Vagal innervation is required for the formation of tertiary lymphoid tissue in colitis.

Tertiary lymphoid tissue (TLT) is lymphoid tissue that forms in adult life as a result of chronic inflammation in a tissue or organ. TLT has been shown to form in a variety of chronic inflammatory diseases, though it is not clear if and how TLT develops in the inflamed colon during inflammatory bowel disease. Here, we show that TLT develops as newly formed lymphoid tissue in the colon following dextran sulphate sodium induced colitis in C57BL/6 mice, where it can be distinguished from the preexisting colonic patches and solitary intestinal lymphoid tissue. TLT in the inflamed colon develops following the expression of lymphoid tissue-inducing chemokines and adhesion molecules, such as CXCL13 and VCAM-1, respectively, which are produced by stromal organizer cells. Surprisingly, this process of TLT formation was independent of the lymphotoxin signaling pathway, but rather under neuronal control, as we demonstrate that selective surgical ablation of vagus nerve innervation inhibits CXCL13 expression and abrogates TLT formation without affecting colitis. Sympathetic neuron denervation does not affect TLT formation. Hence, we reveal that inflammation in the colon induces the formation of TLT, which is controlled by innervation through the vagus nerve.

Splenic autonomic denervation increases inflammatory status but does not aggravate atherosclerotic lesion development.

UNLABELLED: The brain plays a prominent role in the regulation of inflammation. Immune cells are under control of the so-called cholinergic anti-inflammatory reflex, mainly acting via autonomic innervation of the spleen. Activation of this reflex inhibits the secretion of proinflammatory cytokines and may reduce the development of atherosclerosis. Therefore, the aim of this study was to evaluate the effects of selective parasympathetic (Px) and sympathetic (Sx) denervation of the spleen on inflammatory status and atherosclerotic lesion development. Female APOE*3-Leiden.CETP mice, a well-established model for human-like lipid metabolism and atherosclerosis, were fed a cholesterol-containing Western-type diet for 4 wk after which they were subdivided into three groups receiving either splenic Px, splenic Sx, or sham surgery. The mice were subsequently challenged with the same diet for an additional 15 wk. Selective Px increased leukocyte counts (i.e., dendritic cells, B cells, and T cells) in the spleen and increased gene expression of proinflammatory cytokines in the liver and peritoneal leukocytes compared with Sx and sham surgery. Both Px and Sx increased circulating proinflammatory cytokines IL-1ß and IL-6. However, the increased proinflammatory status in denervated mice did not affect atherosclerotic lesion size or lesion composition. CONCLUSION: Predominantly selective Px of the spleen enhances the inflammatory status, which, however, does not aggravate diet-induced atherosclerotic lesion development.

A distinct vagal anti-inflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen.

The cholinergic anti-inflammatory pathway (CAIP) has been proposed as a key mechanism by which the brain, through the vagus nerve, modulates the immune system in the spleen. Vagus nerve stimulation (VNS) reduces intestinal inflammation and improves postoperative ileus. We investigated the neural pathway involved and the cells mediating the anti-inflammatory effect of VNS in the gut. The effect of VNS on intestinal inflammation and transit was investigated in wild-type, splenic denervated and Rag-1 knockout mice. To define the possible role of α7 nicotinic acetylcholine receptor (α7nAChR), we used knockout and bone marrow chimaera mice. Anterograde tracing of vagal efferents, cell sorting and Ca(2+) imaging were used to reveal the intestinal cells targeted by the vagus nerve. VNS attenuates surgery-induced intestinal inflammation and improves postoperative intestinal transit in wild-type, splenic denervated and T-cell-deficient mice. In contrast, VNS is ineffective in α7nAChR knockout mice and α7nAChR-deficient bone marrow chimaera mice. Anterograde labelling fails to detect vagal efferents contacting resident macrophages, but shows close contacts between cholinergic myenteric neurons and resident macrophages expressing α7nAChR. Finally, α7nAChR activation modulates ATP-induced Ca(2+) response in small intestine resident macrophages. We show that the anti-inflammatory effect of the VNS in the intestine is independent of the spleen and T cells. Instead, the vagus nerve interacts with cholinergic myenteric neurons in close contact with the muscularis macrophages. Our data suggest that intestinal muscularis resident macrophages expressing α7nAChR are most likely the ultimate target of the gastrointestinal CAIP.

Inhibition of spleen tyrosine kinase as treatment of postoperative ileus.

