of mAChRs reduced the expression of Aldh1a1 and Aldh1a2 in colonic APCs, resulting in a decrease in colonic pTregs (Fig. 3a-d). In VGx and HVx mice, the expression of c-Fos in the myenteric plexus of the colon was significantly reduced. +Although the number of enteric neurons was confirmed to be lower in VGx and HVx mice than in sham-operated mice, the expression of the transcription factor Hand2, which is required for the terminal differentiation of enteric neurons, was not affected (Fig. 11a-f). Furthermore, in VGx and HVx mice, the intestinal small molecule and peptide neurotransmitters of the parasympathetic nervous system (ACh), but not of the sympathetic nervous system (norepinephrine) or sensory nervous system (calcitonin gene-related peptide [CGRP]), were reduced compared to sham-operated mice (Fig. 11g-h). Because hepatic selective vagotomy and truncal vagotomy primarily reduce local ACh levels in the intestine, we examined whether activation of mAChRs restores the expression and activity of aldehyde dehydrogenase in intestinal APCs. Treatment with bethanechol, an mAChR agonist, restored Aldh1a1 and Aldh1a2 expression in colonic APCs in HVx mice compared with sham-operated mice, whereas treatment with 7-nAchR agonists and antagonists, as well as with 7-nAchR43 gene deletion, had little effect (Fig. 12a-j). Furthermore, bethanechol-treated HVx WT mice, but not mAChR TKO mice, increased the frequency of pTregs, suggesting that the hepatic-brain-gut neural arc stimulates colonic APCs to form the pTreg niche (Fig. 3e; Fig. 12k, l). Taken together, these results support the idea that neural input from hepatic sensory afferents is essential for initiating this vagal-vagal liver-brain-gut neural arc, and that this reflex arc is independent of the sympathetic nervous system and axon reflexes.

[0051] Given that the generation and maintenance of pTregs is highly dependent on the microbiome and metabolites, we investigated the role of the microbiome in the generation and maintenance of pTregs. The gut microbiomes derived from HVx mice and sham-operated control mice did not differ significantly in composition and diversity, and transplantation of fecal bacteria from these mice induced comparable amounts of gut pTregs in germ-free mice (Figures 3 and 13a-e). This suggests that the hepatic-enteric-neuronal arc maintains the pTreg pool, regardless of changes in the gut microbiota and metabolites caused by HVx. Furthermore, intestinal sterilization of HVx mice did not result in the generation of pTregs, particularly microbe-independent pTregs.13 No reduction in pTregs was observed (Fig. 13l, m). Taken together, these data indicate that the liver-brain-gut neural arc maintains basal levels of intestinal pTregs, which are dependent on tonic microbial input.

[0052] Since the liver-brain-gut neural circuitry's role in regulating intestinal pTregs was previously unknown, we investigated whether this is related to the development of colitis. Mice with surgical or chemical transection of the hepatic vagus nerve branch exhibited reduced pTreg frequencies (Fig. 3a, Fig. 8a, b, 5c, d), which resulted in increased susceptibility to DSS- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis (Fig. 4a-c, Fig. 14a-c). Similarly, Rag2 - / - Unlike T cell-deficient mice, HVx-induced colitis was not exacerbated in these mice (Fig. 14d-f). Furthermore, splenectomy was effective in preventing the progression of endotoxemia. 37-39 Unlike the control group, HVx mice 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 their sham-operated counterparts (Figure 14g, h). Furthermore, HVx did not increase the susceptibility of antibiotic-treated or MyD88-deficient mice to DSS-induced colitis (Figure 14i). This suggests that sustained microbial input is necessary to maintain the intestinal 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 agonists (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 intestine from excessive inflammation (Figure 14p).

[0053] Based on the above, this study demonstrated that the extrinsic vagal reflex, which connects the hepatic vagal sensory afferents, the brainstem, the vagal efferents, and enteric neurons, is mediated by mAChR. +These findings reveal interesting activities, such as stimulating APCs and maintaining the peripheral regulatory T cell reservoir. A recent retrospective cohort study reported that patients with new-onset depression are at higher risk of developing IBD. 44 Imbalances in the autonomic nervous system likely contribute to the pathogenesis of IBD. Direct and reciprocal gut-brain neural reflexes controlling appetite, food reward, cancer, fatty liver, Parkinson's disease, and other neurological disorders. 45-48 In addition, our findings provide a unique insight into tissue-specific immune cell adaptation mediated by both the liver and the central nervous system. Impaired function of this liver-brain-gut neural circuitry predisposes the intestine to inflammation. Therefore, the suppression of tumorigenesis by denervation may be due to a reduced number of pTregs in the colon. This study revealed that the liver-brain-gut neural circuitry plays a crucial role in identifying the immunoregulatory niche and fine-tuning the intestinal immune response. Interventions targeting this liver-brain-gut neural arc may be beneficial in the treatment of IBD. 49 It has the potential to have a wide range of applications in the treatment of diseases such as infectious diseases and intestinal cancer.

[0054] [Experimental Method] animal C57BL / 6 (WT) mice, BALB / c mice, and Jcl:Wistar rats were purchased from CLEA Japan (Tokyo, Japan). Five-week-old male germ-free (GF) mice (C57BL / 6 background strain) were purchased from Sankyo Lab Services Co., Ltd. and housed in the GF facility at Keio University School of Medicine. Ly5.1 mice, Foxp3 CreERT2 Mouse, Cx3cr1 GFP / GFP Transgenic (Cx3cr1 gfp ) mice, Rag2 knockout (Rag2 - / - ) mice, Myd88 knockout (Myd88 - / -) mice were obtained from The Jackson Laboratory (Maine, USA). Chrm1 / Chrm2 / Chrm4 triple knockout (mAChR TKO) mice were obtained from the Animal Resources Development Center (Kumamoto, Japan). Wnt1 promoter / enhancer (Wnt1-Cre) was crossed with EGFP reporter mice (CAG-CAT loxP / loxP-EGFP) to generate Wnt1-Cre / Floxed-EGFP double transgenic mice. 50 Foxp3 CreERT2 Mice were transformed into floxed-tdTomato reporter mice 51 Foxp3-reporter mice were obtained by mating with . Six to eight-week-old mice were used for all experiments. All mice were housed under specific pathogen-free conditions in the animal care facility of the Keio University School of Medicine. All experiments were approved by the local animal care committee (Keio University, Tokyo, Japan) and conducted in accordance with institutional guidelines and home office regulations.

