Role of ICAM-1 in the Adhesion of T Cells to Enteric Glia: Perspectives in the Formation of Plexitis in Crohn's Disease.

BACKGROUND & AIMS: The presence of myenteric plexitis in the proximal resection margins is a predictive factor of early postoperative recurrence in Crohn's disease. To decipher the mechanisms leading to their formation, T-cell interactions with enteric neural cells were studied in vitro and in vivo. METHODS: T cells close to myenteric neural cells were retrospectively quantified in ileocolonic resections from 9 control subjects with cancer and 20 patients with Crohn's disease. The mechanisms involved in T-cell adhesion were then investigated in co-cultures of T lymphocytes with enteric glial cells (glia). Finally, the implication of adhesion molecules in the development of plexitis and colitis was studied in vitro but also in vivo in Winnie mice. RESULTS: The mean number of T cells close to glia, but not neurons, was significantly higher in the myenteric ganglia of relapsing patients with Crohn's disease (2.42 ± 0.5) as compared with controls (0.36 ± 0.08, P = .0007). Co-culture experiments showed that exposure to proinflammatory cytokines enhanced T-cell adhesion to glia and increased intercellular adhesion molecule-1 (ICAM-1) expression in glia. We next demonstrated that T-cell adhesion to glia was inhibited by an anti-ICAM-1 antibody. Finally, using the Winnie mouse model of colitis, we showed that the blockage of ICAM-1/lymphocyte function-associated antigen-1 (LFA-1) with lifitegrast reduced colitis severity and decreased T-cell infiltration in the myenteric plexus. CONCLUSIONS: Our present work argues for a role of glia-T-cell interaction in the development of myenteric plexitis through the adhesion molecules ICAM-1/LFA-1 and suggests that deciphering the functional consequences of glia-T-cell interaction is important to understand the mechanisms implicated in the development and recurrence of Crohn's disease.

Clinical and Histopathological Spectrum of Adult Gastrointestinal Inflammatory Neuropathy.

Gastrointestinal inflammatory neuropathy, namely, eosinophilic myenteric ganglioneuronitis (EMG) and lymphocytic ganglioneuronitis (LG), is a form of chronic intestinal pseudo-obstruction and results from the infiltration of the myenteric plexus by eosinophils and lymphocytes, respectively. The literature related to the clinicopathological features of adult inflammatory neuropathy is scarce. We aim to elucidate the clinical and histological details of 7 cases of inflammatory neuropathy (EMG, n = 4, and LG, n = 3) and compare the features of EMG and LG retrospectively. There was no difference between these two entities in terms of clinical, hematological, or biochemical parameters. Histologically, almost all cases (n = 6/7) showed accompanying elements of ganglion cell vacuolization, mesenchymopathy, and partial/complete desmosis in addition to the disease-defining pathology. Besides, all cases of EMG showed infiltration of the inner circular muscle of muscularis propria by eosinophils. Two cases of LG showed additional muscular pathology pertaining to the muscularis propria. Inflammatory infiltration of the myenteric plexus is pathognomonic for the diagnosis of gastrointestinal inflammatory neuropathy although additional features in the form of ganglion cell vacuolization, reduction in the number of ganglia, desmosis, mesenchymopathy, and inflammation of the muscularis propria (eosinophils in EMG) can be seen. The pathologists need proper awareness along with judicious use of special and immunostains for clinching the diagnosis.

Lower esophageal sphincter muscle of patients with achalasia exhibits profound mast cell degranulation.