OBJECTIVE: Intestinal inflammation resulting from manipulation-induced mast cell activation is a crucial mechanism in the pathophysiology of postoperative ileus (POI). Recently it has been shown that spleen tyrosine kinase (Syk) is involved in mast cell degranulation. Therefore, we have evaluated the effect of the Syk-inhibitor GSK compound 143 (GSK143) as potential treatment to shorten POI. DESIGN: In vivo: in a mouse model of POI, the effect of the Syk inhibitor (GSK143) was evaluated on gastrointestinal transit, muscular inflammation and cytokine production. In vitro: the effect of GSK143 and doxantrazole were evaluated on cultured peritoneal mast cells (PMCs) and bone marrow derived macrophages. RESULTS: In vivo: intestinal manipulation resulted in a delay in gastrointestinal transit at t=24 h (Geometric Center (GC): 4.4 ± 0.3). Doxantrazole and GSK143 significantly increased gastrointestinal transit (GC doxantrazole (10 mg/kg): 7.2 ± 0.7; GSK143 (1 mg/kg): 7.6 ± 0.6), reduced inflammation and prevented recruitment of immune cells in the intestinal muscularis. In vitro: in PMCs, substance P (0-90 µM) and trinitrophenyl (0-4 µg/ml) induced a concentration-dependent release of ß-hexosaminidase. Pretreatment with doxantrazole and GSK143 (0.03-10 µM) concentration dependently blocked substance P and trinitrophenyl induced ß-hexosaminidase release. In addition, GSK143 was able to reduce cytokine expression in endotoxin-treated bone marrow derived macrophages in a concentration-dependent manner. CONCLUSIONS: The Syk inhibitor GSK143 reduces macrophage activation and mast cell degranulation in vitro. In addition, it inhibits manipulation-induced intestinal muscular inflammation and restores intestinal transit in mice. These findings suggest that Syk inhibition may be a new tool to shorten POI.

Lipid-rich enteral nutrition regulates mucosal mast cell activation via the vagal anti-inflammatory reflex.

Nutritional stimulation of the cholecystokinin-1 receptor (CCK-1R) and nicotinic acetylcholine receptor (nAChR)-mediated vagal reflex was shown to reduce inflammation and preserve intestinal integrity. Mast cells are important early effectors of the innate immune response; therefore modulation of mucosal mast cells is a potential therapeutic target to control the acute inflammatory response in the intestine. The present study investigates intestinal mast cell responsiveness upon nutritional activation of the vagal anti-inflammatory reflex during acute inflammation. Mucosal mast cell degranulation was induced in C57/Bl6 mice by administration of Salmonella enterica LPS. Lipid-rich enteral feeding prior to LPS significantly decreased circulatory levels of mouse mast cell protease at 30 min post-LPS compared with isocaloric low-lipid nutrition or fasting. CCK-1R blockage reversed the inhibitory effects of lipid-rich feeding, whereas stimulation of the peripheral CCK-1R mimicked nutritional mast cell inhibition. The effects of lipid-rich nutrition were negated by nAChR blockers chlorisondamine and α-bungarotoxin and vagal intestinal denervation. Accordingly, release of ß-hexosaminidase by MC/9 mast cells following LPS or IgE-ovalbumin complexes was dose dependently inhibited by acetylcholine and nicotine. Application of GSK1345038A, a specific agonist of the nAChR α7, in bone marrow-derived mast cells from nAChR ß2-/- and wild types indicated that cholinergic inhibition of mast cells is mediated by the nAChR α7 and is independent of the nAChR ß2. Together, the present study reveals mucosal mast cells as a previously unknown target of the nutritional anti-inflammatory vagal reflex.

Peripheral oscillators: the driving force for food-anticipatory activity.

Food-anticipatory activity (FAA) and especially the food-entrained oscillator (FEO) have driven many scientists to seek their mechanisms and locations. Starting our research on FAA we, possibly like many other scientists, were convinced that clock genes held the key to the location and the underlying mechanisms for FAA. In this review, which is aimed especially at discussing the contribution of the peripheral oscillators, we have put together the accumulating evidence that the clock gene machinery as we know it today is not sufficient to explain food entrainment. We discuss the contribution of three types of oscillating processes: (i) within the suprachiasmatic nucleus (SCN), neurons capable of maintaining a 24-h oscillation in electrical activity driven by a set of clock genes; (ii) oscillations in metabolic genes and clock genes in other parts of the brain and in peripheral organs driven by the SCN or by food, which damp out after a few cycles; (iii) an FEO which, we propose, is a system built up of different oscillatory processes and consisting of an as-yet-unidentified network of central and peripheral structures. In view of the evidence that clock genes and metabolic oscillations are not essential for the persistence of FAA we propose that food entrainment is initiated by a repeated metabolic state of scarcity that drives an oscillating network of brain nuclei in interaction with peripheral oscillators. This complex may constitute the proposed FEO and is distributed in our peripheral organs as well as within the central nervous system.

Daily rhythms in metabolic liver enzymes and plasma glucose require a balance in the autonomic output to the liver.