[0055] Subdiaphragmatic vagotomy and hepatic selective vagotomy Subdiaphragmatic vagotomy was performed bilaterally or unilaterally (left or right) as previously reported (Figure 5c, d). 52 Male mice were anesthetized with a combination of medetomidine, midazolam, and butorphanol. A midline incision was made to expose the upper abdominal organs. The bilateral subdiaphragmatic trunks of the vagus nerve along the esophagus were exposed and transected. In the sham-operated group, these vagus nerve trunks were exposed but not transected. Hepatic selective vagotomy (HVx) was performed as described (Figure 7). 25 The ventral subdiaphragmatic vagus nerve trunk was exposed under anesthesia as described above. Because the common hepatic branch of the vagus nerve forms a neurovascular bundle, this branch was selectively ligated with silk thread and then transected under a microscope. In the sham-operated group, the common hepatic branch was exposed but not transected.

[0056] Selective blockade of hepatic vagal afferents by perivagal application of capsaicin The hepatic branch of the vagus nerve trunk was freed from the surrounding tissue with paraffin paper and then wrapped for 30 minutes with a cotton swab soaked in either vehicle (Tween 80: olive oil = 1:9) or 10 mg / ml capsaicin dissolved in the vehicle solution. After 30 minutes, the cotton swab 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 and superior mesenteric ganglion (CG-SMG) complex was exposed along the superior mesenteric artery and removed. In the sham-operated group, the superior mesenteric artery was exposed but not removed. 53 .

[0058] Intrathecal administration of resiniferatoxin (RTX) and capsaicin TRPV1 in DRG or spinal cord + To perform neuron-targeted ablation, mice were injected intrathecally with 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) using a 25-liter Hamilton syringe with a 28-gauge needle. Control mice received vehicle only. Colonic immune cell phenotypes were analyzed 7 days after injection. TRPV1 expression in the DRG and spinal cord was also analyzed. + Neuronal depletion was confirmed by immunostaining.

[0059] Parabiosis Parabiosis surgery was performed as previously described. 54After shaving the corresponding temporal region of each mouse, a corresponding skin incision was made from the forelimb to the base of the hind limb, 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 connect the parabionts. Two weeks after surgery, the mice were subjected to sham or HVx.

[0060] T cell reconstitution model The T cell reconstitution model was performed as previously described. 54 Rag2 - / - Mice were cultured with FACS-sorted wild-type naive CD4 + CD45Rb hi 3 x 10 cells 5 Each mouse was injected intraperitoneally. The weight of the mouse 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% dextran sulfate sodium (DSS) solution to induce colitis. Mice were weighed daily and visually inspected for diarrhea and rectal bleeding. DAI was assessed blinded across groups (maximum total score 12). Histological activity score (maximum total score 40) was calculated as the sum of three parameters: extent, inflammation, and crypt damage. 55 .

[0062] TNBS-induced colitis model 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was obtained from Sigma-Aldrich. To presensitize 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 presensitization, mice were re-administered intrarectally with 150 μl of 2.5% TNBS in 50% ethanol 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] Antibiotics To evaluate the possible contribution of gut microbiota to the exacerbation of DSS-induced colitis after vagotomy, 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) via nasogastric tube (500 μL / mouse) three times a week for 3 weeks. As a control, an equal volume of distilled water was administered via nasogastric tube.

[0064] In vivo administration of bethanechol, salbutamol, propranolol, methyllycaconitine, and GTS-21 Bethanechol (BETH) was dissolved in water. After 12 hours of surgery, mice were given either water or BETH (300 μg per mouse) daily. 57 To evaluate the effect of adrenergic signaling on colonic Treg homeostasis, salbutamol (β2-agonist) and propranolol (β-blocker) were used. Salbutamol and propranolol were dissolved in PBS. 12 hours after surgery, mice were intraperitoneally injected 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 the maintenance of colonic Tregs was assessed using methyllycaconitine (MLA, an α7 nicotinic acetylcholine receptor antagonist) and GTS-21 (an α7 nicotinic acetylcholine receptor agonist). MLA and GTS-21 were dissolved in PBS. 12 hours after surgery, mice were intraperitoneally injected 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 One microliter of Alexa Fluor 488-labeled wheat germ agglutinin (WGA488) (5 mg / ml) was injected into the liver at 40 spots using a 30-gauge needle attached to a Hamilton syringe. One week after WGA488 injection, mice were perfused first with PBS and then with 4% PFA in PBS. Isolated NG and DRG were post-processed 2 hours later and cryoprotected by immersion in 30% sucrose in PBS for 24 hours. Frozen NG and DRG sections were cut at 6 mm thickness using a cryostat, collected on slides, and immediately dried. Slides were then stored in ProLong PBS containing DAPI. TM Mounted with Diamond Antifade Mountant.