BACKGROUND: Eosinophils and mast cells are key effectors of allergy. When they accumulate in the esophagus, their myoactive, pro-inflammatory, and cytotoxic products potentially could cause achalasia-like motility abnormalities and neuronal degeneration. We hypothesized that there is an allergy-mediated form of achalasia. METHODS: LES muscle samples obtained during Heller myotomy from patients with achalasia or EGJ outflow obstruction (EGJOO) and from organ donor controls were immunostained for tryptase. Eosinophil and mast cell density, and mast cell degranulation were assessed. LES muscle was evaluated by qPCR for genes mediating smooth muscle Ca2+ handling and contraction. KEY RESULTS: There were 13 patients (7 men, median age 59; 10 achalasia, 3 EGJOO) and 7 controls (4 men, median age 42). Eosinophils were infrequent in LES muscle, but mast cells were plentiful. Patients and controls did not differ significantly in LES mast cell density. However, 12 of 13 patients exhibited profound LES mast cell degranulation involving perimysium and myenteric plexus nerves, while only mild degranulation was seen in 2 of 7 controls. Hierarchical clustering analysis of qPCR data revealed two "mototype" LES gene expression patterns, with all type II patients in one mototype, and type I and III patients in the other. CONCLUSIONS & INFERENCES: LES muscle of patients with achalasia or EGJOO exhibits striking mast cell degranulation, and patients with different achalasia manometric phenotypes exhibit different LES patterns of expression for genes mediating Ca2+ handling and muscle contraction. Although these findings are not definitive, they support our hypothesis that achalasia can be allergy-driven.

Colorectal Cancer Invasion and Atrophy of the Enteric Nervous System: Potential Feedback and Impact on Cancer Progression.

Colorectal cancer (CRC) invasion within the large intestine wall results in the replacement of normal tissue architecture by tumour mass. Cancer cells digest the extracellular matrix (ECM) by the release of proteolytic enzymes. The disintegration of matrix ground substance activates several deposited growth factors which stimulate cell proliferation. Stromal (mainly fibroblasts), immune and cancer cells dominate in this area and become involved in a network of multimodal interactions which significantly induce proliferation of colon cancer cells, inhibit their apoptosis and promote their spreading within the local tumour microenvironment. Cancer invasion destroys nerve fibres and neurons of the local enteric nervous system (ENS) and induces subsequent atrophy of the submucosal and myenteric plexuses in areas adjacent to the cancer boundary. Interestingly, the reduction of plexuses' size is accompanied by the increased number of galanin-immunoreactive neurons and increased galanin content in parts of the colon located close to the tumour. Galanin, a neuroprotective peptide, may inhibit the extrinsic pathway of apoptosis and in this way promote cancer cell survival. The possible role of acetylcholine and some ENS neuropeptides was also discussed. Invasion of cancer cells spreads along nerve fibres with the involvement of locally-released neutrophins which promote, via their specific receptors, cancer cell proliferation and pro-survival signalling pathways. Thus, during CRC development cancer cells and neurons of the ENS release many neurotransmitters/neuropeptides which affect key cellular signalling pathways promoting cancer cell proliferation and pro-survival phenotype. The multiple interactions between ENS neurons, cancer cells and other cell types present in the colon wall increase cancer cell invasiveness and have a negative impact on the course of CRC.

Unexpected High Prevalence of Lymphocytic Infiltrates in Myenteric Ganglions in Intestinal Inertia.

Intestinal inertia is a severe form of gut dysmotility that may require surgical resection. Loss of myenteric ganglion cells has been proposed as a possible etiology. Preclinical models have also suggested that virus infection-associated ganglionitis may be an alternative pathogenic factor. We determined to the extent intestinal inertia is associated with the lack of myenteric ganglion cells or ganglionitis using resection specimens from 27 intestinal inertia and 28 colon cancer patients. A hot spot approach with 5 HPFs was used for quantifying inflammatory cells. CD3, CD8, and CD20 immunohistochemistry was used to quantify T and B lymphocytes, along with subtyping the T-lymphocyte population by CD8. None of the intestinal inertia nor control cases showed the absence of myenteric ganglion cells. A total of 15 (55.6%) of the intestinal inertia cases showed inflammatory cell infiltration in the myenteric ganglion cells, compared with only 1 of 28 (3.6%) control cases (P<0.0001 by Fisher exact test). The inertia cases with inflammatory infiltrates were all associated predominantly with lymphocytes, including 3 cases (11.1%) with concurrent eosinophil infiltration, and 1 case (3.7%) with concurrent neutrophil infiltration. Furthermore, all 15 inertia cases with myenteric lymphocytic ganglionitis were associated with T lymphocytes (100%), including 1 case with a subset of concurrent B lymphocytes. The average CD3 count was 3.8 cells/HPF. CD8 immunohistochemical stain showed positive staining in 12 of the 15 cases (80%) with CD8-positive cells ranging from 1 to 8/HPF. In contrast, the only control case with lymphocytic ganglionitis showed mixed B and T lymphocytes and eosinophils. The high prevalence of T-lymphocyte infiltration in the myenteric ganglion in intestinal inertia cases suggests a possible pathogenic role.