Daily variations in plasma glucose concentrations are controlled by the biological clock, located in the suprachiasmatic nucleus. Our previous studies indicated an important role for the sympathetic innervation of the liver in the generation of the daily glucose rhythm. In the present study, we investigated further the role of the autonomic nervous system (ANS) in the genesis of the plasma glucose rhythm. First, we showed that complete removal of the autonomic inputs to the liver did not impair the plasma glucose rhythm or the daily expression of the glucoregulatory enzymes in the liver. Consequently, we studied whether the daily glucose rhythm is driven by the daily feeding activity in denervated animals. Surprisingly, complete denervation combined with a noncircadian feeding schedule also did not abolish the 24-h profile in plasma glucose or all daily rhythms in the gene expression of liver enzymes. These results demonstrate that the mechanisms used by the suprachiasmatic nucleus to control the rhythmic expression of glucose-metabolizing enzymes and the 24-h rhythm in plasma glucose concentrations are highly versatile and the glucose rhythm can be maintained in absence of hepatic ANS input and/or a day/night rhythm in feeding activity. Interestingly, a hepatic sympathectomy or parasympathectomy did abolish the plasma glucose rhythm, demonstrating that a unilateral denervation of the liver is more deleterious to maintaining the rhythmic liver metabolism than a complete removal of both branches. This observation supports the notion that an unbalanced ANS in obesity and diabetes accounts for the disturbed glucose balance in these disorders.

The role of feeding rhythm, adrenal hormones and neuronal inputs in synchronizing daily clock gene rhythms in the liver.

The master clock in the hypothalamic suprachiasmatic nucleus (SCN) is assumed to distribute rhythmic information to the periphery via neural, humoral and/or behavioral connections. Until now, feeding, corticosterone and neural inputs are considered important signals for synchronizing daily rhythms in the liver. In this study, we investigated the necessity of neural inputs as well as of the feeding and adrenal hormone rhythms for maintaining daily hepatic clock gene rhythms. Clock genes kept their daily rhythm when only one of these three signals was disrupted, or when we disrupted hepatic neuronal inputs together with the adrenal hormone rhythm or with the daily feeding rhythm. However, all clock genes studied lost their daily expression rhythm after simultaneous disruption of the feeding and adrenal hormone rhythm. These data indicate that either a daily rhythm of feeding or adrenal hormones should be present to synchronize clock gene rhythms in the liver with the SCN.

Absence of diurnal variation in visceromotor response to colorectal distention in normal Long Evans rats.

BACKGROUND: Enhanced colorectal sensitivity (i.e. visceral hypersensitivity) is thought to be a pathophysiological mechanism in irritable bowel syndrome (IBS). In healthy men a circadian variation in rectal perception to colonic distention was described. Disturbed day and night rhythms, which occur in shift work and trans meridian flights, are associated with the prevalence of IBS. This raises the question whether disruptions of circadian control are responsible for the observed pathology in IBS. Prior to investigating altered rhythmicity in relation to visceral hypersensitivity in a rat model for IBS, it is relevant to establish whether normal rats display circadian variation similar to healthy men.  METHODOLOGY AND FINDINGS: In rodents colorectal distension leads to reproducible contractions of abdominal musculature. We used quantification of this so called visceromotor response (VMR) by electromyography (EMG) to assess visceral sensitivity in rats. We assessed the VMR in normal male Long Evans rats at different time points of the light/dark cycle. Although a control experiment with male maternal separated rats confirmed that intentionally inflicted (i.e. stress induced) changes in VMR can be detected, normal male Long Evans rats showed no variation in VMR along the light/dark cycle in response to colorectal distension. CONCLUSIONS: In the absence of a daily rhythm of colorectal sensitivity in normal control rats it is not possible to investigate possible aberrancies in our rat model for IBS.

MT1 melatonin receptor mRNA tissular localization by PCR amplification.

OBJECTIVES: The pineal gland transduces photoperiodic informations to the neuroendocrine axis through the nocturnally melatonin secretion. This hormonal message plays a major role in the biological rhythm regulation. By autoradiography, more than 130 melatonin putative targets have been reported in the central nervous system (CNS) and in peripheral tissues. However, cross-species consensus concern only a few of them like the suprachiasmatic nuclei (SCN), the master circadian clock, and the pars tuberalis of the pituitary. Recently, MT1 melatonin receptor cDNA have been cloned in several mammals providing us with new tools to investigate its tissular location at the gene level. In the present study, we report a screening for MT1 mRNA by RT-PCR amplification of numerous tissue mRNA. METHOD: mRNA were extracted from a large variety of rat tissues. To semi-quantify the melatonin receptor mRNA expression level, each cDNA was amplified concomitantly with both beta-actin and MT1 specific primers. RESULTS: In central and peripheral tissues previously reported to bind melatonin, strong PCR signals were logically observed. More surprisingly, a vast majority of studied tissues express MT1 mRNA and then might be responsive to melatonin. CONCLUSION: Numerous biological functions express diurnal rhythmicity and internal-synchronization. As, most of them apparently do not receive any out-coming neuronal message from the SCN, endocrine communication was proposed to support biological rhythm synchronization. Our present data strengthen the idea that the nocturnally restricted melatonin secretion could be one internal zeitgeber that putatively distributes the endogenous circadian rhythmicity to all tissues expressing melatonin receptors.