[0066] Electrophysiological recording of sympathetic nerve activity Sympathetic nerve activity was measured as previously described. 57 The common hepatic branch of the vagus nerve, or CG-SMG, was identified and exposed for neural activity measurement. The electrical activity of each nerve was amplified 50,000–100,000 times with a 100–1,000 kHz bandpass filter and monitored on an oscilloscope. The amplified and filtered neural activity was converted to standard pulses using a window discriminator, after which the discharge and electrical background noise were separated. Both the discharge rate and neurogram were sampled using a PowerLab analog-to-digital converter, and recording and data analysis were performed on a computer. Background noise, measured 30–60 min after euthanasia, was subtracted. The neural activity was rectified and integrated, normalized to 100% of baseline neural activity.

[0067] Isolation of mouse colonic lamina propria mononuclear cells Lamina propria mononuclear cells (LPMCs) were isolated as previously described. 6 The dissected colonic mucosa was cut into 5 mm pieces. The tissue was then incubated in CaCl2+ buffer containing 1 mM DTT and 5 μM EDTA. 2+ , Mg 2+After incubation in HBSS-free for 30 minutes at 37°C, the cells were further digested with collagenase and DNase for 45 minutes. Cells were then separated using a Percoll density gradient. The number of viable cells was determined using a Countess II (Thermo Fisher Scientific).

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

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

[0070] Fluorescence-activated cell sorting (FACS) analysis After blocking with anti-mouse CD16 / CD32 antibodies for 20 minutes, the cells were incubated with specific fluorescently labeled monoclonal antibodies at 4°C for 30 minutes, then permeabilized with permeabilization buffer. For Treg staining, intracellular staining was performed 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, and 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 +Colonic APCs were cultured overnight in RPMI-1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin, and then stimulated with muscarinic acid.

[0071] Measurement of aldehyde dehydrogenase activity Aldehyde dehydrogenase (ALDH) activity was measured using an ALDEFLUOR staining kit according to the manufacturer's protocol. 10 μL of ALDH was stained in ALDEFLUOR assay buffer containing activated ALDEFLUOR substrate (final concentration 1.5 μM) with or without the ALDH inhibitor diethylaminobenzaldehyde (DEAB) (final concentration 15 μM). 6 Cells were suspended at a concentration of 1000 cells / ml and incubated for 30 minutes at 37° C. FACS analysis was performed using a BD Biosciences FACS Canto II.

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

[0073] tissue samples Normal intestinal mucosa was collected from the unaffected area of ​​colon 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 colonic LP cells The colon was dissected and cleaned in situ of mesenteric fat and connective tissue. The entire colon was cut into 0.5 cm pieces and digested. These fragments were first washed with HBSS and then incubated in PBS containing 1 mM DTT and 5 mM EDTA for 20 min at 37°C. 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 for 60 min at 37°C. The LP suspension was passed through a 70-μm filter. Cells were then separated using a Percoll density gradient. The interface was collected and washed before staining and cell sorting. For cell sorting, CD45 + CD3 - CD19 - CD56 - HLA - DR hi Human colonic APCs were gated on by sorting cells using a BD FACS Aria-II. Human colonic APCs were cultured overnight in RPMI-1640 containing 10% FSB and 1% penicillin-streptomycin, followed by stimulation with muscarinic acid.

[0075] RNA sequence analysis RNA sequencing (RNA-seq) was performed and analyzed as previously described. 61Total RNA was prepared from approximately 20,000–50,000 cells using TRIzol. Subsequently, mRNA-seq libraries were generated 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) according to the protocol. Libraries were sequenced using Illumina with 150 bp (paired-end reads). To quantify transcript abundance, we used kallisto (v0.44.0, options: -b 100). 62 RNA-seq reads were pseudo-aligned to ENSEMBL transcripts (release 95 GRCm38) using [link missing or illegible]. Expression levels of APC subset signature genes (expressed in at least one sample >1 TPM) with neurotransmitter receptor genes were visualized by creating heatmaps with hierarchically clustered rows and columns (MORPHEUS; https: / / software.broadinstitute.org / morpheus / ) and ternary plots (ggtern v3.1.0).

[0076] Collection of fecal samples and isolation of bacterial DNA Feces were collected from the same mice on postoperative days 0 and 2. 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. DNA samples were purified by treatment with ribonuclease A followed by precipitation with 20% polyethylene glycol solution (PEG 6000 in 2.5 M sodium chloride). The DNA was then centrifuged, washed with 75% ethanol, and dissolved in tris-ethylenediaminetetraacetic acid (tris-EDTA) buffer.

[0077] Sequencing and processing of bacterial 16S rRNA genes in fecal DNA The hypervariable region V3-V4 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 the Miseq Reagent Kit V3 (600 cycles) and a Miseq sequencer (Illumina) according to the manufacturer's instructions. Sequence analysis was performed using the QIIME software package version 1.9.1. 64,65 Paired-end sequences were joined using the fastq-join tool in the ea-utils software package (https: / / doi.org / 10.2174 / 18750 36201307010001). High-quality sequences per sample (15,000) were randomly selected from those that passed the quality filter. Both primer sequences were trimmed using cutadapt (https: / / doi.org / 10.14806 / ej.17.1.200), followed by de novo detection of chimeras using the USEARCH66 algorithm, followed by the UCLUST algorithm. 67Sequences were assigned to operational taxonomic units using a sequence identity threshold of 96%. The GLSEARCH program was used to perform similarity searches against the publicly available 16S (RDP version 10.27 and CORE update 2 September 2012) and NCBI genome databases to assign each operational taxonomic unit. Data were rarefied to 10,000 sequences per sample, as determined by rarefaction curves. The rarefied data were used to determine the relative abundance of community members. UniFrac analysis was performed as previously described. 68 .

[0078] Germ-free mice Fecal samples were collected from sham and HVx mice. They 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 volumes of PBS, and passed through a 100 μm cell strainer. GF mice were orally inoculated with 200 μl of the suspension using a sterile stainless steel needle. After 3 weeks of colonization, the phenotype of colonic immune cells was analyzed.

[0079] Quantitative reverse transcription (qRT-) PCR analysis RNA was isolated and purified from colon tissues 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 to 18S ribosomal mRNA.