A Population of Radio-Resistant Macrophages in the Deep Myenteric Plexus Contributes to Postoperative Ileus Via Toll-Like Receptor 3 Signaling.

Postoperative ileus (POI) is triggered by an innate immune response in the muscularis externa (ME) and is accompanied by bacterial translocation. Bacteria can trigger an innate immune response via toll-like receptor (TLR) activation, but the latter's contribution to POI has been disproved for several TLRs, including TLR2 and TLR4. Herein we investigated the role of double-stranded RNA detection via TLR3 and TIR-domain-containing adapter-inducing interferon-ß (TRIF) signaling pathway in POI. POI was induced by small bowel intestinal manipulation in wt, TRIF-/-, TLR3-/-, type I interferon receptor-/- and interferon-ß reporter mice, all on C57BL/6 background, and POI severity was quantified by gene expression analysis, gastrointestinal transit and leukocyte extravasation into the ME. TRIF/TLR3 deficiency reduced postoperative ME inflammation and prevented POI. With bone marrow transplantation, RNA-sequencing, flow cytometry and immunohistochemistry we revealed a distinct TLR3-expressing radio-resistant MHCIIhiCX3CR1- IBA-1+ resident macrophage population within the deep myenteric plexus. TLR3 deficiency in these cells, but not in MHCIIhiCX3CR1+ macrophages, reduced cytokine expression in POI. While this might not be an exclusive macrophage-privileged pathway, the TLR3/TRIF axis contributes to proinflammatory cytokine production in MHCIIhiCX3CR1- IBA-1+ macrophages during POI. Deficiency in TLR3/TRIF protects mice from POI. These data suggest that TLR3 antagonism may prevent POI in humans.

Neuroprotection and immunomodulation in the gut of parkinsonian mice with a plasmalogen precursor.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. It is typically associated with motor symptoms originating from the degeneration of nigrostriatal dopamine (DA) neurons. Early stages of PD have been associated with an alteration in DA production in intestinal DAergic neurons along with inflammation. Interestingly, decreased serum concentrations of ethanolamine plasmalogens (PlsEtn) have been reported in PD patients. Ethanolamine plasmalogens play a role in vesicular fusion and release during neurotransmission, and store neuroprotective polyunsaturated fatty acids, such as docosahexaenoic acid (DHA) and are strong anti-oxidants, highlighting areas of potential therapeutic interest. Docosahexaenoic acid is known to play important roles in both the central nervous and peripheral systems, in addition to acting as a precursor of several molecules that regulate the resolution of inflammation. The present study investigated the neuroprotective and anti-inflammatory properties of the DHA-containing PlsEtn precursor, PPI-1011, in the intestine of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Treatment with PPI-1011 prevented the MPTP-induced decrease in PlsEtn levels. In addition it prevented the loss of tyrosine hydroxylase (TH) expression and reduced the infiltration of macrophages in the myenteric plexus of MPTP-treated mice. The protective effects of PPI-1011 were observed regardless of whether it was administered pre- or post- MPTP treatment. These results suggest that PPI-1011 has neuroprotective and anti-inflammatory properties in the gut and indicate its potential utility as a treatment for both early and more advanced stages of PD.

Enteric neuron density correlates with clinical features of severe gut dysmotility.