[0080] Histological and immunohistochemical examination 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 in 30% sucrose solution for 24 hours and preserved in OCT compound. 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 (dilutions: PGP9.5 (1 / 1000), pERK1 / 2 (1 / 500), TUBB3 (1 / 200), TRPV1 (1 / 1000), and TH (1 / 1000)) or overnight at 4°C (1 / 200 for IA / IE and TUBB3 (1 / 200)). After washing with PBS, the sections were incubated with Alexa Fluor 488- or Alexa Fluor 647-conjugated secondary antibodies (1 / 400) at room temperature for 2 hours. Tissue samples were observed under 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] Immunohistochemistry of pERK1 / 2 and c-Fos The expression of pEKR1 / 2 and c-Fos was analyzed immunohistochemically as previously reported. 1DSS-treated mice were transcardially perfused under anesthesia with 4% paraformaldehyde and 0.2% picric acid in PBS. The nodule ganglion and brain were harvested and postfixed in the same fixative for 2 hours to overnight at 4°C, followed by incubation in 30% sucrose in phosphate buffer for 48 hours. Longitudinal sections (8 μm) of the 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 freezing microtome. Rabbit polyclonal antibody against pERK1 / 2 (1 / 500) and Alexa 488-conjugated goat anti-rabbit IgG (1 / 500) were used. Fluorescent images were captured 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 immunoreactive for pERK1 / 2 and c-Fos within the NTS were counted.

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

[0083] Measurement of CGRP, ACH, and NE levels in the large intestine Neurotransmitter levels in the colon were measured as previously described. 69-71Colon tissue was washed with PBS and homogenized. The homogenate was centrifuged at 15,000 × g for 10 minutes at 4°C, and the supernatant was collected. Samples were stored at -80°C until use. Protein concentration was measured by BCA assay (Thermo Fisher Scientific). The concentrations of CGRP (Phoenix Pharmaceuticals), acetylcholine (Abcam), and noradrenaline (LsBio) in the homogenate were measured by ELISA.

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

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

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

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

[0088] [Figure description] Figure 1. Possible interactions between APCs and neurons in the intestine. a) Representative immunofluorescence staining of CX3CR1-GFP (green) and β-tubulin III (red) in the mouse colon. b) CD45.2 in Cx3cr1Gfp mice. + TCRβ - Representative CD11c and MHC-II staining of CD3-B220-NK1.1-gated colonic squamous mononuclear cells. cf, Eight-week-old male B6 mice (WT mice) underwent sham surgery (Sham) or truncal vagotomy (VGx). Colonic T cell phenotype and colonic gene expression were analyzed 2 days later (n = 12 / group). c, CD4 in the colonic lamina propria (LP). + Foxp3 in T cells + d, Frequency of Treg cells in the colon. + Expression of RORγt in Tregs. e) Expression of Aldh1a1 and Aldh1a2 mRNA in colonic APCs. f) MHC-II in the colon. + APC (CD45 + TCRβ-CD3-B220-NK1.1-MHC-II + ) ALDH + Left panel, frequency of ALDH in APCs + Histogram of cells. Colon mononuclear cells were incubated with ALDEFLUOR in the absence (solid) or presence (dotted line) of DEAB (an 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 sorted by sorted colonic and splenic APCs by RNA-seq analysis. h, Colonic 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) Tertiary plot of gene expression in colonic CD11b SP, DP, and CD11c SP cells. The color scale indicates 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 for 12 hours 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) (n = 5 / group). k, Expression of Aldh1a1 and Aldh1a2 in colonic APCs from WT and mAChR TKO mice. Colonic APCs were isolated from WT or Chrm1,2,4-deficient (mAChR TKO) mice and treated with 10 μM Mus or left untreated for 12 h (n = 6 / group). l, mRNA levels of ALDH1A1 and ALDH1A2 in human colonic APCs. Colonic APCs were treated with 10 μM Mus or left untreated for 12 h (n = 7 / group). Representative of three independent experiments (a, b, jl) or pooled from 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 the mean ± s.e.m.

[0089] Figure 2 Hepatic vagal sensory afferent pathways are 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: nucleus of the solitary tract; DMV: dorsal motor nucleus of the vagus; AP: occipital lobe; NG: nodose ganglion. a, Representative image of c-Fos immunostaining in the medulla oblongata (upper panel, bar: 200 μm); c-Fos expression counts per section in the NTS and DMV (lower panel). b, Representative image of pERK immunostaining in the nodose ganglion (NG) (upper panel, bar: 100 μm). c, d, Retrograde tracing of WGA. Alexa Fluor 488 immunostaining in the NG 1 week after WGA injection into the liver. + Representative fluorescence images (c) and quantitative images (d) of neurons (green) and DAPI (blue). The white arrow indicates Alexa Fluor 488 in NG. + Neurons are shown. e, WT mice underwent sham, VGx, or hepatic vagotomy (HVx) (n = 9 / group). f, WT mice underwent sham, ventral subdiaphragmatic vagotomy (LVx), or dorsal subdiaphragmatic vagotomy (RVx) (n = 4 / group). e, f, CD4 in the colon 2 days after surgery. + Foxp3 in cells + Cell frequencies. Representative of two independent experiments (a-d, f) or pooled from three independent experiments (e). P values ​​were obtained by unpaired two-tailed Student's t-test (a, b, d) or one-way ANOVA with Tukey's post-hoc test (e, f). Error bars represent the mean ± s.e.m.

[0090] Figure 3. The liver-brain-gut axis regulates colonic Treg homeostasis via APC muscarinic signaling. ad, WT and mAChR TKO mice underwent sham or HVx. e, WT and mAChR TKO mice underwent sham or HVx and were further injected with bethanechol (BETH; ip 300 μg / day) daily. The phenotype of colonic immune cells was analyzed on the second day after surgery (n = 5 / group). a, MHC-II + ALDH in colonic APC +Cell frequency. ALDH + Histograms of cells and colonic APCs (left panel). Quantification (right panel). b) mRNA expression of Aldh1a1 and Aldh1a2 in colonic APCs. c) and e) CD4 + Foxp3 in cells + d, Frequency of Foxp3 cells in the colon + RORγt in Tregs + Cell frequencies. P values ​​were obtained by unpaired one-way ANOVA with Tukey's post-hoc test. Error bars represent the mean ± s.e.m.

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

[0092] Figure 5 Muscarinic signaling in colonic APCs activates Treg induction. a, CX3CR1 in the mouse colon + 3D reconstruction of APC (green) and enteric neurons (purple). b, MHCII + APC (green), intestinal Tuj + Neuron (purple), Foxp3 + Three-dimensional reconstruction of Tregs (yellow). c, d, Anatomy of the vagus nerve trunk transection under the diaphragm (c) and surgical field (d). e, f, Phenotype of colonic T cells in mice after VGx surgery. CD4+ Foxp3 in T cells + The frequency of Treg cells (e) and colonic Foxp3 + RORγt in Tregs + Representative contour plot of pTreg frequency (f). g, Chrm1, Adrb2, Htr7, and Chrna7 mRNA expression in colonic and splenic APCs. h, CD11b-CD11c expression in colonic APCs by FACS. + (CD11c SP), CD11b + CD11c + (DP), CD11b + CD11c - Selection method for the (CD11b SP) subset. i, j, Expression of retinol metabolism-related genes by enteric neuron-derived neurons in colonic APCs was dependent on muscarinic signaling. i, Schematic diagram of the experiment. j, mRNA levels of Aldh1a1 and Aldh1a2 in colonic APCs (n = 6 / group). k, l, Muscarinic signaling in colonic APCs promoted the induction of Tregs. k, Schematic diagram of the experiment. CD4 + Foxp3 in T cells + Treg frequency (left panel). Representative contour plot (right panel). m, n, Intestinal neurospheroid-derived neurons upregulate Foxp3 via activation of muscarinic signaling in colonic APCs. + m, Schematic diagram of the experiment. n, CD4 + Foxp3 in T cells + Treg frequency (left panel). Representative contour plots (right panel). Quantification (n = 6 / group). Representative of two (a, b, i, k) or three (e, f) independent experiments. P values ​​were obtained by one-way ANOVA with Tukey's post-hoc test. Data are presented as mean ± SEM (j, l, n).

[0093] Figure 6 Colitis activates the liver-brain axis. WT mice were subjected to sham or VGx and then administered DSS for 7 days starting on postoperative day 2. The graph shows pooled data from three independent experiments (n = 15 / group). a) Relative body weight change during colitis. ** * 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 were administered DSS or water for 6 days (n=6 / group). d, Representative image of immunofluorescent staining of pERK1 / 2 (green) in the NG (upper panel, bar: 100 μm). Quantification of pERK1 / 2-expressing neurons (lower panel). e, Representative image of c-Fos immunoreactivity in the NTS (left panel, bar: 200 μm). Number of neurons showing c-Fos immunoreactivity (right panel). f, Representative image of immunofluorescent double staining of pERK1 / 2 (green) and PGP9.5 (red) in mouse liver sections. Co-stained areas are indicated in yellow (left panel). Scale bar indicates 10 μm. Quantification of pERK1 / 2 expression sites in PGP9.5-positive nerve fibers (right panel). g. Phosphor-mTOR and total mTOR protein levels in the liver. 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 then underwent sham or HVx 3 days later (n = 6 / group). The phenotype of colonic T cells was analyzed 2 days after surgery. h, CD4 + Foxp3 in T cells + Treg frequency (left panel). Representative contour plot (right panel). i, Foxp3 in the colon. + RORγt in Tregs + Cell frequency. Representative contour plot (left panel). Quantification (right panel). Representative of two independent experiments (di). P values ​​were obtained by unpaired two-tailed Student's t-test (af) and one-way ANOVA with Tukey's post-hoc test (h,i). Data are presented as mean ± SEM (bf, h, i).

[0094] Figure 7. Anatomy of the mouse hepatic vagus nerve. a, Anatomy. b, Surgical field for hepatic vagotomy. c, Schematic diagram showing the firing of the liver-brain-gut nerve arc during colitis. d, The common hepatic branch of the vagus nerve does not contain sympathetic nerves. Surgical field for electrical recording of the hepatic sympathetic nerve (left panel). Electrical activity of the common hepatic branch of the vagus nerve and the hepatic sympathetic nerve (middle panel). Representative images of immunofluorescence staining of tyrosine hydroxylase (TH) in the hepatic branch and DRG (right panel, bar: 100 μm). e, TRPV1 in the hepatic vagus nerve branch 2 days after capsaicin administration. + Fluorescent immunostaining of neurons (bar: 200 μm). f, g, h, Retrograde tracing of WGA. f, g, WT mice were subjected to sham or VGx and then injected with Alexa Fluor 488-labeled WGA on the second day after surgery (n = 3 / group). Alexa Fluor 488 in NG (f) and Th4 DRG (g) 1 week after WGA injection into the liver. + Representative images of neurons (green) and DAPI (blue). (Left panel, bar: 50 μm). h, Alexa Fluor 488 in DRG (Th4-7 and Th13) 1 week after WGA injection into the liver. + Fluorescence images of neurons (green) and DAPI (blue) (bar: 100 μm). Representative values ​​from two independent experiments (dh). P values ​​were obtained by unpaired two-tailed Student's t-test. Data are shown as mean ± SEM (f, g).