Gastrointestinal (GI) symptoms can originate from severe dysmotility due to enteric neuropathies. Current methods used to demonstrate enteric neuropathies are based mainly on classic qualitative histopathological/immunohistochemical evaluation. This study was designed to identify an objective morphometric method for paraffin-embedded tissue samples to quantify the interganglionic distance between neighboring myenteric ganglia immunoreactive for neuron-specific enolase, as well as the number of myenteric and submucosal neuronal cell bodies/ganglion in jejunal specimens of patients with severe GI dysmotility. Jejunal full-thickness biopsies were collected from 32 patients (22 females; 16-77 yr) with well-characterized severe dysmotility and 8 controls (4 females; 47-73 yr). A symptom questionnaire was filled before surgery. Mann-Whitney U test, Kruskal-Wallis coupled with Dunn's posttest and nonparametric linear regression tests were used for analyzing morphometric data and clinical correlations, respectively. Compared with controls, patients with severe dysmotility exhibited a significant increase in myenteric interganglionic distance (P = 0.0005) along with a decrease in the number of myenteric (P < 0.00001) and submucosal (P < 0.0004) neurons. A 50% reduction in the number of submucosal and myenteric neurons correlated with an increased interganglionic distance and severity of dysmotility. Our study proposes a relatively simple tool that can be applied for quantitative evaluation of paraffin sections from patients with severe dysmotility. The finding of an increased interganglionic distance may aid diagnosis and limit the direct quantitative analysis of neurons per ganglion in patients with an interganglionic distance within the control range.NEW & NOTEWORTHY Enteric neuropathies are challenging conditions characterized by a severe impairment of gut physiology, including motility. An accurate, unambiguous assessment of enteric neurons provided by quantitative analysis of routine paraffin sections may help to define neuropathy-related gut dysmotility. We showed that patients with severe gut dysmotility exhibited an increased interganglionic distance associated with a decreased number of myenteric and submucosal neurons, which correlated with symptoms and clinical manifestations of deranged intestinal motility.

Enteric Murine Ganglionitis Induced by Autoimmune CD8 T Cells Mimics Human Gastrointestinal Dysmotility.

Inflammatory bowel diseases frequently cause gastrointestinal dysmotility, suggesting that they may also affect the enteric nervous system. So far, the precise mechanisms that lead to gastrointestinal dysmotility in inflammatory bowel diseases have not been elucidated. To determine the effect of CD8 T cells on gastrointestinal motility, transgenic mice expressing ovalbumin on enteric neurons were generated. In these mice, adoptive transfer of ovalbumin-specific OT-I CD8 T cells induced severe enteric ganglionitis. CD8 T cells homed to submucosal and myenteric plexus neurons, 60% of which were lost, clinically resulting in severely impaired gastrointestinal transition. Anti-interferon-γ treatment rescued neurons by preventing their up-regulation of major histocompatibility complex class I antigen, thus preserving gut motility. These preclinical murine data translated well into human gastrointestinal dysmotility. In a series of 30 colonic biopsy specimens from patients with gastrointestinal dysmotility, CD8 T cell-mediated ganglionitis was detected that was followed by severe loss of enteric neurons (74.8%). Together, the preclinical and clinical data support the concept that autoimmune CD8 T cells play an important pathogenetic role in gastrointestinal dysmotility and may destroy enteric neurons.

Sera with anti-enteric neuronal antibodies from patients with irritable bowel syndrome promote apoptosis in myenteric neurons of guinea pigs and human SH-Sy5Y cells.

BACKGROUND: Sera anti-enteric neuronal antibodies (AENA), neuronal inflammation, and degeneration in myenteric plexus in patients with irritable bowel syndrome (IBS) were reported. Effects of sera AENA in patients with IBS are unclear. METHODS: Patients with IBS met Rome III criteria were enrolled. Controls included healthy subjects (HS) and patients with slow transit functional constipation, inflammatory bowel disease, chronic intestinal pseudo-obstruction, and autoimmune diseases. Indirect immunofluorescence was used to detect AENA. Anti-enteric neuronal antibodies intensities were termed as "1" = weak fluorescence (mild positive); "2" = moderate fluorescence (moderate positive); "3" = very high fluorescence (intensive positive). Intensities of ≥1 were defined as positive and ≥2 were defined as obvious positive. Cultured myenteric neurons of small intestine from guinea pigs and human SH-Sy5Y cells were incubated with fetal bovine serum (FBS), HS sera, or IBS sera with or without AENA. Indirect immunofluorescence with anti-PGP9.5/DAPI/anti-active caspase-3 or TUNEL, Western blot, and flow cytometry were used to detect apoptosis. KEY RESULTS: Overall, 293 patients with IBS were enrolled (41.7 ± 11.5 years). AENA-positive and obvious positive rates in IBS were higher than HS (76.8% vs 33%; 43.7% vs 7%; all P < 0.001). Myenteric neurons incubated with AENA moderate or intensive positive IBS sera showed higher rates of anti-active caspase-3 and TUNEL-positive cells than HS or FBS (20% ± 7.3% and 35% ± 13.3% vs 4.3% ± 1.5% and 0.9% ± 0.4%, respectively; 6.2% ± 2.0% and 10.2% ± 4.6% vs 1.3% ± 1.9% and 0.5%±0.5%, respectively; all P < 0.05). Human SH-Sy5Y cells incubated with AENA moderate or intensive positive IBS sera showed increased cleaved caspase-3 and Bax expression and decreased Bcl-2 expression. Flow cytometry showed apoptosis rates of these two groups were higher than that of AENA mild positive, negative, HS, and FBS (7.6%±0.8% and 10.7%±1.3% vs 5.0%±0.8%, 3.8%±0.3%, 3.4%±0.2% and, 2.8%±0.2%, P < 0.05). CONCLUSIONS AND INFERENCES: The AENA obvious positive rate in patients with IBS was higher than HS, and sera with higher levels of AENA promoted neuronal apoptosis. AENA-mediated neuropathy might exist in a subset of patients with IBS.