[0095] Figure 8. Effect of vagotomy on the maintenance and stability of colonic pTregs. WT mice underwent either sham, VGx, or HVx (n = 9 / group). The phenotype and gene expression of colonic immune cells were analyzed 2 days after surgery. a, Colonic CD4 + Foxp3 in cells + b, Cell frequency. Representative contour plot. c, Foxp3 in colonic LP. + RORγt in Tregs +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). e, f, WT mice underwent sham or HVx. e, f, WT mice underwent sham or HVx, and colonic T cell phenotypes were analyzed at the indicated time points after HVx (n = 9 / group). CD4 in colonic LP. + Foxp3 in T cells + Frequency of cells (e) and Foxp3 in colonic LP + RORγt in Tregs + The frequency of CD4 cells (f). Representative contour plots (left panel) and quantification (right panel) are shown (e, f). g, h, i, CD4 cells in the colon on day 2 after surgery. + Foxp3 in cells + Cell frequency (g, h, i) and colonic Foxp3 + RORγt in Tregs + Cell frequency (g, h). g, B6 mice underwent sham or HVx (n = 10 / group). h, BALB / c mice underwent sham or HVx (n = 4 / group). i, 6-week-old WT rats underwent sham or HVx (n = 4 / group). j, k, Rag2 - / - Mice were subjected to sham or HVx and CD4 + CD45RB hi T cells were transplanted (n = 8 / group). Mice were sacrificed 4 weeks after transplantation, and colonic Treg cells were analyzed. j, CD4 + Foxp3 in cells + Frequency of cells. Representative contour plot (left panel). Quantification (right panel). k, Foxp3 in the colon. + RORγt in Tregs +The frequency of CD4 cells. Representative contour plots (left panel). Quantification (right panel). l. WT mice were subjected to sham or HVx. The phenotype of colonic immune cells was analyzed 2 days later. + IFN-γ in cells + , Gata3 + , and IL-17A + Cell frequencies (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 with Tukey's post hoc test (bf) and unpaired two-tailed Student's t-test (gl). Data are presented as mean ± sem.

[0096] Figure 9 Vagal afferents from the liver, but not the spinal cord, are involved in the maintenance of colonic Treg homeostasis. ae, Corn oil (Oil) or capsaicin (Cap) was applied to the hepatic vagus nerve branch of WT mice. Two days after capsaicin application, phenotypic analysis of colonic T cells and APCs was performed (c, d, n = 16 / group; e, f, n = 8 / group). a, b, Representative fluorescent images of TRPV1 (red) and DAPI (blue) in NG (a) and Th4-DRG (b). The scale bar indicates 100 μm. c, CD4 in the colon. + Foxp3 in cells + Representative contour plots (left panel) and quantification (right panel) of cells. 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 mice were intrathecally injected with capsaicin (gj, n = 5 / group) and resiniferatoxin (RTX) (kp, n = 4 / group). On the 7th day after injection, TRPV1 expression in the spinal cord was significantly increased. + Neural (Th4-7 and Th13) and colonic immune cells were analyzed. g, TRPV1 in the spinal cord + Fluorescent immunohistochemistry of nerves (bar: 200 μm). CD4 in the large intestine + Foxp3 in cells + Cell frequency (h, n) and colonic Foxp3 + RORγt in Tregs + Cell frequency (i, o). k, l, Effect of intrathecal injection of RTX in the DRG (k) and NG (l). Scale bar indicates 100 μm. m, CGRP levels in the colon. Representative of two independent experiments (a-b, g-o) or pooled from two (e) or three (c, d, f) independent experiments. P values ​​were obtained by unpaired two-tailed Student's t-test. Data are shown as mean ± sem.

[0097] Figure 10. Hemidiaphragmatic vagotomy reveals functional asymmetry of the vagus nerve. WT mice underwent sham, ventral (left) subdiaphragmatic vagotomy (LVx), or dorsal (right) subdiaphragmatic vagotomy (RVx) (n = 4 / group). Phenotypic analysis of colonic T cells and APCs was performed 2 days after surgery. a) CD4 + Foxp3 in cells + b, Frequency of Foxp3 cells in colonic LP + RORγt in Tregs + Frequency of cells. 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, Blockade of sympathetic signals via CG / SMG does not affect the maintenance of Tregs in the colon. d, Surgical field of CG / SMG ganglionectomy. e, Electrical activity of the splenic nerve. 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 administration (n = 5 / group). f, i, j, CD4 in the colon (f) and spleen (i, j). + Foxp3 in cells + Cell frequency. g, colonic Foxp3 + RORγt in Tregs + Cell frequency. h, MHC-II + ALDH in colonic APC + After sham or HVx, WT mice were injected daily with vehicle, salbutamol (30 μg / day, ip), or propanol (300 μg / day, ip) for 2 days (n = 6 / group). CD4 cell frequency in the colon 12 hours after the last injection. + Foxp3 in cells + Frequency of cells (k), colon Foxp3 + RORγt in Tregs + Cell(l) frequency, MHC-II + ALDH in colonic APC + O, WT mice underwent sham or HVx. The phenotype of T cells in the large intestine, small intestine, and spleen was analyzed two days after surgery. CD4 + Foxp3 in T cells + Cell frequency (n = 4 / group). Representative values ​​from two independent experiments (a-o). P values ​​were obtained by one-way ANOVA with Tukey's post hoc test (b, c, ko) and unpaired two-tailed Student's t-test (f-j). Data are shown as mean ± sem.

[0098] Figure 11 Effects of VGx and HVx on intrinsic enteric neurons. ac, g: WT mice underwent sham (n=4) or VGx (n=5). df, h: WT mice underwent sham (n=6) or HVx (n=6). Activity of intrinsic enteric neurons was measured two days after surgery. a, d: Representative images of immunofluorescent staining for HuC / D (white) and c-Fos (red) in the large intestine. The scale bar indicates 100 μm. b, e: c-Fos + Quantification of neurons. c, f, Expression of Hand2 mRNA in the large intestine. g, h, Acetylcholine, norepinephrine, and CGRP levels in the large intestine. * and ** * indicates p < 0.05 and p < 0.01, respectively. P values ​​were obtained by unpaired two-tailed Student's t-test. Data are presented as mean ± sem.

[0099] Figure 12. Effect of mAChRs and α7nAChRs on the maintenance of colonic Tregs. ag, WT mice were administered sham or HVx and then injected daily for 2 days with water or bethanechol (BETH; ip 300 μg / day) (ad) or GST-21 (ip 300 μg / day) (eg) (a, b, n = 5 / group). GST-21 (ip 300 μg / day) (eg) (a, b, n = 5 / group; c, d, n = 10 / group; e.g., n = 6 / group). hj, WT mice were administered sham or HVx and then injected daily with water, BETH alone, or BETH alone. + MLA was injected daily for 2 days (n = 6 / group). kl, WT and mAchR TKO mice were injected daily with water or BETH for 2 days (n = 4 / group) after sham or HVx. As shown in Figure 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) Expression of Aldh1a1 and Aldh1a2 mRNA in colonic APCs. c), e), h) CD4+ Foxp3 in cells + Cell frequency d, f, i, k, Foxp3 in the colon + RORγt in Tregs + Cell frequencies. P values ​​were obtained by one-way ANOVA with Tukey's post hoc test. Data are presented as mean ± sem.

[0100] Figure 13. Effect of gut microbiota on colonic Treg maintenance in the liver-brain-gut axis. a-c: WT mice underwent sham or HVx (n = 4 / group). Feces were collected from the same mice before treatment and on day 2 after surgery. a: Microbial α-diversity in feces. b: Principal coordinate analysis (PCoA) based on weighted UniFrac analysis of bacterial community structure (black, pretreatment; 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 assessed 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 on day 21 after inoculation (n = 5 / group). f, g: WT mice underwent sham or HVx and were co-housed for 2 days in our SPF facility. The graph shows pooled data from three independent experiments (n = 14 / group). hk, On day 14 of life, mice underwent sham or HVx (n = 10 / group). The phenotype of colonic immune cells was analyzed two days later (h). l, m, WT mice were treated with Abx-cocktail (metronidazole, vancomycin, ampicillin, neomycin) for 3 weeks and then underwent sham or HVx. The phenotype of colonic T cells was analyzed two 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 + Frequency of cells. e, g, j, m, colon Foxp3 + RORγt in Tregs + Expression of MHC-II +ALDH in colonic APC + Cell frequencies. P values ​​were obtained by one-way ANOVA with Tukey's post hoc test (a, d, e, l, m) or unpaired two-tailed Student's t-test (f, g, ik). Data are presented as mean ± sem.

[0101] Figure 14. Effect of HVx on colitis. ac, WT mice were sensitized with TNBS. Seven days later, the mice underwent sham or HVx and received TNBS intrarectally. The graph shows pooled data from three independent experiments (n = 20 / group). df, 8-week-old male Rag2 mice. - / -Mice underwent sham surgery or HVx and were administered DSS for 7 days starting on postoperative day 2 (n = 12 / group). g, h, Sham-operated and HVx-treated mice were housed together and orally challenged with 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 were sham-operated or HVx-treated and 2 days later orally challenged with 2.0% DSS (w / v) for 7 days. The graph shows pooled data from two independent experiments (n = 10 / group). k, l, Sham-operated and HVx-treated Myd88-deficient mice were orally challenged with 2.0% DSS (w / v) for 7 days (n = 13 / group). mo, Sham-operated and hepatic vein-transection-treated mice were orally challenged with 2.0% DSS (w / v) and treated daily with BETH for 7 days. Graphs show pooled data from two independent experiments (n = 10 / group). a, d, g, l, k, m: Relative body weight change during acute colitis. b, e, h, j, l, n: DAI. c, f, o: Representative HE staining of colon sections (left panel, bar: 200 μm) and histological scores (right panel). Each experiment was repeated at least twice, with similar results. P values ​​were obtained by unpaired, two-tailed Student's t-test. Error bars indicate mean ± s.e.m. p, Schematic diagram of the liver-brain-gut neural arc. A mouse is shown in a supine position. The liver senses the intestinal microenvironment and transmits its sensory input to the left NTS in the brainstem, ultimately transmitting it to the left vagal parasympathetic and enteric neurons. Intestinal APCs activated by the liver-brain-gut neural arc promote ALDH expression and RA synthesis via mAChRs, thereby maintaining a reservoir of peripheral regulatory T cells.

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[0103] [Example 2] [Description of Drawings] Figure 15. Schematic diagram of hepatic vagus nerve stimulation (VHNS) C57BL6 / J mice (male, 10 weeks old) were purchased. After one week of acclimation, the mice underwent laparotomy under inhalation anesthesia, and a cuff electrode (Figure 15a) was placed on the hepatic branch of the vagus nerve (Figure 15b). To ensure that electrical 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, the mice were allowed to recover for approximately one week. The electrodes were then connected to a modular stimulator, and freely moving mice were subjected to electrical stimulation at 10 Hz with a pulse width of 500 μs, with an ON / OFF interval of 10 s and 90 s, for 3 h / day. Mice with electrodes placed on the hepatic branch and connected to the modular stimulator but not stimulated served as a control group. After three consecutive days of electrical stimulation, the mice were sacrificed, and the colons and brains were collected. The proportion of regulatory T cells (Tregs) in the colon was analyzed by FACS (Figure 15c). VHNS increased colonic Tregs. The brain activation areas induced by electrical stimulation were assessed using the neural activation marker cFos. In situ hybridization revealed activation of the left nucleus tractus solitarius (NTS), which is the projection of the hepatic branch of the vagus nerve, the left dorsal motor nucleus of the vagus nerve (DMV), which contains the efferent vagal cell bodies, and the area postrema (AP) (Figure 15d).