Sustained pain hypersensitivity in the stressed colon: Role of mast cell-derived nerve growth factor-mediated enteric synaptic plasticity.

BACKGROUND: Sustained pain hypersensitivity is the hallmark of stressed colon which could be partially explained by central sensitization with synaptic plasticity, the key mechanism of memory. We previously identified that synaptic plasticity of enteric nerve system (ENS) contributed to peripheral pain maintaining in the gut. However, the mechanisms of enteric "memory" formation remain elusive. METHODS: In this study, rats were exposed to water avoidance stress (WAS) or sham stress (SS), with cromolyn sodium or physiological saline injected intraperitoneally 30 minutes before stress every day. The abdominal withdrawal reflex scores, mesenteric afferent nerve activity, enteric neural c-fos expression, and enteric synaptic plasticity were assessed, and mast cell infiltration and degranulation. Furthermore, colonic mucosal mediators-induced enteric synaptic plasticity and the role of mast cell-derived nerve growth factor (NGF), tryptase, and histamine were investigated via ex vivo longitudinal muscle-myenteric plexus (LMMP) organotypic culture. KEY RESULTS: It is shown that mast cell stabilizing inhibited WAS-induced visceral hypersensitivity through enhancing visceral pain threshold, decreasing spontaneous and distention-induced mesenteric afferent firing, and downregulating enteric neural activation (c-fos). Importantly, WAS led to evident enteric synaptic plasticity, but decreased by cromolyn. Water avoidance stress-derived mucosal supernatants markedly enhanced the c-fos expression and enteric synaptic plasticity in LMMP tissues, which could be eliminated by mast cell inhibition or NGF neutralization, but not tryptase or histamine blocking. CONCLUSIONS & INFERENCES: In conclusion, mast cells/NGF pathway may be the key regulator of synaptic plasticity of ENS and participate in the formation of chronic stress-induced sustained visceral hypersensitivity.

Acrylamide-induced alterations in the cocaine- and amphetamine-regulated peptide transcript (CART)-like immunoreactivity within the enteric nervous system of the porcine small intestines.

The main goal of the present study was to determine the influence of low and high doses of acrylamide on CART-like immunoreactivity within the ENS of the porcine small intestines. Investigation was performed on 15 sexually immature female pigs, divided into three groups: control group, where empty gelatin capsules were administered, and two experimental groups, where capsules containing low or high doses of acrylamide were given. After 28days of acrylamide exposure all animals were euthanized with a gradual overdose of anaesthetics. Immediately after euthanasia fragments of the duodenum, jejunum and ileum were collected and fixed. Then, 14µm-thick cryostat sections were processed for routine double-labelling immunofluorescence using primary antisera directed towards a pan neuronal marker - protein gene-product 9.5 (PGP 9.5) and cocaine- and amphetamine-regulated peptide transcript (CART). During the present investigation, CART-LI cell bodies were detected in all types of enteric plexuses of duodenum, jejunum and ileum. Acrylamide intoxication resulted in a significant increase in expression of CART in the intramural neurons of the porcine small intestines, especially in myenteric plexuses. It may suggest participation of CART in neuronal protection and recovery processes within the gut. Moreover, results of the present study suggest that even low doses of acrylamide cause a significant response of ENS neurons.

Myenteric plexuses atrophy in the vicinity of colorectal cancer tissue is not caused by apoptosis or necrosis.

INTRODUCTION: The previously performed studies showed that the presence of colorectal cancer (CRC) tumor is associated with the atrophy of myenteric plexuses in the vicinity of cancer invasion; however, the possible mechanisms of this phenomenon are not known. The aim of the present study was to determine whether the atrophic changes of the enteric nervous system (ENS) within an intestine wall of the CRC patients were caused by apoptosis or necrosis and whether they were associated with changes in the number of galanin-immunore-active (GAL-Ir) neurons. MATERIAL AND METHODS: Samples of the large intestine wall located close to the CRC invasion and control, distally-located part of the colon, were collected from 9 CRC patients. The size of ENS plexuses and the number of neurons were compared. Triple immunofluorescent staining was used to visualize the co-expression of caspase 3 (CASP3) or caspase 8 (CASP8) with GAL and protein gene-product 9.5 (PGP 9.5, panneuronal marker) in the submucosal and myenteric ENS plexuses. The cells expressing myeloperoxidase (MPO, marker of neutrophils) and CD68 (marker of macrophages) were detected by immunohistochemistry around/in myenteric plexuses (MPs) and in the muscularis externa of the colon wall in the vicinity of tumor invasion. RESULTS: Myenteric plexuses in the vicinity of the CRC tissue were significantly smaller and had lower number of neurons per plexus than distantly located plexuses. The number of CASP8- and CASP3-Ir neurons in the ENS plexuses was similar in the colon wall both close to and distally from tumor invasion. The number of CASP8-Ir neurons within MPs located close to CRC invasion was higher than of CASP3-Ir neurons. The percentage of neurons co-expressing CASP8 and GAL in myenteric plexuses close and distantly from tumor was three-fold lower than of those co-expressing CASP3 and GAL. The mean number of neutrophils and macrophages inside and around myenteric plexuses located close to tumor invasion was higher or similar, respectively, as compared with adjacent muscularis externa. CONCLUSIONS: The atrophy of myenteric plexuses in the vicinity of CRC invasion is not caused by apoptosis or necrosis. The differences in the proportions of neurons expressing galanin and the studied caspases suggest as yet unknown role of this neuropeptide in the mechanisms of neuron's atrophy in MPs located close to CRC tumor.

Achalasia--An Autoimmune Inflammatory Disease: A Cross-Sectional Study.

Idiopathic achalasia is a disease of unknown etiology. The loss of myenteric plexus associated with inflammatory infiltrates and autoantibodies support the hypothesis of an autoimmune mechanism. Thirty-two patients diagnosed by high-resolution manometry with achalasia were included. Twenty-six specimens from lower esophageal sphincter muscle were compared with 5 esophagectomy biopsies (control). Immunohistochemical (biopsies) and flow cytometry (peripheral blood) analyses were performed. Circulating anti-myenteric autoantibodies were evaluated by indirect immunofluorescence. Herpes simplex virus-1 (HSV-1) infection was determined by in situ hybridization, RT-PCR, and immunohistochemistry. Histopathological analysis showed capillaritis (51%), plexitis (23%), nerve hypertrophy (16%), venulitis (7%), and fibrosis (3%). Achalasia tissue exhibited an increase in the expression of proteins involved in extracellular matrix turnover, apoptosis, proinflammatory and profibrogenic cytokines, and Tregs and Bregs versus controls (P < 0.001). Circulating Th22/Th17/Th2/Th1 percentage showed a significant increase versus healthy donors (P < 0.01). Type III achalasia patients exhibited the highest inflammatory response versus types I and II. Prevalence of both anti-myenteric antibodies and HSV-1 infection in achalasia patients was 100% versus 0% in controls. Our results suggest that achalasia is a disease with an important local and systemic inflammatory autoimmune component, associated with the presence of specific anti-myenteric autoantibodies, as well as HSV-1 infection.

GPER1-mediated immunomodulation and neuroprotection in the myenteric plexus of a mouse model of Parkinson's disease.