[0104] Figure 16. Electrical stimulation of the hepatic branch of the vagus nerve (VHNS) suppresses the pathology of colitis in mice. C57BL6 / J male mice (10 weeks old) were purchased. After 1 week of acclimation, the mice underwent laparotomy under inhalation anesthesia, and a cuff electrode was placed on the hepatic branch of the vagus nerve. To ensure electrical current flow 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, the mice were allowed a recovery period of approximately 1 week. To induce colitis, the mice were given 2% dextran sulfate sodium (DSS) in water ad libitum. Simultaneously with the start of DSS administration, the electrodes were connected to a modular stimulator, and electrical stimulation was administered to freely moving mice at 10 Hz with a pulse width of 500 μs, with an ON / OFF period of 10 s and 90 s, for 3 h / day. Mice with electrodes placed on the hepatic branch and connected to the modular stimulator but not receiving stimulation served as a control group. Seven days after DSS administration, colons were harvested from the mice, and colitis pathology was assessed using pathological specimens and intestinal length. Body weight was measured over a 7-day period after the onset of colitis. VHNS inhibited weight loss caused by DSS-induced colitis (Fig. 16a). VHNS also inhibited intestinal shortening caused by DSS-induced colitis (Fig. 16b). Pathological specimens showed that VHNS inhibited the loss of colonic epithelial cells caused by colitis (Fig. 16c).

[0105] Figure 17 Two-bottle preference assays using vagotomized mice C57BL6 / J mice (male, 10 weeks old) were purchased. After one week of acclimation, they underwent laparotomy under inhalation anesthesia, and the mouse vagus nerves were transected bilaterally (VGx), left (LVx), or right (RVx) (Fig. 17a). Mice that underwent laparotomy alone (Sham) served as controls. After a recovery period of approximately one week, these mice were used in two-bottle preference assays. Two drinking bottles were prepared: one filled with 600 mM glucose (Glu) and the other with 30 mM acesulfame potassium (Ace K), an artificial sweetener. These were placed in a Rick analysis-based choice preference experiment apparatus (Fig. 17b). The mice were housed in cages in the apparatus from 8 PM to 10 AM, and the number of times they touched the drinking bottles with their mouths was counted. This experiment was repeated for three days for the same individual, and the ratio of sucrose to Ace K in the total number of contacts was calculated for each day. This ratio was evaluated as preference. On the first day of the test (Day 1), the preference for sucrose and Ace K was almost the same in all mice. However, Sham and RVx mice selectively consumed sucrose as the days went by. On the other hand, VGx and LVx mice continued to selectively consume sucrose and Ace K even on Day 3 of the test (Figure 17c).

[0106] Figure 18. Regulation of pulmonary immune cells by the hepatic branch of the vagus nerve C57BL6 / J mice (male, 10 weeks old) were purchased. After one week of acclimation, they underwent laparotomy under inhalation anesthesia, and the hepatic branch of the vagus nerve (HVx) was resected (Figure 18a). Mice that underwent laparotomy only (Sham) served as controls. After a recovery period of approximately 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 pulmonary ILC2 cells increased after HVx (Figure 18b).

[0107] Figure 19 Vagus nerve control of intestinal peristalsis (Figure 19a) C57BL6 / J mice (male, 10 weeks old) were purchased. After one week of acclimation, laparotomy was performed under inhalation anesthesia, and both vagus nerves (HVx) were removed. Mice that underwent laparotomy alone (Sham) served as controls. After a recovery period of approximately one week, intestinal peristalsis was evaluated in these mice. Motility of the entire gastrointestinal tract was evaluated using the intestinal transit time (ITT) test, gastric motility was evaluated using the gastric emptying test, and small intestinal motility was evaluated using the small-bowel (SB) transit test. Vagus nerve removal reduced gastric and small intestinal motility and inhibited gastrointestinal peristalsis. (Fig. 19b) C57BL6 / J mice (male, 10 weeks old) were purchased. After 1 week of acclimation, the mice underwent laparotomy under inhalation anesthesia, and either the proper hepatic branch (HVx) or the gastroduodenal branch (GVx) of the mouse vagus nerve was transected (Fig. 19b). Mice that underwent laparotomy alone (Sham) served as controls. After a recovery period of approximately 1 week, the intestinal peristalsis of these mice was evaluated. The motility of the entire gastrointestinal tract was assessed by the intestinal transit time (ITT) test, gastric motility by the gastric emptying test, small intestinal motility by the small-bowel (SB) transit test, and large intestinal motility by the colonic transit test. Neither HVx nor GVx affected gastric motility. HVx reduced small intestinal motility, whereas GVx reduced large intestinal motility. These results suggest that each branch of the vagus nerve controls peristalsis in different regions of the gastrointestinal tract.

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

[0109] The present invention can be used in pharmaceutical-related industries.

Claims

1. A pharmaceutical composition for increasing the amount of peripheral regulatory T cells (pTreg) in the intestinal tract, characterized in that it contains an agonist of a muscarinic acetylcholine receptor.

2. 2. The pharmaceutical composition according to claim 1, wherein the agonist of the muscarinic acetylcholine receptor is bethanechol, muscarine, pilocarpine, or cevimeline.

3. A therapeutic agent for a disease, comprising the pharmaceutical composition according to claim 1 or 2, which is used to treat a disease caused by an intestinal immune disorder.

4. A therapeutic agent for a disease, comprising the pharmaceutical composition according to claim 1 or 2, which is used to treat inflammatory bowel disease, autoimmune disease, allergy, cancer, or depression.

5. The therapeutic agent for a disease according to claim 3, wherein the disease is inflammatory bowel disease.

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