Lewy pathology affects the gastrointestinal tract in Parkinson's disease (PD) and recent reports suggest a link between the disorder and gut inflammation. In this study, we investigated enteric neuroprotection and macrophage immunomodulation by 17ß-estradiol (E2) and the G protein-coupled estrogen receptor 1 (GPER1) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse PD model. We found that both E2 and the GPER1 agonist G1 are protective against the loss of dopamine myenteric neurons and inhibited enteric macrophage infiltration in MPTP-treated mice. Coadministration of GPER1 antagonist G15, while completely blocking the neuroprotective and anti-inflammatory effects of G1 also partially prevented those of E2. Interestingly, we found that E2 and G1 treatments could directly alter MPTP-mediated immune responses independently from neurodegenerative processes. Analyses of monocyte/macrophage NF-κB and iNOS activation and FACs immunophenotype indicated that 1-methyl-4-phenylpyridinium (MPP(+)) treatment induces a strong immune response in monocytes, comparable to that of canonical challenge by lipopolysaccharide. In these cells, G1 and E2 treatment are equally potent in promoting a shift toward an anti-inflammatory "M2" immunophenotype reducing MPP(+)-induced NF-κB and iNOS activation. Moreover, G15 also antagonized the immunomodulatory effects of G1 in MPP(+)-treated macrophages. Together these data provide the first evidence for the role of GPER1 in enteric immunomodulation and neuroprotection. Considering increasing recognition for myenteric pathology as an early biomarker for PD, these findings provide a valuable contribution for better understanding and targeting of future therapeutic strategies.

The Effect of Ischemia and Reperfusion on Enteric Glial Cells and Contractile Activity in the Ileum.

BACKGROUND: We investigated the effects of ischemia followed by different periods of reperfusion (I/R) on immunoreactive S100ß-positive glial and Hu-immunoreactive neurons co-expressing the P2X2 receptor in the myenteric plexus of the rat ileum. METHODS: The ileal artery was occluded for 35 min with an atraumatic vascular clamp. The animals were killed 24 h, 72 h, and 1 week after ischemia. Sham animals were not submitted to ileal artery occlusion. The relative density, size, and co-localization of P2X2 receptor-expressing cells in relation to S100ß-immunoreactive glial and Hu-immunoreactive neuronal cells were evaluated. Additionally, we analyzed the effects of I/R on gastrointestinal transit and ileum contractile activity. RESULTS: The cellular density of P2X2 receptor and neuronal Hu immunoreactivity/cm(2) decreased after I/R, whereas glial S100ß immunoreactivity/cm(2) increased. No significant differences between sham and I/R groups were observed regarding the perikarya area of Hu-positive neurons. The area of S100ß-immunoreactive glial cells increased by 24.1 % 1 week after I/R compared with the 24 h group. Methylene blue progression along the small intestine decreased (P < 0.05) from 24.5 ± 2.3 % in the sham group to 17.2 ± 2.0 % 1 week post-ischemia. We noted a significant (P < 0.05) decrease in the maximal contraction amplitude triggered by electrical field stimulation in the presence of ATP in preparations submitted to 24 h of I/R. CONCLUSIONS: Changes in the P2X2 receptor density parallel myenteric neuronal loss following I/R of the rat ileum. This, together with the increase in the activated (oversized) glial cells, may contribute to decreased GI motility after I/R.

A spontaneous animal model of intestinal dysmotility evoked by inflammatory nitrergic dysfunction.

BACKGROUND AND AIMS: Recent reports indicate the presence of low grade inflammation in functional gastrointestinal disorders (FGID), in these cases often called "post-inflammatory" FGIDs. However, suitable animal models to study these disorders are not available. The Biobreeding (BB) rat consists of a diabetes-resistant (BBDR) and a diabetes-prone (BBDP) strain. In the diabetes-prone strain, 40-60% of the animals develop diabetes and concomitant nitrergic dysfunction. Our aim was to investigate the occurrence of intestinal inflammation, nitrergic dysfunction and intestinal dysmotility in non-diabetic animals. METHODS: Jejunal inflammation (MPO assay, Hematoxylin&Eosin staining and inducible nitric oxide synthase (iNOS) mRNA expression), in vitro jejunal motility (video analysis) and myenteric neuronal numbers (immunohistochemistry) were assessed in control, normoglycaemic BBDP and diabetic BBDP rats. To study the impact of iNOS inhibition on these parameters, normoglycaemic BBDP rats were treated with aminoguanidine. RESULTS: Compared to control, significant polymorphonuclear (PMN) cell infiltration, enhanced MPO activity, increased iNOS mRNA expression and a decreased ratio of nNOS to Hu-C/D positive neurons were observed in both normoglycaemic and diabetic BBDP rats. Aminoguanidine treatment decreased PMN infiltration, iNOS mRNA expression and MPO activity. Moreover, it restored the ratio of nNOS to Hu-C/D positive nerves in the myenteric plexus and decreased the abnormal jejunal elongation and dilation observed in normoglycaemic BBDP rats. CONCLUSIONS: Aminoguanidine treatment counteracts the inflammation-induced nitrergic dysfunction and prevents dysmotility, both of which are independent of hyperglycaemia in BB rats. Nitrergic dysfunction may contribute to the pathophysiology of "low-grade inflammatory" FGIDs. Normoglycaemic BBDP rats may be considered a suitable animal model to study the pathogenesis of FGIDs.

Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.

BACKGROUND & AIMS: Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. METHODS: We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2(-/-), TLR-4(-/-), TLR-2/4(-/-), MyD88(-/-), MyD88/TLR adaptor molecule 1(-/-), interleukin-1 receptor [IL-1R1](-/-), and interleukin (IL)-18(-/-) mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. RESULTS: Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1(-/-) mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1ß before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1ß were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1ß stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. CONCLUSIONS: IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with the IL-1 signaling pathway are likely to be effective in the treatment of POI.

Endogenous regulation of visceral pain via production of opioids by colitogenic CD4(+) T cells in mice.

BACKGROUND & AIMS: A dysregulated response of CD4(+) T cells against the microbiota contributes to the development of inflammatory bowel disease. Effector CD4(+) T cells, generated in response to microbe-derived antigens, can reduce somatic inflammatory pain through the local release of opioids. We investigated whether colitogenic CD4(+) T cells that accumulate in the inflamed colon also produce opioids and are able to counteract inflammation-induced visceral pain in mice. METHODS: Colitis was induced via transfer of naive CD4(+)CD45RB(high) T cells to immune-deficient mice or by administration of dextran sulfate sodium. Mice without colitis were used as controls. Samples of colon tissue were collected, and production of opioids by immune cells from inflamed intestine was assessed by quantitative polymerase chain reaction and cytofluorometry analyses. The role of intestinal opioid tone in inflammation-induced visceral hypersensitivity was assessed by colorectal distention. RESULTS: In mice with T cell- or dextran sulfate sodium-induced colitis, colitogenic CD4(+) T cells (T-helper 1 and Th17 cells) accumulated in the inflamed intestine and expressed a high level of endogenous opioids. In contrast, macrophages and epithelial cells did not express opioids; opioid synthesis in the myenteric plexus was not altered on induction of inflammation. In mice with colitis, the local release of opioids by colitogenic CD4(+) T cells led to significant reduction of inflammation-associated visceral hypersensitivity. CONCLUSIONS: In mice, colitogenic Th1 and Th17 cells promote intestinal inflammation and colonic tissue damage but have simultaneous opioid-mediated analgesic activity, thereby reducing abdominal pain.

[Participation of peripheral sensory structures of autonomic nervous system in the mechanisms of neuroimmune interactions].

On the basis of the electrophysiological research carried out with immunohystochemical methods on rats, it is found, that introduction of products of mast cell degranulation into blood as well as endogenous release of mast cell mediators with either the compound 48/80 or introduction of egg albumin to presensitized rats, enhances activity of mesenteric afferent nerve fibers. The obtained data provide evidence that intestinal afferents contribute an early signal to the brain regarding potential pathogens. The question of whether or not the intrinsic enteric neurons are involved in these processes, however, has not been carefully studied. So we investigated this problem and found that the enteric neurons express receptor of innate immunity TLR4 and nociceptive vanilloid receptor TRPV1, by means ofimmunohistochemical method. The analysis of neurons distribution revealed that vanilloid receptors are expressed by neurons localized mainly in myenteric plexus whereas TLR4-immunoreactive neurons generally are present in submucous plexus. It is also established coexpression of both receptors in the single intrinsic enteric neuron. In conclusion, our findings indicate that sensory terminals of external afferent fibers as well as intrinsic neurons of intestine can modulate reactions of the organism to endotoxins and thus may be involved in reception of inflammatory and immune responses.